Auditory Midbrain Hypoplasia and Dysmorphology after Prenatal Valproic Acid Exposure

Accepted Manuscript Research Article Auditory midbrain hypoplasia and dysmorphology after prenatal valproic acid exposure Yusra Mansour, Sarah Mangold, Devon Chosky, Randy J Kulesza Jr PII: DOI: Reference:

S0306-4522(18)30740-1 https://doi.org/10.1016/j.neuroscience.2018.11.016 NSC 18738

To appear in:

Neuroscience

Received Date: Revised Date: Accepted Date:

28 July 2018 11 November 2018 12 November 2018

Please cite this article as: Y. Mansour, S. Mangold, D. Chosky, R.J. Kulesza Jr, Auditory midbrain hypoplasia and dysmorphology after prenatal valproic acid exposure, Neuroscience (2018), doi: https://doi.org/10.1016/ j.neuroscience.2018.11.016

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

TITLE:

Auditory midbrain hypoplasia and dysmorphology after prenatal valproic acid exposure

AUTHORS:

Yusra Mansour Sarah Mangold Devon Chosky Randy J Kulesza Jr, PhD

AFFILIATIONS:

Department of Anatomy Lake Erie College of Osteopathic Medicine Erie, PA

CORRESPONDING AUTHOR: Randy J Kulesza Jr., PhD Department of Anatomy Lake Erie College of Osteopathic Medicine 1858 West Grandview Blvd Erie, PA 16504 814-866-8423 [email protected]

1

ABSTRACT Prenatal exposure to the antiepileptic valproic acid (VPA) is associated with an increased risk of autism spectrum disorder (ASD) in humans and is used as an animal model of ASD. The majority of individuals with ASD exhibit adverse reactions to sensory stimuli and auditory dysfunction. Previous studies of animals exposed to VPA reveal abnormal neuronal responses to sound and mapping of sound frequency in the cerebral cortex and hyperactivation, hypoplasia and abnormal neuronal morphology in the cochlear nuclei (CN) and superior olivary complex (SOC). Herein, we examine the neuronal populations in the lateral lemniscus and inferior colliculus in animals exposed in utero to VPA. We used a combination of morphometric techniques, histochemistry and immunofluorescence to examine the nuclei of the lateral lemniscus (NLL) and the central nucleus of the inferior colliculus (CNIC). We found that the VPA exposure resulted in larger neurons in the CNIC and the dorsal nucleus of the lateral lemniscus (DNLL). However, we found that there were significantly fewer neurons throughout all nuclei examined in the auditory brainstem of VPA-exposed animals. Additionally, we found significantly fewer calbindinimmunopositive neurons in the DNLL. VPA exposure had no impact on the proportions of perineuronal nets in the NLL or CNIC. Finally, consistent with our observations in the CN and SOC, VPA exposure resulted in fewer dopaminergic terminals in the CNIC. Together, these results indicate that in utero VPA exposure significantly impacts structure and function of nearly the entire central auditory pathway.

KEYWORDS: inferior colliculus, lateral lemniscus, autism, calbindin

2

LIST OF ABBREVIATIONS + ASD CA CB CI CN CNIC CP D DC DL DNLL E EC IC INLL LL LNTB LSO M MSO MWU NLL NTR P PAG PB PFA PV SOC SPON TH VCN VM VNLL VNTB VPA WFA LPN

immunopositive autism spectrum disorder cerebral aqueduct calbindin confidence interval cochlear nucleus central nucleus of the inferior colliculus commissure of Probst dorsal dorsal cortex dorsolateral dorsal nucleus of the lateral lemniscus embryonic external cortex inferior colliculus intermediate nucleus of the lateral lemniscus lateral lemniscus lateral nucleus of the trapezoid body lateral superior olive medial medial superior olive Mann Whitney U nuclei of the lateral lemniscus neurotrace red postnatal periaqueductal gray phosphate buffer paraformaldehyde parvalbumin superior olivary complex superior paraolivary nucleus tyrosine hydroxylase ventral cochlear nucleus ventromedial ventral nucleus of the lateral lemniscus ventral nucleus of the trapezoid body valproic acid wisteria floribunda agglutinin lateral parabrachial nucleus

