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Subnetwork organization of the superior colliculus and visual system in the mouse brain

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Subnetwork organization of the superior colliculus and visual system in the mouse brain

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SUBNETWORK ORGANIZATION OF THE
SUPERIOR COLLICULUS AND VISUAL SYSTEM
IN THE MOUSE BRAIN

By

Nora Lissett Benavidez

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
NEUROSCIENCE
August 2021

Copyright 2021 Nora Lissett Benavidez
ii
ACKNOWLEDGEMENTS

I have been gifted a beautiful network of family, friends, and colleagues that have all
contributed to the fulfillment of my dissertation odyssey. Whether it was academic,
professional, emotional, mental, physical, or cosmic, the support and encouragement was
influential and so personally meaningful to me. I revere each and all of my connections
in their singular way. I thank them all, and here I deeply thank:

My graduate advisor and mentor: Dr. Hong-Wei Dong.

My guidance committee members: Drs. Alan Watts (Chair), Larry Swanson, Judith
Hirsch, Hong-Wei Dong (PI), Michael Bonaguidi, and Li Zhang.

My mentor: Dr. Michael Bienkowski.

My previous research advisors and mentors: Drs. Gloria Choi, Richard Axel, Linda
Wilbrecht, Moses Lee, Badr Albanna, and David Presti.

My Neuroscience Graduate Program administrators: Deanna Solorzano, Dawn Burke,
Judith Hirsch, Pat Levitt, and everyone in NGP.

My lab mates at the UCLA B.R.A.I.N. Center and at USC: Dr. Houri Hintiryan, Brian
Zingg, Muye Zhu, Marina Fayzullina, Luis Garcia, Ian Bowman, Kaelan Cotter, Seita
Yamashita, Lei Gao, Lin Gou, Darrick Lo, Hyung-Sun Mun, Sarah Ustrell, Tyler Boesen,
iii
Amanda Tugangui, Chunru Cao, Bin Zhang, Nicholas Foster, Neda Khanjani, Laura
Korobkova, Sarvia Aquino, David Lennon, Sana Azam, Josue Diaz, and all colleagues.

My NGP and life friendships: Brian Zingg, Monica Y. Song, Rosa Vasquez, Jannifer Lee,
Ellen DeGennaro, Clarissa Liu, Marlene Becerra, Alicia Quihuis, Kasey Rose, Hector
Montoya, , Selma Sehovic, Glenda Ramirez, Miriam Winthrop, Magali Barba, Sheila
Saunders, Anita van der Zwan, Hannah DeMara, Zachary Murdock, So Young Choi,
Phillip Maire, Eric Hendricks, Eddie Catich, Aida Bareghamyan, and many more.

My family: My mother Rosa Emilia, my father David, my sister Emily Roxann, my sister
Marlyn Janet, my brother Sonny Lee, my brother David and sister-in-law Cindy, my sister
Sophia Raquel, my brother Ezekiel Orlando and sister-in law Maria, my sister Olydia, all
14 of my beloved nieces and nephews, and all my esteemed relatives.

My partner: Adam J. Lundquist.

My influences: Carl Jung, William Blake, Santiago Ramón y Cajal.

“And I thank, in the end, the LSD… It was certainly destined in my fate that I was
supposed to discover this substance… I thank you that you appeared here.”
– Albert Hofmann, Ph.D.
1

1
From speech for 100
th
birthday symposium in Basel, Switzerland. January 2006.
iv
TABLE OF CONTENTS

ACKNOWLEDGEMENTS ___________________________________________________________ ii
LIST OF FIGURES _________________________________________________________________ vi
LIST OF TABLES _________________________________________________________________ viii
LIST OF ABBREVIATIONS ________________________________________________________ ix
ABSTRACT ______________________________________________________________________ xiii

GENERAL INTRODUCTION ____________________________________________ 1
OVERVIEW OF STRUCTURAL FRAMEWORK __________________________________________ 1
CONTEXT OF HYPOTHETICAL FRAMEWORK _________________________________________ 2
1.2.1 SC AND THE VISUAL SYSTEM ______________________________________________________ 2
1.2.2 SENSORIMOTOR AND HIGHER-ORDER ALIGNMENT IN THE SC ___________________________ 3
1.2.3 CORTICO-CORTICAL SUBNETWORKS. _______________________________________________ 5
INTEGRATING STRUCTURAL AND HYPOTHETICAL FRAMEWORKS _______________________ 7

SUBNETWORK ORGANIZATION OF THE INPUTS AND OUTPUTS OF
THE MOUSE SUPERIOR COLLICULUS ________________________________ 11
ABSTRACT ____________________________________________________________________ 11
INTRODUCTION _______________________________________________________________ 12
METHODS (FOR RESULTS PARTS I & II) ____________________________________________ 14
2.3.1 MOUSE CONNECTOME PROJECT METHODOLOGY _____________________________________ 14
2.3.2 2D IMAGING AND INFORMATICS PROCESSING _______________________________________ 22
METHODS (FOR RESULTS PART III) _______________________________________________ 31
2.4.1 3D TISSUE PROCESSING AND IMAGING PROTOCOL ___________________________________ 31
2.4.2 3D RECONSTRUCTIONS, VISUALIZATIONS, AND ANALYSIS OF NEURONAL MORPHOLOGY ___ 32
2.4.3 STATISTICAL ANALYSES ON GROUPS OF RECONSTRUCTIONS ___________________________ 33
RESULTS (PART I) : THE CORTICO-TECTAL PROJECTOME _____________________________ 35
2.5.1 GENERAL STRATEGY FOR DELINEATING THE CORTICO-TECTAL PROJECTOME _______________ 35
2.5.2 VISUAL, SOMATOSENSORY, AND MOTOR CORTEX PROJECTION MAPS IN SC ________________ 41
2.5.3 HIGHER-ORDER ASSOCIATION AND PREFRONTAL CORTICAL PROJECTIONS TO SC ___________ 46
2.5.4 CORTICO-TECTAL PROJECTIONS ARE ORGANIZED AS MODULAR COMMUNITIES _____________ 47
RESULTS (PART II) : BRAIN-WIDE SC CONNECTIVITY ________________________________ 51
2.6.1 BRAIN-WIDE CONNECTIVITY CORROBORATES CORTICO-TECTAL NETWORKS _______________ 51
2.6.2 ZONE-SPECIFIC CONNECTIONS WITH SENSORIMOTOR NUCLEI IN THE LOWER BRAINSTEM _____ 51
2.6.3 HYPOTHALAMIC INTERACTIONS WITH SC __________________________________________ 58
2.6.4 CONNECTIVITY WITHIN CORTICO-TECTO-THALAMIC SUBNETWORKS _____________________ 59
2.6.5 INTERACTIONS BETWEEN CORTICO-TECTAL AND CORTICO-BASAL GANGLIA NETWORKS _____ 62
RESULTS (PART III) : THALAMUS-PROJECTING SC CELLS _____________________________ 67
2.7.1 MORPHOLOGICAL CHARACTERISTICS OF THALAMUS-PROJECTING SC CELLS _______________ 67
DISCUSSION __________________________________________________________________ 73
2.8.1 SUBNETWORKS IMPLICATED IN APPROACH / APPETITIVE BEHAVIORS _____________________ 77
2.8.2 SUBNETWORKS IMPLICATED IN DEFENSIVE / AGGRESSIVE BEHAVIORS ____________________ 77
2.8.3 SUBNETWORKS IMPLICATED IN NAVIGATION AND GOAL-ORIENTED BEHAVIORS ____________ 78
CONCLUSION __________________________________________________________________ 80

v
EXTRASTRIATE CONNECTIVITY OF THE MOUSE LATERAL
DORSAL GENICULATE THALAMUS _________________________________ 81
ABSTRACT ____________________________________________________________________ 81
INTRODUCTION _______________________________________________________________ 82
METHODS ____________________________________________________________________ 87
RESULTS ______________________________________________________________________ 93
3.4.1 VERIFICATION OF LGD AND VIS CORTEX BOUNDARIES BY CYTOARCHITECTURE,
HISTOCHEMICAL/MOLECULAR STAINING, AND CONNECTIVITY PATTERNS ________________ 93
3.4.2 DISTRIBUTION OF TRACER LABELING WITHIN LGD AFTER VISP VERSUS VISAM/PM
COINJECTIONS ________________________________________________________________ 100
3.4.3 DISTRIBUTION OF LABELING WITHIN VIS CORTICAL AREAS AFTER LGD COINJECTIONS _____ 105
3.4.4 DISTRIBUTION OF TRACER LABELING WITHIN VISAL, VISL, AND VISPL AFTER LGD
COINJECTIONS ________________________________________________________________ 110
DISCUSSION _________________________________________________________________ 115
3.5.1 TECHNICAL CONSIDERATIONS ___________________________________________________ 116
3.5.2 LGD CONNECTIONS WITH THE VISP ______________________________________________ 117
3.5.3 EXTRASTRIATE LGD CONNECTIONS WITH VISAM AND VISPM _________________________ 118
3.5.4 EXTRASTRIATE CONNECTIONS OF LGD WITH VISAL, VISL, AND VISPL __________________ 119
3.5.5 RELATIONSHIP TO FUNCTIONAL MAPPING OF THE VISUAL CORTEX ______________________ 120
3.5.6 FUNCTIONAL CONSIDERATIONS RELATED TO BLINDSIGHT _____________________________ 120
CONCLUSION _________________________________________________________________ 121

SUMMARIZING DISCUSSION ________________________________________ 123
SUMMARY OF FINDINGS _______________________________________________________ 123
FUTURE DIRECTIONS __________________________________________________________ 125
TRANSLATIONAL APPLICATIONS ________________________________________________ 129
4.3.1 ADHD AND ASD TREATMENTS _________________________________________________ 130
4.3.2 PSYCHOTHERAPY RESEARCH ____________________________________________________ 131
CONCLUSION _________________________________________________________________ 134

PARTING PERSPECTIVE _________________________________________________________ 135
REFERENCES ____________________________________________________________________ 140
APPENDIX A : SC PROJECTION MAP _____________________________________________ 153
APPENDIX B : MANUSCRIPT INFORMATION _____________________________________ 155
vi
LIST OF FIGURES

FIGURE 1.1 | INTEGRATING CONNECTIVITY MAPS ________________________________________________ 10

FIGURE 2.1 | FLATTENED LAYOUT OF MOUSE CORTEX WITH INJECTION SITES. __________________________ 38
FIGURE 2.2 | EXPERIMENTAL WORKFLOW. _______________________________________________________ 39
FIGURE 2.3 |PROBABILITY DISTRIBUTION PLOTS. _________________________________________________ 40
FIGURE 2.4 | VISUAL, AUDITORY, AND SOMATIC SENSORIMOTOR MAP OF CORTICAL PROJECTIONS TO
SC ZONES. __________________________________________________________________ 42
FIGURE 2.5 | SENSORY RELATED CONNECTIVITY WITH SC. _________________________________________ 45
FIGURE 2.6 | DISTRIBUTION OF HIGHER-ORDER CORTICAL INPUTS ACROSS SC ZONES. __________________ 48
FIGURE 2.7 | HIGHER-ORDER CORTICO-TECTAL ARRAYS. ___________________________________________ 50
FIGURE 2.8 | BRAIN-WIDE CONNECTIVITY OF SC INPUTS AND OUTPUTS. ______________________________ 54
FIGURE 2.9 | INJECTION SITES IN SC. ___________________________________________________________ 55
FIGURE 2.10 | DOWNSTREAM PROJECTIONS FROM DISTINCT SC ZONES TO BRAINSTEM REGIONS. _________ 56
FIGURE 2.11 | TOPOGRAPHIC ORGANIZATION OF BRAIN-WIDE INPUTS AND OUTPUTS OF SC ZONES. _______ 61
FIGURE 2.12 | CORTICO-STRIATAL AND CORTICO-TECTAL PROJECTIONS HAVE CONSERVED
TOPOGRAPHIC ORGANIZATION. _________________________________________________ 64
FIGURE 2.13 | UPPER-LIMB AND OROFACIAL SUBNETWORKS IN SC.CL AND SC.L. _______________________ 66
FIGURE 2.14 | CHARACTERIZATION OF NEURONAL CELL TYPES IN THE SC BASED ON THEIR
ANATOMICAL LOCATIONS, PROJECTION TARGETS AND NEURONAL MORPHOLOGY. ______ 70
FIGURE 2.15 | EXAMPLES OF LAYER-SPECIFIC SC NEURON RECONSTRUCTIONS. ________________________ 71
FIGURE 2.16 | OVERLAPPED RECONSTRUCTIONS OF SC NEURONS AND CORTICO-TECTAL PROJECTIONS. ___ 72
FIGURE 2.17 | SUBNETWORK ORGANIZATION OF SC ZONES. ________________________________________ 76

FIGURE 3.1 | BIDIRECTIONAL CIRCUIT TRACING STRATEGY. ________________________________________ 86
FIGURE 3.2 | MAPPED INJECTION SITE SPREAD THROUGHOUT THE VISUAL CORTEX AND THALAMUS. ______ 92
FIGURE 3.3 | VALIDATION OF LGD/IGL BOUNDARY: CYTOARCHITECTURE, HISTOCHEMICAL MARKERS,
CONNECTIVITY. ______________________________________________________________ 95
FIGURE 3.4 | VALIDATION OF THE VISAM AND VISPM BOUNDARIES BY RORB GENE EXPRESSION. ________ 97
FIGURE 3.5 | VALIDATION OF THE VISAM AND VISPM BOUNDARIES BY VISP AND THALAMIC
CONNECTIVITY. ______________________________________________________________ 99
FIGURE 3.6 | VISP COINJECTIONS SHOW SPECIFIC TOPOGRAPHIC BIDIRECTIONAL CONNECTIVITY
WITHIN LGD. _______________________________________________________________ 102
FIGURE 3.7 | VISAM AND VISPM COINJECTIONS PRODUCE LABELING CLUSTERS ALONG
VENTROMEDIAL LGD BORDER. ________________________________________________ 103
FIGURE 3.8 | ORGANIZATION OF VISP VS. VISAM/PM THALAMOCORTICAL CONNECTIVITY. ____________ 104
vii
FIGURE 3.9 | ANTEROGRADE AND RETROGRADE LABELING PATTERN WITHIN VISP AFTER
COINJECTIONS INTO THE LGD THAT AVOID THE VENTRAL STRIP REGION. _____________ 107
FIGURE 3.10 | ANTEROGRADE AND RETROGRADE LABELING PATTERN WITHIN VISP, VISAM, VISPM
AFTER COINJECTIONS INTO THE LGD THAT ARE LOCATED WITHIN THE VENTRAL
STRIP REGION. ______________________________________________________________ 108
FIGURE 3.11 | ANTEROGRADE AND RETROGRADE LABELING PATTERNS LOCATED WITHIN VISAM AND
VISPM AS DEFINED BY NISSL CYTOARCHITECTURE. _______________________________ 109
FIGURE 3.12 | LGD CONNECTIONS WITH LATERAL EXTRASTRIATE AREAS AND SUBCORTICAL RELAYS
WITH MEDIAL EXTRASTRIATE AREAS. ___________________________________________ 112
FIGURE 3.13 | DISTRIBUTION OF TRACER LABELING IN LGD AFTER COINJECTION INTO THE LATERAL
EXTRASTRIATE AREAS. ________________________________________________________ 113
FIGURE 3.14 |TOPOGRAPHIC ORGANIZATION OF MOUSE LGD CORTICAL CONNECTIVITY. _____________ 114

FIGURE 4.1 | DOWNSTREAM PROJECTIONS OF SNR AND SC TO THE PPN. ____________________________ 127
FIGURE 4.2 | FLATMAPS AND FRACTALS. _______________________________________________________ 136
FIGURE 4.3 | SELF-ASSOCIATION LOOPS. _______________________________________________________ 138

viii
LIST OF TABLES

TABLE 2.1 | INJECTION SITES. _________________________________________________________________ 15
TABLE 2.2 | ANGULAR RANGES FOR EACH SC ZONE. ______________________________________________ 26
TABLE 2.3 | PROPORTION OF LABELING VALUES: SENSORY CORTICES. ________________________________ 27
TABLE 2.4 | PROPORTION OF LABELING VALUES: ASSOCIATION AREAS. _______________________________ 27
TABLE 2.5 | MORPHOMETRIC P-VALUES FOR SC NEURON RECONSTRUCTIONS. ________________________ 34
TABLE 3.1 | LITERATURE ON THALAMOCORTICAL CONNECTIVITY. ___________________________________ 84
TABLE 3.2 | INJECTION SITES. _________________________________________________________________ 89

ix
LIST OF ABBREVIATIONS

TRACERS
AAV, Adeno-associated virus
BDA, Biotinylated dextran amine
CTB, Cholera toxin subunit B
FG, Fluorogold
PHAL, Phaseolus vulgaris leucoagglutinin

BRAIN REGIONS
ACAd, Anterior cingulate area cortex, dorsal part
ACAv, Anterior cingulate area cortex, ventral part
AD, Anterodorsal nucleus thalamus
AId, Agranular insular area, dorsal part
AIv, Agranular insular area, ventral part
AIp, Agranular insular area, posterior part
APN, Anterior pretectal nucleus
AUDd, Auditory cortex, dorsal part
AUDp, Primary auditory cortex
AUDv, Auditory cortex, ventral part
AV, Anteroventral nucleus thalamus
BLA, Basolateral amygdalar nucleus
BLAa, Basolateral amygdalar nucleus, anterior part
BLAp, Basolateral amygdalar nucleus, posterior part
BMA, Basomedial amygdalar nucleus
BMAa, Basomedial amygdalar nucleus, anterior part
BMAp, Basomedial amygdalar nucleus, posterior part
CA1, Field CA1, hippocampal formation
CA3, Field CA3, hippocampal formation
CLA, Claustrum
CM, Centromedial nucleus thalamus
CP, Caudate putamen
CPc.d, Caudate putamen, caudal, dorsal domain
CPi.dl, Caudate putamen, intermediate, dorsolateral domain
CPi.vl, Caudate putamen, intermediate, ventrolateral domain
CPi.vm, Caudate putamen, intermediate, ventromedial domain
CPr, Caudate putamen, rostral
CUN, Cuneate nucleus
DN, Dentate nucleus
DG, Dentate gyrus
DTN, Dorsal tegmental nucleus
ENT, Entorhinal cortex
GRN, Gigantocellular reticular nucleus,
GU, Gustatory cortex
Hb, Habenula nucleus thalamus
x
IAM, Interanteromedial nucleus thalamus
ICe, Inferior colliculus, external part
ILA, Infralimbic area cortex
IO, Inferior olivary nucleus
IRN, Intermediate reticular nucleus
KF, Koelliker-Fuse subnucleus
LA, Lateral amygdalar area
LD, Laterodorsal nucleus thalamus
LG, Lateral geniculate nucleus thalamus
LGd, Lateral geniculate nucleus thalamus, dorsal part
LGv, Lateral geniculate nucleus thalamus, ventral part
LP, Lateroposterior nucleus thalamus
LM, Lateral mammillary nucleus
MARN, Magnocellular reticular nucleus
MD, Mediodorsal nucleus thalamus
MDRNv, Medullary reticular nucleus, ventral part
MOp, Primary motor cortex
MOp-oro, Primary motor cortex, orofacial part
MOp-ul, Primary motor cortex, upper-lum
MOs, Secondary motor cortex
MOs-fef, Secondary motor cortex, frontal eye field
MRN, Midbrain reticular nucleus
MPT, Midbrain pretectal region
ORBl, Orbitofrontal area cortex, lateral part
ORBm, Orbitofrontal area cortex, medial part
ORBvl, Orbitofrontal area cortex, ventrolateral part
PAG, Periaquaductal gray nucleus
PAG.dl, Periaquaductal gray nucleus, dorsolateral part
PAG.vl, Periaquaductal gray nucleus, ventrolateral part
PAR, Parasubiculum
PARN, Parvicellular reticular nucleus
PBG, Parabigeminal nucleus
PBl, Parabrachial nucleus, lateral part
PBmm, Parabrachial nucleus, mediomedial part
PCN, Pericentral nucleus thalamus
PERI, Perirhinal area cortex
PF, Parafascicular nucleus
PF.ll, Parafascicular nucleus, lower limb
PF.m, Parafascicular nucleus, mouth domain
PF.tr, Parafascicular nucleus, trunk domain
PF.ul, Parafascicular nucleus, upper limb domain
PGRNl, Paragigantocellular reticular nucleus, lateral part
PL, Prelimbic area cortex
PP, Peripenduncular nucleus
PPN, Pedunculopontine nucleus
POST, Postsubiculum
PRE, Presubiculum
PRNc, Pontine reticular nucleus, caudal part
PRNr, Pontine reticular nucleus, rostral part
xi
PSV, Principal sensory nucleus of the trigeminal
PTLp, Posterior parietal cortex
RE, Reuniens nucleus thalamus
RSPagl, Retrosplenial area cortex, agranular part
RSPd, Retrosplenial area cortex, dorsal part
RSPv, Retrosplenial area cortex, ventral part
SC, Superior colliculus
SC.m, Superior colliculus, medial zone
SC.cm, Superior colliculus, centromedial zone
SC.cl, Superior colliculus, centrolateral zone
SC.l, Superior colliculus, lateral zone
SC layers,
zo, zonal
sg, superficial grey
op, optic
ig, intermediate grey
iw, intermediate white
dg, deep grey
dw, deep white
SI, Substantia innominate
SNc, Substantia nigra pars compacta
SNr, Substantia nigra pars reticulata
SOCm, Superior olivary complex, medial part
SPFPp, Subparafascicular nucleus, parvicellular part
SPVI, Spinal motor nucleus of trigeminal, interpolar part
SPVOvl, Spinal motor nucleus of trigeminal, oral ventrolateral part
SSp, Somatosensory cortex primary
SSp-bfd, Somatosensory cortex primary, barrel field
SSp-ll, Somatosensory cortex primary, lower limb
SSp-m, Somatosensory cortex primary, mouth
SSp-n, Somatosensory cortex primary, nose
SSp-tr, Somatosensory cortex primary, trunk
SSp-ul, Somatosensory cortex primary, upperlimb
SSs, Somatosensory cortex, supplementary
STN, Subthalamic nucleus
SUB, Subiculum
SUBd, Subiculum dorsal
SUBdd, Subiculum dorsal, dorsal part
SUBdv, Subiculum dorsal, ventral part
SUBv, Subiculum ventral
SUBvv, Subiculum ventral, ventral tip
ProSUB, Prosubiculum
TEa, Temporal association area
TRN, Tegmental reticular nucleus
V, Trigeminal cranial nucleus
VII, Facial motor cranial nucleus
VISC, Visceral cortex
VIS, Visual cortex
VISal, Visual cortex, anterolateral part
xii
VISam, Visual cortex, anteromedial part
VISl, Visual cortex, lateral part
VISp, Visual cortex, primary
VISpl, Visual cortex, posterolateral part
VISpm, Visual cortex, posteromedial part
VM, Ventromedial nucleus thalamus
vmPFC, Ventromedial prefrontal cortex
VMH, Ventromedial hypothalamic nucleus
VMH.c, Ventromedial hypothalamic nucleus, central part
VMH.dm, Ventromedial hypothalamic nucleus, dorsomedial part
VMH.vl, Ventromedial hypothalamic nucleus, ventrolateral part
ZI, Zona incerta
ZI.m, Zona incerta, medial part
ZI.c, Zona incerta, central part
ZI.l, Zona incerta, lateral part

xiii
ABSTRACT

The central nervous system is a wonder to study. It is elegant, efficient, and
phenomenally complex. Santiago Ramon y Cajal discovered and taught us that this
system is composed of neural units with distinct inputs and outputs assembled into
intricate networks of connectivity. He identified the key principle that there is an orderly
flow of information throughout the brain and body that ultimately grants animals the
capacity to translate experiences of the world into neurobiological signals, convert them
into behavioral responses and form meaningful representations. Over a century later, the
ongoing effort to map this highly ordered system has driven the invention of innovative
tools and ambitious large-scale mapping projects. In particular, as part of the Mouse
Connectome Project (MCP), our goal was to assemble a comprehensive wiring map of
the entire mouse brain, a connectome. The overall approach combined an array of
neuroanatomical and neuroinformatics tools to systematically map all circuits,
subnetworks and greater networks throughout the healthy mouse brain model. The
studies in this dissertation on the neuroanatomical mapping of the superior colliculus
and visual thalamus directly contribute toward this effort.

Chapter 2 focuses the organization of the mouse brain superior colliculus (SC). We
constructed a comprehensive map of all cortico-tectal projections and identified four
newly defined collicular zones with differential cortical inputs: medial (SC.m),
centromedial (SC.cm), centrolateral (SC.cl) and lateral (SC.l). We also delineated the
distinct brain-wide input-output organization of each collicular zone to reveal their
subnetwork organization. This study provides a structural basis for understanding the
xiv
critical role of the SC in integrating different sensory modalities (visual, auditory, and
somatic sensory), translating sensory information to motor command, and coordinating
different actions (eyes, orofacial, whiskers, head and neck and limbs) in goal-directed
behavior.

Chapter 3 focuses on the mouse visual system where we have contributed to the
discovery of extrastriate connectivity with the primary thalamic visual relay, the dorsal
geniculate thalamus (LGd). We provide evidence of bidirectional extrastriate
connectivity with the mouse LGd. We found robust reciprocal connectivity of the medial
extrastriate regions with LGd neurons distributed along the ‘ventral strip’ border with
the intergeniculate leaflet (IGL). Overall, our findings support the existence of extrastriate
LGd circuits and provide novel understanding of LGd organization in rodent visual
system.

Chapter 4 provides a summarizing discussion on the main findings within Chapters 2
and 3. I elaborate upon additional neuroanatomical experiments that could be explored
in future pursuits. Additionally, I discuss how an understanding of subnetwork
connectivity offers significant insight in translational studies that are interested in
connectopathies and circuit dynamics of neuropsychiatric disorders and psychotherapy
research. The dissertation concludes with a parting perspective that presents a reflection
on personally-derived symbolic connections with the nervous system.

1

GENERAL INTRODUCTION

OVERVIEW OF STRUCTURAL FRAMEWORK
The brain can be modeled from a set of generalized organizational principles that offer a
tangible grasp of its intricate design
1
. This section briefly introduces (in simplified form)
the structural terms that are frequently referred to throughout all chapters. They are
described in a “bottom-up” approach to illustrate how sensory representations provide
a structural foundation for the understanding of more complex network assembly.
Importantly, these principles also informed the hypothetical framework that was used
for our experimental design and interpretation of results.
The primary senses of vision, audition, and tactile perception are represented in
modality-specific topographic maps throughout the nervous system. Sensory and motor
topographic maps mediate the orderly projection, translation and interpretation of
sensorimotor information across multiple cortical and subcortical brain structures.
Neuroanatomical and functional mapping studies reveal that though maps may be
uniquely represented, the information from the same systems must align and overlap
accurately across structures for efficient signaling. The maintenance and conservation of
these topographic connectivity patterns across structures underlies the organization of
distinct neural circuits. Together, the convergence and integration of highly

2
interconnected group of structures and circuits assemble to form localized subnetworks
that mediate the transduction and modulation of information between structures.
Subnetworks are implicated in specific functional or cognitive states to coordinate
specialized behavioral outputs, and can also be regulated regionally or globally by
various signaling dynamics, such as feedback loops, necessary to update information.
Multiple subnetworks may operate in hierarchical systems and in parallel as part of larger
networks that supervise executive control over brain states and functions
2–5
.

CONTEXT OF HYPOTHETICAL FRAMEWORK
1.2.1 SC AND THE VISUAL SYSTEM
Neuroanatomical research into the organization of superior colliculus (SC) and its
evolutionary homologue, the optic tectum, has been an area of great interest since
pioneering studies in the late 1800s
6
. The SC has largely been studied in primates
7–10
, cats
11

and chick
12
models, and is predominantly recognized for its visuomotor functions that
generate saccades and orienting movements
13,14
. Over the last decade, mice and rodent
species have emerged as prominent models for the neuroanatomical and functional study
of the mammalian visual system
15–20
. As the key hub of orienting responses in the
midbrain with direct retinal and visual cortical inputs, the SC is positioned to facilitate
innate/reflexive responses to visual stimuli (defense, escape, freezing), as well as higher
cognitive responses such as spatial attention
14
and target selection
21
involved in goal-
directed voluntary movements.
By virtue of mediating these critical functions, the SC is also implicated in
attention-related issues that underly the insufficient filtration of sensory stimuli,
dysregulation of neuromodulatory systems involving arousal, and connectopathies from

3
prefrontal cortical areas. As such, it is now a prime translational target for the modulation
and treatment of distractibility in attention-deficit hyperactivity disorders
22
. By
understanding how the SC operates in concert via subnetwork interactions throughout
the brains of healthy animal models, we can better understand how to address the
complications in models of neuropsychiatric disorders. For reference, a few major visual
related structures interconnected with SC include: the lateral geniculate nucleus in the
thalamus; the zona incerta, supraoptic, and retrochiasmatic nuclei in the hypothalamus;
the preoptic and parabigeminal nuclei in the midbrain; the pontine reticular formation;
and the medullary reticular nuclei.

1.2.2 SENSORIMOTOR AND HIGHER-ORDER ALIGNMENT IN THE SC
The SC is an ideal structure to study the relationship between structure and function as
it harbors a unique laminar architecture that facilitates multi-sensory integration to
command complex motor outputs. In addition to receiving direct retinotopic and
visuotopic maps, it also receives auditory and somatosensory topographic maps. Each
sensory map encodes a specialized representation of how stimuli are perceived in the
environment
23,24
. Visual stimuli are represented in a 2D receptive field map oriented with
respect to the central, peripheral, upper and lower quadrants of visual space (in the
superficial SC layers). Auditory stimuli are registered in a spherical coordinate frame by
way of the inferior colliculus (to the intermediate SC layers). And, tactile stimuli and the
somatic sensorimotor map are proportionally in register with the “homunculus”
representation of the body map (in the intermediate and deep SC layers).
This alignment of multiple sensory maps in the SC is a dominating organizational
principle that presumably guides how other information is integrated. Of particular

4
interest here are the extensive inputs from higher-order association cortices such as the
retrosplenial cortex (RSP), anterior cingulate area (ACA), infralimbic area (ILA), and
posterior parietal cortex (PTLp). Many studies have analyzed inputs from individual
cortico-tectal projections, and the findings support the general notion that the SC is
functionally divided into two broad medial and lateral divisions
25
. However, a
comprehensive map or understanding of how all cortical projections to SC has remained
largely unknown, and major unaddressed questions still prevail in the literature.

The main questions I wanted to address were the following: How are all the
neocortexàSC projections organized? How do the higher-order cortices overlay with
sensory maps in SC? Is there a more refined way to delineate and characterize the SC?
What functional roles can be implied?
In order to thoroughly address these questions, I first needed to contextualize and
understand the leading theories on cortical organization in the mouse model
1,3,4,26
. Studies
from our lab’s Mouse Connectome Project (MCP) on the interconnectivity between
cortical regions provided the anatomical foundation to design my approach and
formulate a compelling hypothesis. The 2014 cortico-cortical study revealed that there are
three major overarching subnetworks interacting across the neocortex where each is
comprised of more distinct modules; these include the somatic sensorimotor subnetwork,
the medial subnetwork, and the lateral subnetwork
4
(described further below). From this
perspective, my leading hypothesis postulated that if the virtual majority of cortical
regions projected to the SC, then their input organization in the SC would reflect the
subnetwork organization of the cortico-cortical connections as well. This hypothesis was
supported by the subsequent 2016 MCP study that mapped all the cortical projections to
the caudoputamen (CP; striatum), and revealed that the downstream cortico-striatal

5
projections conserved their cortico-cortical order across smaller topographic domains
27
.
In other words, the terminals from structures in the medial cortico-cortical subnetwork
clustered into distinct domains within the CP, the terminals from structures within the
somatic sensorimotor subnetwork clustered into distinct domains, and so on across all
modules. This provided robust anatomical support that cortico-tectal projections would
not only conserve the cortical subnetwork order, but that they may also be organized into
more discrete domains (or zones) uniquely defined by the overall structure of SC.

1.2.3 CORTICO-CORTICAL SUBNETWORKS.
As introduced in Section 1.1, understanding topographic sensory maps is an essential
foundation to understand the assembly of more complex subnetworks. This section
briefly summarizes the organization of the modules and components within the three
major cortico-cortical subnetworks from Zingg et al. (2014), where the somatic
sensorimotor and medial subnetworks are of the greatest relevance to the SC.
(1) The Somatic Sensorimotor Subnetworks -- consist of four major cortical
components: the primary and supplementary somatosensory and motor regions (SSp,
SSs, MOp, MOs, respectively). These regions are topographically arranged based on body
parts, and are further organized into four functionally distinct, yet highly interconnected,
modules. The four modules are the orofaciopharyngeal (mouth and nose), the upper
limb, the lower limb/trunk, and whisker (barrel-field). Altogether, this subnetwork
organization “allows direct interactions between sensory and motor areas in the absence
of higher-association areas”, and “enables rapid integration of different sensory
modalities for dynamically regulating motor actions.
4
” From previous functional and
anatomical studies, it is known that the SC receives somatic sensorimotor inputs to

6
command downstream motor behaviors via its deep layer outputs. Thus, I hypothesized
that each module from the somatic sensorimotor subnetwork would be topographically
arranged in the SC as well.
(2) The Medial Subnetworks -- consist of two parallel subnetworks along the
medial bank of the neocortex that primarily mediate interactions between sensory areas
and higher-order association areas. The first medial subnetwork predominantly consists
of the visual cortex (VIS) and auditory cortex (AUD) modules that integrate and transmit
information to the ventrolateral orbitofrontal cortex (ORBvl). These areas are densely
interconnected with other higher-order areas like the anterior cingulate area (ACAd,
ACAv), retrosplenial cortex (RSPd, RSPv, RSPagl), and posterior parietal cortex (PTLp),
and with SS and MO areas. Functionally, all of these regions are implicated in orienting,
spatial navigation tasks, and coordinating movements of the eyes, head and body.
Furthermore, the second medial subnetwork includes multisynaptic connections that
relay processed information regarding navigation, spatial navigation and episodic
memory from the hippocampus (SUBd) to the RSPv which then relays to the ACAv and
on to regions in the medial prefrontal cortex (ILA/PL/ORBm). In relation to the SC, it is
well known that the SC is functionally involved in generating orienting responses and
visually coordinated movements; thus, the array of cortico-tectal projections from these
medial subnetworks would hypothetically need to maintain complementary
arrangements in the SC as well.
(3) The Lateral Subnetworks -- consist of two modules, the insular and temporal.
The anterolateral insular (AI) module predominantly integrates information from
visceral (VISC), olfactory (PIRI, TTd), and gustatory (GUS) cortices, and has strong
reciprocal connections with the orofaciopharyngeal subnetwork. The posterolateral
temporal modules further process AI information with temporal association (TEa),

7
perirhinal (PERI), and ectorhinal (ECT) cortices that are involved in object recognition,
perception, self-awareness, and memory. These share strong connections with the medial
VIS, upper limb, lower limb/trunk, and whisker sensorimotor subnetworks. In regard to
their potential integration with the SC, I hypothesized that since the SC is known only for
its VIS, AUD and SS/MO sensory maps, there would likely not be any connections with
the AI lateral subnetwork; however, there could be connections with the temporal lateral
subnetwork. In other words, since the SC is not known to process any direct olfactory or
gustatory information, then it would also not integrate primary sensory information from
PIRI, TTd, GUS or VISC cortices from the AI subnetwork. By contrast, the temporal lateral
network processes object recognition which is also important for the role of SC in
identifying objects for attention and memory, and importantly, it also integrates VIS,
AUD, SS/MO information that could be relevant to the SC.
Overall, the cortico-cortical subnetwork organization summary provides the
essential structural and hypotheses-driven background to understand how the SC is
positioned as a key player in overall networks in brain architecture models.

