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Characterizing response and plasticity in sensory cortices of the Fmr1⁻⁄⁻ mouse

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Characterizing response and plasticity in sensory cortices of the Fmr1⁻⁄⁻ mouse

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Content i

CHARACTERIZING RESPONSE AND PLASTICITY IN SENSORY CORTICES OF
THE FMR1
-/-
MOUSE

by

Megan Arnett

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

MAY 2016

ii
Dedication

For my mom and dad,
who are corresponding authors on any
good thing I've ever done

iii

ACKNOWLEDGEMENTS

This project is a testament to the incredible support and encouragement I have
received from a number of people. My thesis advisor, Dr. Aaron McGee,
accepted me unconditionally into his lab and encouraged me to pursue a
project that was completely outside the scope of his work at the time. His
support and encouragement provided the foundation for all of the work
discussed in this dissertation.

My graduate career has been marked by few constants, but the one stabilizing
force has been my thesis committee. I would like to thank my committee
members, Drs. Judith Hirsch and David McKemy, for their mentorship,
encouragement and feedback. In addition, I am very grateful for the support of
Dr. Krishna Nayak, Alycen Hall and the BELA Fellowship program, which was
one of the most rewarding experiences of my life.

I would also like to thank my family, who are truly the corresponding authors on
this project. They have tolerated my absence from the east coast (and more
amazingly, my visits back) with admirable cheerfulness and unending support.
Thank you to my mom, Denise, for giving me the confidence to do something
I'm passionate about, and my dad, Jim, for encouraging me to be passionate
about something meaningful. And thank you to my bother, Andrew, for his
unending (and occasionally unasked for) supply of perspective, intelligence and
humor. In addition, I am extremely grateful for the support of my SF family, Rita
and Ted Yang, Greg Yang, and Natalie and Lucas Hession.

My greatest teachers in graduate school have been my lab mates, and I'm
extremely grateful for how generously they have shared their time, knowledge,
and bench space with me. In particular, I want to thank Dr. Florian Freudenberg,
who taught me how to work like a scientist, Dr. David Herman, who taught me
how to think like one, and Chao Huang, who (among other things) taught me
how to dress like one. I also want to thank Hilary Dorton, who is a gift from the
lab gods, and Dr. Jennifer Park, who has been my travel partner on this road
from the beginning.

There is a very small part of the population that, despite never having studied
neuroscience, sensory systems or Fragile X Syndrome, knows an inordinate
amount of information about all of the above. These friends of mine have been
iv
my collaborators, cheerleaders and champions, and I'm grateful for the
opportunity to thank them by name. Thank you to Ann Hoang, my hero, and a
constant source of gator-aid, Brittany Johnson, the incarnation of human
kindness, and Laura Vigliotti, who is a very, very funny person.

Finally, I would like to thank my amazingly supportive husband, Nick, for all of
the wonderful things he does, including accepting 'grad school' as an excuse to
avoid housework for the past 7 years. You are ok, sir. I'm so glad to be on our
team.

v

LIST OF FIGURES

2.1 The thin skull surgical prep allows for long-term, in vivo
imaging……..............................................................................................19

2.2 Intrinsic signal optical imaging is a non-invasive method for
measuring evoked activity in the somatosensory cortex………………….21

2.3 Intrinsic signal optical imaging is a non-invasive method for
measuring evoked activity and plasticity in the visual cortex……………..23

2.4 During intrinsic imaging, Fourier analysis extracts the magnitude
of the change in light reflectance corresponding to the
stimulus frequency…………………………………………….……………....24

2.5 The gap cross assay serves as a tactile learning paradigm……………….28

2.6 The visual water task measures visual acuity…………………………….…31

3.1 Intrinsic signal optical imaging reveals changes in sensory
evoked activity in the barrel cortex …………………………………………38

3.2 Evoked activity is increased in the barrel cortex of Fmr1 ko
mice…………………………………………………………………….……….39

3.3 Fmr1 ko learning on the gap cross is distance dependent……………….42

3.4 Fmr1 ko mice display normal learning on the gap cross assay
at shorter distances…………………………………..…..……………...……43

3.5 Fmr1 ko mice display impaired learning on the gap cross assay
at longer distances………………………………………….…………………44

4.1 Cortical response to the deprived eye is diminished following
3 day monocular deprivation in critical period wild type mice……..........54

vi
4.2 Cortical response to the non-deprived eye is potentiated, while
response to the deprived eye is unaltered following 3 day
monocular deprivation in Fmr1 ko mice……….…………..………..……...55

4.3 The magnitude of the ODI shift following 3 day MD is disparate
between Fmr1 ko and WT mice.………………..……..…………….………56

4.4 Diazepam treatment restores deprived eye depression in Fmr1
ko mice following 3 day monocular deprivation.………………..…………60

4.5 Diazepam treatment partially restores the ODI shift in Fmr1 ko
mice….……………….………………..………….………………..…………..61

4.6 The number of parvalbumin expressing cells is unaltered in all
layers of the primary visual cortex in Fmr1 ko mice.……………....………63

4.7 The maturation of visual acuity is unaltered in Fmr1 ko mice.……………65

4.8 Fmr1 ko mice do not display recovery of visual acuity following long
term monocular deprivation.……………………………………………....…67

vii

Table of Contents

DEDICATION.........................................................................................................ii
ACKNOWLEDGEMENTS......................................................................................iii
LIST OF FIGURES...................................................................................................v

CHAPTER ONE: Introduction
Fragile X Syndrome...........................................................................................1
The Fragile X Mental Retardation Protein.........................................................2
The Fmr1
-/-
mouse..............................................................................................5
Disinhibition in the Fmr1
-/-
mouse......................................................................6
The primary somatosensory cortex of the mouse (barrel cortex)......................9
The barrel cortex in the Fmr1
-/-
mouse............................................................11
The primary visual cortex of the mouse...........................................................12
Plasticity in the mouse primary visual cortex and the role of inhibition...........13
Presented Contributions..................................................................................16

CHAPTER TWO: Materials and Methods
Mice and surgical procedures..........................................................................17
Imaging methods.............................................................................................20
Behavioral assays.............................................................................................24
In vitro methods...............................................................................................31

viii
CHAPTER THREE: Characterizing sensory response and learning in
the somatosensory cortex of the Fmr1
-/-
mouse
Introduction.....................................................................................................34
Evoked activity in barrel cortex........................................................................36
Sensory learning on the gap cross assay.........................................................41
Discussion........................................................................................................46

CHAPTER FOUR: Characterizing response and plasticity in the primary
visual cortex of the Fmr1
-/-
mouse
Introduction.....................................................................................................49
Critical period ocular dominance plasticity......................................................51
Diazepam treatment........................................................................................57
Parvalbumin expression in primary visual cortex.............................................61
Recovery of visual acuity following long-term monocular deprivation............64
Discussion........................................................................................................68
CHAPTER FIVE: Conclusions...............................................................................73
REFERENCES.......................................................................................................76

1
CHAPTER ONE: Introduction
1.1 Fragile X Syndrome
Fragile X Syndrome (FXS) is the leading inheritable form of intellectual
disability and the foremost genetic cause of autism (Hagerman et al 2005;
Kaufmann et al. 2004). In the United States, Fragile X syndrome occurs in
approximately 1 in 4,000 males and 1 in 8,000 females.
In addition to cognitive impairment, Fragile X Syndrome is characterized
by an array of atypical responses to sensory stimulation. For example,
hypersensitivity to sensory stimuli is a common symptom of many autism
spectrum disorders, including FXS. During somatosensation in Fragile X
patients, this hypersensitivity manifests as tactile defensiveness and extreme
avoidance of normally neutral tactile stimuli (Baranek et al., 1997). Similarly,
more than 90% of male patients with FXS display heightened sensitivity to visual
stimuli and increased gaze aversion (Merenstein et al., 1996), a further indicator
of increased sensitivity to eye contact (Cohen et al., 1989). In addition, patients
with FXS display elevated responses in the auditory cortex in response to sound,
as measured by the event-related brain potential (ERP) recorded in the
electroencephalography (EEG) (Castren et al., 2003). This hyper-responsiveness
to sensory stimuli is gaining popularity for its proposed role as a potential
trigger for the heightened susceptibility to seizures observed in patients with
2
Fragile X Syndrome. In this population, seizures are reported in 10-20% of
children, typically as benign focal epilepsy of childhood (Berry- Kravis, 2002;
Musumeci et al., 1999).

1.2 The Fragile X Mental Retardation Protein (FMRP)
In most cases, Fragile X Syndrome arises from the expansion of a
trinucleatide repeat sequence in the 5'-untranslated region of the FMR1 gene,
located on the X chromosome. Normally this sequence is repeated 5 to 40 times
in the healthy population, however in individuals with Fragile X syndrome this
sequence is repeated more than 200 times. This expansion results in
transcriptional silencing and subsequent loss of the gene product, fragile X
mental retardation protein (FMRP).
FMRP is expressed in many tissues, most abundantly in the brain, where it
has been detected in neuronal cell bodies, dendrites and synapses (Weiler et al.,
1997). In mammals, the primary isoform of FMRP consists of various RNA-
binding and protein-protein interaction sites, including a novel KH0 motif that
allows it to interact with mRNAs, and two Agenet (Tudor) motifs, which are
thought to function as a site for protein-protein interactions (Myrick et al., 2015a,
2015b). FMRP was first characterized by its function as a regulator of translation,
but more recent research has demonstrated roles for FMRP outside of regulating
3
protein synthesis (Brager and Johnston, 2014). For example, the capacity to aid
protein-protein interactions allows the amino terminus of the FMRP protein to
directly influence elements of ion channel proteins, such as potassium channels
Slack and BK (Brown et al., 2010; Deng et al., 2013), while the carboxyl-terminus
can regulate calcium channel dynamics (Ferron et al., 2014). As a result, FMRP
has the ability to influence cellular excitability and function by engaging directly
with membrane ion channels.
In its role as a regulator of postsynaptic translation, FMRP binds mRNA in
the nucleus, and is believed to facilitate trafficking and regulation of mRNA from
the nucleus to the synapse (Kim et al., 2009). However, FMRP tends to localize
to the postsynaptic areas of dendritic spines, where it selectively binds to and
negatively regulates translation of approximately 4% of the mRNA in the
mammalian brain. A combination of microarray assays and high-throughput
sequencing of RNAs isolated through cross-linking immunoprecipitation have
identified possible FMRP target mRNAs (Brown et al., 2000; Darnell et al., 2011).
Among those identified, the majority of the target mRNAs are significantly
enriched for proteins involved in neuronal and synaptic transmission.
Interestingly, many of the identified FMRP target mRNAs are also localized to
dendrites. The dense, immature dendritic spines found in the brains of FXS
patients and Fmr1 mice, as well as the function and localization of the FMRP
4
target mRNAs could indicate a role for FMRP in dendritic development and
function. Indeed, several of these identified target mRNAs colocalize with FMRP
in dendrites, including PSD-95, which is known to directly associate with FMRP in
dendrites both in vitro and in vivo (Zalfa et al., 2007).
While there is some evidence suggesting that FMRP plays a direct role in
dendritic mRNA transport, most studies indicate that its primary function is
translational regulation of its target mRNAs. FMRP represses translation of
various mRNAs in vitro (Mazroui et al., 2002; Laggerbauer et al., 2001) and an
overall effect of reduced FMRP in vivo is increased protein synthesis (Lu et al.,
2004; Muddashetty et al., 2007; Zalfa et al., 2007). Phosphorylated FMRP
inhibits translation and delays ribosomal translocation, while dephosphorylation
of FMRP upregulates translation (Coffee et al., 2012; Muddashetty et al., 2011;
Ceman et al., 2003). One potential link between synaptic activity and local,
FMRP-mediated translation is the activity dependent, bidirectional regulation of
FMRP phosphorylation by the S6 kinase and protein phosphatase 2A.
In addition to regulating protein translation, there is significant evidence
that FMRP functions as a primary regulator of synaptic plasticity. For instance,
postsynaptic, activity-dependent translation of dendritic mRNA is required for
the expression of numerous forms of synaptic plasticity (Kang and Schuman,
1996; Huber et al., 2000). Therefore, as a primary regulator of dendritic mRNA
5
translation at the synapse, FMRP plays a key role in facilitating synaptic plasticity
(Laggerbauer et al., 2001; Li et al., 2001; Huber et al., 2007). A point mutation in
one FMRP/mRNA binding site is sufficient to evoke plasticity phenotypes
observed in the Fmr1
-/-
mouse (Zang et al., 2009) as well as human patients (De
Boulle et al., 1993), indicating that FMRP regulates plasticity primarily by
suppressing protein translation. Finally, FMRP is bidirectionally regulated by
activity: synaptic activity can trigger its local translation or rapid degradation.
Multiple experimental paradigms correlated with synaptic plasticity have been
shown to cause rapid and transient changes in FMRP levels, including whisker
deflection, enriched environment, complex learning tasks and pharmacological
activation of group 1 metabotropic glutamate receptors (mGluRs) (Weiler et al.,
1997; Irwin et al., 2000; Gabel et al., 2004; Todd et al., 2003). Given its quick
increase in dendrites in response to well-characterized plasticity induction
experiments, as well as its demonstrated role in regulating mRNA translation at
the dendrite, FMRP appears to be functionally well-suited to regulate synaptic
plasticity.

