Differential regulation of anatomical and functional visual plasticity by NgR1

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Content i

Differential Regulation of Anatomical and Functional
Visual Plasticity by NgR1
by
Michael Gideon Frantz

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)

Advisory Committee:
Aaron W. McGee, Ph.D
Michael Jakowec, Ph.D
Huizhong Tao, Ph.D

May, 2016

ii
Table of Contents

Acknowledgements iii
List of Figures v
Abstract 1
Chapter 1: Introduction 3
Chapter 2: Nogo Receptor 1 Limits Ocular Dominance Plasticity but not 33
Turnover of Axonal Boutons in a Model of Amblyopia
Chapter 3: Deletion of NgR1 From Layer IV of Visual Cortex Extends 64
Critical Period Plasticity into Adulthood
Conclusions 84
References 89
Appendix A: Nogo Receptor 1 Limits Tactile Performance 98
Independent of Basal Anatomical Plasticity
Appendix B: Nogo Receptor 1 Limits Ocular Dominance Plasticity 111
but not Turnover of Axonal Boutons in a Model of Amblyopia
Appendix C: Multiple Roles for Nogo Receptor 1 in Visual 122
System Plasticity
Appendix D: Layer IV Restricts the Propagation of Critical 136
Period Plasticity in Visual Cortex Through Nogo Receptor 1

iii
Acknowledgements
When beginning work on the presentation for my final committee meeting, I
noticed that every template style contained the phrase ‘lorem ipsum.’ I knew I’d
seen it before but had no clue what it meant. It turned out to be from an excerpt
of Cicero’s The Purposes of Good and Evil, meaning was ‘Neither is there
anyone who loves, pursues or desires pain itself because it is pain.’ After reading
that, I thought ‘Cicero had obviously never met a graduate student.’ It sure
seems that way when you’re in the trenches, doing surgeries until your eyes blur,
imaging in a dark room for weeks on end, analyzing thousands of blurry images,
or freezing your ass off while you record. Not until starting to write this document
did I begin to feel a sense of accomplishment and the accompanying notion that
I’ve actually earned this distinction. It was a tough journey but I am glad I made it
with a little help from my family and friends.
First and foremost, I need to thank my parents, who’ve worked hard to give
me every opportunity they could. They always ‘encouraged’ me to do well in
school by setting grade quotas for everything from stakeboarding, time to driving
privileges, to university tuition, which was tough at the time but what can I say, it
worked. My father was a huge driving force to get me interested in science. I
have so many experiences of him being involved in my development, including
helping with science fair projects, helping me code a tic-tac-toe game in C++ and
sending me to coding camp at UCI, even giving me the opportunity to write and
file a patent. My mom was always supportive, and I probably would’ve lost my
sanity through graduate school many more times if it weren’t for her. She’s the
iv
one who really taught me how to be an adult and take care of myself. My
bathroom would smell like a barnyard if it weren’t for her.
I have to mention my younger brother Elan, who followed my lead to UCSB
and has done great things since. It’s been wonderful to see him through his
adventures. I wish he was here more than anyone, as he’s currently living in Tel
Aviv.
I also can’t go without thanking my mentor, Dr. Aaron McGee. I was his first
graduate student, and he’s been an excellent teacher for me. His high standards
for data quality and analysis have had a great impact on me far beyond its
application to science within the lab. A big thanks to other members of the lab
present and past: Celeste Stephany, Megan Arnett, Jen Park, Hilary Dorton,
Ryan Kast, Katie Chapman and Shirveen Parivash. I worked closely with Ryan
and Hilary to analyze axonal boutons shown in chapter 2 of this dissertation, they
were definitely critical to getting that project done. I also coauthored a paper with
Jen Park attached in Appendix A.
I had a lot of great support from friends. To mention a few, Bryan Roth,
Lauren Vandenberg, Kyle Trafas, Tommy Grandsaert, Dan Siegal, Rod Kashini,
Sam Cantor, Emerald Yang, Max Echt, and Harrison Sallow have been around
for different parts of my postgraduate career.
Last but definitely not least, I have to thank the wonderful Sami Svrcek. Me
being in grad school has definitely not been easy on her, and she’s been more
supportive and understanding than I could expect of anyone.
v
List of Figures

1.1 Organization of the visual system is conserved among mammals 5
1.2 Aspects of visual system circuitry are conserved among mammals 9
1.3 Several disparate extracellular ligands bind NgR1 27
1.4 The ngr1 functions in neurons within distinct circuits to limit OD plasticity and
improvement in acuity 28
2.1 Experimental design 57
2.2 Turnover of axonal boutons in vivo during adult OD plasticity 58
2.3 Turnover of axonal boutons in vivo during recovery from LTMD 59
2.4 OD plasticity measured with repeated OIS following LTMD 60
2.5 OD plasticity measured with single-unit recordings following LTMD 61
2.6 Laminar analysis of OD plasticity following LTMD 62
3.1 Experimental Design 78
3.2 OD was measured using single-unit recordings following 4D MD 79
3.3 Layer IV is more plastic after 2D MD in ngr1
flx/flx
;scnn1a-cre and WT CP mice 81
3.4 Progression of OD plasticity across layers 83
4.1Application of Prisms in 2PLSM 88
1
Abstract
The visual system is a premier model for studying experience-dependent
plasticity. During development, there’s a period of sensitivity called the ‘critical
period’ when brief monocular deprivation (MD) alters ocular dominance(OD) in
primary visual cortex (V1). There are several factors thought to mediate the
closure of the critical period such as maturation of inhibition, myelination, and
molecular cues. One of these molecular cues is the nogo-66 receptor (NgR1), as
deletion of ngr1 prevents closure of the critical period for OD plasticity and allows
for recovery of visual acuity in adulthood following long-term monocular
deprivation (LTMD).
NgR1’s restriction of neurite outgrowth in vitro led to the hypothesis that NgR1
restricts developmental visual plasticity by restricting the rates of addition and
loss of axonal boutons, as explored in chapter 2. Chronic in-vivo 2-photon laser
scanning microscopy (2PLSM) has become a standard for observing experience-
dependent changes in anatomical plasticity over time in vivo. Various experiential
manipulations have elicited changes in dendritic spine dynamics in different
cortical areas, including visual cortex. However, the effect of altered experience
on axonal bouton dynamics had not been studied prior to the paper published in
chapter 2, where I asked whether 4D MD or recovery of visual acuity was
accompanied by altered axonal bouton dynamics in ngr1-/- mice. I found that
neither alteration in visual experince affected the rates of axonal bouton gain and
loss in V1. This supports a model where anatomical changes correlating with
2
functional visual plasticity are largely postsynaptic while axonal structures remain
relatively stable.
Visual cortex is subdivided into lamina. It remains poorly understood whether
each layer plays a different role in the expression of OD plasticity. In cats, layer
IV is relatively resistant to OD shifts, although this is not true in mice, as shifts in
layer IV have been recorded in as little as 1D. In chapter 3, I examined whether
removing the brakes on plasticity in individual layers using conditional deletion of
ngr1unmasks plasticity in adults. I also examined how shifts in OD progressed in
a laminar fashion. I found that deleting ngr1 in excitatory neurons of layer IV
enabled OD plasticity throughout all layers through a disinhibitory circuit. Further,
in mice lacking ngr1 in layer IV and critical period WT mice, 2D MD produced a
dramatic shift in eye dominance in layer IV with only modest shifts in layers II/III
and V. This data supports a model where plasticity in cortex is regulated in a
feed-forward manner from layer IV.

3
Chapter 1: Introduction
1.1 Introduction to Visual Cortex
The study of the visual system has made enormous contributions to the
understanding of the brain. Discoveries made in the visual system have led to a
multitude of Nobel Prizes, including Allvar Gullstrand in 1911; Ragnar Granit,
Halden Hartline, and George Wald in 1967; and Roger Sperry, David Hubel, and
Torsten Weisel in 1981. Since the correct localization of primary visual cortex, or
V1, to the occipital lobe by Hermann Munk in 1881 (Schiller, 1986), researchers
have capitalized on advances in technology including many exciting optical and
physiological techniques to study vision in a plurality of species including
humans, non-human primates, cats, ferrets, rats, and mice.
In the late 1950s, advances in the manufacture of tungsten microelectrodes
allowed researchers to perform extracellular ‘single-unit’ recordings throughout
the depth of cortex (Hubel, 1957). Using this technology in a series of landmark
studies, David Hubel and Torsten Weisel characterized physiological response
properties of neurons in V1 including simple vs. complex receptive fields,
orientation selectivity, direction selectivity, and binocularity (Hubel, 1962; Hubel
and Wiesel, 1968; Wiesel et al., 1974; Hubel et al., 1977). Their observations
concerning binocularity of neurons in V1 provided a substrate for the use of
visual cortex as a model to study cortical plasticity. Indeed, their discovery that
neurons in the binocular region of V1 of kittens but not adult cats alter their eye
preference, or ocular dominance (OD) in response to occlusion of one eye
4
(Wiesel and Hubel, 1963; Wiesel, 1965; Hubel and Wiesel, 1965, 1970) is a
phenomenon that continues to be studied today.
Although the visual system differs slightly among mammalian model
organisms, the basic architecture of the visual system is conserved (Figure 1.1).
In primates, cats, and mice, visual information enters the nervous system at the
retina, where a proportion of retinal ganglion cells (RGCs) project across the
optic chiasm to eye-specific domains of the contralateral lateral geniculate
nucleus (LGN). Binocular inputs do not converge until they reach cortex. Mouse
eyes are situated on the sides of the head; consequently, a large proportion of
RGC axons cross at the optic chiasm to the contralateral LGN. However, as
primates and cats mainly gaze forward, a much smaller proportion of RGC axons
cross at the optic chiasm.
The study of visual cortex has become a great model to study the
developmental disorder amblyopia. Amblyopia results from abnormal visual
experience such as strabismus early in life and is characterized by deficits in
spatial vision. This prominent visual disorder affects up to 3% of children in the
United States (Webber and Wood, 2005). Amblyopia is easiest to treat in children
and until recently was generally considered untreatable in adults (Li et al.,
2008).This mirrors the developmental profile of other mammals, as shown in
Figure 1.2.

5

Figure 1.1. Organization of the visual system is conserved among
mammals. Axons from projection neurons in the retina with receptive fields
within the binocular visual field (green) converge on the optic chiasm (blue and
yellow lines) and then target the lateral geniculate nucleus (LGN) in each
hemisphere. In mouse, the majority of axons cross the chiasm and the ipsilateral
projection (blue dashed line) is smaller than in predatory mammals such as
primates and cats. Within the LGN, axons from each eye innervate distinct
domains. These domains comprise six layers in primate and three layers in cat,
whereas the ipsilateral eye targets a smaller patch nestled within a larger domain
of the contralateral eye in mouse. The axons from the neurons in LGN then
project to primary visual cortex (V1). (Stephany et al., 2015)

6
1.2Critical Periods in Visual Cortex
As previously mentioned, OD describes the preference of neurons for one
eye or the other. There exists a critical period during postnatal development
when brain circuitry, in this case OD, is sensitive to modification by experience
(Figure 1.2). Closure of one eye, or monocular deprivation (MD), results in a shift
in the preference of neurons in binocular V1 towards the non-deprived eye during
the critical period (Wiesel and Hubel, 1963, 1965; Hubel and Wiesel, 1965a,
1970).The critical period for OD plasticity in mice is accepted to be between P19
to P32 and has been localized to visual cortex (Gordon and Stryker, 1996; Taha
and Stryker, 2002). Both monkeys and cats are more binocular to begin with
owing to their forward-facing eyes, while mice normallyhave acontralateral bias
(Wiesel and Hubel, 1963; LeVay et al., 1980; Gordon and Stryker, 1996) (Figure
1.2A). Despite these differences, brief MD of during the critical period results in a
shift towards the non-deprived eye in all species. The duration of MD required
for a saturating shift in OD is longer in monkeys and cats than the 4D required for
mice (Figure 1.2A).
In primates, cats, and mice, visual acuity also develops during a time
coincident with the critical period for OD plasticity (Figure 1.2B). Various lengths
of deprivation during this period of development are required to induce visual
deficits across model organisms. In cats, prolonged periods of MD early in life
result in blindness through the deprived eye (Giffin and Mitchell, 1978). However,
periods of deprivation as short as 4-6 days at the height of the critical period can
impair visual acuity (Mitchell and Gingras, 1998; Mitchell et al., 2001). In mice,
7
only deprivation throughout the critical period produces a persistent deficit in
visual acuity (Prusky and Douglas, 2003).
It remains unknown whether the decrease in visual acuity caused by LTMD is
dependent on OD plasticity. Both maturation of visual acuity and the onset of the
critical period for OD plasticity require a balance between excitatory and
inhibitory neurotransmission (e/i balance) (Hensch et al., 1998; Hanover et al.,
1999; Fagiolini and Hensch, 2000; Levelt and Hübener, 2012). The onset of the
critical period for OD plasticity and maturation of visual acuity are both delayed
by dark rearing (Fagiolini et al., 1994). Additionally, environmental enrichment
accelerates the onset of the critical period for OD plasticity and development of
visual acuity (Huang et al., 1999; Sale et al., 2004). However, it remains possible
that these types of visual plasticity are separable, as genetic manipulation of fast-
spiking parvalbumin-positive inhibitory interneurons (PV neurons), which are
increasingly implicit in the regulation of visual plasticity, allows for OD plasticity
but not behavioralrecovery of visual acuity (Fagiolini et al., 2004; Katagiri et al.,
2007; Stephany et al., 2014).
In addition to critical periods for plasticity of OD and visual acuity, there
are critical periods for long-term potentiation (LTP) and long-term depression
(LTD) in mouse V1 (Jiang et al., 2007). In white matter to layer IV synapses, LTP
magnitude reaches its nadir around P18, while LTD magnitude does so around
P32. Alternately, layer IV to layer II/III synapses maintain relatively stable
magnitudes of LTP and LTD into adulthood. These results suggest that plasticity
8
terminates sequentially in V1, mirroring feed-forward information flow in cortex
(Jiang et al., 2007).

