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Analysis of Stretch Responses in Mice Lacking Munc18-1 in Proprioceptors

Amr Mohi Wright State University

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ANALYSIS OF STRETCH REFLEX RESPONSES IN MICE LACKING MUNC18-1 IN PROPRIOCEPTORS

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science

By

AMR MOHI B.D.S., Cairo University 2009

2017 Wright State University

WRIGHT STATE UNIVERSITY

GRADUATE SCHOOL

DECEMBER 14, 2017

I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Amr Mohi ENTITLED Analysis of Stretch Reflex Responses in Mice Lacking Munc18-1 in Proprioceptors BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF Master of Science.

______David R. Ladle, Ph.D. Thesis Director

______ERIC S. BENNETT, Ph.D. Department Chair Department of Neuroscience, Cell Biology and Physiology

Committee on Final Examination ______Mark M. Rich, M.D. Ph.D. ______Patrick M. Sonner, Ph.D. ______Barry Milligan, Ph.D. Interim Dean of the Graduate School

Abstract

Mohi, Amr. M.S. Anatomy Program, Department of Neuroscience, Cell Biology, and Physiology, Wright State University, 2017. Analysis of Stretch Reflex Responses In Mice Lacking Munc18-1 In Proprioceptors.

The monosynaptic stretch reflex circuit is one of the simplest circuits of the central nervous system. We studied the connection between Ia proprioceptive afferents and motor neurons that comprise the circuit, by stimulating the dorsal root and recording from the ventral root of the fourth lumbar spinal nerve. In our study, we analyzed the status of neurotransmission in the stretch reflex circuit in postnatal PV-cre; munc18-1 lox/lox conditional mutant mice. Munc18-1 is responsible for synaptic vesicular release in neurons. In the PV-cre; Munc18-1 lox/lox mutants we use, Munc18-1 is only knocked out in proprioceptive afferents and A-beta somatosensory afferents, both of which express parvalbumin (PV). Our results showed that on the first postnatal day (P1), responses evoked from the ventral root are significantly reduced in mutants compared to wild type mice. Similar results were also observed at P6. Our results lead us to conclude that PV- cre; Munc18-1 lox/lox mutant mice lack synaptic vesicular release in proprioceptive afferents after birth as evidenced by a dramatically reduced stretch reflex circuit response analyzed with the electrophysiology experiments in our study.

iii

Table of Contents

I. INTRODUCTION……………………………………………………………...1

II. MATERIALS AND METHODS……………………………………………...8

Mouse Genetics………………………………………………………...... 8

Phenotyping………………………………………………………………8

Tissue Preparation………………………………………………………...8

Electrophysiology………………………………………………………...9

III. RESULTS…………………………………………………………………….11

IV. DISCUSSION………………………………………………………………..14

V. BIBLIOGRAPHY……………………………………………………………..28

iv

LIST OF FIGURES

1. Representative traces from a P6 WT animal demonstrate that ventral root response

amplitude (millivolts) increases with increasing stimulus intensity

(microamperes)…………………………………………………………………..17

2. Sample traces of WT Vs. mutant responses from 4th lumbar ventral root in a WT vs

a mutant mouse at age P6…………………………………………………………19

3. Sample traces of WT Vs. mutant responses from 4th lumbar ventral root in a WT vs

a mutant mouse at age P1…………………………………………………………21

4. Average peak amplitudes of responses in WT (n=4) vs mutant mice (n=4) at age

P6……………………………………………………………………………...…23

5. Average peak amplitudes of responses in WT (n=4) vs mutant mice (n=1) at age

P1………………………………………………………………………………...25

6. Average peak latencies of recordings in WT mice at ages P1 and P6……………27

v

Introduction

Afferent axons carrying different sensory modalities enter the through the dorsal root. These axons relay a variety of information about both the external environment and the internal state of organs and muscles to the central nervous system.

This information is integrated by spinal circuits to generate and also ascends to the brain for conscious perception and processing. One way to classify afferent axon fibers is by axon diameter into Groups I, II, III and IV. Another classification is according to conduction velocity into three categories A, B and C where A-fibers have the highest conduction velocity, subclasses of A group are A-alpha that are also group I.

A-Beta and A-Delta are groups II and III respectively. A-Beta innervates encapsulated cutaneous receptors and unencapsulated cutaneous receptors associated with accessory structures (Meissner, Merkel, Pacinian, etc). A-delta axons innervate nociceptors (sharp pain) and thermoreceptors. B-fibers are less A-fibers in both diameter and conduction speed and represent preganglionic autonomic axons.