1.3 The Fmr1
-/-
mouse
Many of the clinical phenotypes of Fragile X Syndrome are recapitulated
in the predominant animal model of the disease, the Fmr1
-/-
mouse. Fmr1
-/-
mice
6
display abnormal social behaviors (Spencer et al., 2005; Mineur et al., 2006),
learning deficits (Paradee et al., 1999; Peier et al., 2000) and audiogenic seizures
(Musumeci et al., 2000; Chen and Toth, 2001; Yan et al., 2004). In addition,
Fmr1
-/-
display increased sensitivity and altered cortical responses to tactile
stimulation. In the Fmr1
-/-
mouse, single whisker stimulation results in a more
rapid propagation of depolarization in the barrel cortex compared to WT mice.
The same study also found increased sensory-evoked activity in the
somatosensory cortex of Fmr1
-/-
mice in response to forepaw stimulation (Zhang
et al., 2014).

1.4 Disinhibition in the Fmr1
-/-
mouse
The sensory hyperarousal in both the Fmr1
-/-
mouse and in clinical cases
of autism disorders including Fragile X Syndrome, are informed by increasing
evidence of hyperexcitability within cortical networks in the Fmr1
-/-
mouse.
Abnormal excitability from the molecular to the network level has been
demonstrated in the Fmr1
-/-
mouse. While there are currently no studies
providing a direct link between the behavioral changes and the neurological
alterations in the mouse model, there is increasing evidence that elevated
cortical excitability could be an influential factor in the behavioral
hyperexcitability observed in the Fmr1
-/-
mouse.
7
There is mounting evidence that inhibitory transmission is significantly
altered in the Fmr1
-/-
mouse in a brain region and age-dependent manner.
Inhibitory transmission is conducted primarily via the release of the
neurotransmitter GABA at inhibitory synapses, and is a critical component of
cortical network behavior and plasticity. The primary receptors at inhibitory
synapses are ionotropic GABAA receptors and metabotropic GABAB receptors.
In the Fmr1
-/-
mouse both the mRNA and the number of GABAA receptor
subunits are decreased relative to wild type (D'Hulst et al., 2006; Gantois et al.,
2006). In addition, glutamic acid decarboxylase, the rate limiting GABA synthesis
enzyme, is reduced in certain brain regions of the Fmr1
-/-
mouse (Olmos-Serrano
et al., 2010) as is the density of GABAergic synapses (Centonze et al., 2008).
Finally, the number of parvalbumin (PV) expressing inhibitory cells is decreased
in the barrel cortex of Fmr1
-/-
mice compared to WT (Selby et al., 2007). The
functional outcomes of these deficits are difficult to measure and have
differential affects according to brain region. However, given the critical role that
GABA transmission plays, particularly during development, a decrease in such
fundamental components of inhibitory signaling will likely have significant
consequences, and may account for changes observed downstream and at
subsequent developmental time points.
8
It is possible to observe the neural correlates of sensory hypersensitivity in
the Fmr1
-/-
mouse by measuring changes in the response properties of neurons
in sensory cortices. This approach led to the discovery of a decrease in the local
excitatory drive onto fast-spiking inhibitory neurons in layer 4 of the barrel
cortex. The same study also found that layer 4 excitatory neurons display
increased membrane excitability and prolonged UP states in vitro (Gibson et al.,
2008), a result that was later corroborated using cell-attached recordings in vivo
in adult Fmr1
-/-
mice (Hays et al., 2011). Similarly, whole-cell recordings of layer
2/3 neurons in vivo found a 2-fold increase in the probability of neuronal firing
during UP states of Fmr1
-/-
mice compared to WT (Goncalves et al., 2013). In
addition to the increased neuronal excitability observed in Fmr1
-/-
cortical cells,
there is also a state-dependent 3-fold increase in spontaneous firing rates in vivo
(Goncalves et al., 2013).
At the network level, recordings of neuron clusters as well as network
activity provide additional information about the response properties of the
Fmr1
-/-
circuit. Experiments using two-photon Ca
2+
imaging to record the activity
of large ensembles of neurons revealed that cortical circuits of the Fmr1
-/-
mouse
exhibit unusually high activity and abnormally increased network synchrony
during the first 3-4 postnatal weeks (Goncalves et al., 2013). In addition, a
propensity for increased network activity can be revealed through application of
9
GABA antagonists, leading to unusually prolonged bursts of activity in Fmr1
-/-

brain slice compared to WT mice (Hays et al., 2011). As discussed previously in
this text, loss of FMRP results in functional changes in cortical activity as well. In
the Fmr1
-/-
mouse, single whisker stimulation results in a more rapid propagation
of depolarization in the barrel cortex compared to WT mice. The same study
also found increased sensory-evoked spiking activity in the somatosensory
cortex of Fmr1
-/-
mice in response to forepaw stimulation (Zhang et al., 2014).

1.5 The primary somatosensory cortex of the mouse (barrel cortex)
The mouse whisker system is well suited to the study of sensory response
and learning. Information from individual whiskers is carried from the facial pad
through the brainstem and thalamus into the primary somatosensory cortex. At
each synaptic junction, the spatial organization of neurons projecting from
individual whiskers are distinct, and arranged in a pattern mirroring the
arrangement of the facial whiskers. Therefore information from individual
whiskers is conveyed to a corresponding barrelette (brain stem), barreloid
(thalamus) and barrel (cortex) in a well-defined one-to-one relationship, and
neurons in each barrel unit preferentially respond to tactile information from a
particular principle whisker (Petersen, 2007). The organization of these barrels is
easily detected in living tissue and in vitro by observing the optically dense
10
aggregates in layer IV (Woosley & Loos, 1970; Finnerty et al., 1999). The
functional representation of a whisker can also be observed using optical
imaging techniques to visualize activity evoked in layer 2/3 of the barrel cortex
in response to whisker stimulation (Grinvald et al., 1986; Frostig et al., 1990).
The functional organization and accessibility of the whisker tactile system
make it an ideal model to investigate structure-function relationships in a local
cortical network. Rodents move their whiskers to explore their tactile
environment, a deliberate and adaptive behavior known as whisking (Carvell and
SImons, 1995). Because they have poor eyesight, rodents rely heavily on tactile
information from their whiskers to navigate their environment, and both whiskers
and the somatosensory cortex are necessary in numerous behaviors requiring
the vibrissal system (Hutson & Masterton, 1986; Guic-Robles et al., 1992). In
addition, the primary somatosensory cortex provides insight into the anatomical
and topographical components of learning. Ablation of the barrel cortex causes
rats to lose their ability to perform a previously acquired sensory learning task
(Hutson & Masterron, 1986). Interestingly, selective ablation of the supragranular
layers of the barrel cortex prevents rats from learning a perceptual learning task.
However, once training on the task is complete, ablation of these layers does
not affect performance on the task (Friedburg, 1991). Therefore the
supragranular layers of somatosensory cortex are required during the acquisition
11
phase of a learning task, but not in the maintenance of the information once the
task is learned.

1.6 The barrel cortex of the Fmr1
-/-
mouse
In the barrel cortex, FMRP is expressed in response to activity (such as
whisker stimulation) and plays a role in regulating synaptic morphology, network
connectivity and neuronal plasticity. In the adult rat barrel cortex, FMRP
expression is elevated following whisker stimulation as the result of increased
activity-dependent translation of FMRP (Todd & Mack, 2000; Todd et al., 2003).
In addition, FMRP expression in mouse layer IV barrel cortex neurons peaks
during a well-defined period of increased plasticity and synaptogenesis (Harlow
et al., 2010; Daw et al., 2007). In the barrel cortex of the Fmr1
-/-
mouse, loss of
FMRP affects multiple functional and developmental archetypes, including
altering inter- and intra-layer connectivity strength, delaying dendritic spine
maturation and modifying well-characterized forms of neuronal plasticity (Bureau
et al., 2008; Gibson et al., 2009; Nimchinsky et al., 2001; Galvez and
Greenough, 2005; Desai et al., 2006). Some of these changes are discussed in
more detail in the earlier sections related to the disinhibition of the Fmr1
-/-

mouse cortical network.

12
1.7 The primary visual cortex of the mouse
Cells in the mouse primary visual cortex have complex receptive field
properties and respond discriminately to distinctive features of visual stimuli.
Visual information from the retina is initially processed in the lateral geniculate
nucleus (LGN), whereupon it is transmitted via thalamocortical afferents into
layer 4 of the primary visual cortex. Neurons in the mouse visual cortex display
all three of the fundamental properties originally established by Hubel and
Wiesel to describe V1 responses, namely; orientation selectivity, a distinction
between linear and nonlinear cells, and for linear cells, a spatial field consisting
of applicably aligned ON and Off subregions (Niell & Stryker, 2008). In addition,
the receptive field properties of cells in the mouse V1 display layer specific
functions similar to those observed in mammals with more complex vision
(Mangini & Pearlman, 1980; Hubel & Wiesel, 1968). Therefore, despite species
differences in organization and receptive field size, the mouse visual cortex
provides an excellent model for the study of visual processing, as well as for
exploring the mechanisms regulating experience dependent plasticity.
In mice, the lateral position of the eyes results in a relatively narrow
binocular field of vision (Dräger, 1978). As a result, only ~10% of retinal ganglion
cells project to the ipsilateral LGN and cortex. Instead, the vast majority of cells
cross over to the contralateral hemisphere at the optic chiasm (Drager, 1978;
13
Gordon & Stryker, 1996; Drager & Olsen, 1980). However, despite the relatively
sparse input from the ipsilateral eye, the binocular region accounts for about 1/3
of the area of the primary visual cortex. In this area the majority of neurons can
be stimulated by either eye, however there is a pervasive dominance of the
contralateral eye and only ~5% of the cells in this area are driven exclusively by
the ipsilateral eye (Drager, 1975; Metin et al., 1988).