9

Figure 1.2. Aspects of visual system plasticity are conserved among
mammals. (A) Monocular deprivation (MD) during the critical period disrupts eye
dominance. Ocular dominance (OD) histograms plot the distribution of relative
responsiveness of neurons in V1 to a visual stimulus presented independently to
each eye. Neurons increasingly more responsive to the contralateral eye are
categorized with lower numbers (3,2,1) while those with increasing preference for
the ipsilateral eye are binned into higher number categories (5,6,7). Neurons with
equal responsiveness to each eye are categorized as ‘4’. In primates and cats
with normal vision (green bars and eye symbols below) this distribution is
binocular. In mouse, normal vision is biased to the contralateral eye. Closing the
contralateral eye for as briefly as a few days (purple bars and eye symbols
below) shifts eye dominance towards the non-deprived ipsilateral eye. This
plasticity is conserved between species although the magnitude of the OD shift
varies. (B) Visual acuity increases during the critical period (green squares) and
10
closing one eye during this maturation permanently impairs visual acuity. The
resulting acuity following MD is similar to the acuity at the age of deprivation
(black square), although these results are more variable in primate studies. In
mice, deprivation for the duration of the critical period (long-term deprivation,
LTMD) is required to impair acuity. (C) One model for how MD impairs visual
performance in mammals. Discordant vision, such as deprivation or strabismus,
first exaggerates eye dominance as in (A), diminishing responsiveness to the
affected eye in visual cortex. This limited representation of the affected eye
prevents the normal maturation of visual circuits subserving performance, such
as acuity as in (B). After the critical period, these mechanisms of plasticity are no
longer accessible. ‘Reactivating’ developmental visual plasticity otherwise
confined to the critical period is one strategy for rectifying eye dominance and
potentially improving vision through the affected eye. (Stephany et al., 2015)

11
1.3Experience-Dependent Functional Plasticity in Mouse Visual Cortex

Plasticity in mouse primary visual cortex has been dissected using a variety of
behavioral, physiological, and optical methods. While some of them have yielded
slightly different results, they measure different aspects of visual plasticity yet
generally agree on many aspects of visual plasticity.
As previously mentioned, the critical period for OD plasticity was first
characterized in mice using single-unit recordings, which measure spiking activity
of one to a few neurons, showing that 4D MD results in a saturating shift towards
the non-deprived eye (Gordon and Stryker, 1996). Visually evoked potentials
(VEPs), another electrophysiological method, measures subthreshold activity in a
wide population of neurons. The particular advantages of this method include
chronic electrode implantation, and the comparison of contralateral and ipsilateral
response magnitudes, allowing comparison of single-eye inputs.Like single-unit
recordings, brief MD results in a shift in OD during the critical period. 3D MD
results in a depression of deprived eye responses and potentiation of open eye
responses that characterizes critical period OD plasticity (Sawtell et al., 2003;
Pizzorusso et al., 2006).
There still remains some plasticity in the adult mouse, as extended MD from
P40 to P60 results in a small shift in OD by single unit recordings (Antonini et al.,
1999). VEP recordings confirm that beyond the critical period, an additional
length of MD is required for a shift in OD, and that adult OD plasticity lacks
deprived eye depression and is primarily driven by potentiation of the non-
deprived eye (Sawtell et al., 2003).
12
Optical techniques have provided yet another great method to measure visual
plasticity. Described in more detail in Chapter 2 of this manuscript, intrinsic signal
imaging (ISI) measures changes in the reflectance of red light caused by altered
blood oxygenation in cortical microvasculature. Some advantages of this
technique include repeated imaging, mapping of retinotopy, relatively high spatial
resolution, and fast experimental time (Kalatsky and Stryker, 2003; Cang et al.,
2005; Hofer et al., 2005). Much like single unit recordings and VEPs, ISI detects
a decrease in contralateral bias following 4D MD during the critical period (Cang
et al., 2005; Hofer et al., 2005).
These physiological and optical techniques, in addition to some behavioral
measures, have also facilitated the study of visual acuity in mice. Behavioral
acuity is best measured with a forced-choice swimming task, where mice are
trained to find an exit platform from the pool directly under a spatial frequency
grating (Prusky et al., 1999). Using this task, it was determined that MD
throughout the critical period but not before or after results in a persistent deficit
in visual acuity through the deprived eye (Prusky and Douglas, 2003; Stephany
et al., 2014).
VEPs have also been used to measure visual acuity. By this technique, the
developmental profile of visual acuity is similar to that observed behaviorally
(Ridder and McCulloch, 1998; Huang et al., 1999; Pizzorusso et al., 2006).
Similarly, ISI has also been used to measure the development of visual acuity,
and generally agrees with VEP and behavioral data (Heimel et al., 2007). These
behavioral and physiological measures have determined that visual acuity
13
matures from an immature acuity of ~.3 cycles/degree to ~.5 cycles/degree in
adulthood.

14
1.4 Anatomical Plasticity in Visual Cortex
The relationship between function and structure in V1 was first tested in
primates in the 1970s (Hubel and Wiesel, 1972; Wiesel et al., 1974; LeVay et al.,
1975). Subsequent work showed that in layer IV of V1, which receives the
majority of thalamocortical input, neurons are clustered according to eye
preference (OD domains) and are less binocular than neurons in extragranular
layers, and that prolonged MD results in an expansion of non-deprived OD
domains and contraction of deprived OD domains. This was accompanied by
shifts in the binocularity of neurons in V1 (Hubel et al., 1977). Analogous findings
were also confirmed in cat visual cortex (Shatz and Stryker, 1978). Shorter
periods of MD also have effects on the structure of thalamocortical arbors. Both
long-term MD and short-term MD result in a decrease in the total length and
complexity of deprived arbors (Antonini and Stryker, 1993). Although mice the
lack OD domains present in omnivorous and predatory mammals, extended MD
also affects anatomy of thalamocortical arbors. Both 20 and 40 days of MD
beginning P17-19 result in trends toward reduced length and complexity of
thalamocortical arbors serving the deprived eye relative to those serving the non-
deprived eye (Antonini et al., 1999). While large structural changes require
weeks of MD, structural remodeling at boutons of thalamocortical axons may
require as little as 3 days of MD. Indeed, the density of Vglut2 labeling in
thalamocortical boutons is reduced after only 3D MD (Coleman et al., 2010).
In addition to alteration of thalamocortical arbor morphology, MD also affects
the anatomy of cortical neurons at the level of the dendritic spine.Chronic LTMD
15
of juvenile rats results in a decrease in spine density by ~50% throughoutthe
depth of cortex (Pizzorusso et al., 2006; Montey and Quinlan, 2011). In mice, 3D
MD during the critical period both increases spine density and spine motility in
layer V neurons observed in coronal sections (Djurisic et al., 2013).
Chronic in vivo 2-photon microscopy (2PLSM) is a promising technique that
observes the gain, loss, and stability of dendritic spines and axonal boutons
through a chronically implanted cranial window or thinned skull preparation. This
technique has been used to investigate the effects of altered experience, stroke,
and neurodegenerative disease models on dendritic spine dynamics in various
brain regions including barrel, motor, and visual cortex (Trachtenberg et al.,
2002; Tsai et al., 2004; Zuo et al., 2005; De Paola et al., 2006; Brown et al.,
2007, 2009; Spires-Jones et al., 2007; Meyer-Luehmann et al., 2008; Xu et al.,
2009; Hofer et al., 2009; Holtmaat and Svoboda, 2009; Wilbrecht et al., 2010).
Both genes and experience can affect structural synaptic plasticity of dendritic
spines in vivo. In αCaMKII-T286A mutant mice, whisker trimming does not result
in the stabilization of new spines at the borders between barrels as it does in WT
mice (Wilbrecht et al., 2010). Throughout postnatal development, the rates of
addition and deletion and normalized spine density decrease with age in
somatosensory cortex (Holtmaat et al., 2005). In adult mice, the rate of dendritic
spine turnover in visual cortex is relatively stable over time (Holtmaat et al.,
2005). In mature visual cortex, 8D of MD results in a transient doubling of the
rate of spine addition without affecting the rate of spine loss in layer V but not
layer II/III neurons (Hofer et al., 2009) However, performing the same MD longer
16
after cranial window implantation produces much less dramatic results (Hofer et
al., 2009). Thus, the extent to which visual experience affects turnover of
dendritic spines remains unclear.
Much less is known about the turnover and stability of axonal boutons in vivo.
There are three anatomically separable classes of axonal boutons in cortex: A1,
A2, and A3, which anatomically correspond to thalamocortical afferents, layer VI
axon collaterals, and axons of layer V and II/III neurons respectively (De Paola
et al., 2006; Portera-Cailliau et al., 2005). Chapter 2 of this manuscript comprises
the first study to date investigating if and how axonal boutons undergo alterations
in their turnover in response to classic paradigms of experience-dependent
plasticity.

17
1.5 Manipulating Plasticity in Visual Cortex
A significant body of work dissects how the critical period can be manipulated.
Through different experiential, pharmacologic, and genetic methods, the critical
period has been hastened, delayed, protracted, and reopened. In addition,
certain manipulations have also promoted recovery of visual acuity after LTMD.
These experiments shed some light on the maintenance of heightened plasticity
and how recovery of visual function is mediated.
Maturation of inhibitory circuitry is increasingly implicit in the regulation of
plasticity in visual cortex. Cortical inhibition increases throughout the critical
period (Morales et al., 2002). Indeed, one day of MD during the critical period
results in decreased excitatory drive onto inhibitory neurons, resulting in altered
e/i balance (Kuhlman et al., 2013). Perturbing thate/i balance is one method of
altering developmental visual plasticity. Prolonged dark rearing in cats results in
delayed onset of the critical period, likely by delaying the development of γ-
aminobutyric acid (GABA) neurotransmission in visual cortex (Cynader and
Mitchell, 1980; Fosse and Heggelund, 1989). This has been confirmed with
studies in rats and mice. Dark rearing also delays the development of visual
function and OD plasticity by reducing GABAergic transmission in V1 (Morales
et al., 2002; Fagiolini et al., 2004). Indeed, mice lacking a form of glutamic acid
carboxylase (GAD65), an enzyme essential for the synthesis of GABA, lack OD
plasticity from brief MD during the critical period (Hensch et al., 1998b; Morales
et al., 2002). However, critical period plasticity can be induced in V1 of these
mice at any age by increasing cortical inhibition via administration of the
18
benzodiazepine diazepam (Fagiolini and Hensch, 2000; Iwai et al., 2003).
Transplanting the medial ganglionic eminence, the developmental source of
inhibitory interneurons, including PV neurons, also allows for developmental OD
plasticity in adults (Southwell et al., 2010). Brain-derived neurotrophic factor
(BDNF)-overexpressing mice exhibit early maturation of inhibition and a
precocious critical period, indicating that inhibitory tone is a central regulator of
visual plasticity (Hanover et al., 1999; Huang et al., 1999). Even experiential
manipulations like environmental enrichment reactivate developmental OD
plasticity by reducing intacortical inhibition, increased BDNF expression, and
possibly increased serotonin levels (Baroncelli et al., 2010). Conversely, dark
exposure for 10 days during adulthood prior to MD allows for developmental OD
plasticity and is accompanied by a decrease in GABAa neurotransmission (He et
al., 2006). Fluoxetine, a serotonin-specific reuptake inhibitor (SSRI), reduces
intracortical inhibition and restores plasticity in visual cortex (Maya Vetencourt et
al., 2008).Thus, modulation of inhibition by a plurality of methods can affect
developmental plasticity in visual cortex.
Perineuronal nets (PNNs) are another good target for enhancing plasticity in
the adult. Infusion of chondroitinase ABC, which degrades specific sugar
polymers in PNNs, also allows for developmental plasticity in V1 (Pizzorusso et
al., 2002). Additionally, MD during the critical period is accompanied by elevated
proteolytic activity by tissue plasminogen activator (tPA); mice that lack OD
plasticity lack this increase in tPA activity (Mataga et al., 2002). Neural cell-
adhesion molecule and laminin are substrates for plasmin, suggesting that
19
degradation of extracellular matrix plays a role in the expression of OD plasticity
(Mataga et al., 2002).
There have also been genes implicit in regulating visual plasticity. Deleting
the paired immunoglobulin-like receptor B (PirB) results in increased Arc
induction and OD plasticity by ISI (Syken et al., 2006; Djurisic et al., 2013).
Deleting Lynx1, which binds to nicotinic acetylcholine receptors (nAChRs) and is
expressed beginning at the end of the critical period, allows for enhanced OD
plasticity in adults (Morishita et al., 2010; Bukhari et al., 2015). Indeed, there is
elevated tPA activity in V1 of adult lynx1
-/-
following MD, suggesting that Lynx1
regulates plasticity by modulating proteolytic activity (Bukhari et al., 2015). The
Nogo-66 receptor 1 (NgR1), discussed here and in great detail in chapter 1.6,
also gates developmental OD plasticity.
Myelination of visual cortex also coincides with the closure of the critical
period (McGee et al., 2005). Inhibitors of axonal outgrowth, especially myelin-
associated inhibitors (MAIs), in vitro have also been implicated in the closure of
critical period plasticity. Digestion of chondroitin sulfate proteoglycans (CSPGs)
promotes OD plasticity in adult rodents (Schoop and Gardziella, 1997;
Pizzorusso et al., 2002). NgR1 is promising target to mediate the closure of the
critical period, as it binds myelin-associated inhibitors such as myelin-associated
glycoprotein (MAG), oligocyte-myelin glycoprotein (OMGP), and Nogo-A, as well
as CSPGs. The following section of the introduction introduces NgR1, a central
tool for dissecting developmental visual plasticity.