C-fibers are unmyelinated and are the same as group IV representing postganglionic autonomic axons and innervate nociceptors (dull aching pain). The larger the diameter and degree of myelination the faster the conduction velocity of these fibers such that the

A-alpha fibers (Ia and Ib) have the greatest conduction velocity. Proprioceptive fibers are a group of afferent axons that carry information about muscle length, force and position that provides the CNS with feedback about orientation, and movement of the body in

1 space. Groups Ia and II carry information about muscle length from muscle spindles into the spinal cord while group Ib carry information about muscle forces from Golgi tendon bodies back into the spinal cord.

The gray matter of the spinal cord is organized into ten laminae; lamina I is the most posterior and lamina IX is the most anterior. The laminae are organized based on neuron types and functions. The ventral laminae are involved with locomotion as proprioceptive neurons make contacts with interneurons and motor neurons. Afferent connections with motor neurons are either direct (monosynaptic) or indirect via one or more interneurons.

Only groups Ia and II can form monosynaptic circuits with motor neurons (in lamina IX) that mediates the stretch reflex, while smaller diameter afferents like groups III, IV do not make any connections with motor neurons. Groups Ia and II also form synapses with interneurons in lamina XII, but these connections only indirectly affect motor neurons output and are not the focus of our study. As our laboratory studies the spinal cord we pay special attention to the monosynaptic stretch reflex. The stretch reflex is a response to changes in muscle length sensed by Ia or II afferents carrying information from muscle spindles that evoke contraction in homonymous or synergistic motor neurons. The direct connections of spindle afferents with the motor neurons that produce the reflex means the reflex is often termed the “monosynaptic stretch reflex” (H.-H. Chen, Hippenmeyer,

Arber, & Frank, 2003).

Our lab is interested in studying the stretch reflex circuit as a model to understand basic questions in developmental neuroscience. How do circuits form in the central nervous system? How do afferents find their target neurons? What factors and proteins affect the

2 assembly and activity of circuits in the CNS? Many unanswered questions remain to fully understand CNS development.

The classic example of the stretch reflex is the patellar where tapping the patellar tendon distal to the patella causes a change in muscle length and stimulates muscle spindles in the quadriceps femoris muscle group. Information about the stretch is conveyed along afferents back into the spinal cord and communicated by direct synapses on quadriceps motor neurons which causes the quadriceps muscles to contract. In normal patients, contraction of the quadriceps femoris should briefly extend the leg at the knee joint and then return to normal position. In some lesions this reflex is absent or reduced

(Westphal’s sign), while in other lesions it could be aggravated. Generally, the is used to assess the function of the lumbar (L) spinal cord segments L2-L3-L4.

Genes that influence proprioceptive-motor connections and circuit formation in the spinal cord

Neurotrophin-3 (NT3) is needed in order for Ia afferents to grow into the ventral cord and innervate their target muscles (Patel et al., 2003). NT3 is a neurotrophic factor, similar to other factors such as nerve growth factor (NGF) and brain-derived neurotrophic factor

(BDNF), that is expressed in and motor neurons in the spinal cord. NT3 binds to the TrkC receptor, a receptor expressed by proprioceptive DRG neurons. If NT3 is absent, as in NT3 knockout mice, the TrkC-expressing sensory neurons will not

3 survive, and will die by means of apoptosis (Ernfors, Lee, & Jaenisch, 1994). In NT3 deficient mice there is also a lack of normal development. Double knockout mice for NT3 and Bax, which prevents apoptosis of NT3 dependent proprioceptive cells, demonstrate an interesting phenotype where proprioceptive axons only grow into the intermediate zone of the spinal cord and stop there (Patel et al., 2003).