1.8 Plasticity in the mouse primary visual cortex and the role of inhibition
This ocular dominance (OD) observed in mice at all ages is sensitive to
manipulation of the visual environment. Using monocular deprivation (MD) to
deprive one eye for several days results in a shift in the response of neurons in
the binocular zone toward the ipsilateral eye. During the critical period of mouse
development, sensitivity to MD is increased and the shift in cortical activity is
driven by a depression in the response to the contralateral eye (Gordon and
Stryker, 1996). In the adult mouse, longer periods of MD are required to
produce a shift in ocular dominance, and in this case the shift results from a
potentiation in the cortical response to the ipsilateral eye (Niell and Stryker,
2008).
Multiple mechanisms have been suggested to account for the plasticity
observed during both critical period and adult plasticity. Of these, many of the
14
predominant theories focus on the role of inhibitory transmission as a critical
element in the opening and maintenance of normal critical period ocular
dominance plasticity (ODP). The evidence demonstrating the significance of
inhibitory neurons in this process can be broken down to three critical
observations. The first being that a sufficient amount of γ-aminobutyric acid
(GABA) mediated inhibition is necessary to open the critical period, and a brief
increase in this transmission will open it precociously. Glutamic acid
decarboxylase (Gad) 65 is one of two enzymes required to synthesize GABA. In
Gad65-knockout mice GABA levels are significantly reduced and brief MD has
no effect on ocular dominance (Hensch et al., 1998). However, at any age,
briefly enhancing inhibition in the mouse V1 using diazepam, (a GABAA receptor
agonist) restored normal ocular dominance plasticity in the Gad65-knockout
mice (Fagiolini & Hensch, 2000). In addition, diazepam administration in wild-
type mice was sufficient to induce a precocious but otherwise normal critical
period at P15, approximately 1 week before normal critical period induction
(Fagiolini & Hensch, 2000). This finding suggests that increased GABAergic
transmission is necessary and sufficient to open the critical period, by way of
machinery that is already present much earlier in development.
Successive experiments identified the GABAA receptor α1 subunit as
being critical for the opening of critical period ODP. Repeating the previous
15
experiment using knockin mice with diazepam-insensitive GABAA receptor
subunits, researchers found that a precocious critical period could still be
induced via diazepam administration in mice with mutant α2 or α3 receptor
subunits, but not in those with α1 subunits (Fagiolini et al., 2004). This result
indicates that inhibitory neurons known to make contacts with α1 subunit
containing GABA receptors, such as the parvalbumin-expressing (PV) basket
cells, may have an essential function in opening the critical period.
The third line of evidence indicating the importance of inhibition in critical
period plasticity involves the factors that regulate the maturation of inhibitory
transmission. There is significant overlap between the molecular factors that
regulate the development of inhibitory neurons, and those that control the
opening of the critical period. Of these, the most predominant example is brain-
derived neurotrophic factor (BDNF). Transgenic mice over-expressing BDNF in
excitatory neurons displayed early maturation of inhibitory neurons, as well as a
precocious critical period and precipitous development of adult visual acuity
(Hanover et al., 1999; Huang et al., 1999).

16
Presented Contributions
Fragile X Syndrome is characterized by a hypersensitivity to sensory
stimuli, a phenotype that is recapitulated in the mouse model of the disease, the
Fmr1
-/-
mouse. Studies investigating the source of this hypersensitivity in Fmr1
-/-

mice depict a system in which an imbalance of excitation/inhibition result in a
constant state of hyperarousal within cortical networks. However, it is unclear
whether this hyperexcitability manifests as abnormal sensory-evoked activity in
sensory cortices of Fmr1
-/-
mice. To determine whether sensory-evoked
responses are altered in Fmr1
-/-
mice, I measured cortical response to whisker
stimulation in the barrel cortex of Fmr1
-/-
and wild type (WT) mice. In addition, to
establish whether Fmr1
-/-
mice exhibit any deficits in perceptual learning, I
examined the performance of Fmr1
-/-
mice on a whisker-dependent learning
paradigm (Chapter 3). In order to investigate whether the underlying,
hyperexcited state of the network in Fmr1
-/-
mice alters cortical plasticity, I
examined plasticity in the visual cortex of Fmr1
-/-
mice, and examined the
contribution of altered inhibitory transmission to the Fmr1
-/-
phenotype (Chapter
4). To date, this is the first report examining evoked response in the barrel
cortex of Fmr1
-/-
mice, as well as the first to directly study the role of inhibitory
transmission in cortical plasticity of Fmr1
-/-
mice.

17
CHAPTER TWO: Materials and Methods
Mice (FVB background)
FVB wild-type (FVB.129P2-Pde6b+ Tyrc-ch/AntJ; stock# 4828, Jackson
Laboratory) and Fmr1 KO mice (FVB.129P2- Pde6b+ Tyrc-ch Fmr1tm1Cgr/J:
stock# 4624, Jackson Laboratory) were maintained and all experiments
conducted according to protocols approved by the Children’s Hospital Los
Angeles Institutional Animal Care and Use Committee. Mice were anesthetized
by isoflurane inhalation and euthanized by carbon dioxide asphyxiation in
accordance with approved protocols.
Mice were weaned at P20, group housed with same-sex littermates (3–5 per
cage) and food and water were available ad libitum except in gap cross groups.
Mice trained and tested on the gap cross were individually housed at the start of
training through completion of the 6 days of testing. During that period mice
were moderately food restricted as normal chow was allocated on a daily basis
to maintain 90–95% initial body weight. All mice were 12–14 weeks of age at the
time of the study.

Mice (C57B6 Background)
C57B6 wild-type (WT) and Fmr1-/- mice (C57B6 genetic background;
Jackson Laboratory) were maintained and all experiments conducted according
18
to protocols approved by the Children’s Hospital Los Angeles Institutional
Animal Care and Use Committee.
Mice were group housed with same-sex littermates and food and water were
available ad libitum. Critical period mice were P26-28 at the time of the first
imaging session.

Cranial Window Surgery
Male FVB wild-type (WT) and Fmr1 KO mice (12–14 weeks of age) were
used. Mice were anaesthetized with isoflurane (4% induction, 1%–1.5%
maintenance) throughout surgery. Body temperature was maintained with a
biofeedback heatplate (Physitemp). A circular region of the skull over barrel
cortex or visual cortex (depending on experiment) was thinned to allow
visualization of blood vessels at the brain surface without perturbing the
underlying dura. A 3 mm diameter #1 thickness cover glass (Bellco) was placed
on the thinned skull, affixed with cyanoacrylate and sealed with dental acrylic
(Figure 2.1). 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 mg/g body weight) and baytril in water (0.1 mg/ml)
post-surgery. Their water was also supplemented with carprofen (0.025 mg/ml)
19
throughout the imaging series. Animals were given at least 2 days to recover
before intrinsic signal optical imaging.

Monocular Deprivation
Monocular deprivation took place immediately following the first imaging
session, at approximately p27. The eye contralateral to the recording window
was closed for 3 days using a single mattress suture tied with 6-0 polypropylene
monofilament (Prolene 8709H; Ethicon) under brief 1% isoflurane anesthesia.
The knot was sealed with cyanoac- rylate glue. Following 3 days of MD, mice
were 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.
protocols approved by the Children’s Hospital Los Angeles
Institutional Animal Care and Use Committee. Mice were
anesthetized by isoflurane inhalation and euthanized by carbon
dioxide asphyxiation in accordance with approved protocols. The
Children’s Hospital Los Angeles Institutional Animal Care and
Use Committee specifically approved this study. Protocol number
264-12.
Mice were weaned at P20, group housed with same-sex
littermates (3–5 per cage) and food and water were available ad
libitum except in gap cross groups. Mice trained and tested on the
gap cross were individually housed at the start of training through
completion of the 6 days of testing and moderately food restricted
asnormalchowwasallocatedonadailybasistomaintain90–95%
initial body weight. All mice were 12–14 weeks of age at the time
of the study.
The Gap Cross Assay
The gap cross assay was performed with a custom-built robot
(D.H. Herman, manuscript in preparation). In brief, the gap cross
assay system is a closed-loop robotic environment with motor
controlled units and sensing elements. The mouse behaves upon
raised platforms driven by independent linear actuators. The
platforms are equipped with servo-motor doors and positional
sensors.Dataacquisitionandcontrolalgorithmsarebothexecuted
online for real-time dynamic control and offline for more
advanced analysis. Independent linear actuators move the
Plexiglass platforms to generate a range of gap-distances from
nose (,4.5 cm) to whisker (5–8 cm) distances in increments of
0.5 cm. To monitor the location of the mouse, IR motion sensors
are at the back and edge of each platform. Near the edge of each
platform are servo-controlled doors that prevent exploratory
behavior during repositioning of the platforms. The linear motors,
servos, and motion sensors are USB controlled through micro-
controller boards (Arduino Mega 2560 and the Quadstepper
Motor Driver) that feed to a quad-core CPU.
Motor positions are processed on a quad-core CPU using the
Arduino and Matlab programming environments. Platform
position, door status (open/closed) and feeders are real-time
controlled using the Arduino C-based development environment
(ADE). Custom-built feeders delivered a small sugar pellet
Figure1.Fmr1KOmiceexhibitincreasedevokedactivityinprimarysomatosensorycortexduringwhiskerstimulation. (A) Schematic
showing experimental set up of intrinsic optical imaging over primary somatosensory cortex (black circle) during periodic whisker stimulation. (B)
Pictures of the thin skull preparation and example images collected from a wild-type (WT) mouse (left) and Fmr1 KO mouse (right) mouse. Scale
bar=0.4 mm. Rostral (R), Caudal (C), Lateral (L) and Medial (M) coordinates are shown. (C) Representative examples of data collected during a typical
imaging session. Above, a time series of pixel values for the cortical location indicated by the asterisk in the Fmr1 KO in panel B. Below, a fast-fourier
transform (FFT) of the raw trace extracts the magnitude of the change in reflectance (DR/R) corresponding to the frequency of whisker stimulation
(red square). (D) The number of pixels within the region of response withDR/R magnitudes greater than the threshold indicated on the abscissa for
WT (n=10) and Fmr1 KO (n=10) mice. The response to whisker stimulation is elevated in Fmr1 KO mice (WT vs. KO, p=.011; 2-way ANOVA).
doi:10.1371/journal.pone.0109116.g001
Tactile Learning Deficits in Fmr1 KO Mice
PLOS ONE | www.plosone.org 2 October 2014 | Volume 9 | Issue 10 | e109116
protocols approved by the Children’s Hospital Los Angeles
Institutional Animal Care and Use Committee. Mice were
anesthetized by isoflurane inhalation and euthanized by carbon
dioxide asphyxiation in accordance with approved protocols. The
Children’s Hospital Los Angeles Institutional Animal Care and
Use Committee specifically approved this study. Protocol number
264-12.
Mice were weaned at P20, group housed with same-sex
littermates (3–5 per cage) and food and water were available ad
libitum except in gap cross groups. Mice trained and tested on the
gap cross were individually housed at the start of training through
completion of the 6 days of testing and moderately food restricted
asnormalchowwasallocatedonadailybasistomaintain90–95%
initial body weight. All mice were 12–14 weeks of age at the time
of the study.
The Gap Cross Assay
The gap cross assay was performed with a custom-built robot
(D.H. Herman, manuscript in preparation). In brief, the gap cross
assay system is a closed-loop robotic environment with motor
controlled units and sensing elements. The mouse behaves upon
raised platforms driven by independent linear actuators. The
platforms are equipped with servo-motor doors and positional
sensors.Dataacquisitionandcontrolalgorithmsarebothexecuted
online for real-time dynamic control and offline for more
advanced analysis. Independent linear actuators move the
Plexiglass platforms to generate a range of gap-distances from
nose (,4.5 cm) to whisker (5–8 cm) distances in increments of
0.5 cm. To monitor the location of the mouse, IR motion sensors
are at the back and edge of each platform. Near the edge of each
platform are servo-controlled doors that prevent exploratory
behavior during repositioning of the platforms. The linear motors,
servos, and motion sensors are USB controlled through micro-
controller boards (Arduino Mega 2560 and the Quadstepper
Motor Driver) that feed to a quad-core CPU.
Motor positions are processed on a quad-core CPU using the
Arduino and Matlab programming environments. Platform
position, door status (open/closed) and feeders are real-time
controlled using the Arduino C-based development environment
(ADE). Custom-built feeders delivered a small sugar pellet
Figure1.Fmr1KOmiceexhibitincreasedevokedactivityinprimarysomatosensorycortexduringwhiskerstimulation. (A) Schematic
showing experimental set up of intrinsic optical imaging over primary somatosensory cortex (black circle) during periodic whisker stimulation. (B)
Pictures of the thin skull preparation and example images collected from a wild-type (WT) mouse (left) and Fmr1 KO mouse (right) mouse. Scale
bar=0.4 mm. Rostral (R), Caudal (C), Lateral (L) and Medial (M) coordinates are shown. (C) Representative examples of data collected during a typical
imaging session. Above, a time series of pixel values for the cortical location indicated by the asterisk in the Fmr1 KO in panel B. Below, a fast-fourier
transform (FFT) of the raw trace extracts the magnitude of the change in reflectance (DR/R) corresponding to the frequency of whisker stimulation
(red square). (D) The number of pixels within the region of response withDR/R magnitudes greater than the threshold indicated on the abscissa for
WT (n=10) and Fmr1 KO (n=10) mice. The response to whisker stimulation is elevated in Fmr1 KO mice (WT vs. KO, p=.011; 2-way ANOVA).
doi:10.1371/journal.pone.0109116.g001
Tactile Learning Deficits in Fmr1 KO Mice
PLOS ONE | www.plosone.org 2 October 2014 | Volume 9 | Issue 10 | e109116
Figure 2.1. Example images of the thin skull preparation collected from two different
adult mice. Scale bar = 0.4 mm. Rostral (R), Caudal (C), Lateral (L) and Medial (M)
coordinates are shown.