3

INTRODUCTION Autism spectrum disorder (ASD) is a developmental disorder associated with neurological dysfunction and significant abnormalities in social, communicative and behavioral domains (Allen, 1988; Wing, 1997; APA, 2013; CDC.gov, 2017). The most recent estimates indicate that by age 8, 1 in 59 children is diagnosed with ASD and the vast majority of these children are male (CDC.gov, 2017). Hypersensitivity to sensory stimuli is a key sign of ASD (CDC.gov, 2017) and can include adverse reactions to touch, smell, taste, light and sound. Furthermore, the majority of individuals with ASD have some manner of difficulty hearing and understanding speech and vocalizations despite normal or even lower thresholds to non-speech sounds (Greenspan and Wieder, 1997; Tomchek and Dunn; 2007; Gomes et al., 2008; Bolton et al., 2012). Severity of auditory dysfunction in ASD spans a wide spectrum and can range from deafness to hyperacusis, but commonly includes difficulties listening in the presence of background noise and localizing sound sources (Roper et al., 2003; Alcántara et al., 2004; Khalfa et al., 2004; Szelag et al., 2004; Lepistö et al., 2005; Teder-Sälejärvi et al., 2005; Gravel et al., 2006; Tharpe et al., 2006; Russo et al., 2009). Consistent with these functional deficits, individuals with ASD have significantly fewer auditory brainstem neurons and surviving neurons are dysmorphic and abnormally organized (Kulesza and Mangunay, 2008; Kulesza et al., 2011; Lukose et al., 2015). Further, there is evidence from a number of non-invasive tests and clinical observations directly supporting auditory brainstem dysfunction in ASD (Ornitz et al., 1985; Khalfa et al., 2001; Kwon et al., 2007; Tas et al., 2007; Lukose et al., 2013; Erturk et al., 2016; Bennetto et al., 2017) and studies of the auditory brainstem response indicate abnormal brainstem processing in ASD (Skoff et al., 1980; Gillberg et al., 1983; Rumsey et al., 1984; Martineau et al., 1987; Rosenblum et al., 1980; McClelland et al., 1992; Klin, 1993; Maziade et al., 2000; Rosenhall et al., 2003; Roth et al., 2012; Azouz et al., 2014; Källstrand et al., 2010; Miron et al., 2016; Santos et al., 2017). Together, these studies support our primary hypothesis that abnormal brainstem structure and function are the root cause of auditory processing issues in ASD.

4

The precise developmental events that lead to the ASD phenotype are poorly understood. At present, the majority of ASD cases are idiopathic, but many are comorbid with other neurodevelopmental disorders such as Fragile X syndrome (Brown et al., 1982). Additionally, maternal exposure to teratogenic drugs can significantly impact fetal brain development and such exposures are likely responsible for a small proportion of ASD cases. In particular, there is a clear association between ASD and prenatal exposure to the anti-epileptic valproic acid (VPA; Christianson et al., 1994; Rodier et al., 1996). VPA is indicated for acute treatment of manic episodes, complex partial seizures and migraines. VPA usage is not advised during pregnancy as this drug is known to cause neurological side effects in the mother and is associated with facial deformities, hypospadias and neural tube defects in the offspring (DiLiberti et al., 1984). Furthermore, prenatal exposure to VPA is associated with a significant increase in the probability of a later diagnosis of ASD and is included as a risk factor for ASD (Moore et al., 2000; Williams et al., 2001; Rasalam et al., 2005; Koren et al., 2006; Bromley et al., 2013; Christensen et al., 2013). Accordingly, in utero exposure to VPA is used as an animal model of ASD (Rodier et al., 1996).

Animals exposed to VPA in utero demonstrate many autistic-like behaviors including reduced social explorations and predilection for repetitive behaviors (reviewed by Nicolini and Fahnestock, 2018) and demonstrate ataxia on gait tasks (Main and Kulesza, 2017). Additionally, there are numerous gross and neuropathological changes associated with in utero VPA exposure in rodents. VPA-exposed animals have smaller bodies and brains, and delayed gross development of the eye and external ear (Zimmerman et al., 2018). Consistent with clinical and neuropathological observations in ASD, animals exposed to VPA have marked auditory dysfunction (Gandal et al., 2010; Engineer et al., 2014; Anomal et al., 2015; Dubiel and Kulesza, 2016) and hypoplasia of auditory hindbrain centers (Lukose et al., 2011; Zimmerman et al., 2018). More specifically, VPA exposed animals have significantly fewer neurons in the cochlear nucleus (CN) and superior olivary complex (SOC), fewer calbindin-immunopositive (CB+) neurons and reduced dopaminergic inputs to CN and SOC neurons (Lukose et al., 2011; Zimmerman et al., 2018). Additionally, VPA-exposed animals have abnormal activation patterns after exposure to pure tone stimuli 5

(Dubiel and Kulesza, 2016). Specifically, when control animals are exposed to pure tone stimuli, c-Fos immunolabeling is found in narrow tonotopic bands within many auditory nuclei. However, similar stimulation to VPA-exposed animals results in significantly more c-Fos immunolabeled neurons in the CN, SOC and central nucleus of the inferior colliculus (CNIC) and these neurons extend well beyond the characteristic tonotopic bands observed in control animals. Further, in vivo recordings from the auditory cortex in VPA-exposed animals have revealed increased response latencies, decreased phase locking capabilities (Gandal et al., 2010), abnormal mapping of sound frequencies (Anomal et al., 2015) and abnormal responses to vocalizations (Engineer et al., 2014). Together, these results indicate that prenatal VPA exposure in rodents results in significant alterations in structure and function in the central auditory pathways.