INTEGRATING STRUCTURAL AND HYPOTHETICAL
FRAMEWORKS
The MCP has generated multiple in depth neuroanatomical projection maps that identify
newly refined subnetwork organization properties of the mouse neocortex
4
, CP
27
,
hippocampus
28
, basolateral amygdala
29
, medial prefrontal cortex
30
, globus pallidus (GP)
with the substantia nigra (SNr)
31
, and here, the SC
32
and visual thalamus
19
(Figure 1.1).
Each of these large-scale projects was founded on the structural model of an elementary
cerebral network that describes the systems organization of the brain, in particular by the

8
four-subsystems model, triple descending projections from the cerebral hemispheres,
and thalamocortical feedback loops proposed by Swanson & Bota (2010)
1,33
.
To summarize, the four-subsystems model of brain architecture describes how
information is categorized and distributed from the environment into the nervous
system. Information is differentiated and interconnected throughout four main systems
– sensory, motor, cognitive, and state – to interactively control internal and external
information. Each of the four systems is influenced by the signaling dynamics of its own
basic organization, and is mediated by specialized circuits to coordinate functions
throughout the network. Thus, at the onset, we contextualized the input and output
organization of each major brain structure with this model in mind. For the SC in
particular, I conceptualized its connectivity and significant functional roles within each
system in the model based on the literature: sensory (visual, auditory, somatosensory),
motor (eye, head, and neck movements, innate or defensive responses), cognitive
(attention, spatial navigation) and state (arousal). This understanding facilitated the
derivation of relevant functional implications within the Discussion section of Chapter 2.
Furthermore, the studies in this dissertation primarily focus on cortical, tectal,
thalamic, and midbrain regions that are closely interconnected subcortically with the
motor system. We reasoned that any newly derived schema of delineations and
organization of would need to be congruous with these systems, in particular with the
triple descending projections from the cerebral hemispheres, and thalamocortical
feedback loops
33
. Figure 1.1 illustrates an overview of the general interconnectivity of
MCP projects, and is described here as a sequence.
First, cortico-cortical subnetworks (Figure 1.1a) provide topographic projections
to distinct CP domains as demonstrated by results from the cortico-striatal projectome
(Figure 1.1b). The SC is part of the midbrain motor system that receives direct excitatory

9
(glutamatergic) descending projections from layer 5 cortical neurons (Figure 1.1c). Recent
mappings from MCP demonstrate how the descending inhibitory projections from the
CP domains, project topographically to the GP and SNr, and how they connect to
thalamocortical loops as part of the basal-ganglia system (Figure 1.1d). Notably, these
structures are interconnected with other integral systems, such as the limbic system
34
, to
mediate a range of affective and motivated behaviors involving the amygdala,
hippocampus and hypothalamus
26,35
(Figure 1.1e-f). Collectively, the newly refined
circuit delineations from the MCP are corroborated across all studies that validate the
elementary cerebral network to the motor system, and also support the brain-wide
interpretation of SC functional integration.

10

Figure 1.1 | Integrating Connectivity Maps
Summary schematic connectivity diagram of how MCP projectomes and whole-brain connectivity maps
throughout multiple brain structures are part of larger systems of organization. Delineations across
structures and subdivisions are topographically congruous. The right section illustrates how motor system
and basal ganglia-related MCP maps are structurally organized as part of triple descending projections
from the cerebral hemispheres, adapted from Swanson 2012. A) Summary figure of cortico-cortical
subnetworks from Zingg et al., 2014. B) Summary figure of cortico-striatal subnetworks from Hintiryan et
al., 2016. C) Figure of brain-wide SC connectivity from this study. D) Summary diagram of the cortico-basal
ganglia loop model from Foster et al., 2021. The left section illustrates connections between structures
implicated in the limbic system. E) Figure of brain-wide connections with the basolateral amygdalar
complex from Hintiryan et al. 2021. F) Figure of brain-wide connections with the hippocampus from
Bienkowski et al., 2018. This manuscript also includes G) circuit diagram of SUBvv connections with
hypothalamus and amygdala. Maps and connectivity diagrams were assembled by analyzing manual
annotations, and processed through the MCP’s standardized annotation, registration and quantification
pipeline. High quality images and complete figure captions can be found in their original manuscripts
(http://brain.neurobio.ucla.edu/publications/).
______________________________________________________________________________
Striatum
Thalamus
Superior Colliculus
Triple Descending Projections
from Cerebral Hemispheres
Cortex
Pallidum
Motor System Sensory System
GLUTAMATE
GABA
GABA
excitatory inhibitory disinhibitory
Hippocampus
Amygdala
Basal-Ganglia
Hypothalamus
Limbic System
A
B
C
D
E
F
SNr

11

SUBNETWORK ORGANIZATION OF THE
INPUTS AND OUTPUTS OF THE
MOUSE SUPERIOR COLLICULUS

Adapted from Nature Communications (2021) manuscript. See Appendix B.

ABSTRACT
The superior colliculus (SC) is a midbrain structure that receives diverse and robust
cortical inputs to drive a range of cognitive and sensorimotor behaviors. However, it
remains unclear how descending cortical input arising from higher-order association
areas coordinate with SC sensorimotor networks to influence its outputs. In this study,
we constructed a comprehensive map of all cortico-tectal projections and identified four
newly defined collicular zones with differential cortical inputs: medial (SC.m),
centromedial (SC.cm), centrolateral (SC.cl) and lateral (SC.l). Informatics analyses
revealed that cortico-tectal projections are organized as multiple subnetworks that are
consistent with previously identified cortico-cortical and cortico-striatal subnetworks.
Further, we delineated the distinctive brain-wide input/output organization of each
collicular zone, assembled multiple parallel cortico-tecto-thalamic subnetworks, and
identified the somatotopic map in the SC that displays distinguishable spatial properties
from the somatotopic maps in the neocortex and basal ganglia. Finally, we characterized

12
interactions between those cortico-tecto-thalamic and cortico-basal ganglia-thalamic
subnetworks. This study provides a structural basis for understanding the critical role of
the SC in integrating different sensory modalities (visual, auditory, and somatic sensory),
translating sensory information to motor command, and coordinating different actions
(eyes, orofacial, whiskers, head and neck and limbs) in goal-directed behavior.

INTRODUCTION
Cortico-tectal projections from the cerebral cortex to the superior colliculus (SC) relay
information to mediate a range of multimodal and cognitive functions
10,36–38
. In order for
the mammalian SC to coordinate complex behaviors, such as attention, navigation,
defense and decision-making, it requires the alignment and integration of top-down
higher-order cortical information with sensorimotor maps. Topographic cortico-tectal
projections from primary visual, auditory, and motor areas have been well studied;
however, the integration of higher-order association cortical inputs to the SC has not been
thoroughly characterized
10,24,39
. Disruptions in cortico-tectal projections have been
implicated in attention-deficit and autism spectrum disorders
40–44
; thus, it is important to
understand the organizational principles of how the confluence of higher-order cortical
information is integrated in the SC and channeled to downstream structures to regulate
and coordinate behavioral outputs. An anatomical parcellation of cortico-tectal pathways
would offer a resourceful and translational reference map.
At present, the mouse SC is regarded as an emergent structural model for the
rodent visual system and circuit formation that facilitates multisensory integration
16,45
.
Though there has been a prolific rise in rodent functional studies, the connectivity of the
SC is often reviewed and generalized across multiple species to suggest there is little left

13
to understand about its mesoscale connections
9,14,16
. Nonetheless, to date, there is no
complete anatomical map of SC connectivity in mice or any other species
46,47
. Thus, as
part of current advancements within the large collaborative effort of the BRAIN Initiative
Cell Census Network (BICCN) to identify detailed mouse brain anatomy
(https://biccn.org/), we contribute a map of the mouse SC connectome using modern
neuroanatomic viral tracing methods and computational tools. Here, we assembled the
first detailed connectivity map of descending cortical subnetworks to the SC, the cortico-
tectal projectome, and concurrently constructed a brain-wide wiring diagram for the SC.
This resource provides a foundational structural model for the mouse SC that supports
working hypotheses of regionally defined functions (such as flight vs. freezing or
approach vs. aggression), and network- and cell type-specific functions.
In this study, we expand upon the laminar organization of the SC by further
delineating its regional organization. The SC has a distinct composition of seven
alternating fibrous and cellular layers, including three superficial (zo, zonal; sg,
superficial grey; op, optic), two intermediate (ig, intermediate grey; iw, intermediate
white), and two deep layers (dg, deep grey; dw, deep white)
48
. Functional studies often
arbitrarily bisect the SC into two medial and lateral halves
25
; however, these broad
divisions can be refined to reflect how distinct subnetworks of contiguous or segregated
inputs/outputs are organized throughout the SC. To address this, we systematically
characterized layer-specific cortical fiber terminations across the SC to define subregional
zones. Analyses of the cortical-tectal distribution patterns revealed that the SC could be
subdivided into four radially distinct zones that extend along the medial-lateral and
rostral-caudal axes. Each zone is defined by a unique subnetwork of cortical inputs, and
displays a distinct brain-wide input-output organization. The zones are correlated with
specialized functional subnetworks involved in the integration of visual, auditory, and

14
somatosensory modalities, and are highly conserved topographically
4,27
. These four
subnetworks of the SC provide a structural basis for understanding different functional
roles of the SC in coordinating movements of eyes, head and neck, whisker, mouth and
limbs in goal-oriented tasks such as attentive orientation, spatial navigation, and
exploratory (i.e., prey capture), appetitive/approach, consummatory (i.e., chewing,
swallowing and biting) behaviors.

METHODS (FOR RESULTS PARTS I & II)

2.3.1 MOUSE CONNECTOME PROJECT METHODOLOGY
Overview.
Anatomical tracer data was generated as part of the Mouse Connectome Project (MCP).
We systematically and carefully mapped neuronal connectivity of each cortical structure
to determine their input connectivity to the superior colliculus (SC) (for complete
injection site list, see Table 2.1). We used multiple fluorescent tracing strategies with a
combination of classic tract-tracing and viral tracing methods. First, we used a triple
anterograde tracing approach with individual injections of PHAL and GFP- and
tdTomato- expressing adeno-associated viruses (AAV). Additionally, to investigate the
convergence or divergence of axonal fiber pathways either into or out of the SC, we used
a double co-injection method that introduces two different tracer cocktails each
containing one anterograde and one retrograde tracer to simultaneously visualize two
sets of input/output connectivity. Similar MCP experimental methods and online
publication procedures have also been described previously
4,19,27,28
. The data analyzed in
this study were produced through the Brain Initiative Cell Census Network (BICCN).

15
Table 2.1 | Injection Sites.
Table of injection site coordinates and tracer information for all cases used throughout this study. Cases
are identified by case number, injection Site (ROI, region of interest), tracer, tracer type, figure reference,
ARA Level of injection site, and coordinates for the injection site based on the ARA Coordinate Frame.
Cortex cases can be cross-referenced to flat map injection site location in Figure 2.1 (n=44 mice with 86 total
cortical pathways).

Mouse # Pathway # Case ID Injection Site (ROI) Tracer Tracer Type Figure ARA Level ML (X) AP (Y) DV (Z)
1 1 SW110321-04B/A ACAv interm PHAL-488 classic / anterograde 3g, 4b 56 0.4 -0.18 -0.75
2 2 SW110322-03A MOs caudal PHAL-488 classic / anterograde 2g, 4b 58 0.6 -0.38 -0.75
3 SW110322-03A SSp-ul BDA classic / anterograde 4b 58 2.3 -0.38 -1.5
3 4 SW110516-02B/A SSp-n PHAL-488 classic / anterograde 2g, 4b 50 3.1 0.45 -2.7
4 5 SW110613-03B/A RSPd rostral PHAL-488 classic / anterograde 3b, SF4a, 4b 83 0.6 -2.98 -0.25
5 6 SW110614-03B/A RSPd caudal PHAL-488 classic / anterograde 4b 88 0.6 -3.45 -0.25
6 7 SW110615-01B/A RSPagl PHAL-488 classic / anterograde 3b, 3g, 4b 91 0.9 -3.78 -0.25
7 8 SW110615-03B/A RSPv rostral PHAL-488 classic / anterograde 3b, SF4a, 4b 73 0.5 -1.85 -0.75
8 9 SW110808-02A ORBl PHAL-488 classic / anterograde SF4b, 4b 37 1.75 1.95 -3.25
9 10 SW110808-04A ORBm PHAL-488 classic / anterograde 3b, 4b 28 0.6 2.62 -2.4
10 11 SW120125-01A TEa rostral PHAL-488 classic / anterograde 3b, 4b 78 4.25 -2.35 -3.4
11 12 SW120403-02A ACAv rostral PHAL-488 classic / anterograde 3b, SF4a, 4b 44 0.5 1.05 -1.8
12 13 SW120819-04A VISl BDA classic / anterograde 4b 92 3.5 -3.88 -1.75
14 SW120819-04A TEa caudal PHAL-488 classic / anterograde 3b, 4b 94 4.1 -4.08 -2.5
13 15 SW121031-03B/A SSp-tr/ll AAV-tdTomato virus / anterograde 4b 70 1.6 -1.55 -0.75
16 SW121031-03B/A ACAd interm PHAL-647 classic / anterograde 3b, SF4a, 4b 46 0.4 0.85 1.25
17 SW121031-03B/A VISam AAV-GFP virus / anterograde 4b 83 1.6 -2.98 -0.5
14 18 SW121031-04B/A ACAv interm PHAL-647 classic / anterograde 3b, 4b 54 0.4 0.2 -1.6
19 SW121031-04B/A SSp-tr AAV-tdTomato virus / anterograde 2f, 4b 74 2 -1.98 -1
20 SW121031-04B/A AUDp interm AAV-GFP virus / anterograde 2g, 2f, 3g, 4b 84 4.1 -3.1 -2.5
15 21 SW121210-02A MOs medial AAV-GFP virus / anterograde 2g, 4b 49 0.5 0.55 -0.85
16 22 SW121221-02A VISp caudolat BDA classic / anterograde 4b 96 2.75 -4.28 -1.25
23 SW121221-02A VISp caudomed PHAL-488 classic / anterograde 2g, 4b 96 2.5 -4.28 -1
17 24 SW121221-03A VISp caudal BDA classic / anterograde 2g, 4b 95 1.8 -4.12 -0.75
25 SW121221-03A VISp rostral PHAL-488 classic / anterograde 4b 86 5 -3.28 -1.75
18 26 SW151210-01A ACAd rostral AAV-tdTomato virus / anterograde 3b, 3g, 4b 36 0.3 1.85 -1.25
27 SW151210-01A SSs AAV-GFP virus / anterograde 4b 53 3.25 0.15 -3.25
19 28 SW151210-02A VISam AAV-tdTomato virus / anterograde 3e, 3g, 4b 84 1.5 -3.08 -0.75
29 SW151210-02A SSp-tr AAV-GFP virus / anterograde 3e, 4b 74 2 -1.95 -0.7
20 30 SW151211-01A/B VISp AAV-GFP virus / anterograde 3d, 6a, 4b 92 3 -3.88 -1.2
31 SW151211-01A/B ACAd rostral AAV-tdTomato virus / anterograde 3b, 3d, 6a, 4b 37 0.6 -1.75 -1.25
32 SW151211-01A/B RSPv caudal PHAL-647 classic / anterograde 3b, 3d, 3g, 6a, 4b 90 0.5 -3.68 -0.75
21 33 SW151211-02A ACAd caudal AAV-tdTomato virus / anterograde 3e, 4b 58 0.4 -0.38 -1
34 SW151211-02A VISam PHAL-647 classic / anterograde 3e, 4b 86 1.5 -3.28 -0.5
35 SW151211-02A SSp-tr AAV-GFP virus / anterograde 3g, 4b 76 3 -2.15 -1.2
22 36 SW151215-03A PTLp lat AAV-tdTomato virus / anterograde 3b, 4b 82 3.6 -2.88 -1.75
23 37 SW170410-03A SSp-ll AAV-GFP virus / anterograde 3g, 4b 56 2.2 -0.18 -1.25
38 SW170410-03A SSp-ul AAV-tdTomato virus / anterograde 3g, 4b 47 2.5 -0.75 1.5
24 39 SW170410-04A SSp-m AAV-tdTomato virus / anterograde 3g,4b, SF3c 43 2.75 1.15 -2.5
40 SW170410-04A SSp-ul AAV-GFP virus / anterograde 4b, SF3c 49 2.5 0.55 -1.5
25 41 SW171101-01A MOp-oro PHAL-647 classic / anterograde 3g, 4b 34 2.1 2.1 -2.75
42 SW171101-01A MOp-ul AAV-tdTomato virus / anterograde 4b 44 1.5 1.1 -1.5
43 SW171101-01A MOp-bfd AAV-GFP virus / anterograde 3g, 4b 38 1.8 1.65 -1.4
26 44 SW171130-02A MOs lateral AAV-tdTomato virus / anterograde 2d, 4b 44 1.1 1.1 -1.5
45 SW171130-02A MOs lateral AAV-GFP virus / anterograde 2d, 4b 46 1.1 0.85 -1.25
46 SW171130-02A MOp-oro PHAL-647 classic / anterograde 2d, 4b 40 1.4 1.42 -1.6
27 47 SW180117-01A ORBl PHAL-647 classic / anterograde 3b, 4b 35 1.5 2.1 -3.3
48 SW180117-01A MOp-oro AAV-tdTomato virus / anterograde 3g, 4b 35 1.5 2.1 -1.75
49 SW180117-01A MOp-ul AAV-GFP virus / anterograde 3g, 4b 41 1.4 1.35 -1.4
28 50 SW180227-01A ACAd interm PHAL-647 classic / anterograde 3g, 4b 42 0.5 1.25 -1.4
51 SW180227-01A MOs medial AAV-tdTomato virus / anterograde 3g, 4b 40 0.6 1.42 -1.25
29 52 SW180227-02A ACAd interm PHAL-647 classic / anterograde 4b 47 0.3 0.75 -1.2
53 SW180227-02A MOs medial AAV-tdTomato virus / anterograde 4b 47 0.4 0.75 -0.75
30 54 SW180228-03A ACAv caudal AAV-tdTomato virus / anterograde 3b, 4b 58 0.4 -0.38 -1.25
55 SW180302-04A VISal AAV-tdTomato virus / anterograde 4b 91 3.4 -3.88 -1.6
56 SW180302-04A PTLp lat AAV-GFP virus / anterograde 4b 82 3.5 -2.88 -1.65
31 57 SW180302-05A VISp caudolat PHAL-647 classic / anterograde 2c, 4b 94 2.25 -3.88 -1
58 SW180302-05A VISal AAV-tdTomato virus / anterograde 2c, 4b 90 3.4 -3.68 -1.3
59 SW180302-05A PTLp lat AAV-GFP virus / anterograde 2c, 4b 81 3.6 -2.88 -1.75
32 60 SW180424-01A PTLp med AAV-tdTomato virus / anterograde 3b, 4b 77 2.25 -2.55 -6
61 SW180424-01A PTLp med PHAL-488 classic / anterograde 3g, 4b 77 1.5 -2.55 -6
33 62 SW180516-02A MOp-oro PHAL-647 classic / anterograde 4b 32 2 2.25 -2.5
63 SW180516-02A MOp-bfd AAV-tdTomato virus / anterograde 4b 40 1.6 1.42 -1.6
64 SW180516-02A MOp-tr AAV-GFP virus / anterograde 4b 52 1.6 0.25 -1.3
34 65 SW180522-04A VISl/pl AAV-tdTomato virus / anterograde 4b 97 3.5 -4.15 -2
35 66 SW180713-03A VISp AAV-tdTomato virus / anterograde 4b 90 2 -3.98 -0.8
67 SW180713-03A SSp-tr PHAL-647 classic / anterograde 4b 68 2 -1.35 -1.25
68 SW180713-03A AUDd AAV-GFP virus / anterograde 3g 74 4 -1.95 -2.1
36 69 SW180713-04A VISp rostromed AAV-GFP virus / anterograde 3g, 6a, 4b 87 2.25 -3.38 -0.75
70 SW180713-04A SSp-tr AAV-tdTomato virus / anterograde 4b, 6a, SF3c 68 2.5 -1.35 -1.25
71 SW180713-04A AUDd PHAL-647 classic / anterograde 4b, 6a 74 4 -1.95 -2.1
37 72 SW180717-03A PTLp med PHAL-488 classic / anterograde 3f, 4b 76 1.25 -2.15 -0.6
73 SW180717-03A PTLp med AAV-tdTomato virus / anterograde 3f, 4b 76 2.1 -2.15 -0.75
38 74 SW180815-01A VISl AAV-tdTomato virus / anterograde 3g, 4b 92 3.75 -3.88 -2
39 75 SW181109-09A SSp-bfd AAV-GFP virus / anterograde 4b, SF3c 64 3 -0.95 -1.5
76 SW181109-09A SSp-bfd AAV-tdTomato virus / anterograde 4b, SF3c 64 3.5 -0.95 -2
40 77 SW190315-05A PL AAV-tdTomato virus / anterograde 3b, 4b, 6a, SF4b 33 0.4 2.15 -2.3
78 SW190315-05A MOs rostral PHAL-647 classic / anterograde 2e, 4b, 6a 31 1.3 2.35 -1.5
79 SW190315-05A MOp-oro AAV-GFP virus / anterograde 2e, 4b, 6a 31 2 2.35 -3
41 80 SW190315-07A ILA PHAL-647 classic / anterograde 3b, 4b 37 0.5 1.75 -3
42 81 SW190315-09A AUDp rostral AAV-tdTomato virus / anterograde 4b 76 4.1 -2.15 -2.6
82 SW190315-09A AUDp caudal AAV-GFP virus / anterograde 4b 86 4.1 -3.28 -2.3
83 SW190315-09A AUDv PHAL-647 classic / anterograde 4b 70 4.2 -1.56 -3.1
43 84 SW190327-02A ORBvl AAV-GFP virus / anterograde 3b, 3g, 4b, SF4b 33 1.25 2.15 -3.1
44 85 SW190327-04A RSPv interm AAV-GFP virus / anterograde 3b, 4b 88 0.4 -3.45 -0.6
86 SW190327-04A VISpm AAV-tdTomato virus / anterograde 3g, 4b 94 1.25 -4.08 -0.75
Cortex cases with anterograde projections to Superior Colliculus

16
(Table 2.1 continued)

Mouse # Pathway # Case ID Injection Site (ROI) Tracer Tracer Type Figure ARA Level ML (X) AP (Y) DV (Z)
45 87 SW150916-01A SC.m PHAL classic / anterograde 4b 86 0.5 -3.28 -0.4
88 SW150916-01A SC.l BDA classic / anterograde 4b 86 1.1 -3.28 -2.5
89 SW150916-01A SC.m CTB-555 classic / retrograde 4b 86 0.5 -3.28 -0.4
90 SW150916-01A SC.l FG classic / retrograde 4b 86 1.1 -3.28 -2.5
46 91 SW150921-01A SC.m FG classic / retrograde 4b 90 0.3 -3.68 -1.6
47 92 SW160716-01A SC.cl/l PHAL classic / anterograde 4b 100 0.5 -4.65 -1.3
48 93 SW160716-02A SC.cm PHAL classic / anterograde 4b 100 0.3 -4.65 -1.2
94 SW160716-02A SC.cm FG classic / retrograde 4b 86 0.5 -3.28 -2.3
49 95 SW170201-05A SC.m-iw/dg CTB-647 classic / retrograde 4b, 5a 96 0.2 -4.28 -1.75
96 SW170201-05A SC.cl-dg CTB-555 classic / retrograde 4b, 5a 96 0.6 -4.28 -2.25
97 SW170201-05A SC.l-dg CTB-488 classic / retrograde 4b, 5a 96 0.9 -4.28 -2.6
50 98 SW170201-08A SC.cm-ig/iw/dg CTB-555 classic / retrograde 4b, 5a 96 0.6 -4.28 -1.5
99 SW170201-08A SC.l-ig FG classic / retrograde 4b, 5a 96 1.4 -4.28 -2.4
51 100 SW170215-01A SC.cm PHAL classic / anterograde 4b 86 0.5 -3.28 -2.3
52 101 SW170315-01A SC.cl/l AAV-tdTomato virus / anterograde 4b 90 1.5 -3.68 -2
53 102 SW170315-02A SC.cm CTB-647 classic / retrograde 4b 90 0.5 -3.68 -1.5
103 SW170315-02A SC.cl FG classic / retrograde 4b 90 1.5 -3.68 -2.75
54 104 SW170426-04A SC.l AAV-tdTomato virus / anterograde 4b 96 1.1 -4.28 -2.1
55 105 SW170830-01A SC.m CTB-647 classic / retrograde 4b 100 0.3 -4.65 -1.5
56 106 SW170830-02A SC.cm CTB-647 classic / retrograde 4b 100 0.25 -4.65 -1.3
107 SW170830-02A SC.cl CTB-488 classic / retrograde 4b 100 0.75 -4.65 -1.3
57 108 SW171010-01A SC.l-ig/iw AAV-tdTomato virus / anterograde 4a-b, SF6c 90 1 -3.68 -2.8
109 SW171010-01A SC.l-dg PHAL virus / anterograde 4a-b 88 1.25 -3.45 -2.75
110 SW171010-01A SC.cl-dg AAV-GFP virus / anterograde 4a-b 94 1 -4.08 2.5
58 111 SW171010-02A SC.cm AAV-tdTomato virus / anterograde 4a-b, SF6b 90 0.55 -3.68 -2.4
112 SW171010-02A SC.cl AAV-GFP virus / anterograde 4a-b, SF6b 90 0.75 -3.68 -2.5
59 113 SW171010-03A SC.m AAV-tdTomato virus / anterograde 4b 96 0.4 -4.28 -1.6
60 114 SW180614-07A SC.m AAV-tdTomato virus / anterograde 4b 96 0.25 -4.28 -1.2
115 SW180614-07A SC.cl AAV-GFP virus / anterograde 4b 96 0.9 -4.28 -1.6
61 116 SW190619-02A SC.m/cm PHAL classic / anterograde 4a-b 90 0.5 -3.68 -1.5
117 SW190619-02A SC.cl AAV-tdTomato virus / anterograde 4a-b 90 0.8 -3.68 -2.1
62 118 SW190619-04A SC.m-iw/dg PHAL classic / anterograde 4a-b, SF6a 90 0.25 -3.68 -1.8
63 119 SW140603-01A LD FG classic / retrograde SF3a 69 1.2 -1.45 -2.6
64 120 SW140603-03A MD FG classic / retrograde 5c 66 0.3 -1.15 -3.2
65 121 SW140916-04A RE FG classic / retrograde 5c 61 0.2 -0.65 -4.7
66 122 SW141021-02A LP FG classic / retrograde SF3a 77 1.6 -2.25 -2.5
67 123 SW150716-02A SNr.cl PHAL-488 classic / anterograde 5d 84 1.2 -3.08 -5.2
68 124 SW171002-03A SNr.l PHAL-647 classic / anterograde 5d 84 1 -3.08 -5.25
125 SW171002-03A SNr.dl AAV-RFP classic / anterograde 5d 84 1.5 -3.08 -5.1
126 SW171002-03A SNr.dm AAV-GFP classic / anterograde 5d 84 0.75 -3.08 -5.3
69 127 SW171213-02A PF.m FG classic / retrograde 5c 75 0.7 -2.05 -3.75
70 128 SW180508-04A SNr.v PHAL-647 classic / anterograde 5e 84 1.5 -3.08 -5.5
129 SW180508-04A SNr.dl AAV-tdTomato classic / anterograde 5e 84 1.5 -3.08 -5.1
130 SW180508-04A SNr.dm AAV-GFP classic / anterograde 5e 84 1 -3.08 -5.5
71 131 SW190110-01B CPc.dm AAVretro-EF1a-Cre virus / retrograde 6b 57 1.6 -0.28 -2.5
132 SW190110-01B MOs AAV1.CAG-FLEX-tdTom virus / anterograde 6b 47 0.5 0.75 -1
72 133 SW190207-09A SNr.m AAV-RFP virus / anterograde 5d 84 0.75 -3.08 -5.6
73 134 SW190213-01A VM/PCN FG classic / retrograde SF3a 72 0.6 -1.75 -4
74 135 SW190829-03A ZI.medial AAV-RFP virus / anterograde 5b 74 0.6 -1.95 -5
75 136 SW190926-10 PF.ul AAVretro-EF1a-Cre virus / retrograde 7, 4b 75 0.5 -2.05 -3.4
137 SW190926-10 SC.l-ig AAV8-hSyn-FLEX-TVA-P2A-GFP-2A-oG virus / anterograde 7, 4b 90 1.5 -3.68 -2.5
138 SW190926-10 SC.l-ig EnvA G-deleted-rabies-mCherry virus / retrograde 7, 4b 90 1.5 -3.68 -2.5
76 139 SW191011-01A ZI.lateral PHAL-647 classic / anterograde 5b 71 1.75 -1.65 -4.5
77 140 SW191011-02A ZI.central PHAL-647 classic / anterograde 5b 74 1.25 -1.95 -4.3
78 141 SW121220-05A AId BDA classic / anterograde n/a 31 2.5 2.35 -3.75
79 142 SW120403-01A AId BDA classic / anterograde n/a 27 2.3 2.75 -3.75
80 143 SW110321-04B AIp BDA classic / anterograde n/a 56 4.1 -0.18 -4.6
81 144 SW110906-02A AIp PHAL-488 classic / anterograde n/a 64 4.3 -0.95 -4.5
82 145 SW120118-01A AId/v BDA classic / anterograde n/a 44 3.4 1.05 -4.5
83 146 SW110906-02A GU PHAL-488 classic / anterograde n/a 38 2.9 1.65 -3.5
84 147 SW120125-03A GU PHAL-488 classic / anterograde n/a 37 2.75 1.75 -3.3
85 148 SW110905-01A VISC BDA classic / anterograde n/a 54 4 0.02 -4.1
86 149 SW120118-02A VISC BDA classic / anterograde n/a 66 4.4 -1.15 -4
87 150 SW110906-04A ECT BDA classic / anterograde n/a 84 4.4 -3.08 -3.95
88 151 SW180815-02A ENT AAV-tdTomato virus / anterograde n/a 101 4.75 -4.78 -2.75
89 152 SW120403-03A PERI BDA classic / anterograde n/a 87 4.7 -3.38 -3.9
Additional cases used throughout study
Cortex regions which do not send anterograde projections to Superior Colliculus

17
Animal Subjects.
All MCP tract-tracing experiments were performed using 8-week old male C57BL/6J
mice (n=60; Jackson Laboratories) (C57BL/6J IMSR Cat# JAX:000664). Mice had ad
libitum access to food and water and were pair-housed within a temperature- (21-22°C),
humidity- (51%), and light- (12hr: 12hr light/dark cycle) controlled room within the
Zilkha Neurogenetic Institute vivarium. All experiments were performed according to
the regulatory standards set by the National Institutes of Health Guide for the Care and
Use of Laboratory Animals and by the institutional guidelines described by the USC
Institutional Animal Care and Use Committee.

Tracer Injection Experiments.
The MCP uses a variety of combinations of anterograde and retrograde tracers to
simultaneously visualize multiple anatomical pathways within the same Nissl-stained
mouse brain. Triple anterograde tracing experiments involved three separate injections
of 2.5% Phaseolus vulgaris leucoagglutinin (PHAL; Vector Laboratories, Cat# L-1110,
RRID:AB_2336656), and AAVs encoding enhanced green fluorescent protein (AAV-GFP;
AAV2/1.hSynapsin.EGFP.WPRE.bGH; Penn Vector Core; 7×10¹² vg/mL) and tdTomato
(AAV1.CAG.tdtomato.WPRE.SV40; Penn Vector Core; 4×10¹² vg/mL). An anterograde
tracer of 5% biotinylated dextran amine (BDA; Invitrogen) was used in some cases.
Retrograde tracers included cholera toxin subunit B conjugates 647, 555 and 488 (CTb;
AlexaFluor conjugates, 0.25%; Invitrogen), Fluorogold (FG; 1%; Fluorochrome, LLC), and
AAVretro-EF1a-Cre (AAV-retro-Cre; Viral Vector Core; Salk Institute for Biological
Studies; 7×10¹² vg/mL). To provide further details on specific connectivity patterns, we
also performed quadruple retrograde tracer, and rabies/PHAL experiments. Quadruple

18
retrograde tracer experiments involved four different injection sites receiving a unique
injection of either 0.25% CTb-647, CTb-555 CTb-488, 1% FG, or AAV-retro-Cre.
To trace the connectivity of projection defined neurons, a Cre-dependent
anterograde tracing strategy was employed, which involved the delivery of Cre to
projection neurons of interest via an injection of AAVretro-EF1a-Cre into their target
regions. Next, AAV1.CAG.FLEX.eGFP.WPRE.bGH (RRID:Addgene_51502; 1×10¹³
vg/mL) and AAV1.CAG.FLEX.TdTomato.WPRE.bGH (RRID:Addgene_51503) were
delivered to the different Cre-expressing SC neuronal populations. Furthermore, to
retrieve the morphological information from different tectal projection neurons, Gdel-
RV-4tdTomato
49
(2.14e10 infectious units/mL), and Gdel-RV-4eGFP
50
(1.51e11 infectious
units/mL) tracers of 10nl volumes were each injected into two different downstream
targets.
Finally, an AAV-based multi-synaptic tracing method TRIO (tracing the
relationship of inputs and outputs) was used to reveal monosynaptic inputs to projection-
specific SC cell types. First an AAVretro-EF1a-Cre (7×10¹² vg/mL) injection was made in
an SC-projection region. At the same time as the AAVretro-EF1a-Cre injection, a Cre-
dependent AAV expressing TVA (an avian receptor protein) and rabies glycoprotein
(AAV8-hSyn-FLEX-TVA-P2A-mCherry-2A-oG) was injected into the SC (where the
projection neuron cell bodies are located). Following three weeks for Cre recombination
and transgene expression of TVA and glycoprotein, an EnvA-pseudotyped G-deleted
rabies-EGFP (EnvA G-deleted-rabies-GFP; 1.51e11 infectious units/mL) was injected into
the same SC injection site. The EnvA/TVA receptor system allows for rabies infection
only within the specific projection neuron population
51
. Following infection, the
reincorporation of rabies glycoproteins in newly packaged rabies virions within the
projection neurons allows the rabies virus to move trans-synaptically to label all neuronal

19
inputs to the specific projection neuron population, including all cortical and brainstem
inputs.
Though the injections were discrete in volume, some may have spread into
adjacent structures. Anterograde injections in the gustatory (GU), visceral (VISC),
entorhinal (ENT), perirhinal (PERI), ectorhinal (ECT) areas, and claustrum (CLA) did not
produce any evident projections to SC. All cases used and referenced in this study are
listed in Table 2.1. Raw microscopy image files (.vsi format) for all cases can be
downloaded at http://brain.neurobio.ucla.edu/superiorcolliculus/vsi, and opened
through the online Olyvia software (available for free download for Windows PC from
https://www.olympus-lifescience.com/en/support/downloads/).