20
1.6 The Nogo-66 Receptor
The Nogo-66 receptor, or NgR1, was first identified as a receptor for a 66-
amino acid fragment of Nogo-A, a myelin-associated inhibitor of axonal
outgrowth in vitro (Fournier et al., 2001). Deleting this receptor was found to
accelerate functional recovery from spinal cord injury (Kim et al., 2004). NgR1
was soon after discovered to be essential to the closure of the critical period for
OD plasticity (McGee et al., 2005). Deleting NgR1 also allows for recovery of
visual acuity during adulthood after LTMD throughout the critical period and
beyond (Stephany et al., 2014). Although the details of how NgR1 functions to
limit plasticity within the CNS are still being worked out, manipulating NgR1 has
become a useful tool for the study of developmental plasticity.
NgR1 is composed of several leucine-rich repeats (LRRs) followed by a stalk
region and a glycosyl-phosphotidylinositol (GPI) anchor attaching it to the
extracellular cell membrane. In addition to Nogo-66, the LRR region binds MAG
and OMGP (McGee and Strittmatter, 2003). The NgR1 LRR also binds various
other proteins like the membrane-bound form of amyloid precursor protein (APP),
soluble fibroblast growth factors 1 and 2 (FGF1, FGF2), and leucine-rich glioma
inactivated 1 (LGI1) (Park et al., 2006; Lee et al., 2008; Thomas et al., 2010;
Stephany et al., 2015). Additionally, the glycosaminoglycan moiety of some
CSPGs binds the stalk region of NgR1 (Dickendesher et al., 2012).
How the NgR1 signal crosses the plasma membrane is less clear. Because
NgR1 is a GPI-linked protein, it requires additional cooperative machinery to
transduce its signal across the cell membrane (Figure 1.3). Experiments
21
investigating the inhibition of dorsal root ganglion (DRG) neurite outgrowth in
vitro identified tumor necrosis factor receptor 19 (Tnfr19/TAJ/TROY), and its
functional homolog, thelow affinity neurotrophin receptor p75, as possible
coreceptors for NgR1 (Wang et al., 2002; Park et al., 2005). NgR1 and TROY or
p75 are proposed to form a tripartite signaling complex with another coreceptor,
the immunoglobulin domain-containing Nogo receptor interacting protein (Lingo-
1), that activates the intracellular signaling molecule RhoA to inhibit neurite
outgrowth(Mi et al., 2004). The coreceptor p75 is not required to mediate CSPG
signaling through NgR1 (Dickendesher et al., 2012), so different
receptor/coreceptor combinations may be required to transduce signals from
different ligands.
NgR1 is expressed throughout the CNS. In the visual system, NgR1 is
expressed in retina (Wang et al., 2012), LGN (Barrette et al., 2007), and primary
visual cortex (McGee et al., 2005). NgR1 is also detectable at P1 in barrel
cortex, and is present at similar levels in visual cortex between P20 and
adulthood (McGee et al., 2005).
The relative subcellular distribution of NgR1 on the neuronal plasma
membrane remains enigmatic as the receptor has be reported to localize to
axons and and presynaptic terminals, or alternatively enriched in the
somatodendritic compartment and dendritic spines. The first ultrastructural
analysis reported confirms that some immunoreactivity for NgR1 can be
observed both in the presynaptic bouton as well as the post-synaptic membrane
(Wang et al., 2002). There are some reports of NgR1 enrichment in
22
synaptosomal fractions (Lee et al., 2008; Akbik et al., 2013), and in presynaptic
terminals (Lee et al., 2008; Zemmar et al., 2014). However, NgR1 is a GPI-
linked protein, and GPI-linked proteins and synaptosomes are isolated similarly
by sucrose gradients, indicating that NgR1’s appearance in synaptosomal
fractions may be coincidental. Alternately, immunostaining suggests that NgR1
appears adjacent to but not overlapping with synaptic proteins such as PSD95,
GluR2, SV2, and GAD67 (Wills et al., 2011). Thus, more careful study is
required to resolve the localization and enrichment characteristics of NgR1.
As previously mentioned, deleting NgR1 prevents closure of the critical period
for OD plasticity (McGee et al., 2005) and allows for the recovery of visual acuity
during adulthood after LTMD (Stephany et al., 2014). There are a few possible
mechanisms by which NgR1 limits critical period plasticity including the
maturation of e/ibalance, transduction of inhibitory signals in the extracellular
environment, and restricting anatomical plasticity.
Early studies in vitro examined the role of NgR1 in neurite outgrowth. In
dorsal root ganglion (DRG) neurons, signaling through the Nogo-A/NgR1
pathway promotes growth cone collapse (Fournier et al., 2001). NgR1 also limits
regrowth of certain types of axons on a mouse model of spinal cord injury (Kim
et al., 2004). These findings motivated the hypothesis that NgR1 functions to limit
plasticity in the adult by restricting changes in physical connectivity between
neurons. Subsequent studies ask whether NgR1 alters the rates of addition and
deletion of dendritic spines and axonal boutons.
23
It remains unclear if NgR1 restricts structural synaptic plasticity in vivo. In
primary hippocampal neurons, knocking down NgR1 increases dendritic spine
density, indicating that NgR1 plays a role in the rate of formation or stabilization
of dendritic spines (Wills et al., 2011). However, knocking out NgR1 did not alter
spine density in hippocampal slices (Wills et al., 2011). One study reports that in
barrel cortex, constitutive or acute deletion of NgR1 results in higher rates of
addition and deletion of dendritic spines (Akbik et al., 2013). However, a similar
study finds no difference between the rates of addition or deletion of dendritic
spines in barrel cortex with constitutive or acute deletion of NgR1 (Park et al.,
2014). It is still unknown how visual experience affects spine turnover in V1 in
NgR1 mutant mice. Thus, additional study is required to validate how NgR1
affects experience-dependent structural plasticity of dendritic spines.
It’s also possible that NgR1 limits structural plasticity in axons and axonal
boutons to limit plasticity. In cats, ocular dominance is partially dictated by the
arborization of thalamocortical axons in layer IV of V1, resulting in ocular
dominance domains (LeVay et al., 1978). Visual deprivation lasting months
results in shrinkage of domains serving the deprived eye and expansion of those
serving the non-deprived eye (Shatz and Stryker, 1978). In mice, weeks of MD
results in a decrease in the total length of thalamocortical arbors serving the
deprived eye relative to those serving the non-deprived eye (Antonini et al.,
1999). However, critical period OD plasticity occurs on the scale of days
(Gordon and Stryker, 1996). It remains possible, though, that anatomical
plasticity of thalamocortical axons occurs during brief MD, since 3 days of MD
24
reduces the density of thalamocortical synapses in layer IV (Coleman et al.,
2010).
Much like dendritic spines, axonal boutons appear and disappear over time in
vivo (De Paola et al., 2006). However, whether genes or experience affect
turnover of intracortical axonal boutons remains relatively understudied. In barrel
cortex, mice lacking NgR1 are reported to have elevated rates of axonal bouton
addition and loss (Akbik et al., 2013). However, in visual cortex, neither short
term MD of 4-8 days nor eye opening following LTMD alters the rates of addition
or loss of axonal boutons from layer II/III and V excitatory neurons of WT or
NgR1-/- mice (Frantz et al., 2015). In motor cortex, there is rapid deletion of
axonal boutons from axons of somatostatin-positive inhibitory neurons following
training (Chen et al., 2015), though effect of experience on inhibitory axonal
bouton dynamics in visual cortex has not been studied. Thus, further
investigation is required into whether and how NgR1 affects critical period
closure by limiting anatomical plasticity of axonal boutons and dendritic spines.
As mentioned earlier in this manuscript, manipulation of e/i balance alters
sensitivity to MD and can also drive recovery of visual function. Deleting NgR1
constitutively or just in PV neurons using a conditional allele for ngr1 (ngr1
flx
)
reduces excitatory drive onto PV neurons in layer II/III, similar to what is seen
after 1D MD during the critical period (Kuhlman et al., 2013; Stephany et al.,
2014). In layer II/III, both the constitutive knockout and the conditional PV
knockout also show reduced EPSCs in PV neurons and reduced IPSCs in
pyramidal neurons, while EPSCs in pyramidal neurons are unaffected (Stephany
25
et al., 2014). Whether restoring inhibitory tone, perhaps with administration of
diazepam, attenuates OD plasticity and recovery of visual acuity in adult mice
lacking NgR1 is necessary to determine whether NgR1 promotes plasticity by
disinhibition.
As previously mentioned, NgR1 receives inhibitory signals from MAIs and
CSPGs. Although there is little circumstantial evidence tying MAIs to OD
plasticity, no direct studies have been made. Digestion of CSPGs does, however,
promoteOD plasticity in adults (Pizzorusso et al., 2002). Thus it is not clear
exactly which ligands, or combinations of ligands, are responsible for NgR1’s role
in OD plasticity.
It is also possible that NgR1 limits synaptic plasticity to close critical periods
and limit functional recovery. Loss of NgR1 results in increased FGF2-dependent
enhancement of LTP in Shaffer collateral to CA1 synapses in hippocampus (Lee
et al., 2008). Additionally, loss of NgR1 attenuates hippocampal LTD (Lee et al.,
2008). Further, function-blocking antibodies against NgR1 enhance LTP in layer
II/III horizontal fiber pathway of rat primary motor cortex, which was accompanied
by improved motor learning (Zemmar et al., 2014). Since closure of the critical
periods for LTP and LTD in V1 coincide with the opening and closing of the
critical period for OD plasticity respectively (Jiang et al., 2007), it’s possible that
NgR1 influences the balance of LTP and LTD in V1 to close the critical period,
although this hypothesis has not been tested.
NgR1 may affect OD plasticity and the recovery of visual acuity through
different populations of neurons. While constitutive deletion of ngr1 allows for
26
both bidirectional OD plasticity and recovery of visual acuity in adulthood,
deleting ngr1 only in PV neurons allows for OD plasticity in adulthood but not the
recovery of visual acuity (Figure 1.4) (Stephany et al., 2014; Frantz et al., 2015).
In chapter 3 of this dissertation, I will discuss how alternate genetic dissection of
NgR1 allows for OD plasticity in adulthood. This body of work somewhat
complicates the role of NgR1 in limiting plasticity, as it functions alternately in
excitatory and inhibitory neurons to limit OD plasticity in adulthood. One central
theme is the manipulations affecting closure of the critical period alter input of
excitatory neurons in layer IV onto putative PV neurons in layer II/III (Stephany
et al., 2014). This seems to agree with the larger body of evidence suggesting
that manipulating e/i balance is a major key to unlocking developmental plasticity
in the adult.

27

Figure 1.3. Several disparate extracellular ligands bind NgR1. Myelin-
associated glycoprotein (MAG), oligodendrocyte-myelin glycoprotein (Omgp),
and the Nogo-66 region of Nogo-A are ligands for NgR1. These proteins each
bind the leucine-rich repeat (LRR) domain of NgR1. Several members of the
family of chondroitin sulfate proteogylcans (CSPGs) also bind to NgR1. The
sugar-chains on these molecules interact with the stalk region of the receptor. As
NgR1 is attached to the plasma membrane by a lipid anchor, NgR1 is proposed
to transduce a signal from these ligands through one or more transmembrane
‘co-receptors’ such as Lingo, TROY and p75, to activate the small GTPase
RhoA. How this signal may limit anatomical plasticity and/or synaptic plasticity
remains unclear. (Stephany et al., 2015)

28

Figure 1.4.The ngr1 functions in neurons within distinct circuits to limit OD
plasticity and improvement in acuity. (A) Mice lacking ngr1 constitutively
(ngr1-/-), or selectively in PV interneurons (ngr1 f/f;PV-Cre), retain developmental
visual plasticity as adults. During the critical period, 4-days of MD (purple bars
and eye symbols below) shifts the distribution of neuronal eye dominance (green
bars and eye symbols below). This is not observed in adult WT mice. In contrast,
in adult ngr1-/- mice or ngr1 f/f;PV-Cre mice, 4 days of MD continues to shift
ocular dominance. (B) Adult ngr1-/- mice spontaneously recover visual acuity
over seven weeks following LTMD, but WT and ngr1 f/f;PV-Cre mice do not. (C)
A comparison of the facets of visual plasticity present in different genotypes of
mice. This genetic dissection of the expression of ngr1 reveals that OD plasticity
is not sufficient to improve visual acuity. (Stephany et al., 2015)

29
1.7 Brief Summary

Since advances in electrode manufacture in the 1960s, OD in V1 has been a
great model in which to study cortical plasticity. Brief MD during a critical period
for OD plasticity early in life results in a shift in OD favoring the non-deprived eye.
Similarly, abnormal vision early in life in humans results in the developmental
disorder amblyopia. The architecture of the visual system is conserved among
mammals, as are critical periods for OD plasticity, although there are slight
variations in the effects of MD and the durations required. In mice, 4D MD results
in a shift in OD, and MD throughout the critical period is required for a persistent
deficit in visual acuity.
The critical period for OD plasticity in mice has been identified to be between
P19 and P32. There are also critical periods for LTP and LTD in visual cortex,
ending at about P18 and P32 respectively. Studies of the opening and closure of
the critical period have revealed many promising techniques for opening, closing,
and reopening the critical period, including experiential manipulations, alteration
of e/i balance, pharmacologic treatments, and genetic manipulations. One of
these genetic manipulations is the deletion of ngr1. These mice maintain the
capacity for critical period-like OD plasticity well into adulthood, and are now
used to study developmental plasticity outside the constraints of the critical
period.
Functional plasticity in visual cortex is measured by a variety of methods,
including extracellular single-unit recordings, visually-evoked potentials, optical
imaging of intrinsic signals, and now calcium imaging. Although there are
30
differences in the effects on OD by these different measures, they generally
agree on the effects of deprivation during and after the critical period. VEPs and
optical imaging have identified that critical period plasticity differs from adult
plasticity, as critical period plasticity involves a depression of deprived eye
responses while adult plasticity comprises a potentiation of the non-deprived eye.
One promising mechanism for visual plasticity is alterations of anatomical
connectivity. In thalamoctical axons of cats, prolong MD results in reduced
representation from the deprived eye and an expanded representation of the
non-deprived eye. Similarly in mice, prolonged MD results in a reduction in the
length and branching complexity of arbors serving the deprived eye relative to
the non-deprived eye. In cortical neurons, LTMD also results in reduced spine
density throughout cortex. Additionally, MD results in an increase in the rate of
spine gain on apical tufts of layer V but not layer II/III neurons in V1.

31
1.8Presented Contributions
The gene NgR1 represents a powerful tool to study developmental plasticity
outside the confines of the critical period for OD plasticity. Although there have
been multiple studies investigating the turnover and persistence of dendritic
spines in response to experiential manipulations, chapter 2 of this dissertation
represents the first study measuring the turnover and persistence of axonal
boutons in response to experiential manipulation. I measured the effects of OD
plasticity and recovery of visual function on the rates of gain, loss, and
persistence of axonal boutons over about a month of imaging per animal. I also
used both ISI and single-unit recordings to measure the recovery of OD shortly
after eye opening following LTMD, finding that mice lacking ngr1 recover OD
marginally more than WT mice without a corresponding change in axonal bouton
dynamics. I however did identify a slight trend towards an elevated rate of axonal
bouton loss in the first 4D following eye opeining after LTMD.
I also used a conditional allele for ngr1 to examine the contributions from
each layer to limiting cortical plasticity. After deleting ngr1 in layers II/III, IV, V,
and VI individually, I examined OD throughout cortex after 4D MD by single-unit
recordings. I initially found that deleting ngr1 in cortical excitatory neurons or
layer IV excitatory neurons but not in II/III, V, or VI excitatory neurons was
sufficient to prevent closure of the critical period for OD plasticity. Last, I found
that mice lacking ngr1 in layer IV excitatory neurons and critical period mice had
robust shifts in layer IV and minimal shifts in II/III and V following 2D MD. This
32
data supports a model where OD shifts progress through cortex in a manner
paralleling the feed-forward flow of information.