These previous studies have focused on the hindbrain and the auditory cortex and have neglected several large auditory centers in the pons, midbrain and thalamus, namely the nuclei of the lateral lemniscus (NLL), the IC and the medial geniculate complex. Consequently, the impact of prenatal VPA exposure on these auditory centers has not been studied. The IC is an important relay and processing center for both ascending and descending auditory information. In fact, previous estimates of neuronal number in rats indicate that the IC outnumbers subcollicular auditory centers more than five to one (Kulesza et al., 2002). Based on the structural and functional changes that occur in the CN, SOC and auditory cortex after VPA exposure, we hypothesize that the NLL and CNIC are significantly impacted by VPA exposure. Herein, we examine this hypothesis in a repeated in utero exposure model using morphometric techniques, estimates of neuronal number, histochemistry for perineuronal nets (PNNs) and immunofluorescence for the calcium-binding protein calbindin (CB) and tyrosine hydroxylase (TH).

6

METHODS ANIMALS All animal handling procedures were approved by the LECOM IACUC (protocol #16-02). Sprague Dawley rats were raised and maintained in the laboratory vivarium on a 12 hour light/dark cycle with unrestricted access to food and water.

Exposure to VPA was done as previously described (Main and Kulesza, 2017; Zimmerman et al., 2018). Briefly, animals engaged in timed mating and pregnant females were randomly assigned to control or VPA-exposed groups. Pregnant females were fed meals of 3.1 g of peanut butter on the mornings (between 0700 and 0900 hrs) of embryonic days 7 through 12 (E7-12). On E10 and E12, females in the VPA group were fed peanut butter mixed with 800 mg/kg of VPA (figure 1A). Females in the control group received only the peanut butter meals. Both control and VPA-exposed females were permitted to deliver pups without interference (litters were not culled). At P21, litters were weaned and only male pups were included in the experiments described below as gender-specific effects of VPA exposure have been reported (Schneider et al., 2008). This report is based on the study of 11 control male rats (from 5 litters) and 8 VPA-exposed rats (from 3 litters) that ranged in age from P50-64.

FIXATION, SECTIONING AND HISTOLOGY Animals were anesthetized with isoflurane (5% isoflurane in O2 at 1 liter/min) and perfused through the left ventricle with 0.9% NaCl followed by 4% paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer (PB; pH 7.4). Brains were dissected from the skull, trimmed to a block that extended from the dorsal cochlear nucleus through the superior colliculus, weighed and the right side was marked with a rostrocaudal register pin track. Brainstems were stored in PFA in PB (at 4°C) for at least 24 hours.

Approximately 24 hours before sectioning, brainstems were submerged in cryoprotectant (30% sucrose in PFA PB) at room temperature. Brainstems used for morphology and estimation of neuronal number were 7

sectioned in the coronal plane on a freezing microtome at a thickness of 50 μm into 2 alternating series. Series 1 was collected in a rostral to caudal sequence into PB and mounted onto glass slides from gelatin alcohol and dried at room temperature. These sections were stained for Nissl substance with Giemsa (Sigma-Aldrich, St Louis, MO), dehydrated through ascending alcohols, cleared and coverslipped with Permount (ThermoFisher Scientific, Waltham, MA). Sections from series 2 were either processed for myelin staining using a modification of Mahon’s method (Jebb and Woolsey, 1977) or saved for immunofluorescence (see below).