Statistics and Reproducibility.
Reproducibility and control of individual variability of labeling was validated in different
cases with injections in the same locations. No statistical methods were used to pre-
determine sample sizes; our sample sizes are similar to those reported in previous
publications
4,27
. In most cases, anterograde tracing results are cross validated by
retrograde labeling injections at the anterograde fiber terminal fields and vice versa.
Anterograde AAV (or PHAL) tracer injections made into identical locations of a given
cortical area in two different mice resulted in identical projection patterns SC. Several
other injection cases were repeated as further controls and resulted in identical labeling
patterns.

Stereotaxic Surgeries.
On the day of experiment, mice were deeply anesthetized and mounted into a Kopf
stereotaxic apparatus where they were maintained under isoflurane gas anesthesia

20
(Datex-Ohmeda vaporizer). For triple anterograde injection experiments, PHAL was
iontophoretically delivered via glass micropipettes (inner tip diameter 24-32μm) using
alternating 7 sec on/off pulsed positive electrical current (Stoelting Co. current source)
for 10 min, and AAVs were delivered via the same method for 2 min (inner tip diameter
8-12μm). For anterograde/retrograde coinjection experiments, tracer cocktails were
iontophoretically delivered via glass micropipettes (inner tip diameter 28-32μm) using
alternating 7sec on/off pulsed positive electrical current (Stoelting Co. current source)
for 5 (BDA or AAV/FG) or 10 min (PHAL/CTB-647). For quadruple retrograde tracing
experiments, at each injection site, 50nL of the retrograde tracer was individually
pressure-injected via glass micropipettes at a rate of 10nL/min (Drummond Nanoject III).
Rabies injections of 10nL were individually pressure-injected via glass micropipettes
(inner tip diameter 8-12μm). All injections were placed in the right hemisphere. Injection
site coordinates for each surgery case are in Table 2.1 based on the (ML, AP, DV)
coordinate system from the 2008 Allen Brain Reference coronal atlas
52
. Following
injections, incisions were sutured, and mice received analgesic pain reliever and were
returned to their home cages for recovery.

Histology and Immunohistochemical Processing.
After 1-3 weeks post-surgery, each mouse was deeply anesthetized with an overdose of
Euthasol and trans-cardially perfused with 50ml of 0.9% saline solution followed by 50ml
of 4% paraformaldehyde (PFA, pH 9.5). Following extraction, brain tissue was post-fixed
in 4% PFA for 24-48hr at 4°C. Fixed brains were embedded in 3% Type I-B agarose
(Sigma-Aldrich) and sliced into four series of 50μm thick coronal sections using a
Compresstome (VF-700, Precisionary Instruments, Greenville, NC; RRID:SCR_018409)
and stored in cryopreservant at -20°C.

21
For double coinjection experiments, one series of tissue sections was processed for
immunofluorescent tracer localization. For PHAL or AAVretro-EF1a-Cre
immunostaining, sections were placed in a blocking solution containing normal donkey
serum (Vector Laboratories) and Triton X (VWR) for 1 hr. After rinsing in buffer, sections
were incubated in PHAL primary antiserum (1:100 rabbit anti-PHAL antibody (Vector
Laboratories Cat# AS-2300, RRID:AB_2313686)) or AAVretro-EF1a-Cre primary
antiserum (1:100) mixed with donkey serum, Triton X, in KPBS buffer solution for 48-72
hours at 4°C. Sections were then rinsed again in buffer solution and then immersed in
secondary antibody solution (donkey serum, Triton X, and 1:500 donkey anti-mouse IgG
conjugated with Alexa Fluor 488 (Thermo Fisher Scientific Cat# A-31571,
RRID:AB_162542)), or 1:500 donkey anti-rabbit conjugated with CY3 (Jackson
ImmunoResearch Labs Cat# 715-165-151, RRID:AB_2315777) for 3 hrs. BDA
immunofluorescence was visualized using a 647- or 568-conjugated Streptavidin. Finally,
all sections were stained with Nissl Neurotrace 435/455 (Thermo Fisher Cat# N21479) for
2-3 hours to visualize cytoarchitecture. After processing, sections were mounted onto
microscope slides and cover slipped using 65% glycerol.

22
2.3.2 2D IMAGING AND INFORMATICS PROCESSING
Overview.
Complete tissue sections were scanned using a 10X objective lens on an Olympus VS120
slide scanning microscope. Each tracer was visualized using appropriately matched
fluorescent filters and whole tissue section images were stitched from tiled scanning into
VSI image files. For online publication, raw images are corrected for correct left-right
orientation and matched to the nearest Allen Reference Atlas level (ARA)
52
.

Connection Lens Workflow.
An informatics workflow was specifically designed to reliably warp, reconstruct,
annotate and analyze the labeled pathways in a high-throughput fashion through our in-
house image processing software, Connection Lens (Figure 2.2b). Tissue sections from
each analyzed case were assigned and registered to a standard set of 10 corresponding
ARA levels ranging from 84-102. All images shown in this manuscript are from the raw
data of unwarped, unregistered VSI images. Threshold parameters were individually
adjusted for each case and tracer. Adobe Photoshop (RRID:SCR_014199) was used to
correct conspicuous artifacts in the threshold output files. Each color channel was
brightness/contrast adjusted to maximize labeling visibility (Nissl Neurotrace 435/455
is converted to brightfield), and TIFF images are then converted to JPEG file format.

Assessment of Injection Sites and Fibers of Passage.
All cortical injection cases included in this work are, in our judgment, prototypical
representatives of each cortical area. We have demonstrated our targeting accuracy with
respect to injection placement, our attention to injection location, and the fidelity of
labeling patterns derived from injections to the same cortical location in previous

23
studies
4,27
. In the current report, we also demonstrate our injection placement accuracy
and the consistent labeling resulting from injections placed in the same cortical areas as
described in detail in Supplementary Methods for Hintiryan et al., 2016. A combination
of labeling produced from both anterograde and retrograde cases across cortex and SC
was used to generate the input/output connectome diagram (Figure 2.1; Figure 2.9;
Figure 2.11). An important concern when employing automated analysis of connectivity
is the issue of fibers of passage getting annotated as functional connections when in fact
no synapses exist. Pathways devoid of synapses can produce bright labeling that can be
annotated as positive pixels. This is especially relevant with regard to cortico-tectal fibers
that travel from the rostral direction through the SC onto a caudal SC termination site.
For example, ARA 86 level exhibited more labeling from fibers of passage entering
rostrally from both sensorimotor and higher-order cortical areas such as MOp/MOs
(Figure 2.4d), PTLp (Figure 2.6f) and RSPv-rostral (Figure 2.7a). Some pixel fibers had
evident terminal boutons, as determined by comparison and validation with the raw
tissue data, thus these pixels remained for quantification. Pixels that arose from lines of
straight bold fibers (indicative of fibers of passage as validated by raw data for each
section) void of boutons or synapses were removed from analysis. The pixel values that
were indistinguishable from terminals remained as part of the computational analysis.

Defining the SC Custom Atlas and Delineating SC Zones.
The workflow was applied toward analyzing projections from 86 representative cortical
injection cases to sections with characteristic labeling across the far rostral SC (ARA 86),
rostral SC (ARA 90), middle SC (ARA 96) and caudal SC (ARA 100). ARA level 86 is the
furthest rostral where notable fiber terminations were observed. The labeling at level 90
(-3.68mm from bregma) was the target of rich projections from all cortical areas,

24
representative of all cortico-tectal projections, and exhibited the most segregation of
labeling in terms of unique zones. Therefore, it was selected as the representative section
for the rostral SC. The SC at level 86 (-3.28mm from bregma) was the furthest rostral level
we could use for analysis that expressed prominent terminals from a subset of cases.
Therefore, it was selected as the representative section for the far rostral SC. The SC at
levels 96 (-4.28mm from bregma) and 100 (-4.65mm from bregma) displayed
distinguishable labeling with variable degrees of layer- and zone-specific cortical inputs
and far fewer discrete termination fields compared to the rostral SC and were therefore
selected to represent the middle and caudal SC, respectively.
In the Allen Brain Reference Atlas (ARA), the SC spans ARA levels 83 through 104,
which measures out to about 2mm in the rostral-caudal axis. ARA levels 86 through 100
were selected for analysis as they represent the majority of the SC volume across all
dimensions. Specifically, these levels span ~1.4mm in the rostral-caudal axis, and extend
up to 3mm in dorsal-ventral depth and up to 2mm in midline-lateral width. The extreme
rostral levels (83-85) and extreme caudal levels (101-104) span the remaining 0.6mm in
the rostral-caudal axis (only about 0.3mm at each extreme end, respectively), and each
extends only about 1mm in dorsal-ventral depth and 1mm in midline-lateral width.
Based on the dimensions of the SC throughout the reference atlas coordinate frame, the
structural volume between atlas levels 86 through 100 constitutes approximately 90% of
the total volume.
We overlapped reconstructed cortico-tectal fibers from the 86 cases to each custom
SC level (86, 90, 96, and 100), and determined the boundaries of the four zone delineations
based on the average patterns generated. Some cortico-tectal patterns naturally
distributed across more than one zone, though the general boundaries were still
consistent within the four zones. As representative examples, anterograde AAV

25
injections into the right hemispheres of PTLp (red), VISam (orange), ACAd (green) and
MOp (purple) terminate with different laminar and regional patterns in SC (ARA 90)
(Figure 2.2a). PTLp produced ipsilateral terminals in the intermediate layers of SC,
delineating the medial zone (SC.m). VISam produced tiered terminals in the SC
superficial and intermediates layers adjacent to SC.m, delineating the centromedial zone
(SC.cm). ACAd produced tiered terminals in the SC intermediate and deeper layers,
laterally adjacent to the VISam terminal field, delineating the centrolateral zone (SC.cl).
MOp produced terminals targeting the far lateral SC, delineating lateral zone (SC.l).
Together, these representative cortico-tectal projections reveal layer-specific terminals
distributed across four distinct zones along the medial-lateral axis of SC.

Polar Coordinate Analysis and Validation of SC Zone Borders.
We designed a computational method to validate our manual annotation and delineation
of the zone borders in the SC. We refer to this as the polar coordinate analysis. Given the
roughly circular nature of the SC structure relative to the midline, we assigned each pixel
of labeling an x and y coordinate value to plot. This renders the angular position and
distribution of terminal labeling for each cortical case. Reconstructed pixel labeling from
each case was plotted as a probability distribution graph where the y-axis represents the
probability density and the x-axis represent the theta angle in degrees (θ°) where the
labeling is found in the SC. Each case is plotted as a normalized smooth histogram where
the area under the curve equals 1. The height of each plot represents how likely the
cortical region projects to the SC at any given angle of theta (i.e., the y-axis value is the
projection density at each angular position). Notably, cases that have a similar histogram
also have a similar projection profile which facilitates in assessing the reproducibility of
labeling patterns from injection sites at the same ROI.

26
At each ARA level 86, 90, 96, and 100, the midline θ value was set to 90°,
corresponding to beginning of the SC.m zone on the left side of the x-axis (Figure 2.3a).
The θ values decreased in value as they shifted to the right corresponding to the SC.cm,
SC.cl, and SC.l zone at the far right, respectively. Histogram curves of individual cases
with confined projections to each SC zone were plotted on the same graph at each ARA
level (Figure 2.3b). The average distribution plots for each zone at each ARA level were
plotted to analyze the approximate θ° range for each zone (Figure 2.3c). The four average
probability distribution plots show peaks with ranges that closely align with the
manually delineated borders in the custom atlas levels. Each zone was clearly
distinguishable based on the average peaks, with the exception at ARA 100 with SC.cl
and SC.l zones where the structure is much smaller relative to the rest of the SC and the
peaks are mostly overlapping. Importantly, the geometric radial lines do not entirely
reflect the natural curvature of the SC, and our manual borders were delineated based on
the raw image data to reflect the natural shape of the structure. Here in Table 2.2, we
provide a list of the approximate theta (θ°) ranges for each SC zone at each atlas level of
analysis.

Table 2.2 | Angular Ranges for Each SC Zone.
SC.m SC.cm SC.cl SC.l
ARA 86 90-75° 75-60° 60-45° 45-30°
ARA 90 90-75° 75-55° 55-35° 35-10°
ARA 96 90-75° 75-60° 60-45° 45-5°
ARA 100 90-75° 75-60° 60-45° 45-0°

27
Color-Coding, Data Visualization, and Proportional Stacked Bar Charts.
The same color scheme within SC zones was used for consistency of reference throughout
the diagrams: SC.m (red), SC.cm (orange), SC.cl (green), SC.l (purple). In the topographic
map of the brain dorsal view (Figures 2.4g, 2.6b, 2.11e), cortical areas were color-coded
based on their SC zone target. Stacked bar charts for visualization of proportion of
labeling across each SC zone (x-axis) from each cortical ROI (y-axis). Values represent
proportion of pixel density for the selected ROI (n=1 per cortical area) distributed across
each SC zone across all layers. The chart facilitates an overview representation of how
cortico-tectal projections preferentially target specific zones and how they compare
across cortical groups. For complete breakdown of values for zone- and layer-specific
proportion of label bar charts, see Tables 2.3 and 2.4.

______________________________________________________________________________
Table 2.3 | Proportion of labeling values: sensory cortices.
Values represent proportion of pixel density for the selected ROI (n=1 per cortical area) distributed across
each SC zone across all layers. Far right summations correspond to values used in Figure 2.4g.

Table 2.4 | Proportion of labeling values: association areas.
Values represent proportion of pixel density for the selected ROI (n=1 per cortical area) distributed across
each SC zone across all layers. Far right summations correspond to values used in Figure 2.6b.

28
Table 2.3 Table 2.4

SC.m-
zo
SC.m-
sg
SC.m-
op
SC.m-
ig
SC.m-
iw
SC.m-
dg
SC.m-
dw
SC.cm-
zo
SC.cm-
sg
SC.cm-
op
SC.cm-
ig
SC.cm-
iw
SC.cm-
dg
SC.cm-
dw
SC.cl-
zo
SC.cl-
sg
SC.cl-
op
SC.cl-
ig
SC.cl-
iw
SC.cl-
dg
SC.cl-
dw
SC.l-
ig
SC.l-
iw
SC.l-
dg
SC.l-
dw
SC.m SC.cm SC.cl SC.l
VISp_caudal 0.45 0.57 0.08 0.00 0.00 0.00 0.00 0.00 0.05 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.10 0.06 0.00 0.00
VISam 0.00 0.04 0.13 0.22 0.09 0.03 0.00 0.00 0.07 0.15 0.13 0.07 0.03 0.02 0.00 0.02 0.07 0.02 0.02 0.01 0.02 0.00 0.00 0.00 0.02 0.51 0.47 0.16 0.02
VISal 0.00 0.10 0.12 0.04 0.00 0.00 0.00 0.01 0.34 0.50 0.05 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.26 0.90 0.01 0.00
VISp_caudomed 0.00 0.02 0.03 0.01 0.00 0.00 0.00 0.22 0.53 0.30 0.04 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.06 1.09 0.01 0.00
VISp_rostromed 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.05 0.13 0.13 0.03 0.00 0.00 0.00 0.19 0.44 0.20 0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.34 0.85 0.00
AUDp_interm 0.00 0.00 0.00 0.00 0.13 0.07 0.00 0.00 0.00 0.00 0.07 0.20 0.35 0.05 0.00 0.00 0.00 0.00 0.06 0.07 0.02 0.13 0.00 0.02 0.00 0.20 0.67 0.15 0.15
MOs_medial 0.00 0.00 0.02 0.06 0.06 0.01 0.01 0.00 0.00 0.03 0.08 0.05 0.01 0.01 0.00 0.01 0.03 0.13 0.15 0.07 0.06 0.03 0.06 0.08 0.21 0.16 0.18 0.45 0.38
SSp_tr 0.00 0.00 0.01 0.06 0.04 0.00 0.00 0.00 0.00 0.00 0.15 0.05 0.00 0.00 0.00 0.00 0.02 0.25 0.21 0.07 0.01 0.12 0.08 0.08 0.00 0.11 0.20 0.56 0.28
AUDd 0.00 0.00 0.02 0.04 0.00 0.00 0.00 0.00 0.00 0.00 0.18 0.03 0.00 0.00 0.00 0.00 0.00 0.19 0.13 0.06 0.00 0.09 0.24 0.18 0.00 0.06 0.21 0.38 0.51
MOs_caudal 0.00 0.00 0.00 0.02 0.01 0.01 0.02 0.00 0.00 0.00 0.01 0.03 0.01 0.02 0.00 0.00 0.05 0.12 0.14 0.08 0.06 0.04 0.07 0.07 0.42 0.06 0.07 0.45 0.60
MOs_lateral 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.02 0.00 0.00 0.00 0.01 0.00 0.07 0.26 0.10 0.01 0.00 0.25 0.18 0.10 0.18 0.00 0.02 0.45 0.71
SSp_bfd 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.25 0.06 0.00 0.00 0.51 0.30 0.05 0.00 0.00 0.00 0.31 0.86
MOp-bfd 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.11 0.15 0.00 0.00 0.00 0.44 0.43 0.02 0.01 0.00 0.00 0.26 0.90
MOp-tr 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.24 0.11 0.02 0.00 0.00 0.66 0.04 0.09 0.00 0.00 0.00 0.38 0.79
MOs_rostral 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.07 0.00 0.00 0.00 0.81 0.19 0.08 0.00 0.00 0.00 0.08 1.08
SSp_ll 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.02 0.06 0.01 0.00 0.00 0.50 0.27 0.32 0.00 0.00 0.00 0.09 1.09
MOp-oro 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.05 0.01 0.00 0.00 0.50 0.54 0.07 0.00 0.00 0.00 0.06 1.11
SSp_m 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.82 0.32 0.02 0.00 0.00 0.00 0.00 1.16
SSp_n 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.81 0.32 0.04 0.00 0.00 0.00 0.00 1.17
SSp_ul 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.64 0.39 0.14 0.00 0.00 0.00 0.00 1.17
SSs 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.89 0.25 0.03 0.00 0.00 0.00 0.00 1.17
SC.m SC.cm SC.cl SC.l Summations

SC.m-
zo
SC.m-
sg
SC.m-
op
SC.m-
ig
SC.m-
iw
SC.m-
dg
SC.m-
dw
SC.cm-
zo
SC.cm-
sg
SC.cm-
op
SC.cm-
ig
SC.cm-
iw
SC.cm-
dg
SC.cm-
dw
SC.cl-
zo
SC.cl-
sg
SC.cl-
op
SC.cl-
ig
SC.cl-
iw
SC.cl-
dg
SC.cl-
dw
SC.l-
ig
SC.l-
iw
SC.l-
dg
SC.l-
dw
SC.m SC.cm SC.cl SC.l
RSPv_caudal 0.01 0.14 0.40 0.25 0.51 0.25 0.04 0.00 0.04 0.16 0.11 0.21 0.03 0.04 0.00 0.01 0.01 0.02 0.01 0.01 0.05 0.00 0.00 0.00 0.00 1.61 0.61 0.12 0.00
RSPv_interm 0.05 0.28 0.57 0.49 0.19 0.04 0.00 0.05 0.10 0.22 0.18 0.03 0.02 0.03 0.00 0.01 0.01 0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.03 1.64 0.62 0.05 0.03
RSPd_rostral 0.00 0.06 0.30 0.41 0.33 0.06 0.05 0.00 0.02 0.15 0.23 0.30 0.05 0.03 0.00 0.00 0.03 0.01 0.07 0.02 0.04 0.00 0.00 0.01 0.16 1.20 0.79 0.18 0.16
RSPd_caudal 0.00 0.04 0.13 0.22 0.27 0.08 0.00 0.02 0.12 0.25 0.26 0.43 0.22 0.13 0.00 0.01 0.03 0.00 0.03 0.01 0.08 0.00 0.00 0.00 0.00 0.75 1.43 0.16 0.01
RSPagl 0.00 0.02 0.09 0.34 0.37 0.09 0.05 0.00 0.05 0.18 0.28 0.23 0.26 0.19 0.00 0.00 0.01 0.01 0.01 0.04 0.08 0.00 0.00 0.00 0.00 0.98 1.20 0.16 0.00
PTLp_lat 0.00 0.02 0.26 0.58 0.04 0.00 0.00 0.00 0.01 0.12 0.75 0.07 0.00 0.00 0.00 0.00 0.01 0.09 0.17 0.08 0.01 0.01 0.04 0.02 0.05 0.91 0.95 0.37 0.11
TEa_caudal 0.00 0.01 0.06 0.08 0.46 0.25 0.09 0.00 0.01 0.02 0.14 0.21 0.39 0.16 0.00 0.00 0.00 0.03 0.02 0.18 0.23 0.01 0.00 0.00 0.00 0.95 0.92 0.46 0.01
ACAv_caudal 0.00 0.00 0.05 0.30 0.01 0.02 0.01 0.00 0.00 0.16 0.38 0.27 0.21 0.06 0.00 0.02 0.08 0.06 0.28 0.09 0.11 0.00 0.00 0.01 0.23 0.39 1.07 0.63 0.24
ACAv_interm 0.00 0.01 0.05 0.14 0.13 0.10 0.14 0.00 0.01 0.05 0.20 0.26 0.20 0.32 0.00 0.01 0.03 0.04 0.08 0.14 0.23 0.00 0.00 0.03 0.16 0.58 1.03 0.52 0.20
ACAv_rostral 0.00 0.00 0.02 0.08 0.15 0.18 0.15 0.00 0.00 0.01 0.11 0.28 0.26 0.25 0.00 0.00 0.01 0.05 0.14 0.19 0.38 0.00 0.01 0.05 0.01 0.58 0.91 0.77 0.07
RSPv_rostral 0.00 0.00 0.02 0.05 0.01 0.03 0.00 0.00 0.00 0.05 0.30 0.11 0.05 0.03 0.00 0.00 0.04 0.16 0.19 0.19 0.20 0.02 0.01 0.04 0.83 0.11 0.54 0.79 0.90
PTLp_med 0.00 0.00 0.02 0.03 0.36 0.24 0.17 0.00 0.00 0.01 0.03 0.26 0.13 0.18 0.00 0.02 0.07 0.07 0.23 0.20 0.28 0.01 0.01 0.04 0.00 0.82 0.60 0.86 0.06
ACAd_caudal 0.00 0.00 0.00 0.06 0.01 0.02 0.03 0.00 0.00 0.02 0.13 0.10 0.03 0.05 0.00 0.07 0.23 0.21 0.58 0.19 0.29 0.00 0.01 0.04 0.24 0.13 0.34 1.57 0.30
ACAd_interm 0.00 0.00 0.05 0.11 0.05 0.07 0.08 0.00 0.00 0.05 0.15 0.19 0.11 0.32 0.02 0.01 0.10 0.11 0.17 0.17 0.15 0.02 0.06 0.10 0.25 0.36 0.83 0.72 0.42
ACAd_rostral 0.00 0.00 0.00 0.03 0.13 0.06 0.08 0.00 0.00 0.00 0.05 0.27 0.03 0.35 0.00 0.00 0.01 0.10 0.28 0.18 0.10 0.03 0.11 0.28 0.22 0.31 0.70 0.69 0.64
PL 0.00 0.00 0.00 0.02 0.00 0.00 0.00 0.00 0.00 0.02 0.26 0.10 0.17 0.28 0.00 0.00 0.00 0.01 0.25 0.11 0.29 0.00 0.12 0.20 0.52 0.02 0.83 0.65 0.84
ORBm 0.00 0.00 0.01 0.12 0.00 0.00 0.00 0.00 0.00 0.11 0.10 0.12 0.06 0.24 0.00 0.07 0.09 0.07 0.13 0.22 0.50 0.03 0.17 0.32 0.00 0.13 0.62 1.08 0.51
ORBvl 0.00 0.00 0.01 0.04 0.00 0.00 0.00 0.00 0.00 0.02 0.09 0.08 0.03 0.00 0.01 0.00 0.03 0.29 0.41 0.22 0.05 0.06 0.33 0.13 0.55 0.05 0.22 1.01 1.06
ILA 0.00 0.02 0.04 0.09 0.10 0.20 0.08 0.00 0.04 0.09 0.03 0.02 0.01 0.13 0.04 0.12 0.39 0.06 0.05 0.06 0.02 0.13 0.15 0.19 0.26 0.54 0.33 0.74 0.73
TEa_rostral 0.00 0.01 0.09 0.11 0.17 0.20 0.13 0.00 0.00 0.00 0.03 0.14 0.10 0.04 0.00 0.00 0.00 0.03 0.16 0.15 0.16 0.24 0.29 0.23 0.06 0.72 0.30 0.49 0.82
ORBl 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.16 0.11 0.01 0.02 1.00 0.61 0.11 0.14 0.00 0.00 0.30 1.86

SC.m SC.cm SC.cl SC.l Summations

29
Community Detection and Connectivity Matrices.
We further analyzed the annotation data to objectively identify groups of cortical
injection sites that send converging inputs within different SC zones. To perform this final
stage of the neuroinformatics pipeline, we first built an adjacency matrix out of our
annotation data. The graph structure of the data is relatively simple: nodes and
connections are organized as a multi-tree with two levels: the cortex and the four SC
zones. We performed community detection (modularity maximization) on ROI
annotated data. The overlap annotation per each case within a group was aggregated into
a single matrix. Overlap refers to a convergence of terminal fields within the SC between
cortical source areas. The overlap value between a source and target ROI is the ratio of
common labeling among the two ROIs to total source labeling
27
. Once the aggregated
matrix was constructed, we further normalized the data so that the total labelling across
each injection site (typically close in the first place) was adjusted to equal with the
injection site featuring maximum total labeling. On this normalized matrix we applied
the Louvain community detection algorithm at a single scale (gamma 1.0) to the data and
identified clusters of injection sites with similar SC termination fields.
As the result of this greedy algorithm being non-deterministic, we performed 100
separate executions, and subsequently calculated a consensus community structure to
characterize the 100 executions as a single result. Given the modularity of the data (that
is, highly topographic labeling), a modularity optimization algorithm, like the Louvain,
was well suited. However, the element of randomness makes it probable that the
algorithm will reveal a different community structure over multiple runs. To mitigate
this issue, the algorithm was run 1,000 times. The community structure that emerged
most often, which we defined as the community structure mode (borrowing from
statistics) is reported. A mean and standard deviation for the number of communities

30
that was detected across the 1,000 runs was also computed. Subsequently, to aid
visualization we employed the community structure to re-order and color code an
adjacency matrix such that connections were placed close to the diagonal. An
accompanying color-coded SC illustrates the spatial arrangement of the
communities/zones. The code for generating the illustrative color-coded SC employed a
‘winner takes all’ reconstruction of community terminal fields. For each cell, the method
compared overlap data from across injection sites and colored according to the
community with the greatest quantity of labeling.
For matrix visualization, we applied an algorithm to modularize ROIs based on
community assignment, as well as prioritize connections along the diagonal. Such a
visualization provides a high-level overview of the connectivity (Figure
2.6g). Community coloring provides more detail by visually encoding segmentation by
community, and ultimately, injection site (anterograde visuals). To carry out this process,
we developed software to programmatically march through each segmented pixel per
each image. Using the ROI name, the algorithm looked up the corresponding community
assigned during the consensus community step. Using a table containing a color assigned
(by the authors) to each injection site, the algorithm retrieved the injection site associated
with the community and colored the pixel with the corresponding injection site color
value. A subsequent step took advantage of the fact that each pixel was assigned to only
a single community by aggregating all colorized images corresponding to a given atlas
level into a single representative image.

31
METHODS (FOR RESULTS PART III)

2.4.1 3D TISSUE PROCESSING AND IMAGING PROTOCOL
To assess whether thalamic-projection neurons in the 4 SC zones were morphologically
distinct, representative neurons in each zone were labeled via a G-deleted-rabies injection
in the LD (for SC.m/cm), in the RE (for SC.m/cm), and dorsal PF (Figure 2.14a; Figure
2.16). One week was allowed for tracer transport following injections, after which the
animals were perfused. The SHIELD clearing protocol was used for the 3D tissue
processing workflow
53,54
, followed by neuronal reconstructions and statistical analyses.
Mice were transcardially perfused with cold saline and SHIELD perfusion solution. The
brains were extracted and incubated in the SHIELD perfusion solution at 4°C for 48
hours. The perfusion solution was replaced with the SHIELD OFF solution and tissues
were incubated at 4°C for 24 hours. The SHIELD OFF solution was replaced with the
SHIELD ON solution and the tissues were incubated at 37°C for 24 hours. The whole
brain was cut into 400µm sections and were cleared in the SDS buffer at 37°C for 72 hours.
The sections were then washed three times with KPBS and incubated in KPBS at 4°C for
24 hours. Sections were mounted and cover-slipped onto 25x75x1mm glass slides with
an EasyIndex matching solution 100% (LifeCanvas Technologies, #EI-Z1001). Sections
were imaged with a high speed spinning disk confocal microscope (Andor Dragonfly 202;
CR-DFLY-202-2540). 10x magnification (NA 0.40, Olympus) was used to acquire an
overview after which 30x magnification (NA 1.05, Olympus) was used to image through
the SC ipsilateral to the injection site at 1µm z steps. Sections were imaged with four
excitation wavelengths (nm): 405 (blue - Nissl background), 488 (green - Rabies), 561 (red
- Rabies) with respective emission detection wavelengths of 450, 525, and 600.

32
2.4.2 3D RECONSTRUCTIONS, VISUALIZATIONS, AND ANALYSIS OF
NEURONAL MORPHOLOGY
Manual reconstruction of the neurons was performed using Aivia (version.8.8.1,
DRVision) (Figure 2.16), and geometric processing of neuron models, including
smoothing, was performed using the Quantitative Imaging Toolkit (QIT)
NeuronTransform and NeuronFilter modules
55
(http://cabeen.io/qitwiki). The software
neuTube was used to facilitate visualizations of reconstructions
56
. All reconstructions are
being made freely available in the Dong archive on the online NeuroMorpho
57
database
(www.NeuroMorpho.org). Although we wanted to compare projection neurons from
each SC zone to each thalamic area, neurons from each injection were spread across two
to four zones. This allowed us to compare SC neurons in neighboring zones that project
to the same thalamic nucleus, as well as those that project to a different thalamic nucleus.
Three groups were assigned from the RE rabies injected case based on the location of
retrogradely labeled cells in SC: RE-SC.m (n=23), RE-SC.cm (n=27), and RE-SC.cl (n=4).
Two groups were assigned from the LD case: LD-SC.m (n=4), and LD-SC.cm (n=4). Four
groups were assigned from the PF/MPT case: PF/MPT-SC.m (n=10), PF/MPT-SC.cm
(n=6), PF/MPT-SC.cl (n=9), PF/MPT-SC.l (n=5). Cells for this analysis were selected as
representative thalamus projecting SC neurons and are not representative of the entire
population of neurons within the four zones or layers.

33
2.4.3 STATISTICAL ANALYSES ON GROUPS OF RECONSTRUCTIONS
To obtain an overall view of the dendritic morphology of SC projection neurons located
in the SC.m, SC.cm, and SC.cl, we applied the classic Sholl analysis using Fiji (ImageJ,
RRID:SCR_003070). Quantitative morphological parameters characterizing arbor
morphology were obtained from L-Measure
58
, and statistical analyses were performed
using the R computing environment (RStudio Version 1.1.463). Using all of the measured
morphological parameters, principal component analysis (PCA) was run to reduce the
dimensionality and create a 2D scatterplot. The PCA shows the segregation of SC zone-
specific neurons based on the measured features, namely the segregation of SC.m-
PF/MPT from all groups, including SC.m projecting neurons to both RE and LD.
Neurons from SC.cm/cl/l projecting to PF/MPT are completely segregated from all
SC.m projecting groups (Figure 2.14b). We calculated a comprehensive battery of
standard measurements, including the number of bifurcations, contraction, width, and
branch path length, to compare morphological features. Statistically significant
differences in several morphological features were detected across all pairwise
comparisons (Table 2.5). Examples of parameters with significant group differences and
greater loading values influencing the PCA are presented with whisker plots (Figure
2.14c). Two-sided pairwise Wilcoxon rank sum tests were performed, and the parameters
that survived FDR correction for multiple testing with a p-value <0.05 are reported.
Significant differences were detected across several pairwise parameters. Our
experiments used code provided by the authors to generate online diagrams using
NeuronTools (https://github.com/Nevermore520/NeuronTools) and the Wasserstein
metric (https://bitbucket.org/grey_narn/geom_matching/src).

34
Table 2.5 | Morphometric p-values for SC neuron reconstructions.
Table of pairwise comparisons using the Wilcoxon rank sum test. Adjustments for p-value were made for
multiple comparisons using the false discovery rate (fdr) method. P-values are listed for the following
neuronal morphometric parameters: contraction, width, number of bifurcations, branch path length, fractal
dimension, fragmentation, height, number of branches, and height/width ratio.