33
Chapter 2: Nogo Receptor 1 Limits Ocular Dominance Plasticity but not
Turnover of Axonal Boutons in a Model of Amblyopia
Authors: Michael G. Frantz, Ryan J. Kast, Hilary M. Dorton, Katherine S.
Chapman and Aaron W. McGee
Published in Cerebral Cortex on 1-11-2015, doi: 10.1093/cercor/bhv014

Abstract

The formation and stability of dendritic spines on excitatory cortical neurons are
correlated with adult visual plasticity, yet how the formation, loss, and stability of
postsynaptic spines register with that of presynaptic axonal varicosities is
unknown. Monocular deprivation has been demonstrated to increase the rate of
formation of dendritic spines in visual cortex. However, we find that monocular
deprivation does not alter the dynamics of intracortical axonal boutons in visual
cortex of either adult wild-type (WT) mice, or adult NgR1 mutant (ngr1-/-) mice
that retain critical period visual plasticity. Restoring normal vision for a week
following long-term monocular deprivation (LTMD), a model of amblyopia,
partially restores ocular dominance (OD) in WT and ngr1-/- mice but does not
alter the formation or stability of axonal boutons. Both WT and ngr1-/- mice
displayed a rapid return of normal OD within 8 days after LTMD as measured
with optical imaging of intrinsic signals. In contrast, single-unit recordings
revealed that ngr1-/- exhibited greater recovery of OD by 8 days post-LTMD. Our
findings support a model of structural plasticity in which changes in synaptic
connectivity are largely postsynaptic. By contrast, axonal boutons appear to be
remarkably stable during dramatic changes in cortical circuit function.

34
Introduction
In the mammalian neocortex, most excitatory synapses reside on dendritic
spines, small protrusions from the dendritic shaft that contain a postsynaptic
density (Sala and Segal, 2014). The corresponding presynaptic apparatus
harboring synaptic vesicles often forms a varicosity, or bouton, on the axon
(Debanne et al., 2011). Several studies have correlated the rate of formation,
loss, and stability of dendritic spines with experience-dependent plasticity in
sensory cortex (Holtmaat and Svoboda, 2009). These imaging experiments have
focused almost exclusively on dendritic spines of apical dendrites resident in
layer I of both layer V pyramidal neurons, and to a lesser extent II/III pyramidal
neurons. Spine dynamics are altered in somatosensory barrel cortex by several
manipulations that affect the size of receptive fields for associated whiskers,
including chessboard deprivation of whiskers (Trachtenberg et al., 2002).
Similarly, inducing abnormal vision by closing one eye (monocular deprivation,
MD) alters eye dominance and increases spine formation and stability in the
binocular zone of visual cortex of adult mice (Sawtell et al., 2003; Fischer et al.,
2007; Sato and Stryker, 2008; Hofer et al., 2009; Sala and Segal, 2014).
However, while the turnover of axonal boutons is lower than that of dendritic
spines (De Paola et al., 2006; Debanne et al., 2011), whether the dynamics of
these presynaptic structures present in layer I are also responsive to sensory
manipulation or correlated with experience-dependent plasticity is unknown.
Ocular dominance (OD) plasticity is a premier model of experience-
dependent plasticity (Morishita and Hensch, 2008; Holtmaat and Svoboda,
35
2009). The developing visual system is acutely sensitive to the quality of visual
experience during a specified ‘critical period’. In mice, this critical period extends
from approximately postnatal days (P) 19 to 32 (Gordon and Stryker 1996;
Trachtenberg et al. 2002). Monocular deprivation for a few days (3-5 days) during
the critical period shifts the relative ocular dominance towards the non-deprived
eye as a consequence of depression of visual responses to the deprived eye
(Frenkel and Bear, 2004; Sato and Stryker, 2008). After the critical period, longer
periods of MD (5-7 days) are required to induce an OD shift in adult mice
(Sawtell et al. 2003; Sato and Stryker 2008). This adult OD shift results from a
potentiation of non-deprived eye responses and correlates with increased spine
formation and stability (Hofer et al. 2009). However, the magnitude of OD
plasticity varies with the method of measurement. Adult OD plasticity is greater
as measured with visually evoked potentials (VEPs) or optical imaging of intrinsic
signals (OIS), two techniques that reflect a combination of subthreshold and
suprathreshold neural activity, whereas plasticity is less evident with single-unit
electrophysiologic recordings that examine neuronal firing (Morishita and Hensch
2008).
MD lasting a few days induces a transient shift in ocular dominance while
longer durations of MD (long-term MD, LTMD) initiated near the opening of the
critical period results in both a permanent shift in ocular dominance towards the
non-deprived eye and deficits in spatial vision, including lower visual acuity, in
the deprived eye (Mitchell and Sengpiel 2009). In mice, LTMD from P21 to P35 is
sufficient to yield a maximum shift in ocular dominance (Gordon and Stryker
36
1996), and reduce visual acuity from the normal value of approximately 0.5
cycles per degree (cpd) to slightly more than 0.3 cpd. In behavioural tests of
visual acuity, this impairment is permanent (Prusky and Douglas 2003).
Interestingly, mice lacking a functional gene for the neuronal nogo-66
receptor (ngr1) retain critical period OD plasticity as adults (McGee et al. 2005).
These ngr1 mutant mice (ngr1-/-) also spontaneously recover visual acuity
following LTMD (Stephany et al. 2014). Ngr1-/- mice have been reported to
exhibit greater dendritic spine and axonal bouton dynamics in both sensory and
motor cortex (Akbik et al. 2013). However, while ngr1 has been proposed to
determine the low set point for synaptic structural plasticity in adult cortex,
whether ngr1 alters anatomical plasticity associated with experience-dependent
cortical plasticity is not yet known.
To explore the correlation between OD plasticity and the dynamics of axonal
boutons in visual cortex, first we examined the turnover and stability of axonal
boutons in both adult WT and adult ngr1-/- mice during 8 days of MD. Next, we
correlated the dynamics of axonal boutons following LTMD with two different
measurements of OD plasticity, optical imaging of intrinsic signals (OIS) and
single-unit recordings for both genotypes. Ngr1-/- mice displayed greater
recovery of normal eye dominance but neither normal vision, MD, LTMD, nor
binocular vision following LTMD altered the dynamics of axonal boutons in WT or
ngr1-/- mice. We propose that ngr1 limits cortical recovery from LTMD but does
not restrict anatomical plasticity of axonal boutons in visual cortex.

37
Materials and Methods
Mice
All animal procedures were conducted under protocols reviewed and
approved by the Children’s Hospital Los Angeles Institutional Animal Care and
Use Committee.
The constitutive ngr1-/- strain has been described previously (Kim et al.
2004). The ngr1-/- strain was F8 when this line was re-derived. The line was then
backcrossed against C57Bl6 Thy1-EGFP-M transgenic mice (Feng et al., 2000),
obtained from a commercial vendor (The Jackson Laboratory). Mice were group
housed with same-sex littermates and food and water were available ad libitum.

Cranial windows for repeated imaging of axonal boutons in visual cortex
Male and female c57/Bl6 EGFP-M transgenic mice (postnatal (P) 45 and
older) (transgenic line M; Jackson Laboratories) were used. Mice were
anaesthetized with isoflurane and administered dexamethasone (4g/g body
weight) subcutaneously. Body temperature was maintained with a biofeedback
heatpad (Physitemp). Cranial windows were implanted as previously described,
with a minor modifications (Holtmaat et al. 2009). A circular region of the skull
over V1 visual cortex was removed without perturbing the underlying dura. A 2.5
mm diameter #1 thickness cover glass (Bellco) was placed on the dura, affixed
with cyanoacrylate (Krazyglue), and sealed with dental acrylic. A small aluminum
bar with tapped screw holes was embedded into the acrylic to stabilize the
animal for subsequent imaging sessions. Mice received buprenorphine (0.1g/g
38
body weight) for 3 days post-surgery and their water was supplemented with
carprofen (1:2000) throughout the imaging series. Animals were given at least 2
weeks to recover before initiating imaging as cranial windows that were optically
clear at 2 weeks were likely to remain clear for the duration of the experiment.
Cranial windows were implanted between postnatal days 50 and 60. Two-photon
imaging began between postnatal day 64 and 74.

Thinned skull cranial window preparation for intrinsic signal imaging
Mice were anaesthetized with isoflurane and administered dexamethasone
(4g/g body weight) subcutaneously. Body temperature was maintained with a
biofeedback heatpad (Physitemp). A circular region of the skull over V1 visual
cortex was thinned and a 3.0 mm diameter #1 thickness cover glass (Bellco) was
placed on the thinned bone, affixed with cyanoacrylate (Krazyglue), and sealed
with dental acrylic. A small aluminum bar with tapped screw holes was
embedded into the acrylic to stabilize the animal for subsequent imaging
sessions. Animals received buprenorphine (0.1g/g body weight) for three days
post-surgery and their water was supplemented with carprofen (1:2000)
throughout the imaging series. Animals were imaged starting 2-3 days post-
surgery, and then subsequently 2, 4, and 8 days later.

Monocular Deprivation
For 8-day MD, the eye contralateral to the hemisphere of study was closed
using a single mattress suture tied with 6-0 polypropylene monofilament (Prolene
39
8709H, Ethicon) under brief 1% isoflurane anesthesia. The knot was sealed with
cyanoacrylate glue. For LTMD, the eye contralateral to the hemisphere of study
one eye was closed on P23 using a single mattress suture tied with 6-0
polypropylene monofilament (Prolene 8709H, Ethicon) under brief 1% isoflurane
anesthesia and the knot sealed with cyanoacrylate glue. At the conclusion of
LTMD, mice were again briefly anesthetized with isoflurane and the sutures cut
away with fine iridectomy scissors. The eyelids were separated and the eye
flushed with sterile saline solution. The eye was examined under a
stereomicroscope and mice with scarring of the cornea were eliminated from the
study.

Optical imaging of intrinsic signals (OIS)
Imaging was performed as described previously (Cang et al., 2005; Smith
and Trachtenberg, 2007; Sato and Stryker, 2008). Mice were administered
chlorprothixene (1g/g body weight; Sigma) and anesthesia wa s maintained with
isoflurane. To visualize visually-evoked changes in intrinsic signals in V1 visual
cortex, horizontal bar 2 degrees high descended from +40 to -40 degrees of the
mouse’s visual field with a period of 8. This stimulus was repeated 35
consecutive times per experiment.
Green light (530nm + 30nm) was used to visualize cerebral vascularization
and red light (620nm ± 20nm) to image intrinsic signals. The imaging plane was
focused ~200-400m below the pial surface. Images were acquired at 10 fra mes
per second at 1024X1024 pixels per image at 12-bit depth with a high-speed
40
camera (Dalsa 1M60) and custom acquisition and analysis software (C++ and
Matlab). Collected images were spatially binned before the response at the
stimulus frequency was extracted from a complete time series for each pixel by
Fourier analysis (Kalatsky and Stryker, 2003; Cang et al., 2005; Kalatsky et al.,
2005).
To measure ocular dominance vision was occluded for one eye during
imaging with a removable eye patch constructed from electrical tape. At the end
of each trial, the patch was switched to the other eye. Three or more trials were
conducted for each eye. The magnitude of each trial was the average of the top
30% of pixel values within the region of response. Ocular dominance Index (ODI)
was calculated by averaging these trials for each eye and dividing the difference
of the contralateral (C) and ipsilateral (I) responses by their sum (C-I)/(C+I).

Single unit recordings
Recordings were adapted from our previously published methods and were
performed by an investigator unaware of the genotype (McGee et al. 2005).
Recordings were performed with Epoxylite-coated tungsten microelectrodes with
tip resistances of 5-10MΩ (FHC), amplifier (model 3600, A-M systems) and
digitizer (micro1401, Cambridge Electronic Design) under Nembutal (50 mg/kg,
i.p.; Abott)/chlorprothixene (10 mg/kg, i.m; Sigma) anesthesia. Atropine
(20mg/kg, s.c.; Sigma Aldrich) was injected to reduce secretions and the
parasympathetic effects of anesthetic agents, and dexamethasone (4 mg/kg, s.c.;
American Reagent) was administered to reduce cerebral edema. Pure O
2
gas
41
was blown over the nostrils at 1L/min. A craniotomy was made over the left visual
cortex, and agar was applied to enhance recording stability and prevent
desiccation. The eyelids were removed from both eyes and the corneas
protected thereafter by frequent application of silicon oil. Animal temperature was
maintained at 37°C by a homeostatically-controlled heating pad. Heart rate and
oxygen saturation were monitored continuously (Kent Scientific).
The electrophysiological responses for 4-6 cells separated by > 90 m in
depth were recorded for each electrode penetration. In each mouse, 4 to 6
separate penetrations were spaced evenly at least 200 m apart across the
binocular region, defined by a receptive field azimuth of < 25°. Responses were
evoked with 0.1 cycles per degree 95% contrast sinusoidal drifting gratings
presented at six different orientations separated by 30° by custom software
(Matlab). Gratings were presented for two seconds during a four second trial. A
blank trial was also included during which no grating was presented. Each of
these seven stimuli (six orientations and the blank) was presented six times in
random order save that each orientation followed the blank stimuli only once.
Action potentials were identified in recorded traces of neural activity with Spike2
(Cambridge Electronic Design). For each unit, action potentials (APs) were
summed for each orientation. The orientation with the greatest number of APs
was considered the preferred orientation for analysis. Cells in which the number
of APs at the preferred orientation was not at least 50% greater than the blank
were deemed non-responsive and discarded.
42
Cells were assigned to OD categories according to the 7-category scheme of
Hubel and Wiesel (Wiesel and Hubel 1963). To categorize each unit, first the
number of APs for the stimulus blank was subtracted from the number of APs to
preferred orientation for stimuli provided to each eye. Next, the responses to the
contralateral eye (C) and ipsilateral eye (I) were computed as: (C - I)/(C + I) as
described previously (Rittenhouse et al. 1999). This scalar was then binned into
OD categories 1-7 as follows: 1 to .75 = 1, .75 to .45 = 2, .45 to .15 = 3, .15 to -
.15 = 4, -.15 to -.45 = 5, -.45 to -.75 = 6, and -.75 to -1 = 7. To determine the
contralateral bias index (CBI), the number of units in each category were
summed for all mice in a group, and the CBI calculated according to the formula:
CBI = [(n
1
-n
7
) + (2/3)(n
2
-n
6
) + (1/3)(n
3
-n
5
) + N]/2N, where N is the total number of
units and n
x
= number of units with OD scores equal to x (Gordon and Stryker
1996).

Laminar Analysis
Units were categorized as layer II/III, IV or V based on the depth of the
electrode measured from the pial surface. Units recorded between 150 and 349
m were classified as layer II/III, units between 350 and 499 m were classified
and units between 500 and 700 m were classified as layer V.