While loss of NT3 causes cell death or axon growth deficits, elevated NT3 causes disruption in the development of the stretch reflex circuit where afferents from multiple muscles converge on the same motor neurons (Wang, Li, Taylor, Wright, & Frank,

2007). Also proprioceptive afferents express the tyrosine kinase C(trkC) receptor, and fetal muscles express NT3, the major trkC ligand. So proprioceptive afferents depend on their peripheral muscle targets for prenatal development (McMillan Carr & Simpson,

1978). And the removal of limb buds prenatally eliminates most proprioceptive afferents

(Oakley et al., 1997)

4 Early growth response protein 3 (Egr3) is essential for muscle spindle development, and in the Egr3 null mutant mice muscle spindles do not express NT3 and will degenerate progressively after birth (Tourtellotte & Milbrandt, 1998). Interestingly, synaptic transmission of Ia afferents is affected, which diminishes the monosynaptic inputs from

Ia afferents onto motor neurons when compared to WT mice in electrophysiology experiments to test the function of monosynaptic connections. Intramuscular injections of

NT3 restored the afferent connections with motor neurons which shows that the NT3 derived from muscle spindles regulates these connections (H. H. Chen, Tourtellotte, &

Frank, 2002).

Another transcription factor is the protein Runt-related transcription factor 3 (Runx3).

Runx3 is integral to differentiation and proper connectivity of sensory neurons as the level of expression of Runx3 across the spinal cord determines the dorso-ventral position of termination of afferents. Experiments on chick embryos showed cutaneous sensory neurons lacked Runx3 and only reached the dorsal part of the spinal cord without extending further into the intermediate and the ventral regions, while the proprioceptive afferents expressed either low or high Runx3 and extended more ventrally in the spinal cord depending on Runx3 level of expression (A. I. Chen, De Nooij, & Jessell, 2006).

Pecho-Vrieseling and colleagues showed that a recognition system involving expression of the membrane bound protein class 3 semaphorin (Sema3E) by some pools, and its receptor PlexinD1 in proprioceptive afferents determines synaptic specificity in the monosynaptic stretch reflex circuit (Maro, Shen, & Cheng, 2009)

5 (Pecho-Vrieseling et al., 2009). Sema3E acts through a repellant signaling to divert neurons from inappropriate targets. This signaling mechanism guides proprioceptive afferents from a muscle to form direct monosynaptic connection specifically with the same muscle motor neurons and avoid other MN pools in the ventral spinal cord.

Changing the profile of Sema3e-PlexinD1 signaling in sensory or motor neurons results in functional and anatomical rewiring of monosynaptic connections.

Er81 and PEA3 belong to the E26 transformation-specific transcription factor family.

Er81 is expressed by proprioceptive afferents and motor neurons and in Er81 mutant mice there is severe motor discoordination but normal motor neuron pools and muscle spindle formation peripherally, so the problem seems to be with the final step of Ia afferents forming enough connections with motor neurons in the ventral spinal cord

(Arber, Ladle, Lin, Frank, & Jessell, 2000) (Patel et al., 2003). Er81 and PEA3 initially are expressed by all proprioceptive afferents during embryonic development linking them to motor neurons. But in chick embryonic development, separate MN pool express either

Er81 or PEA3 and receive inputs from proprioceptive afferents coming from the same muscles, which have the same pattern of Er81 or PEA3 expression (Lin et al., 1998). This suggested Er81 and PEA3 may play a role in the late steps of development to coordinate connections between Ia neurons and MNs.

Munc18-1 is a neuron-specific protein involved in docking and priming of synaptic and large dense-core vesicles before fusion with the cell membrane (exocytosis) releasing neurotransmitters from presynaptic to postsynaptic neurons. Deletion of munc18-1 in all

6 neurons in mice results in total paralysis and complete block of secretory neurotransmission; munc18-1 mutants die at birth (Verhage, 2000). Analysis of nervous system development in these mice showed that all brain structures initially develop normally in the absence of Munc18-1. Neuronal migration patterns and crucial fiber tracts appear no different than wild type control animals. Synapses are also formed and are anatomically normal. Widespread cell death of CNS, but not PNS neurons, occurs after the initial synaptic development, as many CNS neurons depend on factors released during synaptic transmission for trophic support (Verhage, 2000) (J H Heeroma, Plomp,

Roubos, & Verhage, 2003). Addition of trophic factors extends the viability of munc18-1 deficient CNS neurons in culture (Joost H. Heeroma et al., 2004). A lox-p flanked allele of munc18-1 was recently generated and analyzed by Dr. M. Verhage and colleagues to study the effect of blocked synaptic activity in a genetically defined subset of neurons.

Munc18-1 loxp mice are crossed with parvalbumin-Cre (PV-Cre) animals to restrict deletion of munc18-1 to proprioceptive afferents (Hippenmeyer et al., 2005).