PF/MPT - SC.m RE - SC.m LD - SC.m PF/MPT - SC.cm RE - SC.cm LD - SC.cm PF/MPT - SC.cl RE - SC.cl
RE - SC.m 0.0019 - - - - - - -
LD - SC.m 0.024 0.5253 - - - - - -
PF/MPT - SC.cm 0.9578 0.0064 0.049 - - - - -
RE - SC.cm 0.1386 0.052 0.1172 0.2261 - - - -
LD - SC.cm 0.1439 0.4747 0.2057 0.1412 0.5471 - - -
PF/MPT - SC.cl 0.2981 0.0019 0.0503 0.5471 0.0166 0.093 - -
RE - SC.cl 0.4555 0.1386 0.4747 0.553 0.7076 0.7261 0.1439 -
PF/MPT - SC.l 0.4946 0.0064 0.052 0.7226 0.052 0.093 0.82 0.2981
PF/MPT - SC.m RE - SC.m LD - SC.m PF/MPT - SC.cm RE - SC.cm LD - SC.cm PF/MPT - SC.cl RE - SC.cl
RE - SC.m 0.0058 - - - - - - -
LD - SC.m 0.2038 0.00068 - - - - - -
PF/MPT - SC.cm 0.0844 0.2038 0.01633 - - - - -
RE - SC.cm 0.00057 0.03547 0.00057 0.01554 - - - -
LD - SC.cm 0.73327 0.00738 0.03547 0.02743 0.00057 - - -
PF/MPT - SC.cl 0.00134 0.01633 0.00719 0.01518 0.35848 0.00719 - -
RE - SC.cl 0.00599 0.00068 0.03547 0.01633 0.0058 0.03547 0.04028 -
PF/MPT - SC.l 0.00266 0.00057 0.02381 0.00917 0.00188 0.02381 0.00899 0.2038
PF/MPT - SC.m RE - SC.m LD - SC.m PF/MPT - SC.cm RE - SC.cm LD - SC.cm PF/MPT - SC.cl RE - SC.cl
RE - SC.m 0.0003 - - - - - - -
LD - SC.m 0.02569 0.10155 - - - - - -
PF/MPT - SC.cm 0.01038 0.06615 0.05382 - - - - -
RE - SC.cm 0.00037 0.01038 0.02569 0.67932 - - - -
LD - SC.cm 0.03429 0.02823 0.06615 0.14814 0.47941 - - -
PF/MPT - SC.cl 0.00514 0.8358 0.14988 0.27131 0.12966 0.10489 - -
RE - SC.cl 0.03429 0.06615 0.06615 0.67771 0.8358 0.47941 0.17882 -
PF/MPT - SC.l 0.01546 0.9272 0.44167 0.54313 0.17882 0.17241 0.9185 0.48076
PF/MPT - SC.m RE - SC.m LD - SC.m PF/MPT - SC.cm RE - SC.cm LD - SC.cm PF/MPT - SC.cl RE - SC.cl
RE - SC.m 0.00000072 - - - - - - -
LD - SC.m 0.01028 0.23028 - - - - - -
PF/MPT - SC.cm 0.11847 0.00061 0.04699 - - - - -
RE - SC.cm 0.00000072 0.28913 0.33678 0.00072 - - - -
LD - SC.cm 0.01654 0.03506 0.18701 0.18701 0.06164 - - -
PF/MPT - SC.cl 0.01654 0.00148 0.04699 0.30984 0.00548 0.77508 - -
RE - SC.cl 0.02877 0.51997 0.18701 0.33678 0.82103 0.77143 0.49678 -
PF/MPT - SC.l 0.03796 0.04699 0.18701 0.26623 0.13664 0.90476 0.82103 0.49678
PF/MPT - SC.m RE - SC.m LD - SC.m PF/MPT - SC.cm RE - SC.cm LD - SC.cm PF/MPT - SC.cl RE - SC.cl
RE - SC.m 0.00531 - - - - - - -
LD - SC.m 0.06165 0.88571 - - - - - -
PF/MPT - SC.cm 0.83907 0.01258 0.08571 - - - - -
RE - SC.cm 0.12321 0.0526 0.18775 0.15982 - - - -
LD - SC.cm 0.18153 0.40418 0.42562 0.35604 0.76544 - - -
PF/MPT - SC.cl 0.08397 0.00012 0.02877 0.18775 0.00088 0.04476 - -
RE - SC.cl 0.11424 0.42562 0.76544 0.18701 0.63901 0.88571 0.04476 -
PF/MPT - SC.l 0.14259 0.01258 0.04762 0.35532 0.01258 0.04762 0.43636 0.04762
PF/MPT - SC.m RE - SC.m LD - SC.m PF/MPT - SC.cm RE - SC.cm LD - SC.cm PF/MPT - SC.cl RE - SC.cl
RE - SC.m 0.00000026 - - - - - - -
LD - SC.m 0.00621 0.06137 - - - - - -
PF/MPT - SC.cm 0.8645 0.000025 0.01805 - - - - -
RE - SC.cm 0.00000026 0.00621 0.00629 0.000013 - - - -
LD - SC.cm 0.00621 0.51997 0.15238 0.01805 0.1981 - - -
PF/MPT - SC.cl 0.0626 0.00000064 0.00629 0.02752 0.00000026 0.00629 - -
RE - SC.cl 0.00621 0.64756 0.15238 0.01805 0.31541 0.93782 0.00629 -
PF/MPT - SC.l 0.95305 0.0001 0.02597 0.95305 0.00018 0.02597 0.29763 0.02597
PF/MPT - SC.m RE - SC.m LD - SC.m PF/MPT - SC.cm RE - SC.cm LD - SC.cm PF/MPT - SC.cl RE - SC.cl
RE - SC.m 0.00000026 - - - - - - -
LD - SC.m 0.0048 0.00057 - - - - - -
PF/MPT - SC.cm 0.00327 0.00057 0.02017 - - - - -
RE - SC.cm 0.0000001 2.7E-12 0.00046 0.0265 - - - -
LD - SC.cm 0.0048 0.03979 0.04472 0.05331 0.06077 - - -
PF/MPT - SC.cl 0.00019 0.00103 0.00629 0.30614 0.12993 0.12993 - -
RE - SC.cl 0.0048 0.00057 0.04472 0.5042 0.0048 0.04472 0.51788 -
PF/MPT - SC.l 0.0024 0.05331 0.04762 0.06679 0.33766 0.55556 0.12993 0.03008
PF/MPT - SC.m RE - SC.m LD - SC.m PF/MPT - SC.cm RE - SC.cm LD - SC.cm PF/MPT - SC.cl RE - SC.cl
RE - SC.m 0.00022 - - - - - - -
LD - SC.m 0.01835 0.10961 - - - - - -
PF/MPT - SC.cm 0.00951 0.05037 0.05037 - - - - -
RE - SC.cm 0.00022 0.00951 0.01835 0.66578 - - - -
LD - SC.cm 0.01835 0.01835 0.06367 0.26852 0.64114 - - -
PF/MPT - SC.cl 0.00323 0.74031 0.12302 0.18623 0.12793 0.06367 - -
RE - SC.cl 0.01835 0.05037 0.06367 1 0.95915 0.4047 0.10961 -
PF/MPT - SC.l 0.0156 1 0.48076 0.23764 0.10961 0.10961 0.95915 0.41544
PF/MPT - SC.m RE - SC.m LD - SC.m PF/MPT - SC.cm RE - SC.cm LD - SC.cm PF/MPT - SC.cl RE - SC.cl
RE - SC.m 0.00000039 - - - - - - -
LD - SC.m 0.00527 0.04482 - - - - - -
PF/MPT - SC.cm 0.00527 0.00016 0.01797 - - - - -
RE - SC.cm 0.00000021 0.00527 0.0058 0.00246 - - - -
LD - SC.cm 0.00527 0.00527 0.04114 0.12857 0.04482 - - -
PF/MPT - SC.cl 0.00016 0.01797 0.01185 0.01518 0.84875 0.09375 - -
RE - SC.cl 0.00527 0.09805 0.04114 0.02981 0.93215 0.12857 0.84875 -
PF/MPT - SC.l 0.00343 0.07907 0.79654 0.00974 0.00016 0.02597 0.0045 0.02597
Height
Number of Branches
Height/Width Ratio
Fragmentation
Fractal Dimension
Branch Path Length
Number of Bifurcations
Width
Contraction

35
RESULTS (PART I) : THE CORTICO-TECTAL PROJECTOME

2.5.1 GENERAL STRATEGY FOR DELINEATING THE CORTICO-TECTAL
PROJECTOME
First, we systematically analyzed and annotated the cortico-tectal projection patterns of
86 anterograde injections placed across the entire cortex in adult mice (n= 44) (Figure 2.1).
Single anterograde tracer injections of either Phaseolus vulgaris leucoagglutinin (PHAL),
or adeno-associated viruses expressing green (AAV-GFP), or red (AAV-tdTomato)
fluorescent protein were confined to a single cortical area to produce regional specific
projection terminal patterns in the SC. We also carried out double or triple tracer
injections with combinations of PHAL, AAV-GFP and AAV-tdTomato to determine
direct spatial correlations of axonal terminals arising from different cortical areas.
Notably, though we present a representative sample of cortical regions, the discrete
volume of our tracer injections may also render some regions under sampled. To conduct
systematic analyses, raw microscopic images with tracer-labeled cortico-tectal projections
from each individual experimental case were assigned to their corresponding atlas
template levels from the adult mouse brain Allen Reference Atlas (ARA)
52
(see Methods).
Our manual annotations revealed that each cortical area sent unique efferents to
distinct layers and regions in the SC. Some examples from representative cases include
a) terminals from MOp preferentially targeting the furthest lateral region in SC; b) ACAd
terminals targeted a region more dorsal-medially adjacent to MOp; c) VISam was more
medially adjacent to ACAd; and d) PTLp was more medial to VISam adjacent to the
midline (Figure 2.2a). Terminals from these cases often spanned multiple SC layers, but
displayed distinct layer specificities. These projections reveal four distinctive columns or
zones arranged from the midline to the lateral border of the SC. Comparisons across

36
multiple cases (n=44) reveal a consistent distribution pattern which we qualitatively
delineated into regional zones of our custom SC atlas: SC medial (SC.m), centromedial
(SC.cm), centrolateral (SC.cl) and lateral (SC.l) (Figure 2.2a). Though other patterns and
organization schemas may exist within SC
25
, this distribution pattern as determined by
the cortical inputs, suggests it may be a more refined, compact and anatomically relevant
way to analyze the organization of these circuits. Injection sites and tracer details for all
cases used throughout this study can be found in Table 2.1.
To quantitatively test the prominence of these four zones from cortico-tectal
inputs, we applied computational neuroanatomical methods
4,27
to analyze the
representative cortico-tectal pathways that span the rostral-to-caudal and medial-to-
lateral extent of the cerebral cortex and SC. The selected images for analysis were
registered to their corresponding atlas templates at ARA levels 86, 90, 96 and 100,
representative of the rostral, intermediate and caudal SC levels (Figure 2.3). Anterograde
labels were then thresholded, graphically reconstructed, and overlapped onto the atlas
templates using our in-house Connection Lens software
27
(Figure 2.2b). Pixels that were
verified as fibers of passage and that did not represent axonal terminals were excluded
from quantification analysis.
A polar coordinate analysis was designed and applied to visualize how terminal
pixel densities are distributed around the radial axis of SC (across angular degrees
relative to the midline) (Figure 2.2c). The probability distribution for cortico-tectal
projections was plotted as a smoothed histogram, illustrating the overlap and similarities
of projection profiles for multiple cortical cases within distinct ranges of SC (Figure 2.2d).
Probability distribution plots show individual cortical projections that target distinct
zones (SC.m (n=7), SC.cm (n=4), SC.cl (n=12), and SC.l (n=20)) (Figure 2.3). Average
distributions clearly demonstrate the prominence of four peaks along the medial-to-

37
lateral axis validating the raw data, and the peaks are congruent with the manually
delineated zones of our custom SC atlas (Table 2.4). The peaks are also generally
continuous across the rostral-to-caudal axis, though there may be other methods to
parcellate the SC along this axis. We designated zone-specific layer nomenclature to
facilitate more detailed referencing and quantification of cortical inputs to the SC (Figure
2.2e). We hypothesize that these custom SC atlas zones reflect subnetwork communities
similar to those previously identified in cortico-cortical and cortico-striatal
subnetworks
4,27
.

38

Figure 2.1 | Flattened layout of mouse cortex with injection sites.
Shaded brain region corresponds to same shaded color box with corresponding numbers for each case.
Generated by expanding the length of each cortical area from the coordinate framework on the Allen
Institute Adult Mouse Brain Atlas. Filled circles indicate location of injection site. List of injection sites,
coordinates, and location can be cross-referenced by index number with Table 2.1.
______________________________________________________________________________

39

Figure 2.2 | Experimental Workflow.
a) Raw data examples of cortico-tectal projections targeting distinct zones within SC layers at ARA 90.
Anterograde AAV injections into PTLpàSC.m (red), VISamàSC.cm (orange), ACAdàSC.cl (green) and
MOpàSC.l (purple) terminate with different laminar and nonoverlapping patterns. Together, these
representative cortico-tectal projections reveal layer-specific terminals distributed across four distinct
zones along the medial-lateral axis of SC. Scale bar at injection sites is 500 µm, and 200 µm in lower SC
panels. b) The Connection Lens neuroinformatics workflow: Raw tissue image with anterograde labeling
is assigned to the corresponding standard atlas level (this example is ARA 96). The tissue is warped based
on template atlas borders and reconstructed using in-house neuroinformatics software. Thresholded
images are overlapped onto a custom atlas for zone- and layer-specific registration for pixel quantification.
c) Left: Polar Coordinate analysis method used to quantify angular distribution of thresholded pixel
labeling in SC. Angles represented by theta (θ°) values where midline starts at 90° and ranges toward 0° at
lateral angles. Right: Custom SC atlas with overlay of angular range show coarse alignment of manually
delineated borders in SC. d) Example of probability distribution graph for average distributions of zone-
specific cortico-tectal cases. Peaks are aligned with custom SC borders at ARA 90. SC.m (red) is SW180522-
04A. SC.cm (orange) is SW121221-03A. SC.cl (green) is SW171130-02A. SC.l (purple) is SW170410-04A. e)
Layer- and zone-specific SC nomenclature to facilitate referencing and quantification. Below: zone
delineations across representative ARA levels (86, 90, 96, 100) spanning rostral-to-caudal SC.
______________________________________________________________________________

40

Figure 2.3 |Probability distribution plots.
a) Atlas levels for the SC from rostral to caudal: ARA levels 86, 90, 96, and 100. Left side is polar coordinate
ranges used to quantify angular distribution of thresholded pixel labeling in SC. Angles represented by
theta (θ°) values where midline starts at 90° and ranges toward 0° at lateral angles. Right side is the custom
SC atlas with overlay of angular range showing coarse alignment of manually delineated borders in SC.
Color-code associations: red (SC.m), orange (SC.cm), green (SC.cl), purple (SC.l). b) Probability distribution
plots organized by individual cortical cases that target distinct zones. SC.m, SC.cm, SC.cl, and SC.l. Y-axis
represents pixel density, x-axis represents theta angle from medial to lateral. Columns of panels are
organized by ARA level displaying plots of individual cases that preferentially target to each SC zone. Top
row plots from cases that target SC.m (n=7), next row SC.cm (n=4), next row SC.cl (n=12), and last row SC.l
(n=20). c) Average of probability distribution plots from multiple cases. Average is based on cases from
data in b.
______________________________________________________________________________
SC ARA 96
0°
90°
θ°
30°
60°
SC ARA 86 SC ARA 100 SC ARA 90
Custom Standard Custom Standard Custom Standard Custom Standard
SC.m SC.cm SC.cl SC.l
Theta (θ°) Theta (θ°) Theta (θ°) Theta (θ°)
0°
90°
θ°
30°
60°
0°
90°
θ°
30°
60°
0°
90°
θ°
30°
60°
0°
90°
θ°
30°
60°
0°
90°
θ°
30°
60°
0°
90°
θ°
30°
60°
0°
90°
θ°
30°
60°
a
b
c
Theta distribution per case across SC zones
ARA 96 ARA 86 ARA 100 ARA 90
Theta (θ°) Theta (θ°) Theta (θ°) Theta (θ°)
ARA 96 ARA 86 ARA 100 ARA 90

41
2.5.2 VISUAL, SOMATOSENSORY, AND MOTOR CORTEX PROJECTION MAPS IN
SC
We constructed an anterograde cortico-tectal projection map based on neural inputs
arising from visual, auditory, and somatic sensorimotor areas (Figure 2.4a-b). The
primary visual cortical area (VISp) exclusively targets superficial SC layers, while the
secondary visual areas, including the anterolateral (VISal), lateral (VISl), anteromedial
(VISam), and posteromedial (VISpm), target both the superficial and intermediate layers
(Figure 2.4c; Figure 2.5a). The SC.m and SC.cm receive input from all visual cortices, and
represent the upper central and upper peripheral visual field. The SC.cl receives direct
inputs from the VISp-rostromedial domain that represents the lower central and lower
peripheral field, VISam and auditory cortex. Furthermore, the SC.cl receives dense inputs
from the MOs-medial domain that is equivalent to primate frontal eye field (MOs-fef)
(see Zingg et al., 2014).
From the inherent cytoarchitecture of the SC, the three superficial visual layers do
not extend into the far lateral SC, thereby distinguishing the SC.l from the other three
zones. The SC.l does not receive any visual inputs; instead, it predominantly receives
inputs from all somatic sensorimotor cortical areas (Figure 2.4d-f). These cortical
projections are topographically distributed in a unique somatotopic order. The rostral
SC.l dominantly receives denser inputs from the SSp-m (mouth), SSp-n (nose), and SSp-
ul (upper limb), and SSp-bfd (barrel field). By contrast, the SSp-ll (lower limb) and SSp-
tr (trunk) project more densely to the caudal levels of SC.l with extension into the SC.cl
(Figure 2.5b-c). Similarly, all MOp body domains predominantly project across the SC.l
overlapping with their counterpart SSp inputs. These data suggest that the SC.cl is
distinguished from the SC.l by receiving convergent visual, auditory, and somatic
sensorimotor information.

42
Quantification, represented as a proportion of labeling, illustrates the relative
distribution maps of these sensory cortical areas across each SC zone (Figure 2.4g; Table
2.3). Polar coordinate analysis demonstrates a higher probability of pixel densities of MOs
and SSp-ul inputs in the lateral angle ranges indicated by sharp peaks, segregated from
the sharp peaks of VIS regions in the medial angle ranges (Figure 2.4h).

______________________________________________________________________________

Figure 2.4 | Visual, auditory, and somatic sensorimotor map of cortical projections to SC zones.
a) Color-coded schematic overview of the left visual field and body part topography based on their target
zones in right hemisphere of SC at level 90. Reconstructions of cortico-tectal fibers adjacent. b) Schematic
of injection strategy using triple or double anterograde tracers into cortex, and custom SC atlas levels. c)
VISp, VISal, and PTLp-lat projections to SC.m and SC.cm. SC boundaries in all panels were delineated
based on Nissl-stained cytoarchitecture. Dashed lines correspond to specific layers in each SC level. Scale
bar 200 µm in SC panels. d) Two neighboring caudal MOs regions and a rostral MOp injection send
projections to adjacent, but non-overlapping zones in the SC.cl and SC.l. e) Rostral MOs and rostral-ventral
MOp projections to the SC.l zone. f) SSp-tr and AUDp projections target caudal SC.l zones. g) Stacked bar
chart for visualization of proportion of labeling across each SC zone (x axis) from each cortical ROI (y axis)
for each selected ROI (n=20 cases). Values represent proportion of pixel density for an individual ROI
across each zone (n=1 case per row). See Table 2.3 for zone- and layer-specific values. h) Probability
distribution graphs of thresholded labeling represented by probability density (y axis) across angular
ranges (θ°) in SC (x axis) from atlas level 90. VISam cases show distributions in angular ranges that align
with SC.m and SC.cm zones (n=3). MOs cases show distributions aligned with SC.cl (n=10). SSp-ul cases
show distributions in angular ranges aligned with SC.l (and SC.cl) zones (n=3). Color-code associations:
red (SC.m), orange (SC.cm), green (SC.cl), purple (SC.l). Abbreviations: ACAd, anterior cingulate cortex dorsal
part; ARA, Allen reference atlas; AAV, Adeno-associated virus; AAV-gfp, AAV green fluorescent protein; AAV-rfp,
AAV red fluorescent protein; MOp, primary motor cortex; MOs, secondary motor cortex; PHAL, Phaseolus vulgaris
leucoagglutinin; PTLp, posterior parietal cortex; VISam, visual cortex anterior medial part; SC, superior colliculus;
SC Layers: zo, zonal; sg, superficial grey; op, optic; ig, intermediate grey; iw, intermediate white; dg, deep grey; dw,
deep white; Somatotopic body parts; bfd, barrel field; ll, lower limb; m, mouth; n, nose; tr, trunk; ul, upper limb; SSp,
primary somatosensory cortex; VIS, visual cortex. See complete list of abbreviations.

43

44

45
Figure 2.5 | Sensory related connectivity with SC.
a) Raw data of higher order visual areas VISam and VISal. A PHAL injection into VISam (SW120310-03B)
projects mainly to intermediate layers in SC.cm, with some terminals in superficial layers. An AAV-
tdTomato injection into VISal (SW18080302-04A) projects to superficial layers of SC.m with some fibers in
the intermediate layers. b) Within the cortex, somatic sensorimotor areas are each organized into distinct
highly interconnected subnetworks that integrate the somatotopic body map and project subcortically to
the SC. Body parts include mouth (m), nose (n), upper limb (ul), lower limb (ll), barrel-field (bfd), and trunk
(tr). The dorsal view of the cortical map is color-coded with the dorsal view of the SC body part region it
projects to (consistent with the color-coded zones in the SC custom atlas). This organization is conserved
topographically as illustrated by dorsal view of SC with overlayed homunculus of somatosensory body
representation. c) Injection sites into distinct SSp regions project to adjacent and distinct SC.l or SC.cl zones
across rostral to caudal levels show adjacent body part projections. Scale bars are 200 µm for all SC panels;
500 µm for injection site panels. d) Retrograde labeling of FG-labeled cells in the SC from four separate
injections. Top: injection into the lateroposterior nucleus of thalamus (LP) back-labeled cells across
superficial layers in SC.m and SC.cm zones. Second: injection into the laterodorsal nucleus of thalamus
(LD) back-labeled cells across superficial and intermediate layers predominantly in SC.cm and SC.m, with
sparse cells in SC.cl. Third: injection into the ventromedial nucleus (VM) and paracentral nucleus of
thalamus (PCN) back-labeled cells in the intermediate and deep layers confined to the SC.l zone. Bottom:
FG into principal sensory nucleus of the trigeminal (PSV), labeling is confined to SC.l.
______________________________________________________________________________

46
2.5.3 HIGHER-ORDER ASSOCIATION AND PREFRONTAL CORTICAL
PROJECTIONS TO SC
Next, we systematically mapped cortico-tectal projections of higher-order association
areas along the medial edge of the cortex, including the anterior cingulate (ACA),
retrosplenial (RSP), and posterior parietal (PTLp) (Figure 2.6a). These cortical areas are
highly interconnected to form two medial cortico-cortical subnetworks, which transmit
and integrate visual, auditory, somatic sensory, as well as spatial orientation information
to the prefrontal cortex
4
. Here, we found that each of these cortical areas generate
distinctive projections to different SC zones (Figure 2.6b), which provide a subcortical
structural basis for multimodal cortical inputs to be further integrated with visual,
auditory and somatic sensorimotor information. In particular, the dorsal RSP (RSPd),
agranular RSP (RSPagl), lateral PTLp and ventral ACA (ACAv) generate dense
projections to the SC.m and SC.cm (across the superficial and intermediate gray layers),
while the ventral RSP (RSPv), medial PTLp and dorsal ACA (ACAd) preferentially
innervate the SC.cl. The rostral ACAd provides further inputs to the SC.l (Figure 2.6c-d;
Figure 2.7a). The caudal temporal association area (TEa), which is heavily connected with
the visual and auditory cortical areas
4
, generates projections to the SC.cl, while the rostral
TEa, sharing dense bi-directional connections with the somatic sensorimotor areas
4
,
projects preferentially to the SC.l (Table 2.3).
Finally, the SC also receives inputs from prefrontal areas (PFC), including the
medial (ORBm), lateral (ORBl), and ventrolateral (ORBvl) orbitofrontal, prelimbic (PL)
and infralimbic (ILA) (Figure 2.6b; Figure 2.7b). Axonal projections arising from the
ORBm, PL and ILA are distributed across all four SC zones, while ORBvl and ORBl
preferentially target the SC.cl and SC.l. Consistent with this result, the ORBvl and ORBl
also share bi-directional connections with the somatic sensorimotor areas (MOs, MOp,

47
and SSp) associated with trunk, lower limb and whisker regions
4
. Unlike other cortical
areas that generate unilateral cortico-tectal projections, the ORBl generate bilateral
projections to the SC, although its functional significance remains unclear.

2.5.4 CORTICO-TECTAL PROJECTIONS ARE ORGANIZED AS MODULAR
COMMUNITIES
To visualize cortico-tectal inputs as a weighted connectivity matrix, the thresholded
anterograde labels were overlapped on the layer- and zone-specific custom SC atlas
templates. An implementation of the Louvain algorithm was applied to the connectivity
matrix for modular community detection of cortical injection sites with their terminal
distributions to common SC zones
59,60
(see Methods). Here, we reveal that community
organization is conserved in cortico-tectal subnetworks composed of distinct sensory
information and higher-order associative cortical areas in the four SC zones.
The connectivity matrix demonstrates that the SC constitutes four modular
subnetworks differentiated by its cortical inputs (Figure 2.6g). SC.m and SC.l modular
networks are highly differentiated, whereas SC.cm and SC.cl are distinct, but closely
related. The SC.m community contained predominantly inputs from the primary and
secondary visual areas, as well as all subdivisions of the RSP, which shares bidirectional
connections with all visual areas and receives spatial information through robust inputs
from the dorsal subiculum through RSPv
4,28
. By stark contrast, the SC.l community
contained the majority of inputs from somatic sensorimotor/prefrontal subnetworks
from SSp, SSs, MOs, MOp, and PFC areas.
Both the SC.cm and SC.cl receive highly integrated inputs from cortical areas
within the medial cortico-cortical subnetworks associated with visual, auditory, and

48
spatial orientation
4
. In particular, the SC.cm receives denser direct inputs from the visual
and auditory areas, as well as several higher-order association areas, such as the ACAv,
RSPd, RSPagl, and PTLp-lateral
4
. In contrast, the SC.cl preferentially receives (1)
information associated with the lower central visual field; (2) direct inputs from the MOs-
fef/ACAd, which is involved in controlling eye movement; (3) somatic sensorimotor
inputs associated with trunk (SSp-tr); and (4) higher-order association areas receiving
somatic sensorimotor inputs, such as ORBl, ORBvl, and PTLp-medial. The ORBvl also
receives direct visual information
18
, thereby providing multi-modal inputs to the SC.cl.
This separation of communities suggests functionally distinct processing of visuospatial
information in the SC.m and somatic sensorimotor information in the SC.l with more
multi-modal associative integration processed in the SC.cm and SC.cl.

______________________________________________________________________________
Figure 2.6 | Distribution of higher-order cortical inputs across SC zones.
a) Schematic of higher-order association areas part of medial cortico-cortical subnetworks (Zingg et al.,
2014). Color-coding is consistent with the SC zones, and represents the topography based on cortico-tectal
projection patterns that target distinct zones. Reconstruction is composite of inputs to SC ARA 90. b)
Stacked bar chart for visualization of proportion of labeling across each SC zone (x axis) from each cortical
ROI (y axis) for each selected ROI. Values represent proportion of pixel density for each individual ROI
across each zone (n=1 case per row). See Table 2.4 for zone- and layer-specific values. c) Schematic of
anterograde injection strategy, and custom SC atlas levels. d) Raw data panels. VISp, RSPv and ACAd-
rostral projections to SC. e) ACAd-interm and VISam projections. f) Two PTLp-medial projections
projection to caudal SC levels. g) Visualization of cortico-tectal modular communities within the SC (n=34
cases). Matrices show communities identified by Louvain analysis of cortical injection ROIs (y axis) and SC
zones (x axis). Edges are shaded according to their connectivity labeling, and colored boxes along the
diagonal reflect modular communities identified. Legend on right side is based on the range of normalized
values calculated by the Louvain algorithm. Value 0.25 represents the maximum intensity (black), and
value 0 represents the minimum (white). Color-coded brackets below the matrix communities correspond
to the same color codes in the SC custom atlas zones. Color-code associations: red (SC.m), orange (SC.cm),
green (SC.cl), purple (SC.l).

49

50

Figure 2.7 | Higher-order cortico-tectal arrays.
a) Data array of individual cases from higher order projections to SC. Injection sites from top to bottom:
ACAd-intermediate, ACAv-rostral, RSPd-rostral, RSPv-rostral. b) Three cases with single anterograde
injections into PL (AAV), ORBl (PHAL), and ORBvl with their respective bilateral projections
predominately to SC.cl and SC.l zones at ARA 90. Scale bars are 200 µm.
______________________________________________________________________________

51
RESULTS (PART II) : BRAIN-WIDE SC CONNECTIVITY

2.6.1 BRAIN-WIDE CONNECTIVITY CORROBORATES CORTICO-TECTAL
NETWORKS
After establishing the cortico-tectal projectome, we further investigated brain-wide
connections of the SC in the context of their modular subnetworks. We created an
anterograde projection map from each SC zone to visually summarize these spatial and
topographic innervation patterns (Figure 2.8a). Based on extensive anterograde and
retrograde tracing data with a total of 30 injections spanning the entire SC (Figure 2.9),
we assembled a complete mesoscale connectivity diagram of the mouse SC that illustrates
distinctive input/output organization of all four zones (Figure 2.8b).

2.6.2 ZONE-SPECIFIC CONNECTIONS WITH SENSORIMOTOR NUCLEI IN THE
LOWER BRAINSTEM
The SC relays integrated information as motor commands via direct projections to
brainstem motor and thalamic nuclei
61,62
. First, we found that the SC.l is distinguished
from the other three SC zones by receiving dense inputs from the spinal trigeminal
nucleus interpolar (SPVI) and ventrolateral oral part (SPVOvl) - the primary
somatosensory nuclei processing orofacial and tactile information (such as the jaw fur,
inside the mouth, teeth, vibrissae, nose, and adjacent eye areas
63
) (Figure 2.8; Figure
2.10c), as well as the principle sensory nucleus of the trigeminal (PSV). The SC.l generates
significant projections to the trigeminal motor nucleus (V) and supratrigeminal nucleus
(SUT) essential in controlling jaw movements
64
(Figure 2.8; Figure 2.10b). The SC.l also
generates denser projections to the intermediate reticular nucleus (IRN), parvicellular
reticular nucleus (PARN) and paragigantocellular reticular nucleus (PGRNl). These

52
reticular nuclei contain numerous premotor neurons that are organized into different
central motor pattern generators in controlling orofacial rhythmic motor actions, such as
chewing, licking and swallowing
63,65
, and skilled forelimb movements
66
. Additionally, the
SC.l also projects to the medial parabrachial nucleus (PBm) and Kölliker-Fuse nucleus
(KF), which shares reciprocal connections with the preBötzinger complex and plays an
essential role in the control of breathing, licking and whisking
63,65,67–69
.
Quantification of output projections (Figure 2.10d) reveal that SC.l and SC.cl share
several common targets including, (1) the contralateral facial nucleus (VII, predominantly
its lateral part), (2) the gigantocellular reticular nucleus (GRN, predominantly to the
contralateral side), which contains the highest density of reticulo-spinal neurons
projecting directly to neck motor neurons to control head movements
70
and left-right
directional locomotion
71
, and (3) the midbrain reticular nucleus (MRN) which is
implicated in eye-head coupling during gaze through its connection with the SC
72
.
Additionally, it is important to note that both the SC.l and SC.cl receive direct inputs from
deep cerebellar nuclei (DN, IP and FN), but generate topographically arranged
projections to the inferior olivary complex (IO) that relays climbing fibers to the
cerebellum. Together, these cerebellar related circuits also play an integrative role in
sensory-guided orofacial movements
63
. The IO-cerebellar circuits plays an integrative
role in motor coordination and learning
73
, though the functional role of the triangular
circuits, SC.l/SC.cl à IOàcerebellumàSC.l/SC.cl remain to be investigated.
In comparison to the SC.l, the SC.cl generates denser projections to several
auditory related structures, including the inferior colliculus (all three subdivisions, ICe,
ICd, and ICc, bilaterally with ipsilateral predominance), as well as the cuneiform nucleus
(CUN) and nucleus sagulum (SAG)
74
. The reciprocal connections between the SC and IC
are implicated in auditory defense responses
36
. The SC.m, SC.cm, and SC.cl also generate

53
direct projections to several subdivisions of the midbrain pretectal area, including the
nucleus of the posterior commissure (NPC), olivary pretectal nucleus (OP), nucleus of the
optic tract (NOT), nucleus of the posterior commissure (NPC), as well as the posterior
(PPT) and medial pretectal nuclei (MPT). The pretectum receives direct binocular input
from photosensitive ganglion cells in the retina and is primarily involved in mediating
non-conscious behavioral responses to acute changes in light such as the pupillary light
and optokinetic reflexes
75
. All four SC zones provide significant projections to the anterior
pretectal nucleus (APN), which is implicated in nociceptive guided visual motor actions
and the pathogenesis of central pain syndrome
76
. The SC.m projects directly to the
parabigeminal nucleus (PBG) and this SC→PBG pathway has been implicated in
detecting looming objects and triggering fear responses
77
. Finally, each SC zone projects
densely to the periaqueductal gray (PAG, presumably with topographic projection
patterns), which plays a critical role in coordinating somatic and autonomic reactions in
defensive responses and is a critical region in driving motivation for hunting and
foraging behaviors
36,78,79
. It is important to note that the four SC zones share extensive
connections with brainstem monoaminergic cell groups with different preferences. Each
group receives inputs from the locus coeruleus (LC, noradrenergic) and dorsal raphe (DR,
serotonergic). All SC zones project to the midbrain dopaminergic groups in the SNc and
VTA, and PPN (cholinergic), with the SC.m projecting to the lateral dorsal tegmental area
(LDT, cholinergic). The SC.cm also generates specific projections to the medullar raphe
nuclei (DR, RPA, RO, and RPO) (Figure 2.10d).