Chronic In Vivo Two-Photon Imaging
All imaging was conducted blind to the genotype. Animals were anaesthetized
with isoflurane and body temperature was maintained with a biofeedback heat
43
pad (Physitemp). Images were acquired with a modified Movable Objective
Microscope (MOM) (Sutter Instruments) and 40X 1.0 nA water immersion
objective (Zeiss) using scanimage software (MatLab) (Pologruto et al., 2003).
The light source is a Ti:sapphire tunable laser (Chameleon Ultra II, Coherent)
operating at 910nm. Imaging typically required less than 50mW of power. Image
stacks consisted of sections (512 X 512 pixels) collected in 1m steps. Low -
magnification images (0.56 m/pixel) were taken to visua lize axonal arbors.
These guided the high-magnification images (0.14 m/pixel) collected for bouton
analysis. Care was taken to maintain the same level of fluorescence across
imaging intervals. Animals were imaged every 4 days. Imaging sessions lasted
no more than 2 hours. All images of neuronal structures presented in this study
are best projections from z-stacks after linear contrast adjustment (Image J, NIH
and Photoshop software, Adobe).

Image Analysis for axonal boutons
Boutons were identified and image analysis performed following published
guidelines (De Paola et al. 2006). Boutons were defined as new if they were
three times brighter than the surrounding axon and lost if their fluorescence
decreased to lower than 1.3 times the surrounding axon. All analysis was done
blind to genotype using ImageJ (NIH). Clearly defined boutons present on axons
in the first imaging interval were labeled in ImageJ. In image stacks from
subsequent imaging sessions, experimenters determined whether a labeled
bouton was still present or not, and checked for the appearance of new boutons.
44
Newly added boutons were also tracked throughout subsequent imaging
sessions. An experimenter blind to genotype analyzed the image stack series for
each field. In the case of a discrepancy between the two sets of analysis, a third
experimenter repeated the analysis.

Statistics
Statistical comparisons were performed with Prism software (GraphPad). All
values are reported at the mean + standard error of the mean (SEM). The rates
of addition and loss of axonal boutons were compared with the Kruskal-Wallis
test followed by Dunn’s multiple comparison test(unless otherwise noted) as the
n for these experiments is too small to confirm a normal distribution. The
recovery of ocular dominance by OIS was compared between genotypes by
repeated measures (RM) two-way ANOVA with Bonferroni correction for multiple
comparisons. Comparisons of WT and ngr1-/- ODI values at different times post-
LTMD were compared to the ODI of naïve mice with the Kruskal-Wallis test
followed by Dunn’s multiple comparison test. Electrophysiological measurements
of ocular dominance were compared between genotypes using the Mann-
Whitney test. The distributions of ocular dominance indices of single units were
compared between genotypes using the Kolmogorov-Smirnov (KS) test.

45
Results
Axonal boutons maintain normal dynamics during MD
MD alters dendritic spine density both during the critical period for OD
plasticity and in adulthood (Mataga et al., 2004; Hofer et al., 2009; Djurisic et al.,
2013). In adult mice, 4 days of MD doubles the rate of spine addition in layer I in
the binocular zone of primary visual cortex but does not alter the rate of spine
elimination (Hofer et al. 2009). To test whether axonal boutons in layer I exhibit
similar sensitivity to abnormal visual experience, we repeatedly imaged axonal
structures every 4 days before and during MD in adult WT and ngr1-/- mice
harboring the EGFP-M transgene (Feng et al. 2000) (Figure 2.1).
We employed OIS to identify the binocular zone of primary visual cortex for
repeated two-photon imaging of axons (Fig. 2.1A). This imaging approach
extracts the cortical response to a temporally periodic stimulus by Fourier
analysis and yields the magnitude of this response as the fractional change in
reflectance specific to the frequency of the visual stimulus (ΔR/R) (Fig. 2.1B)
(Kalatsky and Stryker 2003; Cang et al. 2005). The visual stimulus was
presented in the central 20° of the visual field to both eyes to map the binocular
zone for 8-day MD imaging experiments (Fig. 2.1C). The same stimulus was
provided exclusively to the non-deprived eye ipsilateral to the cranial window for
subsequent experiments examining axonal plasticity after LTMD (Fig. 2.1D). Low
magnification images of a field of axons imaged before and after a 4-day interval
reveal that the clarity and contrast of these images were sufficient to reliably
identify both axons and axonal boutons at multiple time points (Fig. 2.1E)
46
First, we imaged axonal boutons in the binocular zone of visual cortex of both
WT and ngr1-/- mice for several consecutive 4-day intervals before MD and then
during 8-days of MD (Figure 2.2). The formation and loss of a small percentage
of axonal boutons was evident over this imaging series (Fig. 2.2A). Prior to MD,
the rates of bouton gain and loss were indistinguishable between WT and ngr1-/-
mice (Fig. 2.2B,C). In contrast to dendritic spine dynamics, MD did not alter the
rate of bouton addition for either WT or ngr1-/- mice (Fig. 2.2B). MD also did not
alter the rate of bouton loss for either genotype (Fig. 2.2C). The stability of new
boutons also was not statistically different. The percentage of new boutons that
were present for a single imaging time point and categorized as ‘transient’,
versus boutons that were present for more than one imagine interval and
considered ‘persistent’, was similar between genotypes and not altered by MD
(Fig. 2.2D).
Axonal boutons maintain normal dynamics during LTMD and following restoration
of normal vision
LTMD results in a permanent impairment of visual acuity in WT mice (Prusky
and Douglas 2003). Ngr1-/- mice display low acuity similar to WT mice in the
week following restoration of normal vision but then exhibit spontaneous
improvement in visual acuity over the next six weeks (Stephany et al. 2014). To
test whether the dynamics of axonal boutons in layer I are altered by the
restoration of normal vision in adulthood after LTMD in either WT or ngr1-/- mice,
we imaged axons in the binocular zone of primary visual cortex contralateral to
the deprived eye for several four-day intervals during and after LTMD (Figure
47
2.3). There was no difference between WT and ngr1-/- mice in the rate of
addition of axonal boutons during LTMD (Fig. 2.3A). The rate of bouton loss in
WT and ngr1-/- was also similar (Fig. 2.3B). Reopening of the deprived eye after
approximately 6 weeks of LTMD did not alter the rate of bouton addition for either
genotype, but did increase the variability in the rate of bouton loss. This resulted
in a trend towards greater bouton loss in the 4 days following eye re-opening, but
this difference did not achieve statistical significance (Fig. 2.3B). In contrast to
the effects of MD on ocular dominance and dendritic spine dynamics, presynaptic
structural plasticity by putative intracortical axons was not altered by either MD or
restoration of vision following LTMD.

WT and ngr1-/- mice recover normal contralateral bias rapidly following LTMD as
assessed with OIS
Improvement in visual acuity in ngr1-/- mice by the previously deprived eye
following LTMD requires several weeks of subsequent normal vision. To examine
the acute response of primary visual cortex to restoration of vision after LTMD in
WT and ngr1-/- mice, we performed chronic OIS to measure ocular dominance in
the binocular visual cortex from immediately following re-opening the deprived
eye to 8 days thereafter (Figure 2.4). The relative strength of the cortical
response to the same visual stimulus provided to the contralateral eye (C) and
ipsilateral eye (I) is reported as the Ocular Dominance Index (ODI) = (C-I)/(C+I)
(Cang et al. 2005). After re-opening the contralateral eye under isoflurane
anesthesia, responses to a visual stimulus presented to this eye were barely
48
detectable in both genotypes of mice, yielding is a negative ODI (Fig. 2.4A,B).
Surprisingly, both WT and ngr1-/- rapidly recovered normal contralateral bias in
4-8 days.
After 2 days of normal vision, both WT and ngr1-/- mice had recovered
significant cortical responsiveness to visual stimulation through the contralateral
eye but the average ODI remained significantly more binocular than naïve mice
(Fig. 2.4B,C). By 4 days post-LTMD, the ODI of WT mice was similar to that of
naïve mice, and while the recovery of ngr1-/- mice was slightly less, the
responses to visual stimulation of the ipsilateral eye decreased for both
genotypes (Fig. 2.4B,D). By 8-day post-LTMD, both WT and ngr1-/- mice were
indistinguishable from naïve mice (Fig. 2.4B).
NgR1-/- mice exhibit recovery of OD by single-unit recording after LTMD
As hemodynamic measures of ocular dominance identify plasticity not
detected with classical single-unit electrophysiologic recording (Morishita and
Hensch 2008), we investigated if ngr1-/- mice display OD plasticity after LTMD
sufficient to restore neuronal firing activity as well (Figure 2.5). We performed
single-unit recordings on WT and ngr1-/- mice receiving LTMD throughout the
critical period and maintained for 6 weeks until the day of the experiment (Day 0)
or after 8 days of normal vision (Day 8) (Fig. 2.5A). On Day 0, the OD histograms
(Fig. 2.5B), the mean of Contralateral Bias Index (CBI) reflecting the overall
distribution of the eye dominance of single-unit activity summed across all layers
of cortex for each mouse (Fig. 2.5C), and the cumulative distribution of ODI
values each (Fig. 2.5D), revealed that WT and ngr1-/- mice both lack normal
49
contralateral bias as a result of LTMD. This deficit was similar between
genotypes (Fig. 2.5B-D), and corresponds to the maximal shift observed with 14
days of LTMD in preceding studies (Gordon and Stryker 1996). However, 8 days
after the restoration of normal vision, WT displayed a modest but statistically
significant shift towards binocularity. Ngr1-/- mice exhibited greater OD plasticity
yielding a larger OD shift back towards the previously deprived contralateral eye.
The OD shift at day 8 was significantly greater in ngr1-/- mice (Fig. 2.5E). We
conclude that although modest OD plasticity is detectable in adult WT mice with
single unit recordings following LTMD, OD plasticity is greater in ngr1-/- mice.
To characterize which layers within visual cortex displayed OD plasticity
following LTMD in both WT and ngr1-/- mice, we categorized our single unit
recordings by depth from the pial surface into layer II/III (100-350m), layer IV
(350-500m), and layer V (500 -700m), and generated both OD histograms and
cumulative distributions of these single-unit responses (Figure 2.6). In WT mice,
OD plasticity largely resulted from a decrease in units strongly favoring the
ipsilateral eye (category 6 and 7) and an increase in units with binocular
responses (category 4) (Fig. 2.6A). By comparison, ngr1-/- mice at 8 days post-
LTMD displayed an increase in the percentage of units with greater responses to
the previously deprived contralateral eye (categories 1-3) (Fig. 2.6A). This
greater OD plasticity was more pronounced in the extragranular of visual cortex,
layers II/III and V, but was also detectable in layer IV. Cumulative distribution
histograms reveal that at 8 days post-LTMD ngr1-/- possess more units with ODI
50
scores greater than zero that correspond to increasing contralateral bias in the
extragranular layers. (Fig. 2.6B).

51
Discussion
Ocular dominance in primary visual cortex is a premier model of how
experience modifies functional and anatomical connectivity in the mammalian
CNS. This plasticity is most prominent during a developmental critical period
(Levelt and Hübener 2012). Within the critical period, short durations of MD shift
the relative responsiveness of cortical neurons towards the non-deprived eye.
However these shifts in ocular dominance are rapidly corrected by restoring
normal vision during the critical period, even if for only brief periods of time
(Schwarzkopf et al., 2007). In contrast, LTMD throughout the critical period
results in a permanent shift in ocular dominance and a sustained deficit in visual
acuity (Morishita and Hensch 2008).
OD plasticity both in the critical period and in the adult is accompanied by
anatomical plasticity. An immunohistochemical study reported that brief MD
during the critical period results in a transient decrease in the density of
synapses formed by thalamocortical axons originating from the lateral geniculate
nucleus (LGN) (Coleman et al. 2010). By comparison, LTMD yields modest
alterations in the length and extent of branch by thalamacortical arbors for both
the deprived and non-deprived eye (Antonini et al. 1999). Postsynaptically, both
MD and LTMD during the critical period alter spine density on pyramidal neurons
(Mataga et al. 2004; Montey and Quinlan 2011; Djurisic et al. 2013), whereas
adult MD increases the rate of spine formation and stability of dendritic spines in
layer I (Hofer et al. 2009).
52
Here we measured the turnover of axonal boutons in layer I with chronic two-
photon in vivo imaging. These axons possessed morphologies consistent with
intracortical axons originating from layer II/III and layer V pyramidal neurons (De
Paola et al. 2006), and likely originate from neurons both within and outside of
visual cortex (Douglas and Martin, 2004). In contrast to dendritic spines in layer
I, the formation, loss, and stability of axonal boutons were unaffected by MD in
WT mice (Figures 2.2 and 2.3). Thus, in WT mice, putative intracortical axonal
boutons did not display the increase in formation and stability observed by
adjacent dendritic spines.
Adult ngr1-/- mice retain developmental visual plasticity (McGee et al. 2005).
A recent study reported that ngr1-/- mice exhibit turnover rates in vivo for
dendritic spines and axonal boutons triple that of WT mice (Akbik et al. 2013).
NgR1 binds to several potential inhibitors of synaptic structural plasticity,
including some chondroitin sulfate proteoglycans (CSPGs) and proteins
associated with myelin membranes, including Nogo-A (reticulon 4a, RTN4a),
myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein
(OMgp) (Mironova and Giger, 2013). These myelin-associated inhibitors also
bind to other receptors such as NgR2 and paired immunoglobulin-like receptor B
(PirB) (Filbin, 2008). However, we have also measured the turnover of dendritic
spines in somatosensory barrel cortex in WT and ngr1-/- mice. We do not
observe the dramatic increase is baseline turnover previously reported. In
contrast, we determined that dendritic spine dynamics are indistinguishable from
53
WT mice (Park et al., 2014). Whether the rates of dendritic spine formation or
loss are altered in NgR2 or PirB mutant mice is not yet known.
We observed both under baseline conditions and during MD that the rates of
bouton addition and loss were similar between genotypes. Statistical analysis of
our measurements of the baseline turnover rate for boutons prior to MD (Figure
2.2) indicates that we would have detected a 40% increase in turnover by as
significant (p < .02), much less than the nearly 150% increase in turnover
previously reported for ngr1-/- mice. Akbik et al. They imaged 541 boutons in
total from 5 ngr1+/- mice and 510 boutons in total from 5 ngr1-/- mice across a
single 14-day interval. In comparison, we examined 1278 boutons from 4 WT
mice and 1253 boutons from 6 ngr1-/- mice across several 4-day intervals.
Although there are some technical differences between the two studies, both
employed the same strain of ngr1-/- mice.
LTMD is a murine model of amblyopia that induces a permanent deficit in
visual acuity in WT mice. Visual acuity spontaneously improves to almost normal
over 7 weeks in ngr1-/- mice following LTMD (Stephany et al. 2014). To
determine if the restoration of vision following LTMD alters axonal plasticity in
visual cortex, we again examined the turnover of axonal boutons in layer I with
chronic two-photon in vivo imaging (Figure 2.3). Reopening the deprived eye did
not alter the rate of bouton gain in either WT or ngr1-/- mice but did increase the
variability in the rate of bouton loss for both genotypes. Thus, anatomical
plasticity by intracortical axons in layer I during the first week of normal vision
post-LTMD did not correlate with the eventual improvement of acuity we observe
54
in ngr1-/- mutant mice. However, length and branching patterns of
thalamacortical axons are altered by LTMD (Antonini et al. 1999). Anatomical
plasticity by these projections mediates the changes in OD columns present in
predatory mammals following similar visual deprivation (LeVay et al. 1980).
Future studies will be required to determine if plasticity by this population of
axons differs between WT and ngr1-/- mice following LTMD.
Given this disparity between axonal and dendritic structural plasticity during
MD, we propose that cortical anatomical plasticity in response to durations of
abnormal visual experience is predominantly postsynaptic in origin and that new
spines preferentially form synaptic contacts with pre-existing axonal boutons.
This model is supported by evidence that newly formed spines contact boutons
with other synapses (Knott et al., 2006). However, several alternatives
interpretations are consistent with the results presented here and cannot be
excluded from consideration. Foremost, new spines may form synaptic contacts
on new boutons present on axons not examined here, such as thalamacortical
axons. Projections from LGN send extensive collaterals into the supragranular
layers, including layer I (Antonini et al. 1999). New spines may synapse
selectively on these thalamic projections, or axons from layer VI neurons that are
also evident in layer I (De Paola et al. 2006). In adult mice, not only sensory
adaptation, but also motor learning, and fear conditioning are associated with
elevated cortical spine dynamics (Trachtenberg et al. 2002; Holtmaat et al. 2006;
Keck et al. 2008; Hofer et al. 2009; Xu et al. 2009; Wilbrecht et al. 2010; Yang et
al. 2010; Lai et al. 2013). New spines associated with motor learning, tone-
55
associated fear conditioning, and fear extinction may also preferentially form
synapses on pre-existing stable intracortical axonal boutons (Cang et al., 2005).
Reopening the deprived eye following LTMD yielded a surprising restoration
of normal contralateral bias as reflected by OIS (Figure 2.4). We employed an
established technique for examining OD plasticity with OIS (Cang et al., 2005;
Kaneko et al., 2008a, 2008b, 2010; Sato and Stryker, 2008; Southwell et al.,
2010). Here mice received LTMD from P23, near the beginning of the critical
period, for six weeks (P23-P60+) and were then imaged repeatedly on the day of
eye opening (day 0) and then at day 2, day 4 and day 8 thereafter. By 8 days
following restoration of normal vision both WT and ngr1-/- mice displayed normal
contralateral bias as revealed by ODI scores near 0.2.
OIS likely represents a combination of both subthreshold and suprathreshold
neuronal responses, although the magnitude of response is correlated with firing
activity in some studies (Hofer et al., 2005; Kaneko et al., 2008b). Similar
experiments examining VEPs have not detected similar OD plasticity with
binocular vision following chronic deprivation (He et al., 2007). However in this
latter study, rats received a more extensive LTMD from eye opening to adulthood
(P70-100). As experience-dependent plasticity in visual cortex begins at eye
opening (Smith and Trachtenberg 2007), perhaps the absence of patterned
vision by the deprived eye contributes to these differing results.
LTMD results in a permanent shift in ocular dominance in WT mice. To
determine if the recovery of contralateral bias we observed with OIS reflected
spike-related output changes in visual cortex we examined both WT and ngr1-/-
56
mice following eye-opening under anesthesia as well as after 8 days of binocular
vision (Figure 5). We observed that ngr1-/- displayed significantly greater OD
plasticity that WT mice. On day 8 following LTMD, ngr1-/- mice exhibited nearly
twice the recovery of contralateral bias as WT mice. However, ocular dominance
remained more binocular than non-deprived mice. This OD plasticity precedes
improvement of visual acuity in ngr1-/- because WT and ngr1-/- mice possess
similar deficits in acuity 7 days after LTMD (Stephany et al. 2014). Categorizing
these single unit recordings into cortical layers by depth from the pial surface
revealed that OD plasticity was evident in the extragranular layers II/III and V OD
plasticity following LTMD is similar to OD plasticity with brief MD during the
critical period (Gordon and Stryker 1996).
In summary, as ngr1-/- mice but not WT mice exhibited partial restoration of
normal eye dominance by as measured with single-unit recordings following
LTMD, we propose that ngr1 limits recovery of cortical responsiveness in a
murine model of amblyopia but does not restrict axonal plasticity in visual cortex.