Parvalbumin (PV) is a calcium binding protein specifically expressed by proprioceptive neurons and is first expressed at E15.5 in mice (Arber et al., 2000). Using this strategy, mutants that lack Munc18-1 expression in proprioceptive sensory neurons from embryonic stages are produced (PV Cre/+; munc18-1 flox/flox). We use these animals in this thesis to block synaptic transmission only in proprioceptive neurons.

7 Materials and methods

All animal experimental procedures were conducted under the approval of the Wright

State University Laboratory Animal Care and Use Committee.

Mouse genetics

A lox-p flanked allele of munc18-1 was used and in order to block synaptic transmission only in proprioceptive neurons, munc18-1 loxp mice are crossed with PV-Cre animals.

The genotype of each animal was confirmed by PCR after the physiology experiment.

Two genotypes of mice were in these experiments: PV Cre/+; munc18-1 flox/+

(controls); PV Cre/+; munc18-1 flox/flox (mutants). We used 7 mutant mice (P1 n=3, P6 n=4) and 8 WT mice (P1 n=4, P6 n=4).

Phenotyping

In our colony, PV-Cre; flox/flox mutants animals survive until weaning, but display motor phenotypes similar to other mouse models that lack proprioceptive sensory input

(Arber et al., 2000) (Dallman & Ladle, 2013). On the day of birth (P0) we used that abnormal motor behavior to identify mutants by being not able to perform body correction when placed on their backs.

Tissue preparation

Neonatal mice from two different age groups were used in this study. Mice in the first group were studied on the first postnatal day (P1). A second group of mice were studied on (P6). Artificial cerebral spinal fluid (ACSF) was prepared before beginning the experiment. The artificial cerebral spinal fluid contained 127 mM of NaCl, 1.9mM of

KCl, 1.2 mM KH2PO4, 1 mM MgSO4 * 7H2O, 26 mM NaHCO3, 16.9 D(+)glucose

8 monohydrate, and 2 mM CaCl2. The ACSF was placed in ice and bubbled with oxygen

(95% O2, 5% CO2) for at least 15 minutes prior to beginning the experiment. Mice were phenotyped on the day of birth (P0). Mice were anesthetized using an ice bath to induce hypothermia. The chest cavity was exposed and a transcardial perfusion with 5 ml of ice- cold, oxygenated ACSF was performed. The animal was then decapitated and eviscerated to expose the vertebral column. The two lower limbs were removed at the hip joint. The preparation was then moved to a dissection chamber with constantly circulating, oxygenated ACSF. A dorsal laminectomy was performed to expose the spinal cord. Once the spinal cord was exposed the dura mater was removed. All ventral and dorsal roots were cut on the left side. On the right side all ventral and dorsal roots were cut except the lumbar root 1 to 5. Dorsal and ventral lumbar roots of L4 were separated at the furthest point to the spinal cord to be used for stimulation and recording respectively in electrophysiology. The preparation was left to warm up to room temperature (20-22 degrees Celsius)

Electrophysiology

Glass pipettes from World Precision Instruments Inc. (4 in. thin wall glass; 1.2mm

OD/0.9mm ID) were sized by fire polishing to fit the dorsal and ventral roots of lumbar

(L4) spinal cord. Extracellular recordings were obtained from ventral root (L4) which was placed in a suction electrode (A-M Systems, Sequim, WA). Responses were recorded with an EX4-400 Quad Channel Differential Amplifier (1,000x gain, 2 Hz low cut, 500 Hz high cut; Dagan, Minneapolis, MN) and digitized at 20 kHz with the use of

WinLTP software (WinLTP, Bristol, UK). Compound action potentials (CAPs) in the ventral root (L4) were evoked by stimulation of afferent fibers in dorsal root (L4).

9 Recordings gradually increased with increasing stimulation intensity until it plateaus, with the majority of intensities eliciting maximum recordings ranging from 60 to 90 micro Amperes.

10 Results

Electrophysiology experiments were performed on isolated spinal cord preparations, by recording from the fourth lumbar ventral root (VRL4) while stimulating the same level dorsal root (DRL4). Recordings from ventral roots include signals from all motor neurons that exit the spinal cord at that level. Hundreds of motor neuron axons are contained in each ventral root and the recorded response is the sum of responses from all motor neurons. These responses include action potentials and subthreshold excitatory postsynaptic potentials (EPSPs). The glass capillary electrode that surrounds the ventral root is sealed against the surface of the ventral spinal cord by suction, thus increasing the electrode resistance and the amplitude of the signals that are measured. This allows us to detect not only action potentials passing along the ventral root, but also EPSPs that are passively conducted along the axon from the motor neuron cell body. Subthreshold

EPSPs from individual motor neurons are too small to be detected with ventral root recordings, but the sum of EPSPs from many motor neurons that are evoked synchronously by stimulation of the dorsal root can be visible.