20
Optical Imaging of Intrinsic Signals in Barrel Cortex
We used Fourier analysis-based intrinsic signal optical imaging to
measure cortical response in the barrel cortex (Kalatsky & Stryker, 2003; Cang et
al., 2005). Mice were administered chlorprothixene (1 mg/g body weight) prior
to imaging and anesthesia was maintained with isoflurane (4% induction, 0.8% -
1.0% maintenance in pure oxygen) delivered through a custom-built nose cone.
To visualize whisker- evoked changes in intrinsic signals in S1 barrel cortex, a
single whisker (e.g. C2) contralateral to the cranial window was deflected
approximately 15 degrees every 20 s, with a 3 Hz sinusoidal pulse train for 3 s
using a piezoelectric actuator controlled by a function generator (GW Instek)
(Figure 2.2). This was repeated 35 consecutive times per trial. Green light (530
nm ± 30 nm) was used to visualize cerebral vascularization and red light (620 nm
± 20 nm) to image intrinsic signals. The imaging plane was focused ~200–400
μm below the pial surface. Images were acquired at 10 frames per second at
102461024 pixels per image at 12-bit depth with a high-speed camera (Dalsa
1M60). Custom acquisition and analysis software (C++ and Matlab) spatially
binned images, and the magnitude of the response (ΔR/R) at the stimulus
frequency was extracted from a complete time series for each pixel by Fourier
analysis (Figure 2.4) (Kalatsky & Stryker, 2003).

21

Optical Imaging of Intrinsic Signals in Primary Visual Cortex
Imaging was performed as previously described (Kalatsky and Stryker,
2003; Smith and Trachtenberg, 2007; Sato and Stryker, 2008). Mice were
administered chlorprothixene (1g/g body weight) and anesthesia was
maintained with isoflurane. The eyes were protected with a thin layer of silicon
oil. For visual stimuli, a horizontal bar (6° in height and 20° in width) drifting
downward through the binocular visual field with a period of 8s was presented
for 280s (i.e., 35 cycles) on a high-refresh-rate monitor positioned 25 cm in front
Figure 2.2. Schematic showing experimental set up of intrinsic optical imaging over
primary somatosensory cortex (red circle) during periodic whisker stimulation.
22
of the animal. Green light (530nm + 30nm) was used to visualize cerebral
vascularization and red light (620nm ± 20nm) to image intrinsic signals. The
imaging plane was focused ~200-400m below the pial surface. Optical images
of visual cortex were acquired continuously over the stimulation period at 10
frames per second with a high-speed camera (Dalsa 1M60) at 1024 x 1024 pixel
by 12-bit resolution. The Fourier component of light reflectance changes
matched to the stimulus frequency was extracted pixel by pixel from the image
stream to generate amplitude and phase maps of cortical intrinsic signals (Figure
2.3). Data about magnitude and retinotopic position of the response at each
pixel was obtained as amplitude and phase values of the Fourier components,
respectively. The boundaries of the central binocular zone was identified by
maps of the response of the ipsilateral eye.
The ocular dominance index (ODI) was calculated as the average of (C -
I)/(C + I ), where C and I represent the response magnitude of the top 1, 5 and
10% of responding pixels to the contralateral and ipsilateral eyes, respectively
(unless otherwise noted, results presented refer to top 1% of responding pixels
in the image). The ODI ranges from +1 to -1, where a positive value indicates a
contralateral bias, and a negative value an ipsilateral bias. Data analysis from
mice was not completed when initial responses to contralateral or ipsilateral eye
23
stimulation resulted in ODI values <0.05, as these were inconsistent with
previous experiments.

Figure 2.3. Schematic showing experimental set up of intrinsic optical imaging over
primary visual cortex (red circle) during periodic visual stimulation.
24

Diazepam Treatment
During the ocular dominance plasticity experiments, diazepam (2mg/kg in
0.9% saline) or saline was administered daily (i.p.) for 4 days starting at P21.
Experimenter was blind to groups for the duration of the experiment.

Gap Cross Assay
The gap cross assay was performed with a custom-built robot (D.H.
Herman, manuscript in preparation). The gap cross assay system is a closed-loop
FFT Raw Trace
Time (s)
Reflectance (R)
100 200 300 400 500 600 700
min = 56980 5.75
5.74
5.72
5.70
5.68
0 -1
1.5
-2
0
0.5
1
x 10
-4
Amplitude
A
B
R/R x 10^-4
Figure 2.4. Representative examples of data collected during a typical imaging
session. (A) A time series of pixel values for the cortical location indicated by the
asterisk in the Fmr1 KO in panel B. (B) a fast-fourier transform (FFT) of the raw trace
extracts the magnitude of the change in reflectance (DR/R) corresponding to the
frequency of whisker stimulation (red square).

25
robotic environment with motor controlled units and sensing elements. The
mouse behaves upon raised platforms driven by independent linear actuators
(Figure 2.5A). The platforms are equipped with servo-motor doors and
positional sensors. Data acquisition and control algorithms are both executed
online for real-time dynamic control and offline for more advanced analysis.
Independent linear actuators move the Plexiglass platforms to generate a range
of gap-distances from nose (4.5 cm) to whisker (5–8 cm) distances in increments
of 0.5 cm. To monitor the location of the mouse, IR motion sensors are at the
back and edge of each platform. Near the edge of each platform are servo-
controlled doors that prevent exploratory behavior during repositioning of the
platforms. The linear motors, servos, and motion sensors are USB controlled
through micro- controller boards (Arduino Mega 2560 and the Quadstepper
Motor Driver) that feed to a quad-core CPU.
Motor positions are processed on a quad-core CPU using the Arduino
and Matlab programming environments. Platform position, door status
(open/closed) and feeders are real-time controlled using the Arduino C-based
development environment (ADE). Custom-built feeders delivered a small sugar
pellet (BioServ, product #F05684) following a successful cross. Motion sensor
data are continuously acquired and pre-processed within ADE and are visualized
and stored in real time (Figure 2.5B). Specifically, sensor activity is encoded as
26
behavioral performance metrics including successful and failed crossing events.
Successful trials are defined as trials in which the mouse approaches the gap
and crosses. Failures are defined as trials in which the mouse approaches the
gap and then retreats back. This information is computed in real-time (Figure
2.5C). Behaviors are segmented into interactive events at the gap and the
system is structured as a two state machine: exploration and adjustment. During
exploration, the motors are disabled and the system continuously acquires
behavioral data through the motion sensors. During adjustment the doors close
to halting exploration and motors reposition the platforms for the next
exploration phase. Transitions between the two states are triggered by
behavioral events (i.e. successful/failed gap-crossing).
Mice were handled for 10 minutes a day for one week prior to beginning
the task. The day before training began, mice were habituated to the gap cross
apparatus. They were placed in the chamber with background white noise (60–
65 dB) for 20 minutes in white light, immediately followed by 20 minutes in the
dark. A bridge was placed over the gap to prevent exploration of the gap and
gap crossing behavior. All training sessions took place in a light-tight enclosure
in the presence of background white noise. Food was provided to the mice at
least one hour after their final training session.
27
Each training session lasted for 20 successful trials or a maximum of 20
minutes. The training lasted a total of 6 days, with 2 sessions per day for a total
of 12 consecutive sessions. Training sessions were separated by at least 6 hours.
All sessions began with a trial at 3.0 cm, the shortest distance tested. The
position of the mouse was tracked with motion sensors placed at the back and
near the edge of each platform. As a mouse traversed the platform, these
sensors recorded its progressive position. A successful trial was identified as any
trial in which the mouse successfully crossed the gap between the home and
target platforms and activated the motion sensor at the back of the target
platform. These trials were rewarded with a 5 mg casein pellet delivered from an
automated feeder. A failed attempt was defined as an attempt in which the
mouse explored the edge of the home platform and returned to the back of the
platform. Following each success or failure, the subsequent gap distance was
determined using an adaptive learning algorithm designed to decrease the
predictability of the next gap distance.
The learning algorithm incorporates the progressive history of successful
crosses during a session. Beginning with the first trial at 3.0 cm, the next gap
distance was chosen randomly from a uniform distribution of distances (in 0.5 cm
increments) 1.0 cm less than the maximum distance crossed in the session (to a
minimum of 3.0 cm) to 1.5 cm greater than the maximum distance crossed in the
28
session (to a maximum distance of 7.0 cm). This process was then repeated
iteratively until 20 successful trials or 20 minutes had elapsed, completing the
session (D.H.Herman, manuscript in preparation).

Motion sensors
#2 #3 #1 #4
Home platform Target platform
motion sensor
1
2
3
4
10 20
3
4
5
6
7
gap distance (cm)
10 20
failed
successful
Trials
Reward
Reward
A
B
C
FIgure 2.5. (A) Schematic of the gap cross learning task. Motion sensors positioned
at four points along the 2 platforms (labeled #1–4) track the mouse as it moves
from the starting platform across a given gap distance to the target platform. (B)
Activation of each sensor (grey box) indicates the position of the mouse. (C)
Successful crosses are defined as the movement of the mouse from the starting
platform to the target platform (green circles). Failures are defined as trials in which
the mouse approaches the edge of the home or target platform and returns to the
back of the home platform (red crosses).
29
Visual Water Task
Visual acuity was estimated with the Visual Water Task (Prusky et al.,
2000; Prusky and Douglas, 2003). Two monitors were positioned at the wide end
of a trapezoidal tank behind clear plexiglass. One monitor displayed a sinusoidal
spatial frequency grating at 95% contrast, while the other displayed an
isoluminant grey screen (Figure 2.6). The luminance of the two monitors was
matched and gamma corrected with computer software (Eye-One Match 3).
Inside the tank, the monitors were separated by a 46cm divider. The spatial
frequency grating was determined relative to the length of this divider. The tank
was filled with water and a hidden platform submerged below the surface of the
water in front of the monitor displaying the grating.
Mice were trained to swim towards the monitor displaying the grating
and hidden platform after a molding phase during which mice gradually learned
to swim from a release chute at the back of the tank towards the monitors. Using
a low spatial frequency (0.1 cycles per degree (cpd)), mice were trained to swim
to the monitor presenting the grating. During the training phase, when a mouse
chose incorrectly, it repeated the trial on the same side until it chose correctly
before it was returned to its home cage. For both the training, and the
subsequent testing phase, mice swam blocks of 10 interleaved trials in groups of
5 for a maximum of 4 blocks of trials per day.
30
During the testing phase, the spatial frequency was increased in small,
sequential increments until an animal consistently fell to 70% accuracy. Starting
at 0.1 cpd, mice had to succeed at three consecutive trials before proceeding to
the next special frequency, which presented one more complete cycle of the
sinusoidal grating. Following the first failure, mice were required to achieve 5
correct trials in a row, or 8 correct trials out of 10 at each spatial frequency
before proceeding to the next higher frequency. Once a mouse failed to
complete 8 correct trials out of 10 at a given spatial frequency, it was briefly
retrained at half that spatial frequency to eliminate any potential ‘side bias’.
Then, testing resumed at the spatial frequency below the original failure. The
threshold for visual acuity was established once a mouse exhibited a consistent
pattern of performance. Acuity thresholds were estimated as the spatial
frequency average from three or more failures at adjacent spatial frequencies.
Throughout the testing phase, any mouse that failed to find the hidden platform
on the first try repeated the trial one more time before it was returned to its
home cage, whether or not it chose correctly the second time.
31