57

Figure 2.1 Experimental design (A) Schematic for the setup of optical imaging
of intrinsic signals (OIS). The magnitude of the cortical responses (R/R) to a
drifting horizontal bar (left) are represented as false-color intensity map for each
pixel (lower right). The region of response is circled. This corresponds to the
binocular zone. Mapping this region onto the surface vasculature (upper right)
identifies the location for chronic two-photon imaging of axonal structures in vivo
relative to reference points of the vasculature. (B) (upper) An example trace of
the changes in reflectance for one pixel located within the binocular zone during
presentation of the stimulus (pixel location is indicated by the asterisk in (A)).
Fourier analysis of this waveform extracts the magnitude (R/R) of the neural
response corresponding to the frequency of the visual stimulus. (C) Experimental
timeline for examining axonal plasticity during MD (D) Experimental timeline for
examining axonal plasticity following LTMD. (E) A field of axons in the binocular
zone imaged with two-photon microscopy at two time points, four days apart.
Scale bar 15 μM. (Frantz et al., 2015)

58

Figure 2.2 Turnover of axonal boutons in vivo during adult OD plasticity.
(A) A field of axons over a 12-day imaging time course. Left, a lower
magnification view is presented. Right, segments of axons repeatedly imaged at
4-day intervals. Filled triangles indicate boutons gained and open triangles
indicate boutons lost. (B) The percentage of boutons gained for each genotype
during two baseline imaging sessions (days 4 and 8) were indistinguishable (WT
4 mice, 1278 boutons; ngr1-/- 6 mice, 1253 boutons) (gained Pre-MD, WT 6.4%
+ 1.1%, n=4, ngr1-/- 7.7% + 1.1%, n = 6, p > .9). The rate of bouton gain was
similar before (days 4 and 8) during MD (days 12 and 16) (WT, pre-MD, 6.4% +
1.1%, 4-day MD, 5.4% + 0.6%, p > .9; ngr1-/-, pre-MD, 7.7% + 1.1%, 4-day MD,
7.6% + 1.7%, p > .9) (C) The percentage of boutons lost for each genotype
during were similar both during two baseline imaging sessions (days 4 and 8)
and MD (days 12 and 16) (WT 4 mice, 1278 boutons; ngr1-/- 6 mice, lost, WT
7.5% + 0.9%, n=4, ngr1-/- 5.5% + 0.9%, n = 6, p > .9; WT, pre-MD, 7.5% +
0.9%, 4-day MD, 8.8% + 1.7%, p > .9; ngr1-/-, pre-MD, 5.5% + 0.9%, 4-day MD,
9.1% + 1.7%, p > .25) (D) The percentage of new boutons that were transient
and present for only one imaging session was similar between genotypes and
conditions (WT vs. ngr1-/-; p > .13, pre-MD vs. post-MD, p > .30). Overall, there
were no significant differences between the rates of bouton gain or loss across
genotype or condition. Statistical comparisons within and between genotypes
and conditions were performed with the Kruskal-Wallis test and Dunn’s correction
for multiple comparisons. (Frantz et al., 2015)

59

Figure 2.3 Turnover of axonal boutons in vivo during recovery from LTMD.
(A) The percentage of boutons gained for WT and ngr1-/- mice during LTMD
(days 4 and 8) and during 8 days of subsequent binocular vision (days 12 and
16) (WT 4 mice, 727 boutons; KO 6 mice, 1411 boutons). Bouton gains were
similar between genotypes and conditions (gains, WT, LTMD, 7.7% + 0.9% vs. 4-
day vision, 6.7% + 1.3%, n=4, p > 0.9; ngr1-/-, LTMD, 7.5% + 0.8% vs. 4-day
vision, 6.3% + 1.2%, n=6, p > 0.9) (B) The percentage of boutons lost for each
genotype during LTMD (days 4 and 8) and during 8 days of subsequent binocular
vision (days 12 and 16). There was a trend towards increased bouton loss during
the first interval after eye opening but it did not reach statistical significance due
to increased variability in the rate of bouton loss (lost, WT, LTMD, 5.4% + 0.8%
vs. 4-day vision, 6.3% + 3.4%, n=4, p > 0.9; ngr1-/-, LTMD, 6.0% + 0.6% vs. 4-
day vision, 10.8% + 2.0%, n=6, p > .09). (C) Turnover of axonal boutons for WT
and ngr1-/- mice. (D) The percentage of new boutons that were transient and
present for only one imaging session was similar between genotypes and
conditions. Statistical comparisons within and between genotypes and conditions
were performed with the Kruskal-Wallis test and Dunn’s correction for multiple
comparisons. (Frantz et al., 2015)

60

Figure 2.4 OD plasticity measured with repeated OIS following LTMD (A) An
example of response magnitude maps for the contralateral (Contra) or ipsilateral
(Ipsi) eye for a WT mouse at eye opening and after 8 days binocular vision.
Anterior (A), Posterior (P), Lateral (M) and Medial (M) orientation of the image
are indicated. Scale bar = 0.4mm (B) Average ODI values for non-deprived
(normal) WT and ngr1-/- mice and WT and ngr1-/- mice post-LTMD at eye
opening and following 2,4, and 8 days of binocular vision, as well the average
ODI of non-deprived WT and ngr1-/- mice. The grey bar represents the range of
ODI values typical adult non-deprived WT (n=4) and ngr1-/- mice (n = 4). Each
mouse was imaged at each time point. The trajectory of recovery of OD in WT
and ngr1-/- mice are similar. (WT, n = 4, 2-days post-LTMD .04 + .06 vs non-
deprived .20 + .01, p < .02; ngr1-/-, n = 4, 2-days post-LTMD .04 + .02 vs non-
deprived .20 + .01, p < .01; WT, 4-days post-LTMD .25 + .04 vs non-deprived
.20 + .01, p > .9; ngr1-/-, 4-days post-LTMD .28 + .04 vs non-deprived .20 + .01,
p > .8; WT, 8-days post-LTMD .28 + .07 vs non-deprived .20 + .01, p > .9; ngr1-
/-, 8-days post-LTMD .28 + .04 vs non-deprived .20 + .01, p > .8; Kruskal-Wallis
test followed by Dunn’s multiple comparison test) (C) The average magnitude of
response to a visual stimulus presented to the contralateral eye of non-deprived
WT and ngr1-/- mice, and at eye-opening and after 2,4, and 8 days of binocular
vision following LTMD. (D) The average magnitude of responses to a visual
stimulus presented to the ipsilateral eye of non-deprived WT and ngr1-/- mice,
and at eye-opening and after 2,4, and 8 days of binocular vision following LTMD.
(Frantz et al., 2015)

61

Figure 2.5 OD plasticity measured with single-unit recordings following
LTMD (A) Schematic of the experimental design. Mice were deprived of vision in
the eye contralateral to the hemisphere of recording at P24. Several weeks later,
single-unit recordings were performed on either during LTMD (day 0) by opening
the eye under anesthesia (left) or 8 days after the restoration of normal vision
(right). (B) OD histograms for WT mice (top) and ngr1-/- mice (bottom) at day 0,
the time of eye opening (left), and after 8 days after re-opening the deprived eye
(right). (C) Contralateral bias index (CBI) values for each mouse (circles). The
horizontal line indicates the mean CBI value for the group. At eye opening after
LTMD, both WT and ngr1-/- mice possessed minor ipsilateral eye dominance
(CBI < 0.5). WT mice displayed a modest but statistically significant increase in
CBI (WT, n = 4, day 0 CBI = .42 + .02; ngr1-/-, n = 4, day 0 CBI = .44 + .03, p <
.02, Mann-Whitney test) whereas ngr1-/- exhibited greater OD plasticity and
higher CBI values (ngr1-/-, day 0 CBI = .44 + .03, n = 4 vs. ngr1-/-, day 8 CBI =
.56 + .01, n = 7; p < .01, Mann-Whitney test). (D) Cumulative distributions of
ODIs of individual units on the day of eye opening (day 0) for WT and ngr1-/-
mice are similar (p > 0.4, K-S test of cumulative distributions of ODI values) (E)
Cumulative distributions of ODIs of individual units after 8 days of binocular
vision. Ngr1-/- mice exhibited greater OD plasticity resulting in greater
contralateral bias than WT mice (WT vs. ngr1-/-, p < .001, K-S test of day 8
cumulative distributions) (Frantz et al., 2015)
62

Figure 2.6 Laminar analysis of OD plasticity following LTMD (A) OD
histograms of units from layer II/III (left), layer IV (center) and layer V (right) from
WT and ngr1-/- mice either during LTMD (day 0) or 8 days after re-opening the
deprived eye. Cumulative distributions of ODIs for units from layer II/III (left),
layer IV (center), and layer V (right) from WT and ngr1-/- mice after 8 days of
normal vision through the deprived eye post-LTMD. Ngr1-/- mice display greater
overall contralateral bias in layer II/III (p<.01), and layer V (p < .05), but not in
63
layer IV on day 8 (p > .27) (K-S test of cumulative distributions of ODI values).
(Frantz et al., 2015)

64
Chapter 3: the Nogo-66 Receptor Regulates Critical Period Closure from
Excitatory Neurons of Layer IV
Authors: Michael G. Frantz, A.W. McGee
Submitted for Publication:

Abstract

Quality of sensory experience sculpts developing neural circuitry. In primary
visual cortex monocular deprivation (MD) of vision in one eye permanently
degrades cortical responsiveness to that eye, a phenomenon known as ocular
dominance (OD) plasticity. This OD plasticity is confined to a developmental
‘critical period’. Yet, the laminar origins of OD plasticity and the cessation of the
critical period have not been deduced. Here we employed a conditional mutant of
ngr1, a gene required for closure of the critical period, to explore how OD
plasticity is governed by and propagates within cortical circuitry.Deletion of ngr1
in excitatory neurons of cortical layer IV prevented closure of the critical period
throughout cortex. Moreover, following brief MD, these mice displayed OD
plasticity most prominently in the granular layer and only minor shifts in the
supra- and infragranular layers that mirrored the progression of OD plasticity in
juvenile WT mice. Our findings support a model where removing molecular
brakes on plasticity in layer IV prevents closure of the critical period, and that
rapid shifts in binocularity of layer IV that feed forward to extragranular layers are
characteristic of critical period OD plasticity.