For each animal, a variety of stimulus intensities were applied in order to determine the intensity required to evoke the maximal ventral root response. Ten trials at each stimulus intensity were recorded at 10 second intervals. The individual trials were averaged together to produce the average response and to minimize noise (Figure 1).

We analyzed mice at two different ages, postnatal day 1 (P1) and P6 (total animals analyzed =15). Seven P1 mice (WT n=4 and KO n=3) and eight P6 mice were included

11 (WT n=4 and KO n=4). For each animal we analyzed the highest response recording using WinLTP software to determine the peak amplitude of the signal in millivolts and the latency of the peak amplitude in milliseconds. WinLTP automatically detects these parameters within a user-defined time window in the trace. It was important to set the limits on the time window appropriately, so that the time window corresponded to the time where we would expect to observe only monosynaptic Ia-motor neuron connections, and to avoid analyzing signals from polysynaptic circuits or slower conducting neurons.

We determined a time window of 5-10 milliseconds after the stimulation was appropriate for the P6 animal group, and for the P1 animal group we analyzed a time window of 5-15 milliseconds, as at this young age the conduction velocity of axons is lower and we should expect longer latencies (Figure 6: average latencies of responses recorded in WT mice in both P1 and P6). The recording starts 10 milliseconds before the stimulation to calculate the baseline electrode potential. We used the same time window for WT and

KO mice within each age group to compare the responses recorded.

In the P6 mice group the WT (n=4) had a peak response that ranged from 0.184 to 0.623 millivolts with an average of 0.42 millivolts and standard deviation 0.264, while the KO

(n=4) the recordings within the same time window ranged from 0.011 to 0.059 millivolts with an average of 0.035 millivolts and standard deviation 0.019. Mean values were compared using and unpaired Student t-test, and P value (P = 0.0273), suggests that the differences are statistically significant (Figures 2 and 4).

12 The same was done to the P1 age group to see if the PV-cre will knock out the Munc18-1 functionally earlier at the first postnatal day and yielded the following results, for the WT mice (n=4) the peak amplitudes within 5-15 milliseconds after stimulation ranged from

0.406 to 0.979 millivolts with an average of 0.7395 millivolts and a standard deviation

0.285, while the KO (n=3) amplitudes in the same time window ranged from 0.008 to

0.034 with an average of 0.018 and standard deviation 0.014. Unpaired student t-test resulted in a P value of 0.0079 which is very statistically significant (Figures 3 and 5).

13 Discussion

The simplest circuit in the central nervous system is the spinal monosynaptic stretch reflex circuit. It is one of the best understood circuits and a simple circuit to study neuronal connections and nervous system development. Several mechanisms, molecules and transcription factors are involved in guiding growing axons from proprioceptive neurons into their proper termination zones, while others only play role in how the neurons connect with spinal motor neurons and interneurons to form functional synapses and circuits. The focus of this study is the neuron specific protein Munc18-1 that is essential for exocytosis of neurotransmitter vesicles. It is known previously that in Munc

18-1 null mutants there is no synaptic transmission and the mice die at birth. In this study we used the sensory-motor circuit (stretch reflex circuit) to validate a mouse model that we had which is PV-cre Munc18-1 loxp, where we only knock out Munc18-1 in

Parvalbumin (PV) positive neurons, and we already know that had severe sensory-motor abnormalities (Dallman & Ladle, 2013). Parvalbumin is expressed in proprioceptive neurons and also in Purkinje cells of the cerebellum but the focus in my study is only proprioceptive afferents. As parvalbumin starts expression during embryonic development day 15.5 (E15.5) (Arber et al., 2000), we have to specifically investigate the time point when Munc18-1 is functionally eliminated from proprioceptive neurons.