54

Figure 2.8 | Brain-wide connectivity of SC inputs and outputs.
a) Color-coded map of anterograde projections from SC zones throughout the brain demonstrating
topographic output patterns. Top left insert is a visual aid representation the anterograde injections placed
in each SC zone, with colors corresponding to the same color of projections throughout the brain. See Table
2.1 for injection site details. See Appendix A for complete array of coronal sections for anterograde
projection map. SC.m (red): SW190619-04A (PHAL), SW190619-02A (PHAL); SC.cm (orange): SW190619-
02A (AAV-tdTomato); SC.cl (green): SW171010-01A (AAV-gfp), SW171010-02A (AAV-gfp); SC.l (purple):
SW171010-01A (AAV-tdTomato), SW171010-01A (PHAL). b) Unweighted wiring map of all inputs and
outputs of superior colliculus zones and layers. Assembled using data from anterograde and retrograde
injections placed in SC to systematically trace outputs inputs distributed from cortex down through the
hindbrain and cerebellar structures. Color-code associations: red (SC.m), orange (SC.cm), green (SC.cl),
purple (SC.l).
______________________________________________________________________________

55

Figure 2.9 | Injection sites in SC.
a) Anterograde injection sites in superior colliculus. b) Retrograde injection sites. The list can be cross-
referenced with Table 2.1 for complete details of injection sites, coordinates, and locations. Color-code
associations: red (SC.m), orange (SC.cm), green (SC.cl), purple (SC.l). Abbreviations: AAV, adeno-associated
virus; AAV-tdT, AAV-tdTomato; AAV-gfp, AAV-green fluorescent protein; ARA, Allen Reference Atlas; BDA,
biotinylated dextran amine; CTB, cholera toxin subunit B conjugates (488, 555, 567); FG, Fluorogold; PHAL,
Phaseolus vulgaris leucoagglutinin; SC, superior colliculus; m, medial zone; cm, centromedial zone; cl, centrolateral
zone; l, lateral zone.
______________________________________________________________________________

ARA 86
ARA 90
ARA 96 ARA 100
ARA 86 ARA 90 ARA 96 ARA 100
a
b
Anterograde Injections in Superior Colliculus
Retrograde Injections in Superior Colliculus
SW190619-04A / PHAL
SW190619-02A / PHAL
SW190619-02A / AAV-tdT
SW171010-02A / AAV-tdT
SW171010-02A / AAV-gfp
SW170315-01A / AAV-tdT
SW171010-01A / AAV-tdT
SW171010-01A / PHAL
SW150921-01A / FG
SW170315-02A / CTB-647
SW170315-02A / FG
SW150916-01A / CTB-555
SW160716-02A / FG
SW150916-01A / FG
SW180614-07A / AAV-tdT
SW171010-03A / AAV-tdT
SW180614-07A / AAV-gfp
SW170426-04A / AAV-tdT
SW171010-01A / AAV-gfp
SW170830-01A / CTB-647
SW170830-02A / CTB-647
SW170830-02A / CTB-488
SW150916-01A / PHAL
SW170215-01A / PHAL
SW150916-01A / BDA
SW160716-02A / PHAL
SW160716-01A / PHAL
SW170201-05A / CTB-647
SW170201-08A / CTB-555
SW170201-05A / CTB-555
SW170201-05A / CTB-488
SW170201-08A / FG
1
3
3
4
5
6
8 9
10
12
13 14
15
17
18
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
1
2
4
5
7 8
9
10
11
12
13
14
m
cm
cl
l
m
cm
cl
l
m
cm
cl
l
m cm
cl
l
m
cm
cl
l
m
cm
cl
l
m
cm
cl
l
m cm
cl
l
7
2
6
11
16

56

Figure 2.10 | Downstream projections from distinct SC zones to brainstem regions.
a) PHAL injection into SC.m-ig show prominent ipsilateral projections to midbrain and hindbrain regions.
b) AAV-tdTomato injection into SC.cm-ig/iw and AAV-gfp injection into SC.cl-ig/iw produce
downstream targets similar brainstem regions, with segregated but adjacent outputs in IO. c) Downstream
projections from AAV-tdTomato injection in SC.l in intermediate white and deep grey layers. See
Supplementary Table 1 for complete list of abbreviations.
(Next page) d) Quantification of four anterograde injections into each SC zone. Plots represent
reconstructed pixel data from output projection terminals of SC throughout the brain. Distributions to
contralateral and ipsilateral brain regions are shown, with brain regions organized based on ontological
hierarchy in the Allen Reference Atlas ( y-axis). Data normalization assigns values based on minimum
threshold pixel value (0.0006) and maximum threshold pixel value (1) which represents the brain region
that receives the highest value of pixels (x-axis).
______________________________________________________________________________

57
(Figure 2.10d)

Medulla
motor-related
Pons
behavioral state-related
Pons
sensory-related
Pons
motor-related
Midbrain
sensory-related
Midbrain
behavioral state-related
Midbrain
motor-related
Hypothalamus
medial zone
Hypothalamus
periventricular zone
Hypothalamus
lateral zone
Thalamus
polymodal association related
Thalamus
sensory-motor related
Pallidum
Medulla
motor-related
Pons
behavioral state-related
Pons
sensory-related
Pons
motor-related
Midbrain
sensory-related
Midbrain
behavioral state-related
Midbrain
motor-related
Hypothalamic
lateral zone
Thalamus
polymodal association related
Thalamus
sensory-motor related
Pallidum
Medulla
behavioral state-related
Medulla
behavioral state-related
Medulla
sensory-related
Pons
motor-related
Pons
behavioral state-related
Pons
sensory-related
Medulla
motor-related
Midbrain
sensory-related
Midbrain
behavioral state-related
Midbrain
motor-related
Hypothalamic
lateral zone
Thalamus
polymodal association related
Thalamus
sensory-motor related
Pallidum
Medulla
behavioral state-related
Hypothalamic
medial zone
Hypothalamic
periventricular zone
Pons
motor-related
Pons
behavioral state-related
Pons
sensory-related
Medulla
motor-related
Midbrain
sensory-related
Midbrain
behavioral state-related
Midbrain
motor-related
Thalamus
polymodal association related
Thalamus
sensory-motor related
Pallidum
Medulla
behavioral state-related
Hypothalamic
medial zone
Hypothalamic
periventricular zone
lateral zone
Medulla
sensory-related
0.0 0.1 0.2 1.0 0.5 0.0 0.1 0.2 1.0 0.5
Normalized Density Normalized Density
SW190614-04A_ch4: SC.m SW171010-02A_ch4: SC.cm SW171010-02A_ch2: SC.cl SW171010-01A_ch2: SC.l
Contralateral Ipsilateral Contralateral Ipsilateral Contralateral Ipsilateral Contralateral Ipsilateral
0.0 0.1 0.2 1.0 0.5 0.0 0.1 0.2 1.0 0.5
Normalized Density Normalized Density
0.0 0.1 0.2 1.0 0.5 0.0 0.1 0.2 1.0 0.5
Normalized Density Normalized Density
0.0 0.1 0.2 1.0 0.5 0.0 0.1 0.2 1.0 0.5
Normalized Density Normalized Density
d

58
2.6.3 HYPOTHALAMIC INTERACTIONS WITH SC
The SC zones also receive topographic inputs from hypothalamic structures, including
the zona incerta (ZI) and ventromedial nucleus of hypothalamus (VMH)
80,81
. The SC.cl
and SC.l intermediate and deep layers receive dense inputs from the ventrolateral part of
the ventromedial hypothalamus (VMH.vl), known to be involved in approach,
appetitive, reproductive and social attack responses
20,82
. Triple retrograde injections into
the SC.m-ig (CTb-647), SC.cl-dg (CTb-555) and SC.l-dg (CTb-488) reveal that the SC.m
receives dense inputs primarily from the VMH.dm, whereas the SC.cl and SC.l receive
more inputs from the VMH.vl. Another experiment comparing the SC.cm (CTb-555) and
SC.l-ig (FG) revealed the SC.cm receives input from the VMH.dm/c, and the SC.l receives
input from VMH.vl. (Figure 2.11a). Interestingly, we also found the found that the SC.cl
and SC.m generate significant projections to the anterior hypothalamic nucleus (AHN).
The AHN and VHMdm share reciprocal connections and are two essential nodes of the
hypothalamic defensive network
83
.
The output patterns of the ZI to SC support the topographic organization of medial
visuomotor and lateral somatic sensorimotor subnetworks. Anterograde injections into
1) the lateral ZI project to SC.m and SC.cm, 2) central ZI projects to SC.cm and SC.cl, and
3) medial ZI projects to SC.l (Figure 2.11b). Interestingly, lateral ZI→SC.m/cm innervate
superficial and intermediate layers, further supporting the intrinsic alignment of
visuomotor subnetworks that also align with our identified cortico-tectal
communities. These findings are congruent with functional networks implicating the
lateral SC in appetitive and approach behaviors
25,84
.

59
2.6.4 CONNECTIVITY WITHIN CORTICO-TECTO-THALAMIC SUBNETWORKS
Thus far, we identified that each SC zone receives convergent inputs from a specific set
of cortical areas and displays distinguishable connections with sensorimotor nuclei in the
brainstem as described above. Numerous studies have described detailed neuronal
connections of the SC with the thalamus
85,86
. Here, we further demonstrate how each of
these newly defined SC zones sends projections back to specific thalamic nuclei that send
ascending projections to the same cortical areas that generate direct projections to the SC,
thus completing the loop within the cortico-tecto-thalamic subnetwork. The SC.m and
SC.cm share reciprocal connections with the dorsal lateral geniculate nucleus (LGd) and
lateroposterior nucleus (LP); and generate projections to the ventral lateral geniculate
nucleus (LGv), laterodorsal nucleus (LD), posterior nucleus (PO), nucleus of reuniens
(RE), anteroventral nucleus (AV) and parafascicular nucleus (PF) (Figure 2.8a). As shown
in Figure 2.8a, color-coded axonal terminals from the SC.m and SC.cm in the LP and LD
are topographically arranged. Injections of two visual-related thalamic nuclei, the LP and
LD, resulted in dense labeling in the SC.m and SC.cm, but with different laminar
specificities (Figure 2.5d). LP-projecting neurons are distributed specifically in the
superficial gray layer (sg); while LD-projecting neurons are distributed primarily in the
intermediate gray layer (ig). Our results also revealed that the SC.m and SC.cm (mostly
intermediate and deep layers) contain dense thalamic projecting neurons that target the
RE (Figure 2.11c). The LP, LD and RE share massive bi-directional connections with the
visual areas and several high order association areas (such as the ACA, RSP, and PTLp)
that innervate the SC.m and SC.cm. Together, these structures are organized into a
cortico-tecto-thalamic network in processing and integrating visual, auditory, and spatial
orientation information to regulate motor outputs associated with goal-directed
behavior.

60
In parallel, we found the SC.cl contained a discrete thalamic projecting neuron that
targeted the LD, PO, and lateral and center parts of the mediodorsal thalamic nucleus
(MDl, MDc) (Figure 2.11c). The MDl shares dense bidirectional connections with the
ACAd and MOs-fef which generate direct projections to the SC.cl. Altogether, these
structures are organized into another distinctive loop as follows: cortico(ACAd and
MOs)-tecto(SC.cl)-thalamo(MDl)-cortical. The SC.l (and its adjacent SC.cl) contains
thalamic projecting neurons that target several somatic sensorimotor related thalamic
nuclei, such as the ventrolateral parafascicular nucleus mouth region (PF.m)
87
,
centromedial (CM), ventral anterolateral nucleus (VAL), the caudal ventromedial nucleus
(VM), and paracentral nucleus (PCN) (Figure 2.8; Figure 2.5d), following a
topographically organized somatotopic order. In particular, the rostral SC.l projects
primarily to the PF.m, which also shares massive bidirectional connections with the
somatic sensorimotor areas (SSp and MOp) associated with the mouth and upper limb
87
.
In parallel, the caudal levels of SC.l and SC.cl send selective projections to the dorsolateral
PF and rostral VM, which also share bidirectional connections also with the somatic
sensorimotor cortical areas associated with the trunk and lower limb. To our knowledge,
these somatotopically organized cortico-tecto-thalamic loops have not been reported
before (Figure 2.11e).

61

Figure 2.11 | Topographic organization of brain-wide inputs and outputs of SC zones.
a) Raw data labeling from two separate cases of retrograde injections into the SC. Top row: CTB (pink)
injection into SC.m, CTB (red) injection into SC.cl, and CTB (green) injection into SC.l. Cells are
topographically retrogradely labeled in VMH domains and cortex. Bottom row: CTB (red) injection in
SC.cm, and FG injection in SC.l. b) Raw connectivity data in SC (all at ARA 90) from three separate
anterograde injections in the ZI. The ZI.l targets SC.m/cm; the ZI.c targets SC.cl, and the ZI.m targets SC.l.
c) Raw connectivity data in SC (all at ARA 90) from three separate FG injections in different thalamic nuclei,
RE (top), MD (middle) or PF.m (bottom), and the respective retrogradely-labeled thalamic projections
neurons, respectively, in SC.m/cm, SC.cl, or SC.l. Cells were distributed heterogeneously across superficial,
intermediate and deep layers, but clustered topographically and preferentially in distinct zones. Scale bar
200 µm in SC panels. d) Labeling produced from anterograde injections into distinct SNr domains project
to SC. e) Topographic organization of mouse SC connectivity. Directional cross at bottom left. The four
zones of the SC are connected either uni- or bi-directionally with cortex, PF, ZI, VMH and SNr. Plus (+)
signs and minus (-) signs indicate excitatory or inhibitory projections, respectively.
______________________________________________________________________________

62
2.6.5 INTERACTIONS BETWEEN CORTICO-TECTAL AND CORTICO-BASAL
GANGLIA NETWORKS
The interactions between the cortico-tectal and basal ganglia system were noted in
previous work but have not been systematically investigated
61,85,88,89
. Firstly, the
comprehensive cortico-tectal projection map, together with our previous cortico-striatal
projection map
27
, allows us to identify a one-to-one correspondence of cortical axonal
terminal fields in the CP and SC arising from individual cortical areas (Figure 2.12a). This
provides an anatomical framework for understanding functional correlations between
these two systems in regulating motor behavior. In fact, the same cortical projection
neurons generate collateral projections to innervate both targets
36,90
. As an example, a
subset of MOs/ACAd cortical projecting neurons (presumably pyramidal tract projection
neurons or PT neurons) generates collateral projections to both the SC.cl and dorsal
domain of caudal CP (CPc.d) (Figure 2.12b-d). Thus, in addition to direct cortico-tectal
projections, cortical information can also reach the SC indirectly through a multi-
synaptic, cortico-basal ganglia-tectal projection pathway.
Each SC zone harbors distinct thalamic projecting neurons, and receives direct
convergent inputs from functionally correlated cortical areas (as aforementioned) as well
as inputs from specific domains within the reticular part of the substantia nigra (SNr), the
motor output node of the basal ganglia subnetworks (see Figure 2.11d-e). Our recent
work demonstrated that the SNr are subdivided into 14 different domains based on their
convergent and divergent inputs from 30 different striatal domains, each of which in turn
receive a specific set of functional relevant cortical inputs
27,87
. The newly defined medial
SNr domain (SNr.m), which receives convergent inputs from the dorsomedial striatum
that in turn receives convergent inputs from the visual cortical areas, ACA, RSP and
PTLp
4,87
, generate dense projections to the SC.m, SC.cm, as well as SC.cl (Figure 2.11c)

63
that align with patterns from cortico-tectal projections from these high order association
areas (see Figure 2.6a). In contrast, the centrolateral domain of the SNr (SNr.cl), which
receives the densest inputs from the dorsolateral striatal domains that are innervated by
the MOp and SSp trunk and lower limb domains, project specifically to the SC.cl. Finally,
the SC.l receives dense inputs from the SNr domain (Figure 2.11c) that is specifically
innervated by the ventrolateral CP domains, which receive convergent somatic
sensorimotor cortical inputs associated with orofacial and upper limb (Figure 2.12a).

64

Figure 2.12 | Cortico-striatal and cortico-tectal projections have conserved topographic organization.
Within the cortex, somatic sensorimotor areas are each organized into distinct highly interconnected
subnetworks that integrate the visual field, somatotopic body map and higher order areas which then
project subcortically to the caudoputamen (CP) into distinct domains
4,27
. Each cortical area sends parallel
descending projections to SC zones and CP domains. a) Three separate triple anterograde cases into
different cortical areas send topographic projections to corresponding caudate putamen (CP) domains and
SC zones. b) Injection site schematic of combinatorial tract tracing method using AAVretro-Cre injection
(in CPc.d at ARA 57) and Cre-dependent AAV-mCherry (in MOs at ARA 47). c) Injection sites in CPc.dm
and MOs within the same mouse brain (SW190110-01B). d) Red anterograde labeling demonstrates a subset
of MOs cortical projecting neurons (presumably pyramidal tract projection neurons or PT neurons)
generate collateral projections specifically to the SC.cl zone and CPc.dm. Scale bars are 200 µm. Color-code
associations: red (SC.m), orange (SC.cm), green (SC.cl), purple (SC.l).
______________________________________________________________________________

65
In parallel, we demonstrate a refined network of organization of parallel channels
relayed through the cortico-tecto-thalamic circuit. This is most evident in somatotopic
maps from SSp and MOp to SC.cl and SC.l zones. The four SC zones project to at least
four domains within the PF, where SC.cl/SC.l→PF.m/ul, SC.cm/cl→PF.tr/ll, and
SC.m→PF.as (Figure 2.11e). Output projections from SNr domains also target the same
somatotopic zone in SC as well as the corresponding domain in PF. To show the
subnetwork interactions of this cortico-tecto-thalamic pathway and reveal mono-synaptic
inputs to the PF-projecting neurons in the SC.l, we used a TRIO-tracing strategy targeting
AAVretro-Cre in dorsomedial PF, and rabies virus and Cre-dependent helper virus in
SC.l
51
(Figure 2.13a-b). As expected, rabies labeled cells that target PF-projecting SC.l
neurons were found in SSp-ul, SSp-m, SSs and MOp-oro cortical areas. Additionally,
neurons were labeled in the dorsolateral SNr, deep cerebellar nuclei (DN) and the spinal
nucleus of the trigeminal interpolar part (SPVI) and ventrolateral oral part (SPVOvl)
associated with processing orofacial and tactile information
91
(Figure 2.13c). These data
provide evidence to suggest the SC.l receives convergent inputs from functionally
correlated cortical areas, subsets of cortico-basal ganglia (i.e., dorsolateral SNr), deep
cerebellar nuclei and brainstem sensorimotor nuclei associated with hand-to-mouth
coordination behaviors. In turn, the SC.l sends projections to the PF, through which
integrated somatic information is sent back to the corresponding cortical areas. Finally,
we observed that all four SC zones directly project to the SNc and VTA, which
presumably generate dopaminergic projections back to the striatum and cortex. The SC.l
also generates direct projections back to multiple components of the basal ganglia,
including the GPe, GPi and STN
61,62,89
. Additional genetic and trans-synaptic experiments
should be pursued to further validate the connections described.

66

Figure 2.13 | Upper-limb and orofacial subnetworks in SC.cl and SC.l.
a) TRIO-tracing strategy targeting AAVretro-EF1a-Cre in PF.ul (PF upper limb), and rabies virus (AAV8-
hSyn-FLEX-TVA-GFP) and Cre-dependent helper virus (EnvA G-del-Rabies-mCherry) in SC.l reveal
mono-synaptic inputs to the PF-projecting neurons in the SC.l. b) Raw images of injection sites in PF.ul,
and SC.l with zoomed in panels of triple labels starter cells in SC (case SW190926-10A). c) Rabies retrograde
labeling in somatic sensorimotor cortical areas MOp and SSp-bfd. Cells labeled in the dorsal and central
lateral substantia nigra reticulata (SNr), the interpolar part of the spinal nucleus of the trigeminal (SPVI)
and dentate nucleus (DN) of the cerebellum. Bottom right corner is a summary diagram showing that
thalamic (PF) projecting neurons in SC.l receive convergent inputs from the somatic sensorimotor cortical
areas (MOp, SSp), subset of the basal ganglia (SNr), somatic sensory nuclei (i.e., SPVI) and deep cerebellar
nuclei (i.e., DN).
______________________________________________________________________________

67
RESULTS (PART III) : THALAMUS-PROJECTING SC CELLS

2.7.1 MORPHOLOGICAL CHARACTERISTICS OF THALAMUS-PROJECTING SC
CELLS
Finally, to test the anatomical basis for how SC neurons integrate inputs from different
sources, we characterized dendritic morphologies of SC neurons. Representative neurons
in each zone were labeled via a G-deleted-rabies injection in the LD (for SC.m/cm), RE
(for SC.m/cm/cl), and dorsal PF (for SC.cm/cl/l) (Figure 2.14). The large injection into
the dorsal PF also partially infected neurons in the midbrain pretectal region (MPT),
which retrogradely labeled cells in the superficial layers of SC.m (we thus refer to this
injection as PF/MPT). Reconstructions of rabies-labeled SC neurons projecting to RE, LD
and PF/MPT allow us to analyze the spatial correlation between cortical projection fields
and dendritic fields
92,93
of SC-projection neurons
58,94
(Figures 2.15 and 2.16). Here,
reconstructed neurons are superimposed above their corresponding SC zones and
receive inputs from visual-related cortical areas (VIS, RSP, and PTLp), and
reconstructions of PF/MPT projecting neurons in SC.cl/SC.l zones overlap with inputs
from somatic sensorimotor subnetworks (see Figure 2.4c, Figure 2.6d-e).
We found significant distinctions in the morphometric features of width, number
of bifurcations, branch path length, and contraction (Figure 2.14c)
49,50
. Notably, based on
the width (overall arbor size) in each zone, cells projecting from SC.m superficial layers
had smaller dendritic fields, and cells from intermediate and deep layers of SC.l were
larger as they transitioned through intermediary sizes in SC.cm and SC.cl, respectively.
This size trend in the zones is further modulated by a second trend displayed by the
projection target, whereby the LD-projecting cells have the smallest arbors, and RE-
projecting cells in the SC.cl and PF/MPT-projecting cells in the SC.l have the largest

68
arbors. Larger dendritic fields imply the ability to integrate from a broader array of
incoming fiber pathways, whereas a narrower arbor reflects a selectively focused input
tuning
20
. These neurons exhibited heterogeneous morphological properties consistent
with the rodent cell types that may contribute to distinct behaviors
20,77,95
.
It is possible that downstream-projecting SC neurons display varying
morphological features than ascending thalamus-projecting SC neurons
77
; nevertheless,
our results suggest that functional analyses of SC neurons involving motor, sensory and
cognitive behaviors can also be addressed in the context of inputs and outputs from
distinct modular networks. Several rodent studies provide entry into genetic and cell-
type specific investigations of functional SC networks
20,96,97
, though more studies are
needed to characterize how multimodal cortical and subcortical information is integrated
at single neuron resolution.
All neuronal morphologies reconstructed in this study along with detailed
experimental metadata are cataloged into a digital library, accessible online at
www.NeuroMorpho.Org
57
.

69

a
b
c

70
Figure 2.14 | Characterization of neuronal cell types in the SC based on their anatomical locations,
projection targets and neuronal morphology.
a) LD projecting neurons in SC labeled with rabies-GFP in SC.m/cm-ig, and RE projecting neurons labeled
with rabies-RFP injected into cells in SC.m/cm-ig/iw/dg. Tissue was processed by the SHIELD clearing
protocol and followed by confocal imaging for 3D-reconstruction. Dendrites from LD and RE-projecting
SC neurons were reconstructed to visualize differences and dendritic arborizations across SC layers and
zones. The LD-projecting SC.m/SC.cm neurons had pyramidal-shaped cell bodies with dendrites that
extended dorsally to the superficial optic layers and within the intermediate layers. Of the RE-projecting
SC.m/cm neurons, some had dendrites that extended into superficial and intermediate layers, with others
that extended into intermediate and deep layers. A subset of neurons extends dendrites horizontally,
suggesting they integrate information within the same layer across zones. Other neurons extend vertically,
suggesting these receive multimodal information integrated within multiple SC layers. b) Principal
component analysis (PCA) shows segregation of zone- and target-specific cells based on measured
morphological features (see Methods). Number of SC projection neuron reconstructions to RE (n=54), LD
(n=8), and PF/MPT (n=31). c) Data are presented as whisker plots, where the center line represents the
median, box limits show the upper and lower quartiles, and the whiskers represent the minimum and
maximum data values. Examples of reports from pairwise tests of morphological parameters that survived
the false discovery rate correction tests. Width measures the overall arbor size of neurons. RE-projecting
cells in SC.cl and PF/MPT-projecting cells in SC.l are more than twice the width of LD-projecting cells in
SC.m. Number of bifurcations compartmentalizes the arborizations into functionally distinct synaptic
integration elements. Branch length represents a ratio between the width and number of bifurcations
measures, and provides an indication of how they co-vary. It also relates to the number of synapses that
can be received on a single computational element. Contraction measures the efficient occupancy of space
to reach as many axonal boutons as possible in nearby and further distances. P-values for all parameters
can be found in Table 2.3 Abbreviations: LD, laterodorsal nucleus; PF/MPT, parafascicular nucleus of
thalamus/midbrain pretectal area; RE, reuniens nucleus of thalamus; SC, superior colliculus; SC layers: zo, zonal; sg,
superficial grey; op, optic; ig, intermediate grey; iw, intermediate white; dg, deep grey; dw, deep white.
______________________________________________________________________________

71

Figure 2.15 | Examples of layer-specific SC neuron reconstructions.
Reconstructed neurons (rendered in neuTube) are organized based on their laminar- and zone-specific
locations in the SC. Color-coding in the legend corresponds to the SC zones. 32 total neurons are displayed.
Complete dataset of 92 reconstructed SC neurons is available online at www.NeuroMorpho.org.
______________________________________________________________________________

72

Figure 2.16 | Overlapped reconstructions of SC neurons and cortico-tectal projections.
Dendrites from LD-, RE-, and PF/PT-projecting SC neurons were reconstructed to visualize differences
and dendritic arborizations across SC layers and zones. Examples of reconstructed neurons were
overlapped onto reconstructions of cortico-tectal projections on the SC atlas. This facilitates analysis of
spatial correlation between cortical projection fields and dendritic fields of SC-projection neurons.
______________________________________________________________________________

73
DISCUSSION
Numerous anatomical and functional studies have demonstrated that the SC plays a
critical role in integrating external environment information and determining the
whereabouts of specific events, such as potential prey or predators, to guide animals’
goal-directed behaviors
98
. The SC receives extensive cortical inputs to facilitate various
functions including spatial attention, navigation, defense, and decision-making
9,13,14,21,36
.
These connections are often divided into two broad functional modules along the medial
and lateral halves
25,99
, yet there is more complexity underlying its organization that has
not been previously shown. Thus, not only have we refined the SC to four anatomically
relevant subdivisions, but we have also revealed they are organized as distinct
subnetworks throughout the brain. Here, we generated a comprehensive anatomical map
of the mouse SC revealing parallel subnetworks correlated with functional
specializations. In particular, we systematically characterized all cortico-tectal pathways
based on the convergence and divergence of cortical fiber terminations across the SC to
create the mouse cortico-tectal projectome. We identified four zone-specific delineations
from medial to lateral in the SC using a combined connectivity and computational
neuroanatomic approach: SC.m, SC.cm, SC.cl and SC.l (Figure 2.2). Each of these zones
also shares distinguished connectivity patterns with different sensorimotor nuclei in the
lower brainstem, hypothalamus and thalamus. Together, they assemble four parallel
neural networks in controlling different sensory integration and motor actions.
This work provides a new conceptual understanding of how cortical and
subcortical subnetworks are superimposed in the SC to guide functionally distinct
responses. For example, for the first time, we characterized distinctive connectivity
features of the SC.l and SC.cl, which were previously collectively grouped into the lateral

74
SC. In regard to somatotopic order, their connectivity pattern clearly reveal a somatotopic
map with the rostral SC.l associated with orofacial and upper limb, while the caudal SC.l
and SC.cl associated with lower limb, and body trunk (Figure 2.5b). This rough rostro-
caudal somatotopic order is similar to that in the cortex
4
, but distinguished from that in
the dorsal striatum
27
, GPe and SNr
87
. The SC.l sits in a unique interface to integrate
somatic sensorimotor information from brainstem sensory nuclei (i.e., SPVO, SPVI),
cortical somatic sensorimotor areas, and cerebellum. In turn, the SC.l sends this
information to the cortex through the tecto-thalamo-cortical pathway on one hand, and
project to the motor nuclei in the lower brainstem (presumably in regulating behavior).
Specifically, we found that the SC.l receives predominantly somatotopic sensory
information from the SPVI and SPVOvl, and somatic sensorimotor cortical areas
associated with orofacial and upper limb area and projects to the brainstem reticular
formation in controlling orofacial movements. In contrast, the SC.cl (1) receives
convergent cortical inputs associated with the lower visual field, and somatosensory
inputs associated with the upper body trunk, (2) receives inputs from VISp-rostromedial
(lower visual field), MOs frontal-eye-field and sends outputs to the MD thalamic nucleus
involved in eye movements, and (3) the SC.cl also projects to the GRN and other
structures that coordinate head movements. These data suggest that the SC.cl plays a
critical role in controlling eye-head coupling and orienting movements during
exploratory behavior (i.e., prey capture)
68
, while the SC.l is involved in controlling
orofacial and upper limb movement in the consummatory phase of behavior (e.g., biting,
chewing).
Next, in regard to the visual field organization in SC.m, SC.cm, and SC.cl, our
results show that each zone receives dense visual inputs, but with different preferences
corresponding to the upper central, upper peripheral, and lower portions of the visual

75
field. We found that the SC.m receives inputs from the VISp-caudal, caudolateral, and
the SC.cm receives inputs from the VISp-rostral and caudomedial. The SC.m receive
denser inputs from the RSPv, suggesting more visuospatial integration; while SC.cm
receives denser inputs from AUDp, suggesting more visuoauditory integration. The
SC.m and SC.cm share connectivity with most cortical and subcortical structures
associated with visual and auditory integration and visuomotor responses to perceive,
localize and recognize objects in the outside world. In addition, both the SC.cm and SC.m
project to the APN, which receives nociceptive inputs and is implicated in central pain
syndrome
76
.
Furthermore, we found each of the four zones are associated with distinct parallel
cortico-tecto-thalamic subnetworks, and with cortico-basal ganglia-thalamic
subnetworks (Figure 2.11e). The continuity in topographic organization throughout
structures within the basal ganglia system and thalamic nuclei with the SC zones suggests
a grand orchestration of compartmentalized integration to facilitate coordinated
behaviors. Altogether, the complete wiring diagram of the SC enables us to generate
functional networks (Figure 2.17) associated with several testable functional hypotheses,
as elaborated below.

76

Figure 2.17 | Subnetwork organization of SC zones.
a) Integration of SC.cl and SC.l zones in visuomotor, somatic sensorimotor, escape and approach
subnetworks. SC.cl/l neurons receive inputs from MO/SS (orofacial, barrel field, upper limb) cortices,
motor-related domains from thalamic nuclei (PF.mouth, PF.upper limb, VM, PCN), sexual approach
behavior (VMH.vl) and orofacial/upper limb brainstem and cerebellar inputs. This input provides
evidence for SC.cl/l zones as distinct somatic sensorimotor subnetworks that mediate a subset of behaviors
distinct from the more visually integrated range of behaviors mediated through SC.m/cm. Our network
analysis reveals the segregation of ventromedial prefrontal cortex (vmPFC) outputs from ORB and ILA
regions to functionally specific SC.cl/l zones. These prefrontal regions also project densely to VMH, ZI and
PAG, with BLA sharing bilateral connections with ILA (Hintiryan et al., Nature Communications, in press).
b) SC.m and SC.cm integration of visual information with spatial-related and head-direction hippocampal
networks (Bienkowski et al., 2018), attention/orienting regions and freezing/defense regions. See complete
abbreviation list.
______________________________________________________________________________
a
b
IC
PBlc
CUN
MARN Pretectal
PG
cMRN PBG
Brainstem/Hindbrain
Amygdala
Thalamus
Basal Ganglia
Hypothalamus
Basal Ganglia
Thalamus
Amygdala
Hypothalamus
Brainstem/Hindbrain
PBmm
IO
APN TRN
IC
VISp
(upper visual field)
VISal/l
VISam/pm
ACAd/v ILA RSPd RSPv
SC.m
SC.cm
CPi.dm
CPc.d
CPi.vm
LG
LP
LD
- - - - - - -
RE
STN
SNc
VMH.dm/c
PAG.vl
ZI
BLA.am
BLA.ac
AD/AV POST
PRE
PAR
ENT
SUBdd
SUBdv
CA1
ProSUB
SNr.v
Spatial Navigation
Freezing/
Defense
Orienting
PPN
LM DTN
CA3
DG
Somatosensory/Motor
Visual/Association
PTLp-lat
CPi.dl
CPi.vl
CPi.vm
Hippocampus
Head Direction
Network
SC.cl SC.l
LAvm
LP
SPFp/PP
Escape/
Attack
VMH.vl
PAG.dl
ZI
BLAp
BMAp
BLA.al
Auditory/Temporal Prefrontal
STN
SNc
SNr.dl
Visuomotor
Appetitive/
Approach
PRNc
SPVOvl
MRN
MRN
PPN
SSp-m
SSp-ul
MOp-oro
MOp-ul
SSp-ll/tr
MOp-ll/tr
MOs
ILA
ORBl
ORBvl
MD
PF.ll/tr
PF.m/ul
VM
PCN
AUD TEa PL ORBm
SSp-bfd
SSp-n
VISp
(lower
visual
field)
ACAd/MOs
MOs-fef

77
2.8.1 SUBNETWORKS IMPLICATED IN APPROACH / APPETITIVE BEHAVIORS
The cortico-tectal lateral zones have functional implications in somatic sensorimotor
movements that mediate appetitive and approach related behaviors
25,100,101
(Figure 2.17a).
The sparse projections arising from ORBm/vl→SC.cl overlap with ACAd/MOs-
fef→SC.cl inputs target the lower nasal and temporal regions of the visual field
represented in SC.cl, which is medially adjacent to the somatic sensorimotor subnetworks
that facilitate spatial orientation near the ground. This suggests that vmPFC→SC
provides prefrontal top-down input to command emotional and decision-making related
responses. The SC receives cortical information both directly through the cortico-tectal
projection pathway and indirectly through the multi-synaptic cortico-striato-nigra-tectal
projection pathway. The lateral PFC projections from ORBl→SC.l inputs align with
barrel-field and oropharyngeal subnetworks in SC.l, thereby guiding decision-making
about relevant stimuli proximal to the ground
100,102
. The combination of these limbic
system inputs may facilitate the integration of valence and motivated emotional
responses in social and appetitive behaviors
83
, though functional experiments are
required to test this hypothesis. Our anatomical findings support that the SC.cl is
involved in eye-head coordination during approach/appetite behavior, including pray
capturing; while the SC.l is involved in sensorimotor actions of orofacial movement
during the consummatory phase (such as chewing, licking, biting).

2.8.2 SUBNETWORKS IMPLICATED IN DEFENSIVE / AGGRESSIVE BEHAVIORS
The cortico-tecto-thalamic subnetwork is directly and indirectly connected with
hypothalamic and amygdalar structures that may coordinate the activation of fear-
related circuitry
83,103,104
. Within the medial cortico-cortical subnetworks, ORB is

78
interconnected with ACAv, ACAd/MOs and PTLp-med (internal body map), which all
project to the SC.cl/l zones, suggesting a coordination of motor outputs for the whole
body to prompt escape and attack reactions (Figure 2.17). Notably, the SC.m and SC.cm
deep layers also receive dense input from the dorsomedial part of the VMH
(VMH.dm/c), known to evoke innate defensive responses and aggressive responses to
predators
77,105,106
. Moreover, these medial SC zones send outputs to the RE, CM and IAM,
which has been shown to be activated during arousal and the presence of visual
threats
86,107
. Our results reveal a ILA→SC.m/l pathway that is consistent with the role of
ILA in mediating fear extinction, risk assessment and evaluation to threat situations
(particularly to looming stimuli/predators)
25,84,101,108
.

2.8.3 SUBNETWORKS IMPLICATED IN NAVIGATION AND GOAL-ORIENTED
BEHAVIORS
As described, RSP, PTLp-lat and VIS prominently align in the medial zones SC.m and
SC.cm (and partially in SC.cl) that process distinct representations of visual information
from the upper peripheral and central regions of the visual field
20,93
(Figure 2.17b). The
RSP→SC presumably contributes to mediating spatial orientation and navigation,
particularly through head-direction cells in RSPv interacting with the CA1dr/subiculum
hippocampal network
28,97,109
. The SC.m/cm, in turn, projects to RE in the thalamus which
also contains head direction-sensitive cells
110
. The ACA is implicated in guiding response
selection based on the value in context, richness, and memory of an environment during
exploratory and foraging behavior
101
. As environments vary during navigation, the
overlapping inputs from ACA→SC contribute to facilitating attention during
coordinated exploratory and evaluative response strategies. Our findings of the

79
visuomotor subnetwork in SC.cl further support this zone as an analogous point of
convergence for frontal-eye-field orienting behaviors in primates and cats
37
. In particular,
the SC.cl further projects to the MRN and GRN, which are involved in coordinating
locomotive and attentive visual behaviors
111,112
. As such, the widespread projections from
ACAd/v to SC.cm, SC.cl and SC.l may provide information regarding the contextual
value of the environment that integrates with multisensory SC neurons processing input
from virtually the entire visual, auditory, and somatotopic fields. Cells in the SC.cl-ig
aligned with acetylcholinesterase-rich patches also project strongly to the MD thalamic
nucleus, which is involved in frontal-eye-field processing
104,113,114
. By incorporating value-
defined inputs across the SC, we can better understand the role of distinct SC zones in
commanding eye movements and goal-oriented motor behaviors
115
.