Immunohistochemistry
Mice were deeply anesthetized with Ketamine HCl (200mg/Kg, Phoenix
pharmaceuticals)/Xylazine (20mg/Kg, Lloyd Laboratories) and transcardially
perfused with phosphate buffered saline (PBS; ChemCruz SC-362299) followed
by a buffered 4% paraformaldehyde (PFA)/PBS (Acros Organics 416780030).
Brains post-fixed overnight in 4% PFA/PBS. Free-floating 40mm sections were cut
Figure 2.6. Schematic diagram of the visual water task. (A) View from above
indicating major components comprising the pool, midline divider, entrance chute
and two LED monitors. Pool is filled with clean water (gray). Upon entering the chute,
mice choose to swim toward the monitor displaying the grating in order to locate the
hidden platform and escape from the water. (B) Front view of monitor screens,
submerged platform and midline divider. Figure adapted with permission courtesy of
Aaron McGee.
32
on a vibrating microtome (Leica VT 1000S) and preserved in PBS containing
0.05% sodium azide (Sigma-Aldrich S8032).
Coronal sections containing visual cortex were washed in Tris-Buffered
Saline (TBS, 50mM Tris-HCl, 150mM NaCl, pH 7.4) (3 X 5 minutes). Sections
were incubated in blocking solution, 3% normal horse serum (NHS; Vector
Laboratories S-2000) in TBS containing 0.1% Triton X-100 (Sigma-Aldrich T9284)
for 1 hour at room temperature (TBS-T). The primary antibody sheep anti-
Parvalbumin (PV) (R&D Systems, AF5058) was diluted in blocking solution to
1mg/mL. In sections primary antibody was diluted together with fluorescein
conjugated Wisteria Floribunda Agglutinin (WFA) (VectorLabs, FL-1351) at
2mg/mL. Sections incubated in primary antibody overnight at 4°C. After
repeated washing in TBS-T (3 X 10 min), sections were incubated in secondary
antibody, Alexa 488- or 594-conjugated donkey anti-sheep (Jackson Immuno
Research) 1:200 in blocking solution, for 1 hour at room temperature. After a
final series of washes (3x 10 min in TBS-T, 1 X 10 min in TBS), sections were
mounted onto SuperFrost Plus slides (Fisher) with Fluoromount G containing
4',6-diamidino-2-phenylindole (DAPI) (Electron Microscopy Science).

33
Analysis of PV cell density
Forty micron thick coronal sections stained with sheep anti-PV primary
antibody (R&D systems) and a donkey anti-sheep Alexa594 secondary antibody
(Jackson ImmunoResearch). Images were captured with a BX-51 microscope,
20x 0.4 NA objective and 12-bit monochrome camera (Retiga EX, QImaging).
DAPI staining was utilized to identify visual cortex prior to capturing images of
PV density. Two images were required to span the distance from the subcortical
white matter to the pial surface. Images were merged with the software
Photoshop following linear contrast adjustment. Data points are the average of
at least three sections from each of three animals for each genotype.

34
CHAPTER THREE: Characterizing sensory response and learning in the
somatosensory cortex of the Fmr1
-/-
mouse

Introduction
3.1 Response and plasticity in the barrel cortex
The whisker to cortex pathway of the mouse is an optimal system for the
study of cortical response to controlled sensory stimuli. Each whisker on the
facial pad is individually represented by a functionally and anatomically distinct
'barrel', within layer IV of the primary somatosensory cortex (Woolsey & Van der
Loos, 1970). The barrels are topographically organized to mirror the
arrangement of whiskers on the facial pad, and neurons within the barrels will
fire in response to displacement of their associated whisker. The area and
amplitude of cortical response in layer IV to whisker deflection increases in
proportion to increasingly larger deflections (Peterson et al., 1999). In addition,
the response properties of individual barrels are sensitive to environmental
manipulations as well. For example, in response to single whisker deprivation,
the area responding to the deprived whisker will shrink, whereas the functional
representation of the surrounding whiskers will increase (Glazewski & Fox, 1996;
Wallace et al, 2001). Interestingly, exposure to enriched/naturalistic environment
will sharpen functional representations in upper cortical layers (II/III), without
affecting receptive fields in layer IV (Polley et al., 2004). These studies
35
demonstrate a direct correlation between sensory experience and functional
representation in the barrel cortex, and indicate the functional significance of
barrel cortex activity in sensory perception.
The heightened sensitivity to sensory stimuli observed in patients with
Fragile X, as well as Fmr1
-/-
mice, may suggest alterations in cortical
responsiveness to sensory stimuli. Supporting this idea, there is significant
evidence suggesting that FMRP plays an important role in mediating response
and plasticity in the barrel cortex. For instance, whisker deflection results in
increased FMRP expression and increased activity-dependent translation of
FMRP (Todd & Mack, 2000; Todd et al., 2003). Similarly, FMRP expression in
mouse layer IV barrel cortex neurons peaks during a well-defined period of
increased plasticity and synaptogenesis (Harlow et al., 2010; Daw et al., 2007)
and critical period plasticity in S1 is altered in Fmr1
-/-
mice (Harlow et la., 2010).
These results suggest that FMRP expression is involved in both the signaling and
plasticity pathways of the barrel cortex.
In addition, Fmr1
-/-
mice display significant alterations of multiple
functional and developmental archetypes in the barrel cortex, including altering
inter- and intra-layer connectivity strength, delaying dendritic spine maturation
and modifying well-characterized forms of neuronal plasticity (Bureau et al.,
36
2008; Gibson et al., 2009; Nimchinsky et al., 2001; Galvez and Greenough,
2005; Desai et al., 2006).
To test whether cortical response to sensory stimuli is altered in the
absence of FMRP, we measured the cortical responses to whisker stimulation in
Fmr1
-/-
and wild-type (WT) control mice. We quantified both the magnitude and
area of response to single whisker deflection in both groups, and found that in
the region of strongest response, the area of response to whisker deflection is
significantly increased in Fmr1
-/-
mice compared to age-matched controls.

Results
3.2 Fmr1
-/-
mice exhibit increased evoked activity in primary somatosensory
cortex during whisker stimulation.
To explore the cortical representations of tactile stimuli in Fmr1
-/-
and WT
mice, we used Fourier analysis-based intrinsic signal optical imaging (ISI) to
measure cortical responses to whisker stimulation. ISI is a non-invasive measure
of tissue reflectance correlated with neural activity (Grinvald et al., 1986; Frostig
et al., 1990; Chen-Bee et al., 2007). This technique produces in vivo images of
cortical activity with high spatial resolution, and has been used to study
functional representation in the barrel cortex of adult and developing mice and
rats (Frostig et al., 1990; Masino and Frostig, 1996; Prakash et al., 1996; Polley
37
et al., 1999). To visualize whisker-evoked changes in intrinsic signals in the barrel
cortex, a single whisker (C2) contralateral to the cranial window was deflected
approximately 15 degrees every 20s with a 3 Hz sinusoidal pulse train. This
deflection was repeated 35 consecutive times for each trial.
In this study we examined the cortical response to stimulation of the C2
whisker for both Fmr1
-/-
and WT mice (Figure 3.1). To measure the whisker
evoked responses, we quantified the number of pixels in each image with a
magnitude at or above a series of sequential, proportionately increasing
thresholds. We found that in Fmr1
-/-
mice (n = 10), the area (pixels) that
responded to a single whisker deflection was significantly increased relative to
WT mice (n = 10) across the highest magnitude of response thresholds (Figure
3.2).
The size of the region of response in WT mice was approximately the size
of barrels observed in slice preparation of mouse cerebral cortex (Lefort et al.,
2009). Thus, Fmr1 mutant mice exhibit abnormally large responses to whisker
stimulation in barrel cortex despite a normal cytoarchitecture (Till et al., 2012).

38

Figure 3.1. Photographs of the thin skull preparation (A) and example images collected from
a wild-type (WT) mouse (left) and Fmr1 KO mouse (right) mouse (B). Scale bar = 0.4 mm.
Rostral (R), Caudal (C), Lateral (L) and Medial (M) coordinates are shown.
39

Figure 3.2. Evoked activity is increased in the barrel cortex of Fmr1 ko mice during whisker
stimulation. The number of pixels within the region of response with ΔR/R magnitudes
greater than the threshold indicated on the abscissa for WT (n = 10) and Fmr1 KO (n = 10)
mice. The response to whisker stimulation is elevated in Fmr1 KO mice (WT vs. KO, p =
.011; 2-way ANOVA).
40
3.3 The Gap Cross task is a whisker-dependent sensory learning paradigm.
The gap-crossing perceptual learning task is the only reported paradigm
that systematically measures unrestrained, spontaneous (i.e. untrained) whisker-
dependent object localization (Carvell and Simons, 1990b; Towal and Hartmann,
2006, 2008; Celikel and Sakmann, 2007; Mitchinson et al., 2007a; Voigts et al.,
2008; Grant et al., 2009a). This task has frequently been used as a naturalistic
paradigm in order to quantify behavioral decision-making correlated with tactile
sensory input (i.e. whisking) in rodents. In the Gap-Cross task, a mouse is placed
upon one of two elevated platforms that are positioned opposite each other,
with a variable gap between them. Either spontaneously or by reward, the
mouse learns to cross the gap by actively exploring the opposing platform with
its whiskers (Hutson and Masterton, 1986; Harris et al., 1999; Jenkinson and
Glickstein, 2000; Celikel and Sakmann, 2007; Voigts et al., 2008).
Lesion studies on blinded rodents have demonstrated that both whiskers
and the barrel cortex are necessary for successful completion of the gap-cross
task, and that behavioral recovery of the task following temporary lesion was
correlated with functional (somatosensory evoked potentials) and metabolic
improvement (Hutson & Masterton,1986; Troncoso et al., 2004). In addition,
genetic or environmental manipulations which cause measurable effects on
41
sensorimotor circuitry also result in reduced gap-crossing performance
ITroncoso et al., 2004; Pang et al., 2011).
These studies provide convincing evidence that gap-crossing behavior is
dependent on barrel cortex activity. Given our results of altered cortical
response in the barrel cortex of Fmr1
-/-
mice, as well as the heightened
behavioral response to sensory stimuli observed in FXS patients and Fmr1
-/-

mice, we set out to determine whether sensory learning is altered in the absence
of FMRP.

3.4 Fmr1
-/-
mice display deficits in whisker-dependent learning on the gap cross
task.
To determine whether Fmr1
-/-
mice display deficits in whisker-dependent
learning, we examined the performance of Fmr1
-/-
(n = 9) and WT mice (n = 6)
over the course of 12 gap-cross learning sessions. As a measurement of
performance with experience, we compared the percent of successful crossing
between the first six sessions (1–6) and the subsequent six sessions (7–12) at all
gap distances tested for WT and Fmr1
-/-
mice (Figure 3.3). Both genotypes
displayed a similar high percentage of successful crosses at distances less than
4.5 cm (Figure 3.4). At these shorter ‘nose-distances’, mice are able to detect
the target platform by touching it with their nose as well as their whiskers. WT
and Fmr1
-/-
mice displayed similar improvement with experience at these ‘nose
42
distances’ (Figure 3.4). In addition, the percent of successful crosses at 'nose
distances' increased significantly in the second half of sessions for both Fmr1
-/-

and WT mice.