While Cre-recombinase should be expressed from the parvalbumin locus beginning at

E15.5, the timing and efficiency of recombination varies depending on the gene to be recombined. In addition to disruption of the Munc18-1 gene, all Munc18-1 protein produced before expression of PV began must be degraded before the neurons are truly functionally deficient in Munc18-1. The timing of this process was unknown for proprioceptive afferents and was the motivation for this study. From the results we have

14 at the first and sixth postnatal days (P1 and P6) the KO mice do not have any monosynaptic responses to stimulating the afferents in the dorsal root, which shows that the PV-cre drived knock out of Munc18-1 can stop synaptic release in proprioceptive afferents as early as the first postnatal day.

It is still unknown if PV-cre drives that knockout before P1, so future experiments can also look further into this between E15.5 and P1. The primary interest of the lab is studying the pattern of proprioceptive afferent connections with motor neurons and interneurons in the spinal cord, and these experiments are most informative postnatally after connections are first formed. Therefore, the postnatal experiments in this thesis establish PV-Cre; munc18-1 flox/flox mutant mice as a valid animal model to study the effects of synaptic transmission on the pattern of synaptic connections in the stretch reflex circuit. Additional future experiments can study the viability of proprioceptive neurons in this animal model to determine if the neurons will degenerate during the first postnatal week. Some evidence recently showed that neurons in Munc18-1 knockout mice have abnormalities in the Golgi which is a key organelle for vesicular trafficking

(Santos, Wierda, Broeke, Toonen, & Verhage, 2017). And as mentioned before trophic support can delay neuronal degeneration (Joost H. Heeroma et al., 2004). So in the PV-

Cre; munc18-1 flox/flox mutant mice, do proprioceptive afferents survive for up to 3 weeks? If so, could this be explained by the fact that they still have trophic supply (i.e.

NT3) from the intact muscle spindles in the periphery? Other future experiments could investigate these possibilities and determine the extent of proprioceptive afferent survival in PV-Cre; munc18-1 flox/flox mutant mice.

15

Figure 1. Representative traces from a P6 WT animal demonstrate that ventral root response amplitude (millivolts) increases with increasing stimulus intensity (in microamperes), until saturation is reached. Note: difference between the last two stimulation intensities, 90 Micro Amperes in orange and 200 Micro Amperes in dark blue and no change in response size. No further stimulation is applied at higher intensities.

16 Ventral root responses as a function of stimulus strength 0.8 mV 0.75 Stimulation intensity 0.7 0.65 0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 -15 -10 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 -0.05 -0.1 ms -0.15 -0.2

17

Figure 2. Sample traces of WT Vs. mutant responses from 4th lumbar ventral root in a WT vs a mutant mouse at age P6. Wild type mouse response represented by blue.

Mutant mouse response represented by red. Responses measured in millivolts (mV).

Dotted lines represent the time window of the analyzed monosynaptic responses (5 to 10 milliseconds from stimulus).

18 VR recordings in P6 mice 0.7 mV 0.6 WT 0.5 KO

0.4

0.3

0.2

0.1

0 -10 0 10 20 30 40 50 60 70 80 90 100 -0.1 ms -0.2

19

Figure 3. Sample traces of WT Vs. mutant responses from 4th lumbar ventral root in a WT vs a mutant mouse at age P1. Wild type mouse response represented by blue.

Mutant mouse response represented by red. Responses measured in millivolts (mV).

Dotted lines represent the time window of the analyzed monosynaptic responses (5 to 15 milliseconds from stimulus).

20 VR recording from P1 mouse 0.7 mV 0.6 WT 0.5 KO 0.4

0.3

0.2

0.1

0 -10 0 10 20 30 40 50 60 70 80 90 100 -0.1 ms -0.2

21 Figure 4. Average peak amplitudes of responses in WT (n=4) vs mutant mice (n=4) at age P6 in millivolts. Error bars represent + or – standard deviation. * represent P value.

22 Peak amplitudes recorded in P6 mice 0.8

0.7 WT 0.6

0.5 KO mV 0.4

0.3

0.2

0.1 * P= 0.0273

0

23

Figure 5. Average peak amplitudes of responses in WT (n=4) vs mutant mice (n=1) at age P1 in millivolts. Error bars represent + or – standard deviation. * represent P value.

24 Peak amplitudes recorded in P1 mice 1.1

1

0.9 WT 0.8 KO 0.7

0.6 mV 0.5

0.4

0.3

0.2

0.1 * P= 0.0079

0

25

Figure 6. Average peak latencies of recordings in WT mice at ages P1 and P6 in milliseconds. Error bars represent standard deviation.

26 Peak Latencies of Monosynaptic Responses in WT mice

P1 ms P6

27 Bibliography

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