80
CONCLUSION
Overall, these results refine previous studies observing sensory topographic distribution
patterns within the SC and provide a comprehensive understanding of how all higher-
order cortical inputs are integrated. To the best of our knowledge, this map of the cortico-
tectal projectome is the first in the mouse model and will thus serve as a useful cross-
species reference
47,48,116
. We have shown the mouse SC can be subdivided into four
columnar zones with distinct connectivity that correlate with functional subnetworks.
For each zone, we characterized the input/output organization and provided working
hypotheses to explore their functional implications. This study advances our
understanding of SC organization within brain-wide subnetworks.
Understanding the neural networks of attention-related visual behaviors in mouse
models has clinical significance in examining aberrant connections of psychopathologies
and neurodevelopmental disorders
40
. In humans, the SC is hypothesized to be a major
locus of interest for potential therapeutic targets in treating hyper-responsivity and
distractibility in attention-deficit hyperactivity disorders
41–44
, and delineating these
subnetwork circuits could facilitate neuromodulatory treatments as discussed in Chapter
4. Ultimately, the cortico-tectal projectome provides an anatomical framework to further
interrogate how individual SC cell-types integrate convergent input from multiple brain
areas and functional modalities for the control of attention and other goal-directed
behaviors.

81

EXTRASTRIATE CONNECTIVITY
OF THE MOUSE LATERAL
DORSAL GENICULATE THALAMUS

Adapted from Journal of Comparative Neurology (2019) publication. See Appendix B.

ABSTRACT
The mammalian visual system is one of the most well-studied brain systems. Visual
information from the retina is relayed to the dorsal lateral geniculate nucleus of the
thalamus (LGd). The LGd then projects topographically to primary visual cortex (VISp)
to mediate visual perception. In this view, the VISp is a critical network hub where visual
information must traverse LGd-VISp circuits to reach higher-order ‘extrastriate’ visual
cortices, which surround the VISp on its medial and lateral borders. However, decades
of conflicting reports in a variety of mammals support or refute the existence of
extrastriate LGd connections that can bypass the VISp. Here, we provide evidence of
bidirectional extrastriate connectivity with the mouse LGd. Using small, discrete
coinjections of anterograde and retrograde tracers within the thalamus and cortex, our
cross-validated approach identified bidirectional thalamocortical connectivity between
LGd and extrastriate visual cortices. We find robust reciprocal connectivity of the medial
extrastriate regions with LGd neurons distributed along the ‘ventral strip’ border with

82
the intergeniculate leaflet. In contrast, LGd input to lateral extrastriate regions is sparse,
but lateral extrastriate regions return stronger descending projections to localized LGd
areas. We show further evidence that axons from lateral extrastriate regions can overlap
onto medial extrastriate-projecting LGd neurons in the ventral strip, providing a putative
subcortical LGd pathway for communication between medial and lateral extrastriate
regions. Overall, our findings support the existence of extrastriate LGd circuits and
provide novel understanding of LGd organization in rodent visual system.

INTRODUCTION
In all mammals, visual information from the retina is projected topographically onto the
dorsal lateral geniculate nucleus of the thalamus (LGd), which in turn projects a
retinotopic map onto the primary visual cortex (VISp, also known as V1 or striate cortex).
The VISp is positioned as a visual gateway to the rest of the cortex and this view is
supported by the loss of vision caused by VISp damage. However, reports of visually-
dependent behavior and visually-evoked activation of extrastriate cortex in cortically-
blind patients has suggested additional neural circuit pathways that convey retinal visual
information to the brain
117
. One putative pathway for these ‘blindsight’ abilities is the
retino-tectal pathway (direct retinal projections to the superior colliculus). Another
possibility is that the LGd projects to ‘secondary’ extrastriate cortical visual areas,
although the existence of extrastriate LGd projections has been controversial
118
.
Conflicting reports of extrastriate LGd connections using different techniques
across a variety of animal species can be found throughout the literature (Table 3.1). Karl
Lashley’s early retrograde degeneration studies in the rat established the historically
predominant view that the VISp is the only recipient of LGd input
119,120
. In 1965,

83
extrastriate LGd connections were first reported in the opossum
121
(contrary to an earlier
study
122
and later in the rabbit
123
), but both studies seemed to gain little notice from the
research field. Instead, two studies in cat would establish that the cat (but not primate)
LGd provided extensive input to VISp as well as multiple extrastriate visual areas
124,125
.
Initially, the cat extrastriate LGd projections were thought to be an exception compared
to other mammals, but both positive
126–147
and negative/absent reports of LGd extrastriate
projections in multiple species led to debate over the next several decades
138,143,148–163
.
With the advent of new tract tracing methods in the 1980’s, the existence of LGd
projections to extrastriate visual cortex became more established in non-human primates.
LGd axons have been reported within V2
126,127,131,140,142
, V4
134,164
, inferotemporal cortex
146

and MT, although some studies reported a lack of fibers in these areas
157
. Non-human
primate extrastriate LGd projection neurons are scattered throughout the interlaminar
and S layers, but are larger in size than typical koniocellular neurons
127,165
. Using V1-
lesioned macaque monkeys, Schmid and colleagues found that reversible inactivation of
the LGd in VISp-lesioned animals eliminated extrastriate cortex fMRI responses and
‘blindsight’ behavior, proposing that LGd extrastriate connections mediate blindsight
and may provide a shortcut for rapid detection in normal vision
166
.

84
Table 3.1 | Literature on thalamocortical connectivity.
Articles which examined thalamocortical connectivity of the LGd and/or VIS cortices are listed in
chronological order. Columns include brief information about the experimental species, methodology,
whether or not the study reported the existence or non-existence of LGd extrastriate connections, and a
brief summary description of the reported findings. Overall, we found 33 reports of positive findings of
extrastriate LGd projections, 17 reports of negative findings, and 4 studies which did not report their
presence or absence (DNR). Different studies have used different nomenclature to describe visual cortices,
but secondary visual cortex is located immediately adjacent and surrounding primary visual cortex in
mammalian species. Generally, primary visual cortex = VISp, V1, area 17, area striata, striate cortex;
secondary visual cortices = V2-V4 and MT, areas 18 (18a and 18b) and 19, area occipitalis, peristriate,
extrastriate (mouse = VISal, VISl, VISpl, VISam, VISpm).

Lashley, 1934 Rat RD after lesions in cortex No No degeneration is evident except after lesions of the area striata
Bodian, 1935 Opossum RD after cortical lesions No LGd degeneration after striate cortex lesions, but no other cortical area
Diamond and Utley, 1963 Opposum RD after striate/peristriate lesions Yes Degeneration in LGd was greater when lesions included both peristriate and striate
Rose and Malis, 1965 Rabbit RD after striate and peristriate lesions Yes Degeneration in ventromedial LGd following medial peristriate lesion
Glickstein et al, 1967 Cat AD after LGd lesions Yes Dense LGd projections to Areas 17, 18, and 19
Wilson and Cragg, 1967 Monkey and Cat AD after thalamic lesions
No (monkey)
Yes (cat)
In monkey, degeneration did not extend beyond the borders of area 17 after LGd lesions. In cat,
degeneration was found in area 17 and area 18 after LGd lesions
Hall and Diamond, 1968 Hedgehog RD after striate and peristriate lesions Yes Degeneration in LGd following extrastriate lesions
Diamond et al, 1970 Tree Shrew RD after striate and peristriate lesions No No degeneration in LGd following peristriate lesions
Niimi and Sprague, 1970 Cat RD after cortical lesions Yes
Lesions of area 18 and 19 produced marked retrograde degeneration in medial interlaminar nucleus
and mild degeneration in other LGd lamina
Benevento and Ebner, 1971 Opossum AD after thalamus lesions No LGd lesions did not produce degeneration anywhere other than striate cortex
Garey and Powell, 1971 Cat and monkey AD after thalamic lesions Yes (cat) No (monkey)
Degeneration was found in areas 17, 18, 19, and the lateral suprasylvian area after lesions of the cat
LGd. In contrast, lesions of monkey LGd produced degeneration that was restricted to area 17
Hubel and Wiesel, 1972 Macaque monkey AD after thalamic lesions No No degeneration was seen outside of the striate cortex
Kaas et al, 1972 Squirrel RD after cortical lesions No No degeneration in LGd following Area 18 or Area 19 lesions
Harting et al, 1973 Tree Shrew AD after thalamic lesions No No degeneration observed outside of Area 17 after LGd lesions
Dräger, 1974 Mouse Trasneuronal AT w/ tritiated Proline into eye Yes Radioactivity in Area 18a, unclear if from LGd or VISp
Colewell, 1975 Rabbit RT and AT via coinjection of HRP and tritiated amino acids DNR Did not report any extrageniculate LGd projections
Hubel, 1975 Tree Shrew Trasneuronal AT w/ tritiated Proline into eye Yes Radioactivity in Area 18a, unclear if from LGd or VISp
Ribak and Peters, 1975 Rat AT w/ tritiated Proline into LGd Yes Radioactivity in Areas 18, and 18a, unclear if from LGd or VISp
Robson and Hall, 1975 Squirrel AT w/ tritiated amino acids into thalamus DNR Did not report any extrageniculate LGd projections
Winfield et al. 1975 Rhesus monkey RT w/ HRP into cortex Yes
Description of results is vague, but in LGd of all brains there has been a well defined band of
labelled cells throughout all laminae in the caudal third of its anteroposterior extent." Figure show an
injection site in Area 18 that produces labeling across all layers at the medial boundary of LGd
Glendenning et al, 1976 Galago bush baby AD after thalamic lesions No No mention of AD anywhere other than area 17
Levay and Gilbert, 1976 Cat AT w/ Tritiated Amino Acids into different LGd lamina Yes
Radioactivity in area 17 produced from injections in the LGd 'A' lamina and radioactivity in areas 17,
18, 19, and the suprasylvian gyrus after injections in the LGd 'C' lamina. Distribution of projection
fibers in area 18 lamina similar to area 17 organization
Peters and Saldanha, 1976 Rat
AD after thalamic lesions and EM reconstruction of synaptic
contacts in cortex
DNR Did not report any extrageniculate LGd projections
Wong-Riley, 1976 Squirrel monkey
RT w/ HRP after injections into dorsolateral perstriate along the
area 18 and area 19 border
yes
HRP-labelled LGd cells occupied a central wedge or sector exclusively in the anterior one- third of
the nucleus. This wedge extended rostrolateral to caudomedially through all of the parvocellular
and magnocellular laminae (see Fig. 3) and corresponded to the peripheral representation of the
horizontal meridian and the immediately adjacent fields 11. Labelled almost all of the medium and
large cells and none of the small neurons
Coleman et al, 1977 Opposum RT w/ HRP afterinjections into cortex No No retrograde labeled LGd neurons after HRP injections into extrastriate cortex
Hughes, 1977 Rat
AT w/ tritiated amino acids after thalamus injections and RT w/
HRP after cortex injections
Yes
Tritiated amino acid injection into LGd produces radioactivity in the medial aspect of Area 18a and
HRP injection into Area 18a results in extensive retrograde labelling in LGd
Karamanlidis and Giolli, 1977 Rabbit Horseradish-Peroxidase (HRP) injections into V1 cortex DNR Did not report any extrageniculate LGd projections
Weber et al, 1977 Grey-Squirrel Trasneuronal AT w/ tritiated Proline into eye Yes Radioactivity in Area 18a, unclear if from LGd or VISp
Gould et al, 1978 Hedgehog AD after thalamic lesions No No degeneration in parastriate cortex following LGd lesions
Dursteler et al, 1979 Hamster RT w/ HRP injected into areas 17, 18a, 18b No
Found HRP-labeled LGd cells after injection into area 18a, but attributed labeling to leakage into
area 17
Karamanlidis et al, 1979 Sheep RT w/ HRP afterinjections into cortex Yes Retrogradely-labeled HRP neurons in LGd after injections in either area striata and area occipitalis
Coleman and Clerici, 1980 Rat RT w/ HRP afterinjections into cortex Yes Scattered, sparse HRP-labeled LGd neurons following injections in Area 18a
Haight et al, 1980 Marsupial brush-tailed possum
AT w/ tritiated amino acids and RT w/ HRP after injections into
cortex
Yes
Overlapping distribution of tritiated amino acid fibers and retrogradely-labeled HRP neurons
within LGd. LGd projections to peristriate similar to genicul-striate projections, preferential to outer
portions of LGd.
Hollander and Halbig,1980 Rabbit
Trasneuronal AT w/ tritiated amino acids into eye and RT w/
HRP after cortex injections
Yes
Tritiated amino acid labeling in area striata and area occipitalis following intraocular injection and
retrograde HRP-labeled neurons in LGD following injection into area striata and area occipitalis
Caviness and Frost, 1980 Mouse AD after thalamic lesions No Degeneration confined to Area 17 after LGd lesions
Dräger, 1981 Reeler mice Trasneuronal AT w/ tritiated amino acids into eye No Radioactivity present in Area 18a, but interpreted as arising from tectum
Benevento and Yoshida, 1981 Macaque monkey
AT w/ tritiated amino acids into LGd and RT w/ HRP into Areas
18 and 19
Yes Interlaminar LGd neurons project to Layer V in a large part of Area 19 and anterior Area 18
Fries, 1981 Macaque monkey HRP into Areas 18 and 19 Yes
After injections in area 18 and 19, HRP-labeled LGd cells were found broadly scattered across LGd
lamina. Numbers of labelled neurons were low and never exceeded 12 per
Coleman and Clerici, 1981 Opposum RT w/ HRP after cortex injections No
No HRP-labeled LGd neurons following injections into Area 18, 19, or anterior and posterior
peristriate area
Yukie and Iwai, 1981 Macaque monkey
RT w/ HRP after injections into striate, prestriate, inferotemporal
and parietal cortices
Yes
HRP labeled LGd cells projecting to peristriate show unique distribution and morphology compared
to striate-projecting LGd neurons
Benevento and Standage, 1982 Macaque monkey RT w/ wheat germ conjugated HRP into V2 and MT No No retrograde labeling in the LGd after injections into V2 and MT
Towns et al, 1982 Rabbit
AT w/ tritiated amino acids into LGd and RT w/ HRP into striate
and occipital cortex
Yes
Radioactivity in occipital cortex, confirmed by retrogradely-labeled HRP neurons in LGd after
occipital cortex injection
Bullier and Kennedy, 1983 Macaque monkey RT w/ Fast Blue and Diamidino Yellow into V2 Yes Scattered retrograde labeling within interlaminar and S layers
Raczkowski and Rosenquist, 1983 Cat
RT w/ HRP and AT w/ tritiated Leucine in multiple visual
cortices
Yes LGd projects to areas 17, 18, 19, and suprasylvian areas
Kennedy and Bullier, 1985 Macaque monkey RT w/ Fast Blue and Diamidino Yellow into V1 and V2 Yes
V1-projecting LGd neurons were densely packed within lamina whereas V2-projecting LGd neurons
were rare, more scattered, and located within interlaminar and S layers
Lysakowski et al, 1988 Macaque monkey RT w/ Granular Blue and Nuclear Yellow into V1 and V4 Yes
V4-projecting LGd neurons were sparse compared to V1-projecting LGd neurons. No
collateralization of input from LGd to V1 and V4
Tanaka et al, 1990 Macaque monkey RT w/ HRP after cortex injections Yes Sparse numbers of V4-projecting LGd neurons in the interlaminar region
Sanderson et al, 1991 Rat RT w/ WGA-HRP after cortex injections Yes Minor retrograde labeling (less than 5%) in LGd after injections in AM, AL, and LM
Hernandez-Gonzalez et al, 1994 Macaque monkey RT w/ multiple retrograde tracers into inferior temporal cortex Yes
Sparse numbers of LGd interlaminar neurons project to inferior temporal cortex, appear similar to
population of other extrastriate LGd neurons
Sincich et al, 2004 Macaque monkey RT w/ Cholera Toxin B subunit in area MT and WGA-HRP in V1 Yes
MT-projecting LGd neurons were found scattered throughout the intercalated layers. One-third of
MT-projecting LGN cells in the intercalated layers were CAMK2 negative, suggesting a
non-koniocellular cell-type
Oh et al, 2014 Mouse AT w/ fluorescent AAV vectors Yes
LGd sends output to VISp, VISal, and VISl and LGd receives input from VISam, VISp, VISal, VISl, and
the temporal association area
Ajina et al, 2015 Human Diffusion weighted MRI tractography Yes
White matter tracts between LGd and area MT were found in blindsight positive patients and
age-matched controls. Blindsight negative patients with V1 damage were shown to have impaired
or absent LGd-MT fiber tracts
Extrastriate LGd
Connections?
Article Animal Model Method Summary of Findings

85
Compared to the non-human primate, the rodent LGd has been significantly less
studied. However, the popularity of the mouse as a genetic model has renewed interest
in rodent vision
167,168
, particularly toward discovering parallel visual processing streams
through the LGd
169–171
. A major obstacle to understanding the organization of the visual
system in rodents has been structural differences in the visual thalamus and cortex.
Notably, the lack of a laminar structure to the rodent LGd hinders a clear understanding
of LGd cell type organization. In addition, it is unclear how extrastriate visual cortex
regions in the mouse are homologous to the more complex non-human primate. A recent
mouse connectomics study reported multiple LGd extrastriate connections, but did not
specifically address this topic
172
. As part of our Mouse Connectome Project
(www.mouseconnectome.org), we have performed double anterograde/retrograde
tracer coinjections across the cortex, including VISp and the medially (VISam, VISpm)
and laterally adjacent extrastriate VIS areas (VISal, VISl, and VISpl)
52
. To understand VIS
thalamocortical connectivity and investigate LGd extrastriate connectivity in the mouse,
we have targeted anterograde/retrograde tracer coinjections into VIS cortices and small
subregions of the LGd thalamus. By simultaneously visualizing input/output
connectivity, this approach can address bidirectional LGd thalamocortical connectivity
with two cross-validated datasets (Figure 3.1).

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Figure 3.1 | Bidirectional circuit tracing strategy.
To examine bidirectional connectivity between the LGd and VIS cortices, we iontophoretically coinjected
pairs of anterograde and retrograde tracers (PHAL/CTB or BDA/FG) into the LGd and visual cortices
(VISp, medial extrastriate VIS areas (VISam, VISpm), and lateral extrastriate visual areas (VISal, VISl,
VISpl)). (left) Coinjection sites within the VIS cortices produced anterogradely-labeled fibers (green) and
retrogradely-labeled cell bodies (magenta) within the LGd. Conversely, LGd coinjections produced
anterogradely-labeled fibers (green) and retrogradely-labeled cell bodies (magenta) in the VIS cortices
(right). In this way, the same connection can be labeled by an anterograde tracer or a retrograde tracer and
both datasets are cross-validated by the different tracer type (i.e., anterogradely-labeled LGd fibers in the
visual cortices are confirmed by retrograde labeling in the LGd after VIS cortex injection). BDA, biotinylated
dextran amine; CTB, cholera toxin subunit B; FG, Fluorogold; LGd, dorsal lateral geniculate thalamic nucleus; PHAL,
Phaseolus vulgaris leucoagglutinin; VISam, anteromedial visual cortex; VISal, anterolateral visual cortex, VISl,
lateral visual cortex, VISp, primary visual cortex; VISpl, posterolateral visual cortex; VISpm, posteromedial visual
cortex.
_____________________________________________________________________________________________

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METHODS
Mouse Connectome Methodology Overview.
Mouse Connectome Project (MCP) tract-tracing data was generated within the Center for
Integrative Connectomics (CIC) at the University of Southern California (USC) Mark and
Mary Stevens Neuroimaging and Informatics Institute. MCP experimental methods and
online publication procedures have been described previously
27,173
. All MCP tract-tracing
experiments were performed using 8-week old male C57BL/6J mice (n=26; Jackson
Laboratories). Mice had ad libitum access to food and water and were pair-housed within
a temperature- (21-22°C), humidity- (51%), and light- (12hr:12hr light/dark cycle)
controlled room within the Zilkha Neurogenetic Institute vivarium. All experiments were
performed according to the regulatory standards set by the National Institutes of Health
Guide for the Care and Use of Laboratory Animals and by the institutional guidelines
described by the USC Institutional Animal Care and Use Committee.

Tracer injection experiments.
The MCP uses a variety of combinations of anterograde and retrograde tracers to
simultaneously visualize multiple anatomical pathways within the same Nissl-stained
mouse brain. The standard experimental approach is a double coinjection of paired
anterograde (2.5% Phaseolus vulgaris leucoagglutinin (PHAL; Vector Laboratories, Cat#
L-1110, RRID:AB_2336656), 5% biotinylated dextran amine (BDA; Invitrogen) and
retrograde (0.25% Alexa Fluor 647 conjugated cholera toxin subunit b (CTB-647;
Invitrogen) or 1% Fluorogold (FG; Fluorochrome, LLC)) tracer into two different brain
regions. Additionally, quadruple retrograde experiments are performed using 1% FG
and 0.25% Alexa Fluor-488, -555, and -647 conjugated CTB tracers (CTB-488, CTB-555,

88
CTB-647) injected into four distinct brain regions. In this study, we describe coinjection
experiments in the LGd and visual cortex (VISp, VISam, VISpm, VISal, and VISl) as well
as a quadruple retrograde tracer experiment in the visual cortices.

Stereotaxic Surgeries.
On the day of experiment, mice are deeply anesthetized and mounted into a Kopf
stereotaxic apparatus where they are maintained under isoflurane gas anesthesia (Datex-
Ohmeda vaporizer). For anterograde/retrograde coinjection experiments, tracer cocktails
were iontophoretically delivered via glass micropipettes (outer tip diameter 15-30μm)
using alternating 7 s on/off pulsed positive electrical current (Stoelting Co. current
source) for 5 (BDA or AAV/FG) or 10 min (PHAL/CTB-647). For quadruple retrograde
tracing experiments, 50nl of retrograde tracers were individually pressure-injected via
glass micropipettes at a rate of 10nl/min. All injections were placed in the right
hemisphere. Injection site coordinates for each surgery case are on the Mouse
Connectome Project iConnectome viewer (www.MouseConnectome.org). Table 3.2 lists
the injection site coordinates for each tracer based on the (ML, AP, DV) coordinate system
from the 2008 Allen Brain Reference coronal atlas
52
. Following injections, incisions were
sutured and mice received analgesic pain reliever and were returned to their home cages
for recovery.

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Table 3.2 | Injection Sites.
All injection sites are listed by targeted brain region (alphabetical order). For each unique animal case ID,
the table lists target brain region, injected tracer molecules, and the stereotaxic coordinate location and
corresponding ARA level of the injection site center. All image data is hosted online through our
iConnectome viewer at www.mouseconnectome.org and can be found by querying the case number or
target brain region.

Mouse Case ID Injection Location Tracers ML (X) AP (Y) DV (Z)
SW130619-01B LGd PHAL + CTB-647 2.1 -2.355 -2.75
SW130724-04A LGd PHAL + CTB-647 2.25 -2.055 -2.8
SW130724-03A LGd PHAL + CTB-647 2.25 -2.155 -2.8
SW130625-01A LGd PHAL + CTB-647 2.25 -2.48 -2.75
SW130625-02A/B LGd PHAL + CTB-647 2.1 -2.155 -2.5
SW141020-04B LGd BDA + CTB-555 2.1 -2.355 -2.75
SW141020-01B LGd CTB-555 2.25 -2.055 -2.6
SW140827-01A LP PHAL + CTB-647 1.75 -2.255 -2.6
SW180302-07A VISal PHAL 3.5 -3.08 -1.25
SW170927-03A VISal PHAL 3.75 -3.68 -1.75
SW170822-07A VISal PHAL + CTB-647 3.6 -3.78 -1.5
SW170822-06A VISl PHAL + CTB-647 3.75 -3.98 -1.7
SW170822-05A VISl PHAL + CTB-647 3.75 -4.08 -1.7
SW170822-04A VISl PHAL + CTB-647 3.75 -4.15 -1.8
SW121220-02A VISp CTB-488 2.6 -4.155 -0.75
SW121220-02A VISpm CTB-647 1.5 -4.155 -0.5
SW121220-02A VISam FG 1.7 -3.28 -0.5
SW121220-02A VISp CTB-555 2.8 -3.28 -0.5
SW111004-02A VISam FG 1.6 -3.48 -0.4
SW111004-02A VISal PHAL + CTB-647 3.5 -3.18 -1.25
SW120125-02A VISam PHAL + CTB-647 1.6 -3.38 -0.4
SW111004-03A VISpm FG + BDA 1.5 -3.98 -0.4
SW121222-01A VISpm FG + BDA 1.4 -4.38 -0.5
SW111004-01A/B VISp FG + BDA 3.1 -3.48 -0.6
SW110808-03A VISp PHAL + CTB-647 2.75 -3.78 -1
SW110808-01A VISp PHAL + CTB-647 1.8 -4.45 -0.75
SW121221-02A VISp FG + BDA 3.2 -4.38 -1.2
SW121221-02A VISp PHAL + CTB-647 2.2 -4.38 -0.5
SW121221-03A VISp PHAL + CTB-647 2.5 -3.38 -0.4
SW121221-03A VISp FG + BDA 2.75 -4.28 -1.2
SW110615-03A VISpm FG + BDA 1.4 -4.655 -0.4
SW180809-01A VISam FG 1.75 -3.38 -0.5
SW151211-02A VISam PHAL 1.75 -3.28 -0.4

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Histology and Immunohistochemical Processing.
After 1-2 weeks post-surgery, each mouse was deeply anesthetized with an overdose of
sodium pentobarbital and trans-cardially perfused with 50ml of 0.9% saline solution
followed by 50ml of 4% paraformaldehyde (PFA, pH 9.5). Following extraction, brain
tissue was postfixed in 4% PFA for 24-48hrs at 4°C. Fixed brains were embedded in 3%
Type I-B agarose (Sigma-Aldrich) and sliced into four series of 50μm thick coronal
sections using a Compresstome (VF-700, Precisionary Instruments, Greenville, NC) and
stored in cryopreservant at -20°C.
For double coinjection experiments, one series of tissue sections was processed for
immunofluorescent tracer localization. BDA immunofluorescence was visualized using
a 647- or 568-conjugated Streptavidin. For PHAL immunostaining, sections were placed
in a blocking solution containing normal donkey serum (Vector Laboratories) and Triton-
X (VWR) for 1hr. After rinsing in buffer, sections were incubated in PHAL primary
antiserum (donkey serum, Triton-X, 1:100 rabbit-anti-PHAL antibody (Vector
Laboratories Cat# AS-2300, RRID:AB_2313686) in KPBS buffer solution) for 48-72hrs. at
4°C. Sections were then rinsed again in buffer solution and then immersed in secondary
antibody solution (donkey serum, Triton-X, and 1:500 donkey anti-rabbit IgG conjugated
with Alexa Fluor 488 (ThermoFisherScientific Cat# A-21206, RRID:AB_2535792)) for 3hrs.
For parvalbumin and Neuropeptide Y immunostaining, sections were placed in a
blocking solution containing normal donkey serum and Triton X (VWR) for 1 hr. After
rinsing in buffer, sections were incubated in parvalbumin primary antiserum (donkey
serum, Triton X, 1:500 mouse monoclonal-anti-parvalbumin antibody (Sigma-Aldrich
Cat# P3088, RRID:AB_477329) in KPBS buffer solution), and Neuropeptide Y primary
antiserum (1:500 rabbit-anti-Neuropeptide Y (ImmunoStar Cat# 22940,
RRID:AB_2307354) for 48-72 hrs. at 4°C. Sections were then rinsed again in buffer solution

91
and then immersed in secondary antibody solution (donkey serum, Triton-X, and 1:500
donkey anti-mouse IgG conjugated with Alexa Fluor 488 (Thermo Fisher Scientific Cat#
A-21202, RRID:AB_141607) , and 1:500 donkey anti-rabbit conjugated with CY3 (Jackson
ImmunoResearch Labs Cat# 715-165-151, RRID:AB_2315777)) for 3 hrs. Finally, all
sections were stained with Neurotrace 435/455 (Thermo Fisher Cat# N21479) for 2-3 hrs.
to visualize cytoarchitecture. After processing, sections were mounted onto microscope
slides and coverslipped using 65% glycerol.

Imaging, Online Data Publication, and Open Image Data Access.
Complete tissue sections were scanned using a 10X objective lens on an Olympus VS120
slide scanning microscope. Each tracer was visualized using appropriately-matched
fluorescent filters and whole tissue section images were stitched from tiled scanning into
VSI image files. For online publication, raw images are corrected for correct left-right
orientation and matched to the nearest Allen Reference Atlas level (ARA; Dong, 2008).
VSI image files are converted to TIFF file format and warped and registered to fit ARA
atlas levels (all images shown in this manuscript are from unwarped, unregistered VSI
images). Each color channel is brightness/contrast adjusted to maximize labeling
visibility (NeuroTrace 435/455 is converted to brightfield) and TIFF images are then
converted to JPEG2000 file format for online publication. Microscope images of one tissue
series from each case are registered to the ARA and published online and can be viewed
using the MCP iConnectome viewer (www.MouseConnectome.org) (note: manuscript
images are raw and unregistered). iConnectome data from this study (and more) can be
found by querying injection site location (VISp, VISam, LGd, etc.) or experimental case
number (located at the bottom of figure images in this manuscript and Figure 3.2; ex.
SW130619-01B).

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Figure 3.2 | Mapped injection site spread throughout the visual cortex and thalamus.
For LGd (a) and cortical injections (b), the size and shape of each tracer injection site was mapped onto
ARA sections using one 200μm-interval tissue series (ARA atlas numbers listed below each section).
Borders between the VISp and medial (VISam/VISpm) and lateral extrastriate areas (VISal, VISl, VISpl) are
demarcated by red and blue arrowheads, respectively. The boundaries of the LGd and neighboring LP,
IGL, and LGv are labeled at the far right and apply to each consecutive rostrocaudal level. In most cortical
injection cases, retrograde injection sites are spherical in shape with a 400-500μm diameter spreading across
2-3 rostrocaudal levels. Anterograde injection spread was typically smaller than retrograde spread when
coinjected together. In LGd-injected experiments, coinjection sites are spherical in shape with a 200-300μm
diameter spreading across 2-3 rostrocaudal levels and small enough to not completely fill the LGd (most
of the injection site is concentrated at the center with minimal spread across levels).Tracer name is listed
under each case number and color-coded to injection site mapped color.
______________________________________________________________________________

93
RESULTS
Overall, all tracer experiments into the cortex produced 8 anterograde and 9 retrograde
VISp injection sites, 2 anterograde and 3 retrograde VISam injection sites, 3 anterograde
and 4 retrograde VISpm injection sites, 4 anterograde and 2 retrograde VISal injection
sites, and 3 anterograde and 3 retrograde VISl injection sites. Double coinjection
experiments in the LGd produced 9 anterograde and 10 retrograde sites. An additional 2
anterograde/retrograde coinjections were made into the LP for comparison. Microscopy
images of the entire tissue series for each experimental case are available online at
www.MouseConnectome.org. The spread of each coinjection site (except LP) was
mapped onto ARA sections and the overall distribution of the injection sites covered a
large amount of the whole VIS cortices and LGd (Figure 3.2).

3.4.1 VERIFICATION OF LGD AND VIS CORTEX BOUNDARIES BY
CYTOARCHITECTURE, HISTOCHEMICAL/MOLECULAR STAINING, AND
CONNECTIVITY PATTERNS
A critical factor in interpreting anatomical tract tracing data is the accurate verification of
injection site and tracer labeling locations within neuroanatomical structures. Directly
registering experimental tissue sections to standardized brain atlases (such as the ARA)
can be difficult due to variability in histological processing across multiple animals (i.e.,
oblique tissue sectioning angles, tissue warping, etc.). Therefore, we used Nissl
cytoarchitecture, histochemical markers, and distinct connectivity patterns to validate the
boundaries of the LGd and VIS cortex before evaluating neuroanatomical tracer data.
The LGd is located in the dorsolateral thalamus and bordered ventrally by the
intergeniculate leaflet (IGL), and medially by the lateral posterior thalamus (LP) and
external medullary lamina white matter (em). Examination of Nissl cytoarchitecture can

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clearly distinguish these boundaries (Figure 3.3a,e). The ventral boundary between the
LGd and IGL can be distinguished by the tangential orientation of the IGL cell bodies
relative to the more uniform shape of the LGd and LGv neuron cell bodies. Previous
studies in the rat have found multiple neurochemical markers that demarcate the
LGd/IGL border
174
. Immunohistochemical staining for parvalbumin and Neuropeptide
Y in the mouse revealed a similar distribution that is consistent with the LGd/IGL
boundary. Parvalbumin staining is notably dense throughout much of the thalamus
including the LGd and LGv (Figure 3.3b,f; note LGv cell bodies as well), but absent from
the IGL. In contrast, Neuropeptide Y-positive axon fibers and cell bodies are present in
the IGL, but not the LGd (Figure 3.3c,g). Together, the parvalbumin and Neuropeptide Y
provide complementary staining patterns that distinguish the IGL from the LGd and
LGv. In addition, coinjection of PHAL/CTB into the IGL produces an axonal terminal
field and CTB retrograde labeling within the contralateral IGL that clearly distinguishes
the LGd/IGL boundary (Figure 3.3i-k). Coinjections into the IGL also produce
characteristically strong anterograde labeling of the retino-hypothalamic pathway that
terminates in the suprachiasmatic nucleus (SCN; Figure 3.3l). The retino-hypothalamic
pathway is unique to IGL neurons and thus can be used to identify if LGd coinjections
spread into the adjacent IGL. Together, the Nissl cytoarchitecture, neurochemical
staining, and IGL connectivity provide strong agreement for the ventral LGd/IGL
border.

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Figure 3.3 | Validation of LGd/IGL boundary: cytoarchitecture, histochemical markers, connectivity.
(a-d) LGd boundaries in all figures were delineated based on Nissl cytoarchitecture, which corresponds
well with histochemical staining of parvalbumin (PV) and neuropeptide Y (NPY). (e-h) Focusing on the
ventral LGd/IGL boundary (area outlined by yellow box in (a)), IGL neuronal cell bodies are oriented
transversely in contrast to the uniform shape of LGd cell bodies (yellow arrows demarcate the dorsolateral
and ventromedial corners of IGL). This distinct cytoarchitecture corresponds directly with the
complementary absence of PV and presence of NPY staining within the IGL. (i) PHAL/CTB coinjection
into the IGL (SW130521-03A) produces retrogradely-labeled cell bodies and anterogradely-labeled fibers
that are contained within the contralateral IGL (j-k; arrows in k point to CTB-labeled IGL cell bodies). (l)
Injections that target the IGL are easily distinguished from LGd coinjections by labeling of the geniculo-
hypothalamic pathway to the suprachiasmatic nucleus (SCN) and a distinct absence of ascending fibers to
cortex. 3V, third ventricle; IGL, intergeniculate leaflet; LGd, dorsal lateral geniculate thalamic nucleus; LGv, ventral;
NPY, Neuropeptide Y; PV, parvalbumin; och, optic chiasm; SCN, suprachiasmatic nucleus.
______________________________________________________________________________

96
To confirm the ARA boundaries of the VISp with the medial and lateral
extrastriate regions, we examined gene expression, cytoarchitecture, and other
connectivity data that would identify the VISam/VISpm and VISal/VISl/VISpl from the
VISp. First, VISam and VISpm have a thinner layer 4 compared to the laterally-adjacent
VISp and the medially-adjacent agranular retrosplenial cortex (RSP) that does not contain
layer 4. Rorb gene expression has been used as a genetic marker for layer 4 and is densely
expressed in layer 4 neurons across the cortex with more sparse expression in layer 5
175
.
Rorb gene expression in the online Allen Brain Atlas in situ hybridization image database
provides a clear demarcation of the VISam and VISpm boundaries that is in strong
agreement with the ARA boundary delineation (Figure 3.4).