Figure 3.3. Fmr1
-/-
mouse improvement on the gap cross is distance dependent. The
percent successful crosses averaged across the first six sessions and subsequent six
sessions across gap distances ranging from 3.0 cm to 6.0 cm for both wild-type mice
(black lines, n = 6) and Fmr1 KO mice (blue lines, n = 9). For each distance, the line
marker on the left is the average success rate of the first six sessions and the
connected line marker on the right is the average success rate of the subsequent six
sessions. Error bars represent standard error of the mean.

43

Figure 3.4. Fmr1
-/-
mice display normal learning on the gap cross assay at shorter,
'nose' distances. At shorter ‘nose’ distances, both wild-type (WT) and Fmr1 KO mice
(KO) improve to a greater percentage of successful crosses between the average of the
first six sessions (WT, grey, KO light blue) and the last six sessions (WT, black, KO dark
blue). This improvement is statistically significant (WT, p = .007; n = 6; KO, p,.001, n = 9;
WT; two-way ANOVA). Error bars represent standard error of the mean.
(B) Average improvement for WT and KO mice at shorter ‘nose’ distances. Both WT
and Fmr1 KO mice improved an average of more than 15% at these ‘nose’ distances.

44

Figure 3.5. Fmr1
-/-
mice display impaired learning on the gap cross assay at longer,
whisker dependent distances. (A) At whisker- dependent distances, wild-type mice (WT)
improve between early sessions (grey line) and subsequent sessions (black line) despite
the lower overall success rate at increasing gap distances. However, KO mice do not
display significant improvement between early sessions (light blue line) and later
sessions (dark blue line) (WT; p = .002, n = 6, KO; p = .14; n = 9, two-way ANOVA). (B)
Average improvement for WT and KO mice at shorter ‘nose’ distances and longer
‘whisker’ distances. WT mice display significantly greater improvement at whisker-
dependent distances than KO mice (p = .02, two-tailed t-test).

45
In contrast to shorter distances, at longer distances (5.0cm, 5.5cm, 6.0cm)
mice rely exclusively on information from their whiskers to detect the target
platform. Overall, performance declines with increasing gap distance (Figure 3.3).
At the longer, 'whisker' distances, WT mice improved significantly with
experience, and the percentage improvement was similar in magnitude to that
observed at ‘nose distances’ (Figure 3.5A). Interestingly, Fmr1
-/-
mice did not
display significant improvement at these whisker-dependent gap distances
(Figure 3.5A), and the minimal amount of improvement displayed was
significantly less than that of WT controls (p = .02) (Figure 3.5B). Similarities
between the two groups in the total number of trials as well as the number of
successful crossings for all distances suggest that these deficits were not due to
differences in mobility, exploration or motivation on the task (Table 3.1). Thus,
Fmr1
-/-
mice exhibit a deficit in tactile learning that correlates with abnormal
cortical sensory representation of whiskers in barrel cortex.

Table 3.1 Average number of successful and attempted crossings across all
sessions.
Fmr1
-/-
WT
Total number of
successful crossings
195 ± 27 (SEM) 183 ± 29 (SEM)
Total number of trials 336 ± 28 (SEM) 369 ± 24 (SEM)

46
Discussion
Although sensory hypersensitivity is a prominent characteristic of Fragile
X Syndrome, observed in both patients and the Fmr1
-/-
mouse, relatively little
research has focused on locating the source of this hypersensitivity in the
sensory pathway. Here, we have identified the primary somatosensory cortex as
an early cortical location in which sensory responsiveness is altered in the Fmr1
-/-

mouse. Although Fmr1
-/-
mice have normal barrel cortex cytoarchitecture (Till et
al., 2012), the area of response to whisker deflection was significantly increased
in these mice compared to WT controls. Thus, the exaggerated response to
whisker stimulation observed in Fmr1
-/-
mice is more likely the result of altered
network responsiveness than any anatomical differences between groups.
However, it's unclear whether the information coming from the periphery/ earlier
in the sensory pathway in Fmr1
-/-
mice is altered. Future studies that investigate
activity in the thalamus, or experiments that evaluate peripheral responses (such
as measuring number of whisks necessary for decision-making on the gap cross
task), could provide more insight into whether the hyperexcitability observed in
the cortex results from local alterations or those occurring earlier in the
processing pathway.
In addition, in this study we also explore the consequences of loss of
FMRP on sensory learning, by comparing the performance of Fmr1
-/-
and WT
47
mice on a whisker-dependent sensory learning paradigm. Although we found no
difference in the initial performance between groups, the improvement of the
Fmr1
-/-
mice at whisker-dependent distances is significantly decreased compared
to the controls. As the gap cross is a whisker and barrel cortex dependent task,
this suggests a somatosensory specific deficit in sensory learning associated with
loss of FMRP. Thus, the aberrant cortical responses to whisker stimulation we
observed in the Fmr1
-/-
mice correlate with a deficit in a whisker-dependent and
barrel cortex-dependent tactile learning task. However, this perceptual learning
task is not exclusively reliant on cerebral cortex or the somatosensory system.
Distance detection and object localization integrates motor and sensory activity
of both subcortical and cortical circuitry (O'Conner et al., 2010; Huber et al.,
2013). As a result, whether the exaggerated whisker representations in barrel
cortex contribute to the deficits in tactile learning in Fmr1
-/-
mice, or whether this
learning impairment results from aberrant neural circuitry elsewhere in the brain
is unclear at present. We considered whether the increased anxiety-like
behaviors exhibited by Fmr1
-/-
mice might contribute to their deficits in the gap
cross assay (Spencer et al., 2005; Moy et al., 2009). However, as both the initial
performance and improvement of Fmr1
-/-
mice at shorter ‘nose’ distances was
similar to WT mice, as well as their initial performance at longer ‘whisker’
distances, we propose that the anxiety-like behaviors of Fmr1
-/-
mice are unlikely
48
to be a major contributor to the deficit observed at whisker-dependent gap
distances.
These studies have come the closest to date in correlating a physiological
change in cortical response properties with a behavioral deficit in the Fmr1
-/-
mouse. Future studies could elucidate the relationship between altered sensory
processing and performance on the gap cross task in the Fmr1
-/-
mouse by
examining the effect of pharmacological intervention on both whisker-evoked
response and gap cross performance. An intervention that addressed one or
both alterations would provide tremendous insight into the mechanisms driving
the Fmr1
-/-
mouse phenotype, as well as potential drug targets to help alleviate
the tactile hypersensitivity and/or deficits in learning in FXS patients.

49
CHAPTER FOUR: Characterizing response and plasticity in the primary visual
cortex of the Fmr1
-/-
mouse

Introduction
4.1 Critical period plasticity in the mouse primary visual cortex
The mouse primary visual cortex is a preeminent model for the study of
experience dependent plasticity (Wiesel, 1982). The classic paradigm for this
type of plasticity in the visual cortex is ocular dominance (OD) plasticity. In this
paradigm, the relative cortical response to stimulation of either eye is described
as "ocular dominance", and the point where retinotopically matched inputs from
the two eyes converges onto a common postsynaptic neuron in primary visual
cortex serves as a foundation for binocular vision. In mice, only ~10% of retinal
ganglion cells project to the ipsilateral LGN and cortex, instead the majority
cross over to the contralateral hemisphere at the optic chiasm (Drager, 1978;
Gordon & Stryker, 1996; Drager & Olsen, 1980). Despite the relatively sparse
input from the ipsilateral eye, the binocular region accounts for approximately
1/3 of the area of the primary visual cortex. In this region the majority of neurons
respond to either eye, however there is a pervasive dominance of the
contralateral eye, and only ~5% of the cells in this area are driven exclusively by
the ipsilateral eye (Drager, 1975; Metin et al., 1988).
50
During the early stages of visual cortical development there exists a brief,
well-defined period during which the impact of sensory input on nervous system
organization is particularly strong. This 'critical period' of ocular dominance
plasticity in mice takes place from (P21-P35). During this time, briefly depriving
vision in one eye (monocular deprivation) shifts the response properties of
binocular cortical neurons such that their response to the deprived eye will
significantly diminish (Gordon & Stryker, 1996; Antonini et al., 1999; Frenkel &
Bear, 2004). Interestingly, ocular dominance plasticity is also present in the adult
visual cortex, however it is distinguished from critical period ODP in several
ways. First, adult ODP is a slower process, requiring a longer duration of MD to
produce a significant shift in cortical response. In addition, adult ODP is
characterized by an increase in the response to the open-eye, with little to no
effect on the cortical response to the deprived eye (Sawtell et al., 2003; Pham et
al., 2004; Sato & Stryker, 2008).
A variety of mechanisms have been suggested to account for critical
period and adult ocular dominance plasticity. Of these, many of the leading
theories emphasize the role of inhibitory transmission as an essential component
in the opening and maintenance of normal critical period ocular dominance
plasticity (ODP). Notably, several studies have shown that inhibitory transmission
is necessary and sufficient for the opening of the critical period of ODP at any
51
point in the lifespan of a mouse, starting at P15 (Hensch et al., 1998; Fagiolini &
Hensch, 2000; Fagiolini et al., 2004).
Given the significant evidence of reduced inhibitory transmission in
sensory cortices of the Fmr1
-/-
mouse, we set out to determine whether ocular
dominance plasticity was disrupted in the Fmr1
-/-
mouse.

Results
4.2 Critical period plasticity is altered in Fmr1
-/-
mice
To examine cortical representations of visual stimuli in Fmr1
-/-
and WT
mice we used Fourier analysis-based intrinsic signal optical imaging to measure
visual response and plasticity in the binocular region of the visual cortex
(Kalatsky & Stryker, 2003; Cang et al., 2005). A 6 x 20° floating bar was
presented to each eye individually and a CCD camera oriented over primary
visual cortex acquired images of reflected light at 610nm. At each pixel location,
changes in reflectivity corresponding to the stimulus frequency was extracted by
Fourier analysis, and the magnitude of cortical response (Δ R/R) was extracted as
the amplitude value of the Fourier component. The ocular dominance index
(ODI) was calculated using the ratios of response magnitudes to the two eyes
across the highest responding pixels in the response area.
52
We first compared the effect of 3 day MD on the responsiveness of the
visual cortex in critical period Fmr1
-/-
(FX) and wild type (WT) mice. Prior to MD,
in WT mice (n = 6) the magnitude of the contralateral-eye response was ~ 40%
larger than the response to the ipsilateral eye, giving rise to an OD index of
~0.17 (± 0.04) (Figure 4.1A). In Fmr1
-/-
mice (n = 9) prior to MD, the magnitude
of the contralateral eye was ~ 60% greater than the response to the ipsilateral
eye, leading to an OD index of ~0.24 (± 0.04) (Figure 4.2A).

Three days of monocular deprivation of the contralateral eye, initiated
just before the peak of the critical period (P27), resulted in significant shifts in
the ODI for both groups. In the WT group, the response to the contralateral eye
decreased significantly, while the response to the ipsilateral eye was unaltered
(Figure 4.1). The decrease in cortical responsiveness to the contralateral eye led
to a post-MD OD index of ~ -0.17 (± 0.07) in the WT mice (for WT mice pre v.
post: ODI shift p = .0313, contra eye magnitude p = .0313, ipsi eye magnitude
p = .99) (Figure 4.1, 4.3).
In contrast, in Fmr1
-/-
mice 3 day MD of the contralateral eye had no
effect on the cortical response to the contralateral eye. However, it led to a
significant increase in the response to the ipsilateral (non-deprived) eye (Figure
4.2). The increase in cortical response to the ipsilateral eye resulted in a post-
53
MD OD index of ~0.09 (± 0.04) in Fmr1
-/-
mice (for Fmr1-/- mice pre v. post MD:
ODI shift p = .0183, contra eye magnitude p = .7690, ipsi eye magnitude p =
.0047) (Figure 4.3).