65
Introduction
Ocular dominance (OD) plasticity is a premier model in which to study
experience-dependent plasticity. Pronounced OD plasticity is restricted to a
‘critical period’ where brief monocular deprivation (MD) drastically alters OD in
primary visual cortex (V1). It has been confirmed that OD plasticity is cortical, not
thalamic in nature (Taha and Stryker 2002). Cortex is subdivided into 6 layers,
yet how these layers regulate and express OD plasticity in V1 is not known. In
cats, thalamocortical axons terminate in layer IV, giving rise to domains
dominated mostly by one eye or the other (Shatz and Stryker 1978). Periods of
deprivation as short as 24 hours during the critical period in kittens alters the
binocularity of extragranular layers without affecting layer IV (Trachtenberg et al.,
2000). Further, kittens deprived of vision in one eye at the time of eye opening for
8.5-11 months display stronger responses to the deprived eye in layer IV than
layer II/III or V/VI (Shatz and Stryker 1978). MD for 3 months, even in cats aged
8-11 months, shifts OD in extragranular layers but not layer IV (Daw et al.,
1992). Alternately, layer IV does not segregate into eye-specific domains in mice
(Drager et al., 1978). The distribution of OD in mouse layer IV neurons is similar
to extragranular layers (Gordon and Stryker 1996). Additionally, 4D MD is
sufficient to shift OD in all layers of mouse V1 (Gordon and Stryker 1996). In fact,
OD shifts in layer IV have been observed after as little as 1D MD (Liu et al.,
2008). Blocking plasticity in layer II/III or silencing polysynaptic activity in cortex
does not block OD shifts in layer IV after 3D MD, suggesting that layer IV does
66
not require instructive information from other layers in mice (Liu et al., 2008;
Khibnik et al., 2010).
In mice, OD plasticity in visual cortex doesn’t occur after the closure of the
critical period (Gordon and Stryker 1996; Morishita and Hensch 2008). Deletion
of NgR1 allows for critical period-like OD plasticity in adulthood (McGee et al.,
2005), as well as recovery of OD and visual acuity after LTMD (Frantz et al.,
2015, Stephany et al., 2014). However, the details of how NgR1 regulates the
closure of the critical period remain unknown. Here, we test whether NgR1
deletion in one or more layers of excitatory neurons in cortex is sufficient for a
phenocopy of the constitutive NgR1 knockout and critical period wild type (WT)
mice.
We report that deleting ngr1 in cortical excitatory neurons permits OD
plasticity in adulthood from 4D MD. Further, we found that deleting ngr1 in
excitatory neurons of layer IV also prevents closure of the critical period for OD
plasticity. Last, protracted 2D MD in both mice lacking ngr1 in layer IV excitatory
neurons and wild-type critical period (WT CP) mice indicates that layer IV is
sensitive to MD before other cortical layers in mice with developmental OD
plasticity.

67
Materials and Methods
Mice
All animal procedures were conducted under protocols reviewed and
approved by the Children’s Hospital Los Angeles Institutional Animal Care and
Use Committee.
The constitutive NgR1 mutant (NgR1-/-) and the conditional NgR1 mutant
(ngr1
flx/flx
) were generous gifts of Dr. Stephen Strittmatter, Yale University School
of Medicine (Kim et al., 2004; Wang et al., 2011). Both strains had been
repeatedly backcrossed onto the C57BL6background. The NgR1 -/- strain was
F8 and the ngr1
flx/flx
was at leastF6 when these mice were re-derived (The
Jackson Laboratory). Subsequently, the ngr1
flx/flx
was backcrossed onto the
C57BL6 backgroundto F8.The CamKIIa-Cre (B6.Cg-Tg(Camk2a-Cre)T29-1Stl/J,
stock # 005359) (Tsien et al., 1996), scnn1a-Cre Scnn1a-Cre (B6;c3-Tg(Scnn1a-
Cre)3Aibs/J, stock # 009613) (Madisen et al., 2010) , rbp4a-Cre (Tg(Rbp4-
Cre)KL100Gsat/Mmucd, stock # 031125-UCD) (Gong et al., 2003), and ntsr1-cre
(B6.FVB(Cg)-Tg(Ntsr1-Cre)Gn220Gsat/Mmucd, stock # 030648-UCD) (Gong et
al., 2007) were obtained from the Jackson Laboratory. Genotyping was
performed using custom primer sets for PCR amplification. Experiments were
performed on both male and female mice.

In-Utero Electroporation (IUEP)
ngr1
flx/flx
females were bread with ngr1
flx/flx
males for one night only and
separated the following morning. Pups of gravid females underwent IUEP on
68
E16.5. A glass pipette was backfilled with a pCAG;Cre-GFP plasmid
(concentration .5-1 µg/µL).
We induced anesthesia in gravid females with .4% isoflurane, and used .1-
.15% isoflurane to maintain anesthesia. After shaving the belly, the subject was
stabilized on a heating pad. The area of incision was sterilized using alternating
applications of a chlorhexidine scrub and solution, then numbed with lidocaine. A
1-2 cm incision was made in the skin and muscle overlying the uterus. Pups were
removed one wing at a time and kept moist and warm with 37˚ sterile saline. The
left lateral ventricle was injected with .5-1 µg of plasmid. Then using a 3-paddle
approach (dal Maschio et al., 2012), an electric field was applied to pull the
plasmid into dividing neurons in occipital ventricular zone.

Immunohistochemistry
Mice were anesthetized with ___ mg/kg ketamine and ___ mg/kg xylazine.
After losing reflex response to toe pinch, mice were transcardially perfused with
4% paraformaldahide. Brains were then removed and post-fixed overnight. Visual
cortex was cut into 50 µm slices. Slices were bathed in block (1:1000). After 3x10
minute washes in .1% PBST, slices were incubated in dk-anti-rb 488 (1:200?).
Sections were washed 3x10 minutes in .1% PBST, then mounted on slides with
fluoromount w/ dapi.

69
Monocular Deprivation
For 4-day MD, the eye contralateral to the hemisphere of study was closed
using a single mattress suture tied with 6-0 polypropylene monofilament (Prolene
8709H, Ethicon) under brief 1% isoflurane anesthesia. The knot was sealed with
cyanoacrylate glue. Before electrophysiological recording, the eyelids were
separated and the eye flushed with sterile saline solution. The eye was examined
under a stereomicroscope and mice with scarring of the cornea were eliminated
from the study.

Single unit recordings
Recordings were adapted from our previously published methods and were
performed by an investigator unaware of the genotype (McGee et al. 2005).
Recordings were performed with Epoxylite-coated tungsten microelectrodes with
tip resistances of 5-10MΩ (FHC), amplifier (model 3600, A-M systems) and
digitizer (micro1401, Cambridge Electronic Design) under isoflurane (.05-.1%)
mg/kg anestheis. Chlorprothixene (.2 mg/kg, i.m; Sigma) was administered to
reduce the % isoflurane required for anestheia. Dexamethasone (4 mg/kg, s.c.;
American Reagent) was also administered to reduce cerebral edema. O
2
gas
with .05-1% isoflurane was blown over the nostrils at 1L/min. A craniotomy was
made over the left visual cortex, and agar was applied to enhance recording
stability and prevent desiccation. The eyelids were removed from both eyes and
the corneas protected thereafter by frequent application of silicon oil. Animal
temperature was maintained at 37°C by a homeostatically-controlled heating
70
pad. Heart rate and oxygen saturation were monitored continuously (Kent
Scientific).
The electrophysiological responses for 4-6 units separated by > 90 m in
depth were recorded for each electrode penetration. In each mouse, 4 to 6
separate penetrations were spaced evenly at least 200 m apart across the
binocular region, defined by a receptive field azimuth of < 25°. Responses were
evoked with 0.1 cycles per degree 95% contrast sinusoidal drifting gratings
presented at six different orientations separated by 30° by custom software
(Matlab). Gratings were presented for two seconds during a four second trial. A
blank trial was also included during which no grating was presented. Each of
these seven stimuli (six orientations and the blank) was presented six times in
random order save that each orientation followed the blank stimuli only once.
Action potentials were identified in recorded traces of neural activity with Spike2
(Cambridge Electronic Design). For each unit, action potentials (APs) were
summed for each orientation. The orientation with the greatest number of APs
was considered the preferred orientation for analysis. Units in which the number
of APs at the preferred orientation was not at least 50% greater than the blank
were deemed non-responsive and discarded.
Units were assigned to OD categories according to the 7-category scheme of
Hubel and Wiesel (Wiesel and Hubel 1963). To categorize each unit, first the
number of APs for the stimulus blank was subtracted from the number of APs to
preferred orientation for stimuli provided to each eye. Next, the responses to the
contralateral eye (C) and ipsilateral eye (I) were computed as: (C - I)/(C + I) as
71
described previously (Rittenhouse et al. 1999). This scalar was then binned into
OD categories 1-7 as follows: 1 to .75 = 1, .75 to .45 = 2, .45 to .15 = 3, .15 to -
.15 = 4, -.15 to -.45 = 5, -.45 to -.75 = 6, and -.75 to -1 = 7. To determine the
contralateral bias index (CBI), the number of units in each category were
summed for all mice in a group, and the CBI calculated according to the formula:
CBI = [(n
1
-n
7
) + (2/3)(n
2
-n
6
) + (1/3)(n
3
-n
5
) + N]/2N, where N is the total number of
units and n
x
= number of units with OD scores equal to x (Gordon and Stryker
1996).

Laminar Analysis
Units were categorized as layer II/III, IV or V based on the depth of the
electrode measured from the pial surface. Units recorded between 150 and 349
m were classified as layer II/III, units between 350 and 499 m were classified
and units between 500 and 700 m were classified as layer V.

Statistics
Statistical comparisons were performed with Prism software (GraphPad). All
values are reported at the mean + standard error of the mean (SEM). A Kruskal-
Wallis test with Dunn’s correction for multiple comparisons was used for
comparisons of CBIs between genotypes. Kolmogorov-Smirnoff tests were used
to compare between two cumulative distributions, while Freidman test with
Dunn’s correction for multiple comparisons was used for comparisons between 3
or more cumulative distributions.
72

Results
First, we used the ngr1
flx
allele (Figure 3.1A) in combination with IUEP and
numerous cre lines to delete NgR1 from cortical excitatory neurons, then
subsequently in each layer of cortex. In the presence of cre recombinase, the
main coding sequence of the ngr1 gene is removed and GFP is expressed
instead. To delete NgR1 in cortical excitatory neurons, we used the CamKIIa-cre
line on an ngr1
flx/flx
background to delete NgR1 in layer II/III, we performed IUEP
of a pCAG;GFP-cre plasmid at E16.5; and to delete NgR1 in layers IV, V, and VI,
we crossed scnn1a-cre, rbp4a-cre, and ntsr1-cre respectively onto the ngr1
flx/flx

background (Figure 3.1B). Because GFP expression from the ngr1
flx
allele is not
easily detectible, we amplified the signal by staining against GFP. Staining
confirmed that recombination occurred as expected in each individual group.
We then performed single-unit recordings in mice lacking NgR1 from cortical
excitatory neurons (ngr1
flx/flx
;CamKIIa-cre), layer II/III (ngr1
flx/flx
+ E16.5 IUEP),
layer IV (ngr1
flx/flx
;scnn1a-cre), layer V (ngr1
flx/flx
;rbp4a-cre), layer VI
(ngr1
flx/flx
;ntsr1-cre), and ngr1
flx/flx
mice after 4D MD (Figure 3.2). Interestingly, we
found 4D MD was sufficient to elicit an OD shift in ngr1
flx/flx
;CamKIIa-cre mice.
Further, 4D MD also resulted in OD shifts inngr1
flx/flx
;scnn1a-cre but not ngr1
flx/flx

+ E16.5 IUEP, ngr1
flx/flx
;rbp4a-cre, or ngr1
flx/flx
;ntsr1-cre mice (Figure 2B). This
indicates deletion of ngr1 in cortical excitatory neurons is sufficient to prevent the
closure of the critical period for OD plasticity, much like the constitutive ngr1
knockout. Moreover, deletion of ngr1 in layer IV imbues sensitivity to 4D MD in
adulthood as well.
73
To determine whether the plasticity observed in ngr1
flx/flx
;CamKIIa-creand
ngr1
flx/flx
;scnn1a-cre mice was even throughout the depth of cortex, we examined
OD plasticity in individual cortical layers after 4D MD (Figure 3.2C). We
categorized units based on depth of recording and categorized them as layer
II/III, layer IV, or layer V units. We found that 4D MD did not resultOD shifts of
any layer in ngr1
flx/flx
+ E16.5 IUEP, ngr1
flx/flx
;rbp4a-cre, or ngr1
flx/flx
;ntsr1-cre
mice. This indicates that NgR1 does not function in a layer-autonomous manner.
4D MD did however result in OD shifts in all layers of cortex in ngr1
flx/flx
;CamKIIa-
creand ngr1
flx/flx
;scnn1a-cre mice. This suggests that excitatory neurons in layer
IV gate OD plasticity throughout the depth of cortex.
We then wanted to know whether the progression of OD plasticity is similar
among cortical layers in ngr1
flx/flx
;scnn1a-cre mice. Additionally, we asked
whether this plasticity mirrors that seen in wild type critical period mice. We
performed single-unit recordings after 2D MD on WT CP and adult (>P60)
ngr1
flx/flx
;scnn1a-cre mice to measure OD (Figure 3.3A). 2D MD resulted in shifts
towards binocularity in both WT CP and ngr1
flx/flx
;scnn1a-cre mice (Figure 3.3B).
Next, we performed laminar analysis to determine how OD shifts progressed
across cortical layers. There were modest but statistically significant shifts in
layer II/III, IV, and V (Figure 3.3C). We then compared OD distributions between
layers II/III, IV, and V of WT CP and ngr1
flx/flx
;scnn1a-cre mice (Figure 3.3D). We
found that layer IV distributions were more binocular than layer II/III or V, while
there was no difference in the distributions of units from layer II/III and V. This
indicates the progression of OD plasticity initiates in layer IV and propagates to
74
layer II/III and V in the subsequent days of MD. Figure 3.4 shows the
progression of OD plasticity by layer in ngr1
flx/flx
;scnn1a-cre and WT CP mice.
The median ODI of layer IV neurons is more binocular than those of layer II/III
and V in both ngr1
flx/flx
;scnn1a-cre and WT CP mice.