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Figure 3.4 | Validation of the VISam and VISpm boundaries by Rorb gene expression.
The VISam and VISpm can be distinguished from the medially-adjacent RSP and laterally-adjacent VISp
by distinct changes in layer 4. Layer 4 is absent from RSP and thicker in VISp compared to VISam and
VISpm. To clearly visualize layer 4, we compared our boundaries to a layer 4 gene expression marker (Rorb)
in the Allen Brain Atlas in situ hybridization database ( http://mouse.brain-map.org/), ARA atlas sections
on left, corresponding Rorb in situ hybridization on right). As expected, Rorb expression is robust in VISp
layer 4 with more sparse expression in layer 5. In the ARA-defined VISam and VISpm, Rorb expression is
notably weaker, in a thinner lamina compared to VISp, and abruptly ends at the RSP border across all
rostrocaudal levels. Rorb, RAR-related orphan receptor beta.
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98
For further verification of extrastriate boundaries, we analyzed extrastriate
connectivity with VISp and thalamus. In the mouse, a defining characteristic of
extrastriate visual areas are topographic connection with VISp (Wang & Burkhalter,
2007). In case SW121221-02A, we performed a double coinjection of BDA/FG into the
lateral VISp and PHAL/CTB into the medial VISp (Figure 3.5a). Both coinjections
produced topographically-organized columns of anterograde and retrograde labeling
within the ARA-defined VISam and VISal. Furthermore, these connectivity patterns are
consistent with the definition of extrastriate regions as demonstrated by Wang and
Burkhalter in which extrastriate regions receive topographic maps of VISp input
(although it is currently unclear how to directly relate the entire coronal ARA delineation
to the flattened laminar surface sectioning used by Wang and Burkhalter
18
. In addition,
‘higher order’ thalamic nuclei such as the LP project more densely to the extrastriate
visual areas than the VISp and show different laminar projection patterns
176
. In case
SW141021-02A, we coinjected BDA/FG into the caudal medial LP while PHAL/CTB was
coinjected into the LGd (Figure 3.5b). As expected, the anterograde and retrograde
labeling patterns from the LP and LGd injection sites clearly distinguish the extrastriate
visual areas. LP coinjection produced robust axon terminal fields in layer 4 of the
ipsilateral extrastriate visual areas and more minor fiber labeling in VISp layer 5a.
Retrogradely-labeled LP-projecting neurons were densely distributed in layers 5, 6a, and
6b in extrastriate areas whereas retrograde labeling in VISp was limited to layers 5 and
6b.

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Figure 3.5 | Validation of the VISam and VISpm boundaries by VISp and thalamic connectivity.
VISp can be distinguished from extrastriate visual areas by differences in anatomical connectivity. (a) A
double coinjection into the medial and lateral parts of VISp reveals the bidirectional topographic
connections in the VISam and VISal. BDA/FG coinjection into lateral VISp produced a column of
anterogradely-labeled fibers (red) and retrogradely-labeled neurons (yellow) in the medial parts of VISam
and VISal. PHAL/CTB coinjection into the medial VISp produced a column of anterogradely-labeled fibers
(green) and retrogradely-labeled neurons (magenta) in the lateral parts of VISam and VISal. Together, the
full set of topographic connections are within the VISam and VISal boundaries as demarcated in the ARA
(bottom). (b) Double coinjection into the thalamus also reveals bidirectional connectivity features that are
unique to extrastriate regions compared to VISp and are consistent with ARA boundaries. BDA/FG
coinjection into caudal medial part of LP produced thick anterogradely-labeled fiber terminal fields
(magenta) in layer 4 of VISam and VISal whereas only superficial layer 5 was innervated in VISp. In
addition, retrogradely-labeled neurons were located in layers 5, 6a, and 6b in VISam and VISal whereas
retrograde labeling was absent from VISp layer 6a. Coinjection of PHAL/CTB into the LGd produced
anterogradely-labeled fiber terminal fields (green) in VISp layer 4 and retrogradely-labeled neurons (red)
in layer 6a that were mostly localized to the VISp region. Together, the set of connections show clear
differences in connectivity that distinguish the extrastriate visual areas from the VISp.
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100
3.4.2 DISTRIBUTION OF TRACER LABELING WITHIN LGD AFTER VISP VERSUS
VISAM/PM COINJECTIONS
Each VISp coinjection produced a highly overlapped distribution of ipsilaterally
clustered anterograde and retrograde labeling within the LGd (Figure 3.6). Tracer labeled
clusters extended across multiple rostrocaudal levels of the LGd, reflecting the spherical
spread of the VIS cortex injection site. As expected, the overall rostrocaudal and
mediolateral locations of the VISp injection sites demonstrated that the clusters of
labeling within the LGd were topographically-organized. As shown by the labeling
distribution in Figure 3.6, injection sites which were located in the posterior VISp
produced retrograde labeling along the superficial, lateral edge of the LGd. In contrast,
more anterior VISp injections sites retrogradely labeled LGd neurons closer to the deep,
medial LGd border. In the mediolateral direction, lateral VISp injection sites produced
labeling clusters close to the dorsal LGd border (adjacent to the lateral posterior thalamic
nucleus (LP)), whereas more medial VISp injection sites retrogradely-labeled LGd
neurons more ventrally. However, coinjections within the most medial parts of VISp did
not produce labeling at the most ventral part of the LGd (adjacent to the intergeniculate
leaflet, IGL) as would be expected based on the principles of topographic organization.
Instead, coinjections into the VISam and VISpm resulted in distributed tracer
labeling within the LGd along a ventral strip adjacent to the IGL border (Figure 3.7).
Similar to tracer labeling following VISp injections, coinjections within the VISam and
VISpm produced small, localized clusters of anterograde and retrograde labeling within
the LGd. Mapping the distribution of retrogradely-labeled LGd neurons produced by the
array of injection sites along the rostrocaudal extent of the VISam and VISpm determined
that posterior VISpm injections retrogradely-labeled neurons at the most superficial,
ventrolateral part of the LGd. Coinjection sites which were located progressively more

101
rostral through the VISam produced labeled neuron clusters more ventromedially along
the LGd ventral strip adjacent to IGL.
To compare the distribution of VISp and VISam/pm labeling within the same animal,
we used a quadruple retrograde tracing approach and individually injected four
retrograde tracers into the anterior VISp, posterior VISp, VISam, and VISpm (Figure
3.8a). As expected, four distinct clusters of retrograde labeling were observed within the
LGd in a rectangular orientation consistent with the injection site placement. Notably, the
rectangular orientation of the retrograde labeling changes at different LGd rostrocaudal
levels. At mid-rostrocaudal LGd levels, the LGd is split relatively evenly by the midline
between anterior VISp/VISam and posterior VISp/VISpm cortex labeling. However, this
anterior/posterior midline shifts laterally in anterior LGd levels and medially in posterior
LGd levels, suggesting that posterior VIS cortices are more represented at anterior LGd
levels and vice versa. Across all of our experiments, mapping retrograde labeling patterns
produced by VISp vs. VISam/pm coinjection sites across all LGd levels shows two
distinct distributions within the LGd (Figure 3.8b). Finally, Neuropeptide Y (NPY) and
parvalbumin (PV) immunostaining confirmed that retrogradely-labeled VISam-
projecting neurons are located within the boundaries of the LGd directly adjacent to the
IGL (Figure 3.8c). Overall, the distribution of labeling following VISam and VISpm
coinjections suggests that LGd thalamocortical topography extends across the VISp
border and into the VISam/pm.

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Figure 3.6 | VISp coinjections show specific topographic bidirectional connectivity within LGd.
Five anterograde/retrograde tracer coinjections into different parts of the VISp are shown (columns).
Coinjection site center is shown at the top of each column below the corresponding ARA level. For each
injection case, there are dense clusters of overlapping anterogradely-labeled fibers/retrogradely-labeled
cell bodies at each rostrocaudal level of the LGd (rows). Coinjection sites that are located more caudal
within the VISp produced tracer labeling clusters that are distributed along the superficial LGd (lateral)
whereas bidirectional labeling clusters from more rostral VISp coinjections is in deeper parts of the LGd
(medial). Coinjection sites that are located in more lateral VISp produced labeling clusters near the
dorsomedial LGd/LP border whereas more medial VISp coinjections are distributed more ventromedial
parts of LGd.
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Figure 3.7 | VISam and VISpm coinjections produce labeling clusters along ventromedial LGd border.
Six anterograde/retrograde tracer coinjections into VISam or VISpm are shown (columns). Coinjection site
center is shown at the top of each column below the corresponding ARA level. Similar to VISp coinjections,
VISam and VISpm coinjections produce dense clusters of overlapping anterogradely-labeled
fibers/retrogradely-labeled cell bodies that are located in a small strip along the ventromedial LGd border
adjacent to the IGL at each rostrocaudal level of the LGd (rows). Labeled clusters from VISam coinjections
are distributed closer to the ventromedial corner of the LGd whereas labeled clusters from VISpm
coinjections are distributed closer to the ventrolateral corner of the LGd.
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Figure 3.8 | Organization of VISp vs. VISam/pm thalamocortical connectivity.
Quadruple retrograde tracer experiment to simultaneously view LGd thalamocortical connectivity with
VISp, VISam, and VISpm. (a) A FG injection into the VISam (yellow) with CTB-555 injection into the
anterior VISp (red) at the same rostrocaudal level. Within the same mouse, CTB-647 injection into the
VISpm (magenta) and CTB-488 injection (green) into the posterior VISp at the same rostrocaudal level.
(below) The distribution of the four retrograde tracers within the LGd alongside representative ARA levels
are shown. Within the rostral LGd, labeling from the posterior VIS cortices has a greater representation
whereas anterior VIS cortices are more represented at caudal LGd levels. At the middle LGd level, the
anterior/posterior axis is almost perfectly symmetrical within the LGd. Note, the relative spacing of all 4
tracer injection sites in the cortex is relatively maintained within the LGd. (b) Mapping of retrograde
labeling across all injection cases reveals that VISam/pm labeling are distributed uniquely along the ventral
strip of the LGd whereas VISp-projecting neurons are absent from the ventromedial border (except a small
area of the ventromedial LGd corner at middle rostrocaudal levels), but are located throughout the rest of
the LGd. Retrograde injection site centers were mapped onto ARA sections and the distribution and relative
density of retrograde labeling in the LGd was mapped with Adobe Photoshop. (c) Parvalbumin (PV) and
Neuropeptide Y (NPY) staining confirmed that retrogradely-labeled VISam-projecting neurons are located
within the LGd directly adjacent to the IGL boundary.
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3.4.3 DISTRIBUTION OF LABELING WITHIN VIS CORTICAL AREAS AFTER LGD
COINJECTIONS
While LGd tracer labeling from cortical injection sites appears to demonstrate
extrastriate-projecting LGd neurons, it is possible that LGd labeling after VISam/VISpm
injection sites was caused by spread into the adjacent medial VISp.
To cross validate our findings based on cortical injection sites, we placed small
iontophoretic coinjections within distinct LGd subregions (Figures. 3.9, 3.10). Although
the ventral strip region of LGd is too small of a target to place an entirely restricted tracer
deposit, coinjection sites that overlap the ventral strip region should produce tracer
labeling within the VISp that spreads beyond the medial border into the VISam/VISpm.
In contrast, LGd coinjection sites that do not spread into the ventral strip region should
produce VISp tracer labeling patterns that do not cross the border into VISam/VISpm
and are distributed according to topographic organization.
Indeed, each LGd coinjection site produced labeling within distinct parts of VISp,
consistent with a discrete placement of tracer deposit in a small area rather than the whole
LGd (Figures. 3.9, 3.10). All LGd coinjections produced dense anterograde labeling
within cortical layer 4 with lesser labeling extending into layers 1 and 6 and robust
retrograde labeling within cortical layer 6a. For example, SW130619-01 contains a
coinjection site that is located in the central part of LGd and produces a column of PHAL
and CTB labeling that is similarly central along the rostrocaudal VISp (Figure 3.9).
Comparatively, cases SW141021-01A and SW141021-02A contain coinjection sites within
LGd closer to the IGL border but do not spread into the ventral strip region. In the cortex,
tracer labeling is distributed more medially within the VISp and extend completely up to
the VISam/VISpm border.

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In contrast, LGd coinjection sites that were located within the ventral strip region
produced tracer labeling that was distributed in the medial VISp and also in the VISam
and VISpm at multiple rostrocaudal levels (Figure 3.10). For comparison, SW140827-01A
contains a coinjection within the caudal lateral LP that produces labeling that is
distributed along the VISam/VISpm rostrocaudal axis and outlines the location of the
VISam/VISpm.
To confirm the VISam and VISpm boundaries with the extent of layer 4, we
analyzed the Nissl cytoarchitecture in our tracer-labeled tissue sections and again found
strong agreement of the distribution of tracer labeling within the VISam and VISpm
(Figure 3.11). After both LGd ventral strip coinjection (SW130724-04A) and caudal medial
LP coinjection (SW140827-01A), PHAL-labeled fiber distribution is centered in layer 4
throughout the VISam and VISpm to the medial end of layer 4 where it meets the RSP.

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Figure 3.9 | Anterograde and retrograde labeling pattern within VISp after coinjections into the LGd
that avoid the ventral strip region.
Three coinjections of anterograde/retrograde tracer into different subregions of the LGd (columns) that
avoid the ventral strip region produce anterogradely-labeled fibers and retrograde labeling in the VISp,
but not the VISam/pm (rows). For each column, LGd tracer coinjection site is shown at the top with
adjacent ARA atlas level. For each row across, the distribution of labeling throughout the rostrocaudal VIS
cortices is shown (representative ARA level shown at left). In all cases, retrogradely-labeled neurons are
distributed within layer 6 while anterogradely-labeled fibers are distributed in layers 1, 4 and 6 with the
greatest density in layer 4. In case SW130619-01B, the LGd coinjection in the center of the LGd produces
anterogradely-labeled fibers and retrogradely-labeled neurons along the rostrocaudal VISp that are located
in a relatively central mediolateral position. Cases SW141021-01A and SW141021-02A both contain
coinjection sites in the ventromedial half of the LGd but do not spread into the ventral strip area of LGd
that borders the IGL. Both of these cases produced anterograde and retrograde labeling in the medial VISp
adjacent to the border with VISam/VISpm. ARA, Allen Reference Atlas; BDA, biotinylated dextran amine; CTB,
cholera toxin subunit B; FG, Fluorogold; IGL, intergeniculate leaflet; LGd, dorsal lateral geniculate thalamic nucleus;
LP, lateral posterior thalamic nucleus; PHAL, Phaseolus vulgaris leucoagglutinin; VISam, anteromedial visual
cortex; VISp, primary visual cortex; VISpl, posterolateral visual cortex; VISpm, posteromedial visual cortex.
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Figure 3.10 | Anterograde and retrograde labeling pattern within VISp, VISam, VISpm after
coinjections into the LGd that are located within the ventral strip region.
A coinjection into the caudolateral LP near the border of the LGd and three coinjections into the ventral
strip region of the LGd are shown (columns). The caudolateral LP coinjection produced anterogradely-
labeled fibers and retrogradely-labeled cell bodies specifically within the VISam/pm that clearly identifies
the VISam/pm boundary with VISp as demarcated by the representative ARA sections (rows). In
comparison, the three LGd ventral strip coinjections have tracer labeling within the VISp that also crosses
the boundary into the VISam and/or VISpm at multiple rostrocaudal levels. In all cases, retrogradely-
labeled neurons are distributed within layer 6 while anterogradely-labeled fibers are distributed in layers
1, 4 and 6 with the greatest density in layer 4. Tissues sections that are not available at the relevant ARA
level are omitted with a black square. ARA, Allen Reference Atlas; CTB, cholera toxin subunit B; IGL,
intergeniculate leaflet; LGd, dorsal lateral geniculate thalamic nucleus; LP, lateral posterior thalamic nucleus; PHAL,
Phaseolus vulgaris leucoagglutinin; VISam, anteromedial visual cortex; VISp, primary visual cortex; VISpm,
posteromedial visual cortex.
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Figure 3.11 | Anterograde and retrograde labeling patterns located within VISam and VISpm as
defined by Nissl cytoarchitecture.
The thin layer 4 of VISam and VISpm, as well as other cortical layers, can be identified by Nissl
cytoarchitecture within our tracer-labeled tissue sections (injection sites shown on far left, Rorb gene
expression on left, Nissl-only shown in the middle, Nissl plus tracer labeling on the right). At ARA levels
88 and 93, tracer labeling from an LP (SW140827-01A; a) and ventral strip LGd coinjection (SW130724-04A,
b) are both localized within the VISam and VISpm. Anterogradely-labeled LGd fibers densely innervate
layer 4 whereas robust numbers of retrogradely-labeled cortico-thalamic neurons are distributed within
layer 6. ARA, Allen Reference Atlas; BDA, biotinylated dextran amine; CTB, cholera toxin subunit B; ec, external
capsule; FG, Fluorogold; IGL, intergeniculate leaflet; LGd, dorsal lateral geniculate thalamic nucleus; LP, lateral
posterior thalamic nucleus; PHAL, Phaseolus vulgaris leucoagglutinin; Rorb, RAR-related orphan receptor beta;
RSP, retrosplenial cortex; VISam, anteromedial visual cortex; VISp, primary visual cortex; VISpm, posteromedial
visual cortex.
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3.4.4 DISTRIBUTION OF TRACER LABELING WITHIN VISAL, VISL, AND VISPL
AFTER LGD COINJECTIONS
Following LGd coinjections, anterograde and retrograde labeling was observed in VISal,
VISl, and VISpl (Figure 3.12A). Regardless of coinjection site location within LGd, sparse
anterogradely-labeled terminal fields were distributed throughout VISal, VISl, and VISpl
layer 4 whereas more robust numbers of retrogradely-labeled neurons were in VISal,
VISl, and VISpl layer 6. In contrast to the presence of anterograde labeling in the lateral
extrastriate areas after LGd coinjections, lateral extrastriate coinjections produced very
few retrogradely-labeled neurons (Figure 3.12b). The rare few lateral extrastriate-
projecting LGd neurons appeared scattered and inconsistent with the location of the
descending fiber termination. Consistent with descending lateral extrastriate projections
to LGd, tracer coinjection into different levels of the VISal or VISl anterogradely-labeled
terminal fields in subregions of the LGd. Descending lateral extrastriate projections to the
LGd were not as discrete as VISp projections, but in total, lateral extrastriate projections
appear to innervate the entire LGd, including the VISam/VISpm-projecting ventral strip
region (Figure 3.13). In case SW111004-02A, double coinjection of BDA/FG into the
VISam and PHAL/CTB into the VISal revealed that, in addition to direct connections
between the VISam and VISal, VISal anterogradely-labeled fibers overlap with
retrogradely-labeled VISam-projecting LGd neurons, suggesting a subcortical circuit
between the medial and lateral extrastriate areas via the LGd ventral strip region (Figure
3.12c).

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112
Figure 3.12 | LGd connections with lateral extrastriate areas and subcortical relays with medial
extrastriate areas.
Anterograde and retrograde coinjection into all parts of the LGd produced sparse layer 4 anterograde
labeling and robust layer 6 retrograde labeling in the lateral extrastriate visual areas. In SW130724-03A,
PHAL/CTB coinjection into the LGd ventral strip region (injection site shown in Figure 10) produced
sparse anterograde and robust retrograde labeling in the VISal, VISl, and VISpl that is segregated from the
denser terminal field in VISp/VISam (image on right is magnified view of yellow rectangle). This
anterograde and retrograde labeling pattern was consistently observed in all LGd coinjection cases and
within VISal, VISl, and VISpl. (b) Coinjections into the lateral extrastriate areas produced labeling within
LGd that confirms the LGd coinjection data. In SW111004-02A, PHAL/CTB coinjection into the VISal
reveals anterogradely-labeled PHAL terminal fields (green) and a few scattered retrogradely-labeled CTB
neurons (magenta, demarcated by arrows; image on right is magnified view of yellow rectangle). For
additional examples of lateral extrastriate coinjection tracer labeling within rostrocaudal LGd levels, see
Figure 13. (c) In case SW1110004-02A, we also coinjected BDA/FG into the VISam (BDA labeling too weak)
in addition to PHAL/CTB in the VISam (top left). The accuracy of the two coinjection sites was clear as
CTB injection into the VISal produced retrograde labeling in VISam layer 2/3 which overlapped the FG
injection site (middle left) and FG injection in the VISam produced retrograde labeling in VISal layer 2/3
that overlapped the CTB injection site (bottom left). Note the injection sites in these images appear larger
due to oversaturation necessary to visualize the dimmer retrograde labeling in the same section. Within
the LGd of SW111004-02A, PHAL-labeled VISal terminal fields overlapped with retrogradely-labeled
VISam-projecting LGd neurons (middle column, image on bottom right is magnified view of yellow
rectangle). Overall, this data suggests a novel pathway for communication between the medial and lateral
extrastriate visual areas via a subcortical pathway (red) through the LGd ventral strip (top right diagram).
ARA, Allen Reference Atlas; BDA, biotinylated dextran amine; CTB, cholera toxin subunit B; FG, Fluorogold; IGL,
intergeniculate leaflet; LGd, dorsal lateral geniculate thalamic nucleus; LP, lateral posterior thalamic nucleus; PHAL,
phaseolus vulgaris leucoagglutinin; VISam, anteromedial visual cortex; VISal, anterolateral visual cortex, VISl,
lateral visual cortex, VISp, primary visual cortex.
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Figure 3.13 | Distribution of tracer labeling in LGd after coinjection into the lateral extrastriate areas.
Five coinjections of anterograde/retrograde tracer into lateral extrastriate areas at different rostrocaudal
levels (columns) are shown with their labeling patterns in different rostrocaudal LGd levels (rows). For
each column, extrastriate area tracer coinjection site is shown at the top with adjacent ARA atlas level (VISal
= SW180302-07A, SW111004-02A, SW170822-07A; VISl = SW170822-06A and SW170822-05A). For each row
across, the distribution of labeling throughout the rostrocaudal LGd is shown (representative ARA level
shown at left). Each experimental case shows relatively discrete anterogradely-labeled PHAL terminal
fields in different parts of LGd across multiple rostrocaudal levels. A few retrogradely-labeled LGd
neurons were observed in SW111004-02A.
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Figure 3.14 |Topographic Organization of Mouse LGd Cortical Connectivity.
The four quadrants of the VISp (see directional cross at bottom right; anteromedial (dark red),
posteromedial (dark blue), anterolateral (yellow), posterolateral (green)) are bidirectionally-connected with
LGd neurons in corresponding regions on the left (three LGd atlas level shown, related to directional cross
at left). Connections of the VISam (red) and VISpm (blue) with the ventral strip region of LGd are a natural
extension of the LGd/VISp topography. In contrast, VISal/l/pl connections with the LGd are primarily
corticothalamic and terminate within LGd subregions (including the ventral strip region). Ascending LGd
connections with the VISal/l/pl are likely scattered intermittently throughout the LGd and are not shown.
A, anterior; L, lateral; LGd, dorsal lateral geniculate thalamic nucleus; M, medial; P, posterior; VISam, anteromedial
visual cortex; VISal, anterolateral visual cortex, VISl, lateral visual cortex, VISp, primary visual cortex; VISpl,
posterolateral visual cortex; VISpm, posteromedial visual cortex.
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115
DISCUSSION
The results of this study support the existence of LGd extrastriate projections in the
mouse and further extend on these findings to describe the overall thalamocortical
organization of the LGd (Figure 3.14). We have confirmed the previous report of LGd
output to VISal and VISl as well as LGd input from VISam, VISal, and VISl (but not from
the temporal association area)
172
. In addition, we found evidence for LGd output to the
VISam and bidirectional connectivity with the VISpm and VISpl. Our bidirectional circuit
tracing strategy cross-validates the LGd extrastriate projections through localization of
anterogradely-labeled LGd fibers within extrastriate regions and retrogradely-labeled
extrastriate-projecting LGd neurons. Through this method, we characterized VIS
cortex/LGd bidirectional connections both by laminar cell body location and projection
fiber distribution. Overall, we provide evidence that neurons within a ventral LGd
subregion provide bidirectional topographic connections with the medial extrastriate
regions. In contrast, lateral extrastriate regions receive sparse non-topographic input
from LGd but provide robust descending input to the ventral LGd region, including the
medial extrastriate-projecting LGd neurons in the LGd ventral strip. Altogether, this
reveals a potential disynaptic pathway of VISal→LGd→VISam, which suggests that the
LGd may serve as interface for a unidirectional interaction of two extrastriatal areas
(VISal and VISam), although functional significance of this pathway remain to be
clarified.
Our finding of an extrastriate-projecting ventral strip region adds novel
understanding to the organization of the rodent LGd which can be divided into a core
and superficial shell region and contains three morphologically-distinct and
electrophysiologically-distinct cell types (‘X-like’, ‘Y-like’, and ‘W-like’) for parallel visual

116
processing
177,178
. Notably, LGd neurons with ‘X-like’ electrophysiological and
morphological characteristics were shown to be strongly distributed in an area that
coincides with the VISam/VISpm-projecting LGd ventral strip region
177
. In contrast, the
superficial LGd shell region contains higher numbers of ‘W-like’ cells and is distinctly
identified from the core region by input from the superior colliculus and direction-
selective retinal ganglion cells
177–179
. Recently, LGd neurons in the superficial shell region
have been shown to be anterior or posterior direction-selective allowing for horizontal-
axis motion selectivity
180
. Interestingly, the most lateral part of the ventral strip LGd
region containing VISpm-projecting neurons appears to intersect with the superficial
shell region, suggesting that VISpm could receive input from both ‘X-like’ and ‘W-like’
LGd neurons (also superior collicular and direction-selective visual information),
whereas VISam may only receive input from ‘X-like’ LGd neurons.

3.5.1 TECHNICAL CONSIDERATIONS
There are many important technical considerations for the interpretation of anatomical
tracer data both in this study and previous investigations of extrastriate LGd connections
(Table 3.1). First, intact and/or damaged axons passing through the injection site, but not
synapsing, can take up some retrograde tracer molecules and lead to false interpretations
of connectivity. This is an important consideration as the LGd is transected by several
large thalamocortical axon fiber bundles. Retrograde tracer molecules have shown
different affinities for uptake by intact or damaged axons of passage (notably Fast Blue
and WGA-HRP). Many of the previous extrastriate LGd investigations used WGA-HRP
which is taken up by axons of passage, but can also spread trans-neuronally in both
anterograde and retrograde direction. The retrograde tracers used in this study, FG and

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CTB, are taken up by axons of passage to a lesser degree compared to WGA-HRP. In our
coinjection data, we sometimes observed that CTB, but not FG, was sometimes taken up
by passing fibers after LGd injection (although FG can be taken up by glia along
projection pathways). In these cases, CTB-labeled neurons were densely labeled in the
region of VISp that coincided with anterograde thalamocortical labeling with sparser,
lightly labeled CTB neurons scattered across VISp. Importantly, our anterograde
injections into the VISp could confirm the specificity of VISp terminations within LGd.
VISp AAV injections often labeled axons passing through the LGd in addition to a more
distinct terminal field (note, PHAL does not strongly label axon bundles). Overall, our
bidirectional tracing strategy can mitigate these limitations of anatomical tracers by
labeling the same pathways in two different ways and providing a way to cross-validate
the data interpretation. Thalamocortical axons can be retrogradely-labeled by cortical
injections or anterogradely-labeled by thalamic injections, thus providing two
corroborating datasets.

3.5.2 LGD CONNECTIONS WITH THE VISP
LGd axon fibers primarily target VISp layers 1, 4, and 6 and LGd neurons receive input
from VISp layer 6 neurons within defined topographic areas. The size and spherical shape
of labeling clusters in LGd after VISp injection is likely a reflection of the size and shape
of the coinjection site. Interestingly, coinjection sites that were located along the medial
border of the VISp did not produce labeling within the ventral strip region of the LGd
adjacent to the IGL border. Labeling in this ventral strip area was found only after
coinjection sites that were centered in the VISam or VISpm.

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3.5.3 EXTRASTRIATE LGD CONNECTIONS WITH VISAM AND VISPM
Coinjections of anterograde and retrograde tracer into the VISam and VISpm produced
clustered labeling within the ventral strip region of the LGd in a topographic manner.
One possible interpretation of this result could be that the coinjection sites in VISam and
VISpm spread into the laterally adjacent medial VISp area and LGd labeling in the ventral
strip region is actually from VISp. However, our data suggests multiple points of
evidence to the contrary. First, coinjections into the medial VISp do not produce labeling
within the ventral strip LGd region. Second, coinjection sites which are located near the
medial RSP border of the VISam/VISpm (away from the VISp) produced tracer labeling
within the LGd ventral strip. Third, LGd coinjection sites that are centered in or spread
into the LGd ventral strip region produce anterograde and retrograde labeling within the
VISam and VISpm. Fourth, LGd coinjection sites that are located near to, but do not
spread into, the LGd ventral strip region produced anterograde and retrograde labeling
within the medial VISp that is immediately adjacent to the VISam/VISpm border.
Another possible interpretation could be that the boundaries of the VISam and
VISpm are not accurate and the VISp extends further medially then shown in the ARA.
Neuroanatomical atlases provide good approximations, but oblique histological tissue
sectioning can produce incongruencies when comparing experimental data to atlas
sections. Our analysis of gene expression, cytoarchitecture, and other connectivity
patterns all confirmed the accuracy of the VISp and extrastriate area boundaries as shown
in the ARA.

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3.5.4 EXTRASTRIATE CONNECTIONS OF LGD WITH VISAL, VISL, AND VISPL
In all our LGd coinjection cases, we observed sparse anterogradely-labeled fibers in layer
4 and relatively more dense numbers of retrogradely-labeled neurons in layer 6 in the
lateral extrastriate regions. Lateral extrastriate coinjections confirmed the descending
lateral extrastriate axon fibers with each coinjection terminating in different subregions
of LGd. Notably the distribution of VISal and VISl fibers within the ventral parts of the
LGd is entirely different from the adjacent lateral VISp whose fibers target the dorsal
parts of LGd. Therefore, our VISal and VISl anterograde labeling cannot be the result of
coinjection site spread into the VISp.
In contrast, the ascending LGd projection to lateral extrastriate areas was more
difficult to verify as very few retrogradely-labeled neurons were present in the LGd after
lateral extrastriate coinjections and the distribution of those rare cells appeared to be
randomly scattered. One possibility could be that the fiber labeling is so sparse and the
coinjection sites are so small, that only a few cells project within the area of retrograde
tracer uptake. In support of this, many of the studies that reported positive findings of
extrastriate LGd projections in the larger non-human primate brain reported that
retrograde labeling from lateral extrastriate regions was ‘sparse’ and ‘scattered’ with one
non-human primate study reporting retrogradely-labeled neurons numbering ~20
neurons per section (Table 3.1). To test this, we performed a quadruple retrograde
injection experiment along the rostrocaudal axis of the lateral extrastriate regions to
maximize the likelihood of retrograde labeling (data not shown). The quadruple
retrograde tracing approach did manage to retrogradely-label more neurons, but the total
number of retrogradely-labeled neurons was still underwhelming (<5 total neurons).
Overall, our interpretation is that there is a minor scattered subpopulation of LGd
neurons that project to lateral extrastriate visual areas. In turn, the lateral extrastriate

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visual areas send dense descending projections to topographic parts of LGd, including
the ventral strip, suggesting a subcortical LGd pathway for lateral extrastriate influence
on medial extrastriate regions.

3.5.5 RELATIONSHIP TO FUNCTIONAL MAPPING OF THE VISUAL CORTEX
In contrast to the ARA cytoarchitectonic parcellation of visual areas, several previous
studies have parcellated the VISp and extrastriate visual cortices based on topographic
VISp connectivity
18
and functional measures of retinotopy
181,182
. These studies determined
the existence of nine extrastriate visual areas surrounding the VISp with distinct
physiological characteristics. Although it is difficult to directly compare functional
imaging in the horizontal plane with coronal anatomical sections, many of these visual
areas appear to be overlapped with other well-defined brain regions, including the
posterior parietal cortex (PTLp) and post-rhinal cortex, which are known to process a
variety of multimodal sensory and motor stimuli
183,184
. Using cyto- and chemo-
architectonic evidence confirmed by thalamic connectivity, Hovde and colleagues
defined the borders of the parietal cortex and repeated the Wang and Burkhalter
experiments to show that visual area RL was largely contained within the PTLp
185
. In
addition, parts of visual areas A and AM were also found to be included in the parietal
cortex. This study raises important questions about how extrastriate visual areas should
be defined since visual areas like RL may not only process visual information.

3.5.6 FUNCTIONAL CONSIDERATIONS RELATED TO BLINDSIGHT
The results of this study provide evidence for LGd extrastriate connections in the mouse
that could underlie blindsight. In the absence of a known LGd extrastriate connection,

121
regions such as the superior colliculus, pulvinar, or LP have been proposed to mediate
blindsight capabilities. However, several recent studies have shown that the LGd is
critical to blindsight. LGd inactivation in VISp lesioned mice has been shown to reduce
extrastriate activity and eliminate blindsight behavior
166
. Human imaging studies have
suggested that LGd projections to extrastriate area MT are the critical anatomical
substrate to blindsight
186–188
. Area MT in humans (also known as V5 in primates) is part
of the dorsal visual-processing stream and plays an important role in motion perception
as MT neurons are tuned to the speed and direction of moving visual stimuli
189
. Could
mouse VISam or VISpm be homologous to area MT in humans since both receive LGd
input? In support of this idea, network analysis of mouse extrastriate regions identified
VISam and VISpm as members of the dorsal stream
190
and physiological studies have
suggested VISpm may play a role in object tracking
181
. Further experiments are needed to
investigate these similarities between mouse VISam/pm and human area MT.

CONCLUSION
The existence of extrastriate LGd connectivity has been debated for decades in a variety
of different animal models. We provide evidence that the mouse LGd has connections
with both the medial and lateral extrastriate regions. VISam- and VISpm-projecting LGd
neurons are located along a ventral strip region adjacent to the IGL border. These findings
provide new insight into the organization of the rodent LGd and its thalamocortical
connections.