54

Figure 4.1. Cortical response to the contralateral eye is diminished following 3
day MD in critical period WT mice. (A) Magnitude of response (delta R/R) of
contralateral eye and ipsilateral eye in wild type mice in response to visual
stimulus pre and post 3 day MD (n = 6 WT, Wilcoxen test of contra eye
magnitude pre v. post MD p = .0313, ipsi eye magnitude p = .99). (B) Example
images collected from WT mice pre and post 3 day MD. Scale bar = 0.4 mm.
Medial (M) and Caudal (C) coordinates are shown.
55

Figure 4.2. Cortical response to the ipsilateral eye is potentiatiated, while
contralateral eye response is unchanged following 3 day MD in critical period
Fmr1-/- mice. (A) Magnitude of response (delta R/R) of contralateral eye and
ipsilateral eye in Fmr1-/- mice in response to visual stimulus pre and post 3 day
MD (n = 9 FX, paired t test of pre v. post contra eye magnitude p = .7690, pre
v. post ipsi eye magnitude p = .0047). (B) Example images collected from FX
mice pre and post 3 day MD. Scale bar = 0.4 mm. Medial (M) and Caudal (C)
coordinates are shown.
56

The decrease in contralateral eye responsiveness observed in the WT
mice, and subsequent shift in ocular dominance, is consistent with previous
reports of the effects of monocular deprivation during the critical period
(Gordon & Stryker, 1996; Frenkel & Bear, 2004; Cang et al., 2005; Sato &
Stryker, 2008). However, the potentiation of the response to the ipsilateral eye
(and lack of alteration in the contralateral eye) observed in the Fmr1
-/-
mice is
atypical, and more closely resembles the plasticity observed in adult mice
Figure 4.3. The magnitude of the ODI shift following 3 day MD is disparate
between Fmr1 ko and WT mice. Ocular dominance index (ODI) values for wild
type (WT) and Fmr1-/- mice (FX) pre and post 3 day MD (n = 6 WT, n = 9 FXS,
Wilcoxen test of WT *p = .0313, paired t test of FX *p = .0183). Two-way RM
ANOVA **p = .04
57
following 7 days of monocular deprivation (Sawtell et al., 2003; Pham et al.,
2004; Hofer et al., 2006; Sato & Stryker, 2008). To compare the magnitude of
the shift in OD index between WT and FX mice, we tested the pre and post ODI
values for both groups using a two-way RM ANOVA. This analysis revealed a
statistically significant interaction between genotype and MD (p = 0.04),
reflecting the smaller ODI shift in Fmr1
-/-
mice and demonstrating that the
magnitude of the effect of MD differs significantly between genotypes. Thus,
while the direction of the shift in ODI following MD is similar between
genotypes, the magnitude, and more importantly, the driving force behind the
shift is aberrant in the Fmr1
-/-
mouse.
These results demonstrate a deficit in experience-dependent plasticity in
sensory cortices of the Fmr1
-/-
mouse.

4.3 Brief diazepam treatment during the initiation of the critical period partially
rescues critical period plasticity in Fmr1
-/-
mice
The altered critical period plasticity observed in the Fmr1
-/-
mouse is
supported by a previous report describing ipsilateral eye potentiation in the
Fmr1
-/-
mouse in response to 3 day monocular deprivation (Dolen et al., 2007).
To determine whether the altered inhibitory transmission described in the Fmr1
-/-

mouse could play a role in mediating this phenotype, we treated Fmr1
-/-
mice
58
with the GABA agonist diazepam for four days starting at P21. Following
diazepam (or saline) treatment, we compared the effect of 3 day MD on the
responsiveness of the visual cortex during the critical period in Fmr1
-/-
mice.
Prior to MD, there was no significant difference in the ODI values for the
diazepam (DZ) or saline (SAL) treated Fmr1
-/-
mice (FX pre-MD ODI = 0.24 ±
0.03, SAL pre-MD ODI = 0.23 ± 0.03, DZ ODI = .244 ± 0.03). In addition, there
was no significant difference in the pre-MD average magnitude values of the
contralateral or ipsilateral eye for the DZ treated or Saline + FX groups (for some
analysis, saline treated and untreated FX mice are grouped together, indicated
as FX + SAL) (contra eye mag; FX + SAL = 0.2416 ± 0.03, DZ = 0.24 ± 0.03).
Therefore DZ treatment had no effect on the pre-MD response magnitudes for
either eye.
Three days of monocular deprivation of the contralateral eye, initiated
just before the peak of the critical period (P27) in the diazepam and saline
treated Fmr1
-/-
mice resulted in significant shifts in the ODI for both groups. In
the saline treated group we saw no significant difference from the Fmr1
-/-

untreated mice. Namely, we saw a potentiation of the ipsilateral eye response
and no change in the cortical response to the contralateral eye. In contrast, in
the diazepam treated Fmr1
-/-
mice, 3 day MD of the contralateral eye resulted in
a significant depression of the cortical response to the contralateral eye, similar
59
to that observed in the WT mice following 3 day MD (Figure 4.4). However, the
potentiation of the ipsilateral eye response displayed in the untreated/saline
treated Fmr1
-/-
mice was maintained in the diazapam treated group (Fmr1
-/-
DZ
mouse pre v. post MD: ODI shift p = .0002, contra eye magnitude p = .0027,
ipsi eye magnitude p = .0045). Since there was no significant difference pre-MD
for the ODI, contralateral eye or ipsilateral eye magnitude in the DZ treated
mice compared to un-treated or saline treated Fmr1
-/-
mice (Figure 4.4, 4.5), the
effect of diazepam treatment is not the result of altered baseline cortical
responsiveness, but instead appears to be specific to the mechanisms regulating
critical period plasticity. Therefore, in the Fmr1
-/-
mouse, brief diazepam
treatment at the initiation of the critical period rescued contralateral cortical
plasticity in response to MD, but did not affect the potentiation of the cortical
response to the ipsilateral eye observed in untreated Fmr1
-/-
mice.

60

Figure 4.4. Diazepam treatment restores contralateral eye depression in Fmr1-
/- mice following 3 day MD. (A) Magnitude of response (delta R/R) of
contralateral eye and ipsilateral eye in diazepam treated Fmr1-/- mice in
response to visual stimulus pre and post 3 day MD (n = 14 DZ, paired t test of
pre v. post contra eye magnitude p = 0.0027, pre v. post ipsi eye magnitude p
= 0.0045). (B) Example images collected from DZ treated FX mice pre and post
3 day MD. Scale bar = 0.4 mm. Medial (M) and Caudal (C) coordinates are
shown.
61

4.4 The number of parvalbumin (PV) expressing cells is unaltered in the primary
visual cortex of Fmr1-/- mice.
Our results suggest that abnormal inhibitory transmission may play a role
in mediating the alterations in critical period OD plasticity observed in Fmr1
-/-

mice. These results are consistent with previous studies describing changes in
Figure 4.5. Diazepam treatment partially restores ODI shift in Fmr1-/- mice. Ocular
dominance index (ODI) values for wild type (WT), Fmr1-/- mice (FX) + saline treated
mice (FX + SAL) and diazepam treated Fmr1-/- mice. ODI values for pre and post 3
day MD (n = 6 WT, n = 14 FX + SAL, n = 14 DZ, Wilcoxen test of WT *p = 0.0313,
paired t test of FX + SAL *p = 0.0046, paired t test of DZ *p = 0.0002).

62
the inhibitory system in Fmr1
-/-
mice, including one study that describes a 40%
decrease in the number of parvalbumin (PV) expressing inhibitory neurons in the
somatosensory cortex of Fmr1
-/-
mice (Selby et al., 2007). Therefore, we set out
to determine whether there are any alterations in PV cell numbers in the visual
cortex of the Fmr1
-/-
mouse.
To determine whether Fmr1
-/-
mice display layer specific alterations in PV
cell distribution in the primary visual cortex, we used DAPI staining to identify
layer specific cytoarchitecture, and counted the number of PV expressing cells
within each layer in critical period Fmr1
-/-
mice and WT mice (Figure 4.6A).
We found no significant difference in the number of PV expressing
neurons between critical period Fmr1
-/
mice (n = 5) and WT mice (n = 5) (Figure
4.6B). Similar to previous reports, we found differences in PV cell expression
between layers, but these changes were consistent across genotypes. Therefore,
loss of FMRP does not alter the number of PV expressing neurons in the visual
cortex of Fmr1
-/-
mice.
63

Figure 4.6. The number of parvalbumin expressing cells is unaltered in all layers of
the primary visual cortex Fmr1-/- mice. (A) Example images Ocular dominance index
(ODI) values for wild type (WT), Fmr1-/- mice (FX) + saline treated mice (FX + SAL)
and diazepam treated Fmr1-/- mice. ODI values for pre and post 3 day MD (n = 6
WT, n = 14 FX + SAL, n = 14 DZ, Wilcoxen test of WT *p = 0.0313, paired t test of
FX + SAL *p = 0.0046, paired t test of DZ *p = 0.0002).

64
4.5 Recovery of visual acuity following long-term monocular deprivation (LTMD)
is correlated with cortical excitatory/inhibitory balance
While a brief period of monocular deprivation is sufficient to shift ocular
dominance in both critical period and adult mice, longer duration MD initiated
during the critical period results in a permanent loss of visual acuity in the
deprived eye (Prusky et al., 2000; Prusky and Douglas, 2003). Previous studies
have demonstrated that disrupting the normal excitatory/inhibitory balance in
visual cortex is sufficient to enhance recovery of visual acuity following long-term
monocular deprivation (LTMD). Specifically, interventions such as Fluoxetine
treatment and enriched environment are known to promote recovery of acuity in
adult mice through a reduction of intracortical inhibition (Maya Ventencourt et
al., 2008; Sale et al., 2007). Given the evidence of altered E/I balance in the
Fmr1
-/-
mouse (Paluszkiewicz et al., 2011; Contractor et al., 2015), we set out to
determine whether recovery of visual acuity following LTMD is altered in the
absence of FMRP.

4.6 Maturation of visual acuity is normal in Fmr1
-/-
mice
To establish whether Fmr1
-/-
mice display normal development of visual
acuity we first evaluated their performance on a behavioral assay, the visual
water task (Prusky et al., 2000). This assay is a direct measure of visual function
65
that allows us to determine the threshold of mouse visual acuity. By evaluating
visual acuity in Fmr1
-/-
mice every 5 days from P25 to P45 we were able to
develop a timeline of visual acuity maturation in Fmr1
-/
mice (Figure 4.7).
We found that at each developmental time point the visual acuity
displayed by Fmr1
-/-
mice was similar to that displayed by WT mice. Therefore,
loss of FMRP does not affect the development of visual acuity, and Fmr1
-/-
mice
reach normal adult levels of acuity by age P45.

Figure 4.7. Maturation of visual acuity is unaltered in Fmr1-/- mice. Average acuity
measured through both eyes for Fmr1-/- mice (n = 7) every 5 days from P25 to P45
with the visual water task. Error bars indicate SD. Gray region indicates previously
reported standard deviation for acuity in WT mice.