75
Discussion
Previous studies determined that constitutive deletion of ngr1 prevents
closure of the critical period for OD plasticity (McGee et al., 2005; Stephany et
al., 2014). It is already known that deleting ngr1 in fast-spiking parvalbumin-
positive inhibitory interneurons allows for OD plasticity in adulthood, much like
the constitutive knockout (Stephany et al., 2014). We first deleted ngr1 in cortical
excitatory neurons, and found this too allowed for OD plasticity during adulthood,
indicating that NgR1 may act in multiple roles to restrict plasticity at the close of
the critical period.
We also found that restricting ngr1 deletion to excitatory neurons of layer IV
also unmasks critical period plasticity during adulthood. This data suggests that
removing the brakes on plasticity in layer IV is sufficient to prevent closure of the
critical period. Moreover, we observed that brief 2D MD resulted in robust shifts
in layer IV with only minor shifts in layer II/III and V. This finding supports a model
where OD plasticity is expressed sequentially in a laminar fashion that parallels
the flow of information in cortex. Layer IV receives the majority of thalamocortical
input (Drager et al., 1978), thus shifts first. Layer IV projects mostly to layer II/III
and then layer V (Olivas et al., 2011). In concurrence with this model, our
observations indicate that an additional 2D of MD results in shifts in extragranular
layers as well.
The range of laminar-specific effects of brief MD across species suggests
that OD plasticity begins and is most pronounced where eye-specific inputs
converge onto binocular neurons. In mice, there are a large proportion of
76
binocular neurons in layer IV (Gordon and Stryker, 1996) while in cats, neurons
in layer IV generally respond to one eye or the other, except in the transitional
regions between eye-specific domains (Shatz and Stryker 1978; Trachtenberg et
al., 2000). Thus, it’s possible that laminar sensitivity of OD to MD arises where
monocular inputs converge onto binocular neurons.

77

Figure 3.1.Experimental Design. (A) Generation and function of the
ngr1
flx
allele. Recombination occurs in the presence of cre recombinase, driving
EGFP expression. (B) Layer-specific deletion of NgR1. (From left to right) E16.5
IUEP of a cre-GFP plasmid drives recombination in layer II/III; ngr1
flx/flx
;scnn1a-
cre drives recombination in layer IV; ngr1
flx/flx
;rbp4a-cre drives recombination in
layer V; and ngr1
flx/flx
;ntsr1-cre drives recombination in layer VI. Recombination in
layer IV, V, and VI drives weak GFP expression, thus sections corresponding to
these layers were immunostained against GFP.

78

Figure 3.2.OD was measured using single-unit recordings following 4D MD.
(A) A schematic of the experimental design. Mice were allowed to age until at
least P60, then deprived of vision in the eye contralateral to the recorded
hemisphere for 4D. (B) CBI values for each group. Bars represent mean CBI for
each group +/- SEM. Data points represent individual CBIs from mice in that
group. 4D MD resulted in shifts only in ngr1
flx/flx
;scnn1a-cre and
ngr1
flx/flx
;CamKIIa-cre mice (ngr1
flx/flx
4D MD, CBI = .71 +/- .02, n = 8 vs.
ngr1
flx/flx
;scnn1a-cre 4D MD, CBI = .45 +/- .03, n = 8, p = .023; ngr1
flx/flx
4D MD,
CBI = .71 +/- .02, n = 8 vs. ngr1
flx/flx
;CamKIIa-cre 4D MD, CBI = .37 +/- .03, n = 8,
p = .003, Kruskal-Wallis test with Dunn’s correction for multiple comparisons). (C)
Laminar analysis of OD following 4D MD. Cumulative distributions represent all
units in each group from layer II/III (left), layer IV (center) and layer V (right). 4D
MD resulted in reduced contralateral bias within all layers in ngr1
flx/flx
;scnn1a-cre
and ngr1
flx/flx
;CamKIIa-cre mice (ngr1
flx/flx
;scnn1a-cre vs. ngr1
flx/flx
: II/III p < .0001,
IV p < .0001, V p = .0009; ngr1
flx/flx
;CamKIIa-cre vs. ngr1
flx/flx
: II/III p < .0001, IV p
79
< .0001, V p < .0001;Friedman test with Dunn’s correction for multiple
comparisons).
80

81
Figure 3.3.Layer IV is more plastic after 2D MD in ngr1
flx/flx
;scnn1a-creand
WT CP mice.(A) A schematic of the experimental design. flx/flx;scnn1a-cre mice
were allowed to age until at least P60, then deprived of vision in the eye
contralateral to the recorded hemisphere for 2D. Critical period mice were
deprived of vision for 2D during the critical period (P19-P32). (B) CBI values for
each group. Bars represent mean CBI for each group +/- SEM. Data points
represent CBIs of individual mice. 2D MD resulted in small but noticeable shifts in
both WT CP and ngr1
flx/flx
;scnn1a-cre mice (WT CP ND, CBI = .74, +/- .06, n = 6
vs. WT CP 2D MD, CBI = .61 +/- .11, n = 7, p = .035, Mann-Whitney test;
ngr1
flx/flx
;scnn1a-creND, CBI = .71 +/- .07, n = 5 vs. ngr1
flx/flx
;scnn1a-cre2D MD,
CBI = .57 +/- .11, n = 6, p = .0519, Mann-Whitney test). (C) Laminar analysis of
OD following 2D MD. 2D MD resulted in shifts in all layers of cortex after 2D MD
compared to ND mice in CP mice and ngr1
flx/flx
;scnn1a-creadults. (CP ND vs 2D
MD, layer II/III p = .012, layer IV p < .0001, layer V p = .026, Kolmogorov-
Smirnoff test; ngr1
flx/flx
;scnn1a-creND vs 2D MD, II/III p = .011, layer IV p < .0001,
layer V p = .0016, Kolmogorov-Smirnoff test). (D) After 2D MD, layer IV is more
binocular than layer II/III or layer V in both WT CP and adult ngr1
flx/flx
;scnn1a-cre
mice (WT CP 2D MD: layer II/III vs. IV p < .0001, layer II/III vs. V p = .15, layer IV
vs. V p < .0001, Friedman test with Dunn’s correction for multiple comparisons;
ngr1
flx/flx
;scnn1a-cre2D MD: layer II/III vs. IV p < .0001, layer II/III vs. V p = .50,
layer IV vs. V p < .0001, Friedman test with Dunn’s correction for multiple
comparisons).

82

Figure 3.4. Progression of OD plasticity across layers. Differing lengths of
MD have different effects on different layers. By 2D MD, ODI of both
ngr1
flx/flx
;scnn1a-cre and WT CP mice is binocular in layer IV and still contra-
biased in layers II/III and V.

83
Chapter 4: Conclusions
The results in Chapter 2 show that NgR1 deletion allows for greater recover
of OD following LTMD by single-unit recordings, although one week of normal
vision is not sufficient for recovery to normal OD. In adulthood, 4D MD is
sufficient for a saturating shift towards the non-deprived eye in ngr1-/- mice
(McGee et al., 2005; Stephany et al., 2014). Thus, it is likely that the mechanisms
that drive OD plasticity from brief MD and those operating to restore normal OD
after LTMD are different.
By intrinsic signal imaging, both WT and ngr1-/- mice recover normal OD after
4D of normal vision, mostly by potentiation of the previously deprived
contralateral eye response. Again, adult OD plasticity is characterized by
potentiation of non-deprived eye responses (Sawtell et al., 2003). It’s possible
that OD shifts driven by potentiation are possible favoring the previously deprived
eye. These observations support a model where recover of OD is driven mainly
by potentiation of responses from the previously-deprived contralateral eye.
Last, we observed no significant differences in the rates of gain, loss, or
persistence of boutons on intracortical axons in layer I of visual cortex during 4D
MD or during recovery from LTMD in WT or ngr1-/- mice. We did see a trend
towards an increase in the rate of axonal bouton loss 4D after eye opening
following LTMD in ngr1-/- but not WT mice, however this did not reach
significance. If further study confirms this trend, my interpretation would be that
alterations in presynaptic connectivity of neurons in V1 predict recovery of visual
function. There are alterations in the rate of dendritic spine gain during MD in
84
adult mice (Hofer et al., 2009). The data presented in Chapter 2 supports a
model where anatomical changes associated with OD plasticity are largely
postsynaptic.
There is anatomical plasticity due to deprivation during development. In both
cats and mice, the total length and branching complexity of thalamocortical
arbors serving the deprived eye is decreased (Antonini and Stryker, 1993;
Antonini et al., 1999). However it’s difficult to say whether axons from neurons in
V1 are remodeled in response to deprivation. One drawback of current methods
of 2PLSM structural imaging is neuronal labeling. There are two main genetic
tools used in structural imaging: the EGFP-m and the YFP-h lines (Feng et al.,
2000). Driven by the thy-1 promoter, both these transgenic lines express EGFP
or YFP in a subset of layer II/III and V cortical neurons. When studying axons,
one cannot trace the axon back to the cell body, so the only way to classify
neurons is based on anatomy (De Paola et al., 2006). Thus, the study of axons
is prevented from looking at cell-autonomous effects of experience. My study of
boutons in visual cortex comprises 1000-1500 boutons per condition. Although
one can follow an apical dendrite down to the cell body, only the most thorough
studies count up to 150-200 spines/neuron, and no more than a few thousand
spines per group.
Given that there are thousands of spines per neuron on apical and basal
dendrites and about 100,000 neurons/mm
2
and 7.2x10
8
synapses/mm
2
in cortex
(Schüz and Palm, 1989), it is my opinion that current measurements do not
sufficiently sample spines and boutons to reliably measure the effects of
85
experience on structural dynamics. To truly learn about how experience affects
anatomical plasticity, the field must develop methods to label specific neuronal
subtypes and automated spine and bouton counting methods. One challenge
facing the field until recently was that top-down imaging only allows high-
resolution capture of the top 50-100 microns of cortex. By using a prism,
researchers can now image the full depth of cortex (Figure 4.1). This method is
currently difficult to implement and expensive but promises to teach us more
about how anatomical plasticity occurs throughout the depth of cortex.
The results in Chapter 3 show that deleting ngr1 in cortical excitatory neurons
prevents closure of the critical period for OD plasticity. Previous work shows that
preventing plasticity by inhibiting protein synthesis in cortex but not thalamus
blocks OD plasticity during the critical period. Together, these results confirm that
OD plasticity is a cortical phenomenon (Taha and Stryker 2002).
Wedemonstrated that OD plasticity is gated by NgR1 in scnn1a-expressing
neurons in layer IV. Not only did deleting ngr1 in these neurons allow for OD
plasticity across all layers of cortex; it resulted in dramatically more plasticity in
layer IV after 2D MD, similar to WT CP mice that we tested. This suggests that
during the critical period, plasticity begins in layer IV and propagates through
cortex in a manner that parallels the flow of feed-forward excitatory connectivity.
Interestingly, in cats extragranular layers are more plastic than layer IV (Shatz
and Stryker 1978; Daw et al., 1992; Trachtenberg et al., 2000). However, while
neurons in cat layer IV are mostly responsive to one eye or the other, mouse
layer IV neurons are just as binocular as other layers (Shatz and Stryker 1978;
86
Gordon and Stryker 1996). This supports a model where OD plasticity is gated
where monocular inputs converge onto binocular neurons.
I believe the hypothesis that layer IV gates OD plasticity requires further
investigation. As mentioned in the introduction, dark exposure reactivates the
critical period and decreases GABAa neurotransmission (He et al., 2006). It’s
possible that this occurs through a decrease in layer IV excitatory input onto layer
II/III PV neurons (Kuhlman et al., 2013). Mice lacking ngr1 also have a decrease
in layer IV excitatory drive onto PV neurons in layer II/III (Stephany et al., 2014).
My hypothesis is that extended suppression of layer IV activity, and by extension
reduction in excitatory drive onto layer II/III PV neurons, will reopen the critical
period. To accomplish this, I would selectively expresshM
4
D-inactivating G
i
-
coupled DREADD receptors using a stop-floxed AAV on an scnn1a-cre mouse or
IUEP of a DREADD construct into layer IV. By administering clozapine N-oxide
(CNO) in adulthood twice daily for ~10D, I would persistently reduce activity in
layer IV. Following CNO administration, I would perform 4D MD and measure OD
plasticity with single-unit recordings. I predict that mice treated with CNO will
exhibit OD plasticity from 4D MD, while untreated mice will not. Further, I predict
that CNO-treated mice will exhibit a decrease in excitatory drive onto PV
neurons, similar to ngr1-/- and critical period mice. This will provide definitive
proof that layer IV gates OD plasticity in V1.

87

Figure 4.1.Application of Prisms in 2PLSM. Green neurons represent layer V
neurons labeled by EGFP-m or YFP-h. (A) Top-down imaging allows imaging of
only the top 50-100 microns of cortex. (B) By using a prism, we can now image
through the depth of cortex, allowing imaging of basal and apical dendrites of
neurons in each layer.

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Abstract (if available)

Abstract The visual system is a premier model for studying experience-dependent plasticity. During development, there’s a period of sensitivity called the ‘critical period’ when brief monocular deprivation (MD) alters ocular dominance (OD) in primary visual cortex (V1). There are several factors thought to mediate the closure of the critical period such as maturation of inhibition, myelination, and molecular cues. One of these molecular cues is the nogo-66 receptor (NgR1), as deletion of ngr1 prevents closure of the critical period for OD plasticity and allows for recovery of visual acuity in adulthood following long-term monocular deprivation (LTMD). ❧ NgR1’s restriction of neurite outgrowth in vitro led to the hypothesis that NgR1 restricts developmental visual plasticity by restricting the rates of addition and loss of axonal boutons, as explored in chapter 2. Chronic in-vivo 2-photon laser scanning microscopy (2PLSM) has become a standard for observing experience-dependent changes in anatomical plasticity over time in vivo. Various experiential manipulations have elicited changes in dendritic spine dynamics in different cortical areas, including visual cortex. However, the effect of altered experience on axonal bouton dynamics had not been studied prior to the paper published in chapter 2, where I asked whether 4D MD or recovery of visual acuity was accompanied by altered axonal bouton dynamics in ngr1-/- mice. I found that neither alteration in visual experince affected the rates of axonal bouton gain and loss in V1. This supports a model where anatomical changes correlating with functional visual plasticity are largely postsynaptic while axonal structures remain relatively stable. ❧ Visual cortex is subdivided into lamina. It remains poorly understood whether each layer plays a different role in the expression of OD plasticity. In cats, layer IV is relatively resistant to OD shifts, although this is not true in mice, as shifts in layer IV have been recorded in as little as 1D. In chapter 3, I examined whether removing the brakes on plasticity in individual layers using conditional deletion of ngr1 unmasks plasticity in adults. I also examined how shifts in OD progressed in a laminar fashion. I found that deleting ngr1 in excitatory neurons of layer IV enabled OD plasticity throughout all layers through a disinhibitory circuit. Further, in mice lacking ngr1 in layer IV and critical period WT mice, 2D MD produced a dramatic shift in eye dominance in layer IV with only modest shifts in layers II/III and V. This data supports a model where plasticity in cortex is regulated in a feed-forward manner from layer IV.

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Core Title Differential regulation of anatomical and functional visual plasticity by NgR1

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Tag amblyopia,anatomical plasticity,axonal boutons,binocular,dendritic spines,lamina,laminar,layer,layer II/III,layer IV,layer V,mouse,neocortex,neuroplasticity,OAI-PMH Harvest,ocular dominance,plasticity,two-photon microscopy,vision,visual cortex

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