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123

SUMMARIZING DISCUSSION

SUMMARY OF FINDINGS
These combined works demonstrate new ways to consistently delineate the superior
colliculus and visual thalamus to better understand their sensory anatomical connections
throughout the brain. In Chapter 2, we provided a comprehensive resource to understand
the organization of the SC in a way that had not been previously characterized. Following
our well-established connectivity-based parcellation strategy, we delineated the SC into
four columnar zones based on combinatorial manual annotation and quantitative
computational analysis of extensive cortico-tectal projections (Figure 2.2 and 2.3). Axonal
terminals arising from different cortical areas displayed complex convergent and
divergent patterns, and its preferential targeting into distinct zones subdivided along the
medial-lateral axis: SC.m, SC.cm, SC.cl, and SC.l. The distinct input-output organization
of these newly defined zones of the SC reveals novel properties of how they are involved
in different aspects of sensory integration and motor control. For example, in the
literature, the SC.cl and SC.l are lumped together into one lateral division; however, for
the first time, we clearly characterize unique input-output patterns of this large lateral
division to reveal two SC columns, where the SC.cl is one critical network node in
controlling eye-head coordination, while the SC.l (which does not receive direct visual

124
inputs) is more specifically involved in somatic sensorimotor behavior, such as
coordination of mouth, whisker, and forelimb.
Discovery of the new zone delineations supports the hypothesis that in addition
the inherent horizontal laminar organization of the SC, there is another level of vertical
columnar (zone) organization of connectivity that may facilitate higher-order and
multisensory integration. The zones are congruent with intrinsic collicular connections
that integrate information from dorsal-to-ventral across superficial (visual), intermediate
and deep (auditory and somatosensory) layers
23,191–193
, a principle of organization
comparable to cortical columns within the neocortex. This suggests that the cortico-tectal
zones reflect functional columns that can potentially be further subdivided into more
narrow units of layer integration. Interestingly, studies on the postnatal development of
the mouse cortex and SC reveal that the cortex must first codify multisensory cues formed
from specific environmental contexts before they can effectively have a cortico-tectal
influence on multisensory SC neurons
194
. These studies suggest that higher-order
associative inputs specifically target sensory-defined areas in SC to relay top-down
information, and the zones may help refine that transference developmentally. Thus, by
considering the zone organization of the SC as part of the inherent schema of
organization, we can trace the logic of cortico-tectal projections arising from multimodal
and higher-order association areas, such as the PTLp, ORB, RSP, TEa, ACA, ILA, and PL,
and further explore their functions within relevant zones and subnetworks.
In Chapter 3, we validated a long contended debate on the existence of extrastriate,
secondary visual cortical connections with the LGd. This work provides evidence that the
mouse LGd has connections with both the medial and lateral extrastriate regions. We
found that coinjections of anterograde and retrograde tracer into the VISp did not
produce labeling within the ventral strip region of the LGd adjacent to the IGL border.

125
VISam and VISpm did however produce clustered labeling within the ventral strip region
in a topographic manner, with VISal/l/pl producing only sparse labeling. Ultimately, we
demonstrated that even famously canonical pathways of sensory relays can still be
systematically refined by examining the cytoarchitecture, connectivity patterns, and
molecular/histochemical makers to reveal blind spots on our existing maps.

FUTURE DIRECTIONS
A key interest from the SC study was to explore the downstream connectivity between
the SC with nuclei in the basal ganglia, in particular the interactions of cortico-tectal-
thalamic loops in relation to cortico-basal ganglia-thalamic loops. By doing so, we could
better understand how these two parallel circuits interact within specific nodes
195
.
Specifically, the two output nodes of these two systems, the SNr and SC, are able to
directly interact with each other: the SNr projects to the SC, while the SC projects back to
the SNc (SC→ SNc→ CP→ SNr→ SC)
196,197
. A preliminary characterization of convergent
and divergent patterns of motor outputs of these two systems is shown by axonal
projections arising from the SC and SNr (Figure 4.1a). Notably, both the SC and SNr relay
topographic outputs to the PF and the pedunculopontine nucleus (PPN) (Figure 4.1b).
Triple anterograde injections into visual association domains in ventromedial SNr reveal
outputs to dorsal PPN and SC.m/cm/cl, zones which also project to dorsal PPN. By
contrast, anterograde injections into dorsal and dorsolateral SNr domains produce
outputs to ventral PPN and SC.l, while SC.l also projects to ventral PPN (Figure 4.1c). To
further demonstrate the topographically distinct somatotopic outputs of the SC and SNr,
a Cre-dependent tracing method was used to compare the collateral projections from PF-
projecting SC neurons and PF-projecting SNr neurons (Figure 4.1d). This topographic

126
organization is consistent with studies revealing the SC and PPN synapse onto the same
PF neuron dendrites
198
. Studies have shown that a disinhibition from SNr onto
glutamatergic projecting neurons in SC that project to PF are critically involved in
controlling epileptic seizures
85
. These coordinated outputs are necessary for the control
of balanced movement; thus, it is important to further interrogate and understand how
they are integrated subcortically
61,199
. In general, our work leaves open multiple
experimental avenues that could be explored to provide more genetic and circuit-specific
insights into the functions of these new delineations. The neuroanatomical data alone
does not reveal underlying biological mechanisms of synaptic connections, genetic
markers, or comprehensive cell-type interrogation across each SC zone or within the
ventral strip of LGd. Thus, some general example approaches for further systematic
analyses to refine more detailed connections are proposed here.
Firstly, additional TRIO studies (such as those in Figure 2.13) could be used to
reveal monosynaptic connections of topographically defined circuits
51
. For example, in
this study, we demonstrate the upper-limb (ul)/orofacial subnetwork that targeted the
ventrolateral PF (mouth/ul domain, with AAVretro-Cre) and SC.l (with Rabies and
Rabies Helper virus). This ultimately revealed monosynaptic Rabies inputs à SC.l à
PF.ul from the SNr lateral (ul/orofacial domains), MOs and SSp upper limb regions, and
brainstem connections with facial motor nuclei. One could apply this technique to reveal
cortex, thalamus, midbrain and brainstem regions involved in any given subnetwork,
such as: brain-wide inputs that project to SC.cl then to PF dorsolateral (involved in lower-
limb/trunk subnetworks); inputs that project to SC.cm then to RE (involved in spatial
processing); inputs that project to SC.cm/cl then to LP (involved in multisensory
processing); and so on. Overall, this method offers refined insight into convergent inputs
to each SC zone.

127

Figure 4.1 | Downstream projections of SNr and SC to the PPN.
a) Anterograde projection from SC.cm project ipsilaterally to PBG. SC.l neurons project to PBG and
contralaterally to PPNd. b) Triple anterograde projections from dorsal motor domains in SNr target SC.l
and dominantly to the ipsilateral PPNd with sparse terminals in PPNv. c) Case demonstrating the dorsal
medial and lateral SNr outputs target SC.l and PPNd similar to case in b. By contrast, ventral medial SNr
targets SC.cl, SC.cm and PPNv. d) AAV-Cre anterograde tracing shows that PF-projecting SC.l neurons
primarily target PPNd bilaterally, whereas PF-projecting SNr neurons densely target the ipsilateral PPNv.
Output fibers descending from SC.l to brainstem nuclei are predominantly contralateral or bilateral,
whereas SNr outputs terminate predominantly only target ipsilateral domains. Abbreviations: PBG,
parabigeminal nucleus, PF, parafascicular nucleus; PPN, pedunculopontine nucleus; PPNd, dorsal part; PPNv,
ventral part; SC, superior colliculus; SC.cm, centromedial zone; SC.cl, centrolateral zone; SC.l, lateral zone; SNr,
substantia nigra pars reticulata.
______________________________________________________________________________

128
As a complementary approach to TRIO experiments, since the SC does not send
direct projections back to cortex, we can take advantage of the unidirectional cortico-
tectal pathways and apply a recently developed AAV anterograde transsynaptic tracing
strategy
36
. To complement the VIS à SC and AUD à SC findings from the original study,
one could use the strategy in each of the higher-order cortical areas to characterize the
input-defined downstream projections of SC cells. For example, in an Ai14-tdTomato
transgenic mouse line (which only expresses tdTomato in the presence of Cre), inject a
high titer injection of AAV1-Cre into the RSPd, and after 2 weeks the Cre will spread
transneuronally into a subset of downstream target neurons in the SC.m and SC.cm. A
second AAV-Cre-dependent-GFP virus is then injected into either the SC.m or SC.cm,
and will express GFP exclusively in those transneuronally Cre-infected SC cells. GFP-
expressing axons will project downstream to all brain regions from SC cells that
specifically receive inputs from RSPd (and compare them to RSPv, ACAd, PTLp, etc.).
This robust method will reveal the divergent characteristics of input-defined projections
to each SC zone.
Furthermore, these experiments can be combined with other transgenic mouse
lines to further characterize subsets of cortico-tectal pathways and reveal more cell-type
specific circuit properties. Several Cre-recombinase mouse lines have experimental
support within SC studies, including Vglut2-Cre, GAD2-Cre, Ntsr1-GN29-Cre, Grp-
KH288-Cre, and PV-ires-Cre. At present, there exist at least six additional candidate
transgenic Cre-mouse lines (www.gensat.org) with gene expression in SC, including
Cx3cr1-Cre, Gabrr3-Cre, Lypd1-Cre, Pdzk1ip1-Cre, Vipr2-Cre. Combinations of Cre
transgenic mouse lines with anterograde and retrograde viral tracing techniques can be
used to study cell-type specific circuits in SC. These experiments can be studied in 2D, or
could undergo whole-brain/thick-section tissue clearing for confocal Dragonfly or

129
Lightsheet imaging data to characterize high-resolution morphological features of
genetically-defined SC neurons.
From a computational angle, we could apply the matrix analysis of cortico-tectal
subnetworks (from Figure 2.6g) to the SC-downstream connectivity analysis (from
Figure 2.10d) and compare how the community modules cluster together. The systematic
registration process of all inputs and output projections from each SC zone could be
quantified and tested with the Louvain community detection algorithm to reveal
additional properties of brain-wide subnetworks of SC. Specifically, it would be
intriguing to analyze more detailed Cortex à SC à Brainstem pathways, or Cortex à SC
à Visual Thalamus pathways, in the context of functionally implicated subnetworks
identified above.

TRANSLATIONAL APPLICATIONS
Toward the goal of treating and healing neurodegenerative diseases, connectopathies
and psychological disorders in the brain, I resonate with the mission statement of the
BRAIN Initiative (http://braininitiative.nih.gov/), which acknowledges that we must
first “understand how these circuits work to capture the full sense of what is happening in the
healthy brain-and what goes awry in disease.” In addition to the functional hypotheses and
implications proposed in Chapter sections 2.8 and 3.5, I am excited to explore the
potential contributions of this work to translational and psychotherapy research.

130
4.3.1 ADHD AND ASD TREATMENTS
Attention is essential for animals and humans to filter overwhelming sensory input and
guide behavior. Understanding the neural networks of attention-related visual behaviors
in mouse models has clinical significance in examining aberrant connections of
psychiatric and neurodevelopmental disorders, such as in attention deficit hyperactivity
disorder (ADHD) and autistic spectrum disorders (ASD). ASD symptoms include
attention deficits due to an increased inability to filter out sensory information
40
. In
humans, the SC is hypothesized to be a major locus of interest for potential therapeutic
targets in treating hyper-responsivity and distractibility in ADHD
44
. Human studies
supporting this reveal that disconnections in prefrontal cortex projections to SC lead to
increased distractibility and increased error-rate in saccade-tasks
42
. Pharmacological
studies emphasize the importance of targeting neuromodulatory cell-types in SC to
dampen sensory responsivity and promote focused attention
43
. In particular, the widely
used ADHD medication, D-amphetamine, was found to reduce activity from sensory
hyper-responsiveness in SC neurons thereby reducing overall distractibility in an ADHD
mouse model
22,200
. With the circuit maps of visual and attention-specific subnetworks
offered in Chapter 2, future studies can explore how different drugs interact within the
SC circuits to modulate attention dynamics and regulate the behavioral state of arousal.
Of particular interest would be to test effects of other stimulant medications that may
influence collicular function or dysfunction in these disorders. These include inputs to
the SC from neurotransmitter producing structures like the substantia nigra pars
reticulata (dopamine), and dorsal raphe nucleus (serotonin) transporter or receptor
systems. Treatments could potentially be tailored based on circuit- and cell type- specific
interactions with SC, with the hope of improving symptoms and helping patients
attentively focus on their goals.

131
4.3.2 PSYCHOTHERAPY RESEARCH
Historically, research into psychoactive compounds, or psychedelic drugs, thrived in
research centers during the 1950-1960s, and displayed significant potential for
psychotherapy and neuroscience
201
. The unwarranted criminalization of these
compounds into Schedule I illegal drugs by the government in 1970 resulted in a
profound loss of basic research and progress toward treatments for major illnesses
202
.
Recently, psychedelic research and psychotherapy have reentered the scientific sphere
across global collaborations in a monumental way
202,203
. They offer improved
pharmaceutical and therapeutic potential toward greater healing for those suffering from
severe mental health afflictions, such as depression, post-traumatic stress-disorder
(PTSD), anxiety, suicide, alcoholism, and substance abuse. Here, I will review some
network interactions influenced by the psychoactive compounds 3,4-methylene-dioxy-
meth-amphetamine (MDMA) and lysergic-acid diethlymide-25 (LSD), and some
interesting considerations in relation to this dissertation.
Studies have demonstrated that the monoamine psychedelic compounds (MDMA
and LSD) predominantly target serotonergic, dopaminergic, and noradrenergic
transporters by acting as neurotransmitter agonists, or reuptake inhibitors, with unique
binding mechanisms
204
. Of particular interest in this discussion are the brains circuits
influenced by the 5-HT
2A
serotonin (5-hydroxytryptamine [5-HT]) receptor with selective
agonism of these compounds. 5-HT production from the dorsal raphae nucleus (DR) is
projected broadly throughout the brain, notably to the prefrontal cortex (PFC; cognitive,
logical processing), hippocampus (HPF; long-term memory storage), amygdala (AMY;
fear processing), hypothalamus (HYP; oxytocin release), and basal ganglia (decision
making). Imaging studies in PTSD patients highlight reduced activity in the PFC and
HPF, and a hyperactive AMY. Characteristics of this network dynamic manifest as

132
debilitating fear of processing difficult emotions and memories that inhibit patients from
overcoming traumatic experiences. Recent studies through the Multidisciplinary
Association for Psychedelic Studies (MAPS) report the significant and robust attenuation
of PTSD symptoms through MDMA-assisted psychotherapy after an 18 week
treatment
205,206
. Doblin et al., (2021) suggest the neurological effects of MDMA on PTSD
patients are an increase in PFC activity concurrent with increased connectivity between
HPF and AMY that refrain the transference of traumatic memories to HPF for long-term
storage.
The MCP’s neuroanatomical mapping studies in mouse models could provide
foundational maps to refine the basal ganglia circuits and limbic system subnetworks that
are influenced by these psychoactive compounds (see Figure 1.1). Interestingly, layer 5
cortical neurons from mPFC (medial) and lPFC (lateral) regions express 5-HT
2A
serotonin
receptors; these neurons also constitute the cortico-fugal projections to the brainstem that
include the SC. In Chapter 2, we found that the ACA, PL and MOs-frontal-eye-field (fef)
regions of the mPFC
30
predominantly target the SC.cm and SC.cl zones implicated in
visuomotor integration; and the ILA from mPFC and ORBm/l/vl from lPFC
predominantly target SC.cl and SC.l somatic sensorimotor networks. In particular, it
would be intriguing to compare connectivity properties (density, weight) between the
healthy C57BL/6 mouse cortico-tectal pathways with those from a mouse model of PTSD
for MDMA treatment (perhaps combined with a novel form of mouse-specific “therapy”
sessions in an enriched, safe environment). Importantly, the DRN also projects directly
to the superficial layer visual SC neurons that harbor the 5-HT
1
serotonin receptor
subtype (receptive to MDMA, but not LSD)
207
. Behavioral studies employing DREADs
methodologies in a transgenic 5-HT mouse line could also explore the selective activation
or inactivation of these pathways and their functional implications
208
.

133
Finally, it would be interesting to investigate the role of LSD throughout the visual
circuits discussed in Chapters 2 and 3. Among many attributes, LSD is characterized by
its visual hallucinatory effects, emotional arousal, and as a treatment for depression,
alcoholism and nicotine addiction
203
. Notably, LSD is also prominently involved in the
activation of 5-HT
2A
serotonin receptors in the LGd, visual cortices (VIS), PFC regions
(MOs-fef, ACAd/v), and the claustrum
203
. Based on the interconnectivity between the
retina-LGd-VIS-SC subnetwork, it can be logically hypothesized that LSD-activation
throughout the circuit would directly affect the perception of the visual field and alter
spatial attention capacities that contribute to the hallucinatory effects (perhaps through
askew sensory filtration mechanisms, or by engaging other visual centers active during
dream states).
Another exciting direction is the synthesis of psychoactive analogs and
psychedelic-inspired drugs that can provide antidepressant actions with minimal to no
hallucinatory effects, thereby facilitating another form of medicinal treatment.
Ultimately, I believe there is abundant potential in the current applications and future
directions of research in medicinal psychedelic drugs and their dynamic network
interactions in the brain. This section concludes with a famously quoted observation by
Dr. Stanislov Grof that provides an insightful perspective on the scope of psychedelics:
“What the telescope was for astronomy, and the microscope was for biology,
psychedelics will be for the understanding of the human mind.”

134
CONCLUSION
This dissertation is a manifestation of my humbling contributions to the neuroscience and
neuroanatomy fields. I experienced a singular training opportunity through the USC
Neuroscience Graduate Program in Dr. Hongwei Dong’s laboratory to research the
fundamental organization of the brain alongside a phenomenal team of scientists. Here,
I traced through neural millions of years of evolution in neural circuits over the course of
six years; I delineated an intriguing subset of cortical and subcortical circuits, and
provided new insights into their subnetwork connectivity; I gained fundamental practice
in patience by parcellating pixels and pixels of patterns; and I developed a deeper
appreciation of how our brain and body are beautifully bound in biology. My concluding
ambition is that this corpus offers constructive new findings for the scientific community,
and that it makes my family, friends and mentors proud.

135
PARTING PERSPECTIVE

The structural depth of the nervous system.
Reflecting on how far the field has progressed in brain mapping conjures a sense of
astonishment and awe. Each study referenced throughout this work from the late 1800s
until now has contributed a meaningful pixel to the big picture. Modern methodologies
(e.g., 3D-imaging microscopes, neuroinformatics tools, and reconstruction software) now
offer the field improved visualizations to comprehend the dynamic and multi-resolution
scales of neural structures. From the evolution of early flatmap diagrams
209
to the current
layouts offer an unprecedented rendering of advanced connectomic design
3
; it facilitates
greater structural and conceptual apprehension of depth in the nervous system (Figure
4.2A-C). A notable comparison that comes to mind when considering the potential scale
of the nervous system is the Mandelbrot Set. The mathematician Benoit Mandelbrot
discovered this set of complex numbers derived from a simple iterative function
210
. This
seemingly simple equation produced a sequence of numbers from z=0 to infinity, but
could only render limited graphical representations (Figure 4.2D). Not until the
invention of sufficiently powerful IBM computers in the late 1980s, could mathematicians
behold the fractal iterations of infinite magnitude bound within this set (Figure 4.2E,F).
Various renditions of the Mandelbrot set have now been generated with mesmerizing
imagery, and the underlying mathematical principles have been found in chaotic systems
that are also used to model neural network dynamics. As our technology advances, our
visual representations will also improve in resolution to encompass the potentially
infinite scale of detail in the nervous system. This prospect is one of thrilling anticipation.

136

Figure 4.2 | Flatmaps and Fractals.
A) Connectome flatmap illustration from Nauta & Karten (1970) publication. Image can be compared to
newer renditions of flatmaps using modern vector graphics in panel B. It is also juxtaposed to panel D
which shows the first (computationally-limited) rendition of the Mandelbrot set. Little did the math fields
know that the technical leap from A to E would pioneer new fields of fractal geometry and a foundation
for Chaos theory
211
. B) Connectome flatmap illustration from Bota, Sporns & Swanson (2015) publication
with color-coded white matter connections in the rat nervous system. This newer flatmap rendition can be
compared to the updated Mandelbrot set graph in panel E which was only accessible by the development
of better computers. This comparison between B and E is meant to represent the critical role of technological
advancements in our ability to accurately represent complex structures so we may update our models the
systems as well. The importance is further emphasized in the zoomed crops in C and F. This comparison
illustrates the depth of scale and structural dynamics that reveal more intricate details. In the brain as with
fractal geometry, we learn that the more we zoom in, a more complex system is unveiled. As technology
advances, we will also develop new tools to render new representations of the brain and update our current
models. C is cropped from the downloadable flatmap PDF at http://larrywswanson.com/?page_id=1148.
F is cropped from a black and white video of the MS at https://vimeo.com/96951099.

Disclaimer: I do not own any of these images. Please note that all images and copyrights belong to their original
owners. No copyright infringement intended. Using for academic purposes only.
_____________________________________________________________________________________________
A C B
D F E

137
Sensory maps and associative loops
Sensory perceptions are among the most real things we can experience; they not only
provide meaningful representations, but they can engender a sense of purpose. An
intrigue throughout my studies on cortico-tectal connectivity was the notion of
connecting the new (neocortex; cortical) with the ancient (tectum, subcortical). The
evolutionary development of these structures is directly reflective of their functional roles
in processing sensory information.
The dorsal tectum (or SC) is a midbrain structure that preceded cortical evolution
and development by 200-300 million years
212
. The tectum is fundamental for reflexive
reactions, sensorimotor integration, and survival. It is a primitive yet complex structure
that integrates and aligns stimuli for efficient commands and robust outputs of
coordinated behavior. The neocortex is associative and psychomotor. It also integrates
and aligns stimuli, with the added capacity to make associations between stimuli, and
execute developed mechanisms ‘for self-preservation and maintenance’ of organisms.
This is reflected anatomically in brain structures like the primary and supplementary
somatosensory and motor cortices that encode topographic maps (Figure 4.3). The
transference of sensory maps as they are relayed to higher level structures compute
iterative associations as they feedback onto themselves and integrate further rostrally to
the frontal cortices through continual integration and feedback loops. Anatomically, the
homunculus of the human body map of the motor and somatosensory cortex illustrates
the proportional differences of cortical real estate dedicated to processing different body
parts. Allegorically, the unilateral hemisphere homunculus resembles a circular self-
ingesting creature (Figure 4.3A), and the bilateral hemispheres resemble a lemniscate
figure of the same creature in an infinite loop of associations (Figure 4.3B).

138

Figure 4.3 | Self-association loops.
A) Topographic and proportional representation of sensory processing map unilaterally in the human
cortex. The adjacent illustration of the homunculus. Image from http://cnx.org/content/col11496/1.6/.
B) A bilateral cortical homunculus illustrating the differences between somatosensory representations (left)
and motor representations (right). Image from Google stock images. C) Illustration of the Ouroboros
symbol, dating back to Egypt in 16
th
century BC. Image from Google search. D) Lemniscate representation
of the Ouroboros reflective of association loops between cortices in a similar shape. Image from
https://www.tokenrock.com/explain-ouroboros-70.html.

Disclaimer: I do not own any of these images. Please note that all images and copyrights belong to their original
owners. No copyright infringement intended. Using for academic purposes only.
_____________________________________________________________________________________________
Lips
Foot
Toes
Genitals
Trunk
Leg
Hip
Neck
Head
Shoulder
Arm
Elbow
Forearm
Wrist
Hand
Little
Ring
Middle
Index
Thumb
Eye
Nose
Face
Teeth,
gums
and jaw
Tongue
Pharynx
A B
C D

139
Our species has survived through the cyclic renewal of self-association through the
senses, cognitive enhancements, and psychological developments of identity. This is
symbolically weaved in the Ouroboros, an ancient mythological serpent that represents
the cycle of life, perpetual renewal, and an infinite feedback loop (Figure 4.3C-D). The
serpent is also embedded in our genetic ancestry as a predator, and triggers innate and
involuntary fear responses. Fittingly, the ancient “reptilian brain” is comprised of basal
ganglia loops that control core nervous system functions, and is evidence of the primal
foundations of our complex lineage. The student of Carl Jung, Eric Neumann, describes
the Ouroboros as a “representation of the pre-ego, a ‘down-state’ depicting the
undifferentiated infancy experience of both mankind and the individual child.” This
representation is consistent with the cycles of psychological development that every
human uniquely experiences in their lifetime, from infancy to adolescence to adulthood
toward individuation. A literary example of this lifecycle is narrated through a creation
story in religious text. The serpent is introduced as a main figure tempting the first
humans to consume forbidden fruit (knowledge) that ultimately triggered self-
consciousness of the naked body. Here, we see a direct juxtaposition of the Ouroboros
symbol as a predecessor to the embodiment of self-awareness in the Homunculus (by
way of the neocortex). Altogether, I perceive our capacity for self-association as a virtue
of our psychological evolution and survival throughout dynamic environments. The
potential for biological expansion and transcendence of consciousness is still largely
untapped in our cortical adolescence and cognitive development. I believe the
relationship between our ancient origins and our current timeline should remain at the
forefront of evolution as our species progresses toward a technology-driven future. The
imminent integration of neurobiology and artificial intelligence will transform our
concepts of self, as alluded to in Figure 4.2.

140
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APPENDIX A : SC PROJECTION MAP

Complete array of coronal sections from SC anterograde projection map sections cropped in Figure 2.8a.
ARA levels 56-128. Injection site correspondence: SC.m (red): SW190619-04A (PHAL), SW190619-02A
(PHAL); SC.cm (orange): SW190619-02A (AAV-tdTomato); SC.cl (green): SW171010-01A (AAV-gfp),
SW171010-02A (AAV-gfp); SC.l (purple): SW171010-01A (AAV-tdTomato), SW171010-01A (PHAL).

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APPENDIX B : MANUSCRIPT INFORMATION

Manuscript adapted for Chapter 2:
Organization of the inputs and outputs of the mouse superior colliculus.
Nature Communications (2021) Manuscript accepted June 2021 (in press).
(First preprint submission on bioRxiv: https://doi.org/10.1101/2020.03.24.00677)

Nora L. Benavidez
1-3
, Michael S. Bienkowski
2
, Muye Zhu
2,3
, Luis H. Garcia
2,3
, Marina
Fayzullina
2,3
, Lei Gao
2,3
, Ian Bowman
2,3
, Lin Gou
2,3
, Neda Khanjani
2
, Kaelan R. Cotter
2,3
,
Laura Korobkova
1,2
, Marlene Becerra
2
, Chunru Cao
2,3
, Monica Y. Song
1-3
, Bin Zhang
2,3
,
Seita Yamashita
2,3
, Amanda J. Tugangui
2,3
, Brian Zingg
2,3
, Kasey Rose
1
, Darrick Lo
2,3
,
Nicholas N. Foster
2,3
, Sarvia Aquino
2
, Tyler Boesen
2,3
, Hyun-Seung Mun
2,3
, Ian R.
Wickersham
4
, Giorgio A. Ascoli
5
, Houri Hintiryan
2,3
& Hong-Wei Dong
2,3

1
Neuroscience Graduate Program, University of Southern California, Los Angeles, CA.
2
Stevens Neuroimaging and Informatics Institute, Laboratory of Neuro Imaging, Keck
School of Medicine, University of Southern California, Los Angeles, CA.
3
Current affiliation: UCLA Brain Research & Artificial Intelligence Nexus, Department
of Neurobiology, David Geffen School of Medicine, University of California Los
Angeles, Los Angeles, CA.
4
McGovern Institute for Brain Research, Massachusetts Institute of Technology,
Cambridge, MA.
5
Krasnow Institute for Advanced Study, George Mason University, Fairfax, VA.

Author Contributions: N.L.B. conceived, designed and managed the project with
guidance from H.-W.D and M.S.B. N.L.B. and H.-W.D. wrote the manuscript. N.L.B.
created all figures and performed manual analysis of all raw image data, including the
connectivity annotation, created the SC custom atlas, and prepared figures for
publication. M.Z., L.G. and I.B. wrote the code for computational network analysis
throughout the manuscript with design input from N.L.B. L.G. programmed matrices,
stacked bar charts, and anterograde maps. M.Z. programmed the polar coordinate
analyses and plots. N.L.B., L.G., L.G., M.B., M.Y.S., N.F.F., S.A, C.C., and K.R.
performed stereotaxic surgeries and tissue processing to generate anatomical
connectivity data. N.L.B., M.F., and N.K. registered cases on Connection Lens for
analysis. K.C. helped implement the SC custom atlas on Outspector. N.L.B., M.S.B. and
H.W.D. constructed the neural networks diagrams. N.L.B., N.K., and B.Z. processed
tissue clearing and 3D imaging protocols. L.K. traced all 3D reconstructed neurons. L.K.
and G.A.A. performed statistical analysis of morphological data. S.Y. and A.J.T.
managed the iConnectome website and created online informatics and visualization
tools. N.K., M.F., D.J., D.L., T.B., and C.M. performed image processing for image data
uploaded to the iConnectome viewer. I.R.W. provided the rabies viruses. H.-W.D.,
M.S.B., B.Z., G.A.A., H.H. offered constructive guidance for the manuscript edits.

This work was supported by NIH/NRSA F31EY029569 (N.L.B.), NIH/NIMH R01
MH094360 (H.-W.D.), NIH/NIMH U01 MH114829 (H.-W.D.), NIH/NCI U01
CA198932-01 (H.-W.D), NIH/NIMH U19 (JE/EC) and NIH/NIMH U19 MH114821
(J.H./P.A.).

156

Data Availability: All data used in this study are available from the corresponding
author upon reasonable request. Anatomical tracer image data is available through our
iConnectome viewer as part of the Mouse Connectome Project at USC
(http://www.mouseconnectome.org). All reconstructions are being made freely
available in the Dong archive of www.NeuroMorpho.Org. We used various software
to navigate images and data including, FIJI/ImageJ (v1.53a), neuTube (v1.0z), Adobe
Photoshop (v21.2.2), Aivia (v8.8.1, DRVision), and RStudio (v1.1.463).

Code Availability: All custom code that developed for data analysis are available at
GitHub repositories: https://github.com/lhgarciaa/sc_project,
https://github.com/lhgarciaa/sc_analysis.
Code used for other data analyses are freely accessible. The Brain Connectivity Toolbox
(BCT) was employed for the Louvain algorithm implementation:
https://sites.google.com/site/bctnet/.
For matrix visualization, the grid_communities algorithm for matrix visualizations:
https://github.com/aestrivex/bctpy and https://sites.google.com/site/bctnet/.
Geometric processing of neuron models was performed using the Quantitative Imaging
Toolkit (QIT): http://cabeen.io/qitwiki. In addition, we have used several open-source
Python packages: scipy (v0.17.0), matplotlib (v1.5.1), ipython (v2.4.1), ipython-genutils
(v0.2.0), jupyter (v1.0.0), jupyter-client (v5.2.3), jupyter-console (v5.2.0), jupyter-core
(v4.4.0), pandas (v0.23.3), sympy (v1.2), nose (v1.3.7), pydot (v1.2.3), bctpy (v0.5.0),
pyflakes (v1.1.0), pandas (v0.23.3), cycler (v0.9.0), plotly (v3.0.0).

Manuscript adapted for Chapter 3:
Extrastriate connectivity of the mouse lateral dorsal geniculate thalamus.
Journal of Comparative Neurology (2019) https://doi.org/10.1002/cne.24627

Michael S. Bienkowski, Nora L. Benavidez, Kevin Wu, Lin Gou, Marlene Becerra,
Hong-Wei Dong

Stevens Neuroimaging and Informatics Institute, Laboratory of Neuro Imaging, Keck
School of Medicine, University of Southern California, Los Angeles, CA.

Author Contributions: M.S.B. conceived, designed and managed the project. M.S.B.
and N.L.B. wrote and edited the manuscript. M.S.B. and N.L.B. performed manual
analysis of all raw image data and prepared figures for publication. M.S.B., N.L.B.,
K.W., L.G. and M.B. performed stereotaxic surgeries, immunohistochemistry, and
microscopic imaging to generate anatomical connectivity data. H.-W.D. provided the
research resources and constructive guidance for the manuscript edits.

This work was supported by National Institute of Mental Health grants R01
MH094360-01A1 (H.-W.D.), U01 MH114829-01 (H.-W.D.), F32 MH107071-02 (M.S.B.),
and National Eye Institute grant F31 EY029569-01 (N.L.B.).

157
Awarded Fellowships that supported this research:
NIH Eye Institute NRSA F31 Fellowship EY029569-01 (2019-2021)
NIH T32 NGP Training Fellowship (2017-2018)

Google Scholar link to complete list of publications from the Author:
https://scholar.google.com/citations?user=wkoB_KYAAAAJ&hl=en

Abstract (if available)

Abstract The central nervous system is a wonder to study. It is elegant, efficient, and phenomenally complex. Santiago Ramon y Cajal discovered and taught us that this system is composed of neural units with distinct inputs and outputs assembled into intricate networks of connectivity. He identified the key principle that there is an orderly flow of information throughout the brain and body that ultimately grants animals the capacity to translate experiences of the world into neurobiological signals, convert them into behavioral responses and form meaningful representations. Over a century later, the ongoing effort to map this highly ordered system has driven the invention of innovative tools and ambitious large-scale mapping projects. In particular, as part of the Mouse Connectome Project (MCP), our goal was to assemble a comprehensive wiring map of the entire mouse brain, a connectome. The overall approach combined an array of neuroanatomical and neuroinformatics tools to systematically map all circuits, subnetworks and greater networks throughout the healthy mouse brain model. The studies in this dissertation on the neuroanatomical mapping of the superior colliculus and visual thalamus directly contribute toward this effort. ❧ Chapter 2 focuses the organization of the mouse brain superior colliculus (SC). We constructed a comprehensive map of all cortico-tectal projections and identified four newly defined collicular zones with differential cortical inputs: medial (SC.m), centromedial (SC.cm), centrolateral (SC.cl) and lateral (SC.l). We also delineated the distinct brain-wide input-output organization of each collicular zone to reveal their subnetwork organization. This study provides a structural basis for understanding the critical role of the SC in integrating different sensory modalities (visual, auditory, and somatic sensory), translating sensory information to motor command, and coordinating different actions (eyes, orofacial, whiskers, head and neck and limbs) in goal-directed behavior. ❧ Chapter 3 focuses on the mouse visual system where we have contributed to the discovery of extrastriate connectivity with the primary thalamic visual relay, the dorsal geniculate thalamus (LGd). We provide evidence of bidirectional extrastriate connectivity with the mouse LGd. We found robust reciprocal connectivity of the medial extrastriate regions with LGd neurons distributed along the ‘ventral strip’ border with the intergeniculate leaflet (IGL). Overall, our findings support the existence of extrastriate LGd circuits and provide novel understanding of LGd organization in rodent visual system. ❧ Chapter 4 provides a summarizing discussion on the main findings within Chapters 2 and 3. I elaborate upon additional neuroanatomical experiments that could be explored in future pursuits. Additionally, I discuss how an understanding of subnetwork connectivity offers significant insight in translational studies that are interested in connectopathies and circuit dynamics of neuropsychiatric disorders and psychotherapy research. The dissertation concludes with a parting perspective that presents a reflection on personally-derived symbolic connections with the nervous system.

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Creator Benavidez, Nora Lissett (author)

Core Title Subnetwork organization of the superior colliculus and visual system in the mouse brain

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Degree Conferral Date 2021-08

Publication Date 07/26/2022

Defense Date 06/10/2021

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag connectome,cortex,neural circuitry,neuroanatomy,OAI-PMH Harvest,superior colliculus,topography

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Advisor Watts, Alan (committee chair), Dong, Hong-Wei (committee member), Hirsch, Judith (committee member), Swanson, Larry (committee member), Zhang, Li (committee member)

Creator Email Benavidez.nora@gmail.com,Nlbenavi@usc.edu

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