66
4.7. Altered inhibitory transmission in Fmr1
-/-
mice does not result in enhanced
recovery of visual acuity following LTMD
To evaluate whether the changes observed in OD plasticity and inhibitory
transmission in Fmr1
-/-
mice are correlated with altered recovery of visual acuity
following LTMD, we tested visual acuity of the deprived eye in Fmr1
-/-
mice.
At age P24, one eye was sutured and remained closed for a period of 3
weeks (P24-45). At the conclusion of LTMD, the suture was removed and the eye
was checked for signs of scarring of the cornea/other physical damage. Seven
weeks after the removal of the suture, the visual acuity of the deprived eye was
evaluated for all mice. Following training, mice were tested on the visual water
task and the acuity thresholds were recorded as the spatial frequency from three
failures at adjacent spatial frequencies.
Seven weeks after restoring normal vision following LTMD, Fmr1
-/-
mice (n
= 7) displayed diminished visual acuity in the deprived eye (Figure 4.7). For
reference, average effects of LTMD on visual acuity in WT mice from a previous
study are used with permission in Figure 4.8. The average spatial frequency
threshold for three consecutive trials was recorded for all Fmr1
-/-
mice.
Interestingly, seven weeks after normal vision is restored WT mice display some
recovery of visual acuity (Figure 4.7, light gray shaded region) compared to WT
mice tested 7 days after restoration of vision (Figure 4.7 dark gray shaded
67
region). However, seven weeks after normal vision is restored, visual acuity in
Fmr1
-/
mice is similar to that of WT mice 7 days after vision restoration. These
results may suggest that Fmr1
--/
mice have less recovery of visual acuity than WT
mice, although further studies comparing Fmr1
-/-
mice visual acuity 7 days v. 7
weeks post LTMD are required.

Figure 4.8. Fmr1-/- mice do not display recovery of visual acuity following LTMD.
Average acuity measured through the deprived eye for Fmr1-/- mice (n = 7) seven
weeks after vision was restored following LTMD. Error bars indicate SD. Dark gray
region indicates previously reported acuity in WT mice 7 days after restoration of
vision following LTMD. Lighter gray region indicates previously reported acuity in WT
mice 7 weeks after restoration of vision following LTMD.

68
Discussion
There is significant behavioral and physiological evidence demonstrating
hyperexcitability in the Fmr1
-/-
network. However, it is difficult to determine the
consequences that cortical hyperexcitability may have on necessary functions of
these systems, such as the ability to effectively encode and adapt to sensory
information. The deficits observed in ocular dominance plasticity in the Fmr1
-/-

mouse reveal a functional consequence of abnormal transmission and/or
processing of sensory information early in the visual stream. The loss of critical
period plasticity, combined with the evidence of decreased inhibitory
transmission in Fmr1
-/-
mice, led us to investigate whether altered inhibitory
transmission contributed to the Fmr1
-/-
phenotype.
Consistent with previous reports, we observed a deficit in critical period
plasticity in the Fmr1
-/-
mouse. Interestingly, critical period Fmr1
-/-
mice display
'adult' plasticity, namely, a potentiation of the cortical response to the non-
deprived eye, in response to brief monocular deprivation. This is notable, as
previous studies have proposed that potentiation of the non-deprived eye
response develops as a consequence of the depression of the response to the
deprived eye (Sawtelle et al., 2003; Frenkel & Bear, 2004). In critical period
Fmr1
-/-
mice, we observed no change in the response to the deprived eye,
indicating that the mechanisms driving potentiation of the non-deprived eye
69
response can proceed independent of those that drive contralateral eye
depression.
Because increased GABAergic transmission is necessary and sufficient to
induce the critical period of ocular dominance plasticity (Hensch et al., 1998;
Faiolini & Hensch, 2000), we set out to determine whether altered inhibitory
transmission in in Fmr1
-/-
mice could contribute to the loss of critical period
plasticity. We found that brief diazepam treatment at the initiation of the critical
period partially rescued critical period plasticity in Fmr1
-/-
mice, such that
depression of cortical response to the deprived eye was restored in treated
mice. Interestingly, diazepam treatment did not affect the potentiation of the
ipsilateral eye response observed in untreated/saline treated Fmr1
-/-
mice. These
results provide further indication that the mechanisms driving the two cortical
responses to brief MD (deprived eye depression and non-deprived eye
potentiation) function independent of one another, as it is possible to influence
one without altering the other.
Importantly, these results also indicate that the machinery necessary to
drive contralateral eye depression is in place in the Fmr1
-/-
network, therefore
the deficit likely results from a loss of activation rather than a lack of capacity. In
addition, our findings suggest that altered inhibitory transmission in Fmr1
-/-
mice
could lead to disruptions in how the network adapts to experience. Deficits in
70
cortical plasticity early in the sensory processing stream could have significant
effects in higher cortical processing areas. From a therapeutic perspective, our
results suggest that brief, timely intervention in the form of augmented
inhibitory transmission could facilitate mechanisms of plasticity that may
otherwise be aberrant in the clinical form of Fragile X Syndrome. Future studies
examining the effect of diazepam treatment in Fmr1
-/-
mice at different ages
could determine whether there is any maturation of plasticity in the Fmr1
-/-
visual
cortex, and the role of inhibitory transmission in regulating the process.
A previous report describes a significant reduction in the number of
parvalbumin (PV) expressing inhibitory neurons in sensory cortices of the Fmr1
-/-

mice (Selby et al. 2007). However, we found no change in the number of PV
expressing cells in the visual cortex of Fmr1
-/-
mice compared to WT mice. Given
that the literature, (as well as our own experiments) indicate that deficient
inhibitory transmission contributes to the Fmr1
-/-
phenotype, we suggest that
future studies examine the expression of other inhibitory cell types, as well as
the rate and strength of connectivity between excitatory and inhibitory cells in
the visual cortex of Fmr1
-/-
mice.
In addition to altered critical period plasticity, we observe a deficit in
recovery of visual acuity in Fmr1
-/-
mice following long-term monocular
deprivation. The results of this study are preliminary and require future
71
investigation examining the visual acuity of Fmr1
-/-
mice immediately following
vision restoration. Recovery of visual acuity and ocular dominance plasticity are
known to be regulated by distinct mechanisms (Stephany et al., 2014), however,
given the deficits in plasticity observed in Fmr1
-/-
mice, these results may
indicate that while distinct, some of the mechanisms driving these two forms of
plasticity are conserved between systems. Recovery of visual acuity is known to
be age-dependent, wherein LTMD performed in adult animals does not affect
acuity (Prusky and Douglas, 2003). Therefore, our results may suggest that in
Fmr1
-/-
mice, recovery of visual acuity is impeded because either the critical
period closes during the deprivation, or the system lacks the mechanisms
necessary to drive recovery. Interestingly, at P25, non-deprived Fmr1
-/-
mouse
visual acuity is ~0.26 (± 0.025) cycles/degree. In Fmr1
-/-
mice that experience
LTMD (starting at P24) the visual acuity at 7 weeks post vision restoration ~0.22
(± 0.04) cycles/degree. The uniformity between these two values may suggest
that the maturation of visual acuity is halted by LTMD in Fmr1
-/-
mice, and at the
point when vision is restored the system lacks the plasticity necessary to
facilitate recovery of acuity. However, future studies would first need to
determine whether visual acuity is similar in Fmr1
-/-
mice 7 days and 7 weeks
post eye opening. If Fmr1
-/-
mice display a consistent lack of recovery, it would
be interesting to evaluate whether the critical period for recovery is attenuated
72
or aberrant in these mice. In addition, examining the effect of brief
pharmacological intervention (such as diazepam treatment) in Fmr1
-/-
mice would
provide insight into the mechanisms regulating recovery of visual acuity
following long term monocular deprivation.

73
CHAPTER FIVE: Conclusions
Cortical hyperexcitability in autism spectrum disorders such as Fragile X
Syndrome is thought to contribute to many of the behavioral phenotypes
associated with the disorder. Despite our increasing understanding of the
behavioral phenotypes and underlying state of the network in Fmr1
-/-
mice, the
manner in which the Fmr1
-/-
network responds to and encodes new information
remains unclear. In the investigations described in this dissertation, I
characterized the evoked response and plasticity in two sensory cortices of the
Fmr1
-/-
mouse. Together these investigations contribute to our knowledge
regarding the Fmr1
-/-
phenotype, as well as our understanding of the
mechanisms regulating plasticity in sensory cortices.
In Chapter 3 I described the increased evoked response to sensory
stimulation in the somatosensory cortex of Fmr1
-/-
mice, as well as a learning
deficit in a whisker-dependent learning paradigm. Increased cortical
responsiveness to sensory stimuli is concurrent with the predominant theory of
disrupted excitatory/inhibitory balance in autism spectrum disorders such as
FXS. Although far from complete, the experiments presented in Chapter 3 have
gone the farthest to date in exploring the relationship between behavioral
defects and cortical dysfunction in Fmr1
-/-
mice, and come the closest to
providing a full-circle (network-to-behavior) description of the hyperexcitability
74
phenotype in this model. This study is also among the first to identify abnormal
sensory-evoked network activity in primary sensory cortices of the Fmr1
-/-
mouse.
These results identify the primary somatosensory cortex as an early cortical
location wherein hyperexcitability in Fmr1
-/-
mice manifests as enhanced sensory-
evoked response.
In Chapter 4 I discussed the abnormal ocular dominance plasticity
observed in the primary visual cortex of critical period Fmr1
-/-
mice, and
explored the influence of altered inhibitory transmission on this phenotype. I
found that briefly increasing GABAergic transmission (via the GABA agonist
diazepam) partially rescued critical period plasticity in Fmr1
-/-
mice. These results
also demonstrated that mechanisms driving the two forms of ODP observed in
mice (depression of the deprived eye and potentiation of the non-deprived eye)
operate autonomously and can be independently influenced.
Despite the evidence for altered inhibitory transmission, I found no
significant difference in the number of parvalbumin (PV) expressing neurons in
the visual cortex of Fmr1
-/-
mice compared to WT. These results suggest that
future studies should examine the expression of other inhibitory cell types, as
well as the rate and strength of connectivity between excitatory and inhibitory
cells in the visual cortex of Fmr1
-/-
mice.
75
At the conclusion of Chapter 4 I described a preliminary experiment
investigating recovery of visual acuity in Fmr1
-/-
mice following long-term
monocular deprivation (LTMD). The results suggest a deficit in the recovery of
visual acuity in Fmr1
-/-
mice following restoration of vision, however future
studies are required to determine the nature and extent of this deficit.
The experiments described in this dissertation demonstrate several
contexts in which altered excitability in the Fmr1
-/-
mouse manifests as abnormal
sensory-evoked response and plasticity in sensory cortices. These results identify
functional consequences of previously described deficits in Fmr1
-/-
mice, as well
as provide insight into mechanisms regulating response and plasticity in sensory
cortices.

76
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Abstract (if available)

Abstract Fragile X Syndrome (FXS) is the leading inheritable form of intellectual disability and the foremost genetic cause of autism. In the United States, Fragile X syndrome occurs in approximately 1 in 4,000 males and 1 in 8,000 females. In addition to cognitive impairment, Fragile X Syndrome is characterized by an array of atypical responses to sensory stimulation. For example, hypersensitivity to sensory stimuli is a common symptom of many autism spectrum disorders, including FXS. Cortical hyperexcitability in autism spectrum disorders such as Fragile X Syndrome is thought to contribute to many of the behavioral phenotypes associated with the disorder, including abnormal response to sensory stimulation. This study identifies abnormal evoked response and plasticity in two sensory cortices of the Fmr1⁻⁄⁻ mouse, providing insight into the relationship between behavioral defects and cortical dysfunction in Fmr1⁻⁄⁻ mice. In addition, this study suggests that altered inhibitory transmission in the Fmr1⁻⁄⁻ mouse cortical network may contribute to altered plasticity observed during sensory cortex development. These results identify alterations in cortical response and suggest functional consequences of these deficits in Fmr1⁻⁄⁻ mice, as well as providing insight into mechanisms regulating response and plasticity in sensory cortices.

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Creator Arnett, Megan (author)

Core Title Characterizing response and plasticity in sensory cortices of the Fmr1⁻⁄⁻ mouse

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 04/22/2016

Defense Date 12/11/2015

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

Tag cortical plasticity,fragile X syndrome,mouse visual system,mouse whisker system,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor McGee, Aaron (committee chair), Hirsch, Judith (committee member), McKemy, David (committee member)

Creator Email marnett@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-239899

Unique identifier UC11276369

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Legacy Identifier etd-ArnettMega-4348.pdf

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Document Type Dissertation

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Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

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