PERIKARYA MORPHOLOGY The morphology of neuronal cell bodies in the NLL and CNIC were compared from control and VPAexposed animals (n=4 each, 3 control litters and 2 VPA litters). For the CNIC, at least 200 neurons were traced per animal; for the NLL at least 35 neurons were traced per animal. The NLL were identified in accordance with previous descriptions (Kelly et al., 2009; Ito and Oliver, 2010) and the CNIC was designated as per Loftus (Loftus et al., 2008). Perikarya were traced from Giemsa-stained sections by an observer blind to experimental condition. Neuronal profiles were traced using a 60x objective (800x final magnification) on an Olympus BX45 microscope. Neuronal profiles were digitized and analyzed with ImageJ (1.5g). The total number of neuronal profiles analyzed is provided in table 1. For neuronal profiles in the CNIC, the angle of the long axis was also analyzed. Reference arrows for this measurement are provided in figure 2A. CNIC neurons were sampled and analyzed according to location within the CNIC (dorsal, central and ventral thirds). The equation below was used to calculate a circularity measurement for all neuronal profiles: 4π ∗ Area Perimeter 2 Categorization of perikaryon shape was made using objective, morphometric criteria as described previously (Beebe et al., 2014; Kulesza, 2014; Lukose et al., 2015; Ruby et al., 2015; Foran et al., 2017). A profile was classified as round/oval if the circularity value was > 0.60. Neuronal profiles were

8

categorized as fusiform (i.e. elongated or spindle-shaped) if the major axis was at least three times longer than the minor axis. Profiles that did not fit the criteria for round/oval or fusiform were classified as stellate and were characteristically triangular or star-shaped.

NEURONAL ESTIMATES The total number of neurons in the NLL and CNIC were estimated in control and VPA-exposed animals (right and left sides, n=3 animals each) by a researcher blind to condition. For each region of interest, cell body density was estimated by counting neuronal profiles in evenly-spaced sections along the rostrocaudal extent of each nucleus. These neuronal counts were corrected to account for profile splitting using the equation below (Konigsmark, 1970; Thompson and Brenowitz, 2005; Kulesza, 2008; Wagoner and Kulesza, 2009; Kulesza et al., 2011; Lukose et al., 2011; 2015; Foran et al., 2017): N=n

t t + 2a

In this equation, N is the corrected profile count, n is the number of counted profiles, t is thickness of the tissue sections (50 µm) and “a” is the square root of r2 - (k/2)2. In calculation of “a” we used the nucleolus correction factor (Konigsmark, 1970). In this expression, r is the mean nucleoli radius and k is the diameter of observed nucleoli fragments. This results in a correction factor of 0.92. The density of neuronal cell bodies was calculated by dividing the corrected neuron counts (N) by a calculated nucleus volume. The total number of neurons in each nucleus was finally estimated by multiplying neuronal density by estimated nucleus volume (Thompson and Brenowitz, 2005). This method for estimation of neuron produces results statistically similar to stereological techniques (Thompson and Brenowitz, 2005; Kulesza, 2007; Zimmerman et al., 2018).

IMMUNOFLUORESCENCE Brains from 5 control animals (2 litters) and 5 VPA-exposed animals (from 3 litters) were processed for immunofluorescence. Brains were frozen sectioned at a thickness of 50 µm into 3 wells and control and

9

VPA-exposed tissue were processed in parallel. Additional tissue sections were included from series 2 (see above). Sections from all 3 wells were rinsed in PB and incubated in a solution of 1% normal donkey serum (NDS; AbCam, Cambridge, MA) and 0.5% Triton-X for 1 hour at room temperature. Sections from wells 1 and 2 were incubated overnight (>20 hours) in 1% NDS and primary antisera (well 1: rabbit anti-calbindin [CB], 1:1000, AbCam [ab112]; well 2: rabbit anti-tyrosine hydroxylase [TH], 1:1000, AbCam, [ab112]). These sections were rinsed three times in PB and then incubated for at least 2 hours in secondary antisera (1:100; goat anti-rabbit DyLight 488, Vector Labs, Burlingame, CA). Sections from well 3 were processed for wisteria floribunda agglutinin (WFA) histochemistry with a 2 hour incubation in fluorescein-tagged WFA (WFA; 20 µg/ml; Vector Laboratories). Sections from all three wells were rinsed, incubated in a 1:100 solution of Neurotrace Red (NTR; a fluorescent Nissl stain) for 20 minutes, rinsed, mounted onto glass slides from PB, allowed to air-dry and coverslipped with Entellan. Tissue sections processed without primary antisera failed to reveal any fluorescent signal. Photomicrographs were taken with a DP71 digital camera on an Olympus CKX41 microscope or a Leica TCS SP5 confocal microscope.

Estimating the number of CB+ positive neurons or WFA PNNs in each nucleus was done by counting CB+ neurons or WFA PNN ensheathed neurons and then dividing this by the total number of Nisslstained neurons (revealed with NTR). The total number of neurons counted for these analyses are provided in table 1; these counts are based on multiple values/tissue sections from individual animals. Additionally, the density of TH immunoreactive (IR) punctate profiles was counted in the NLL and CNIC. These counts were made from overlayed images with both TH and NTR labeling using a 100 µm x 100 µm counting frame. The number of TH-IR puncta was converted to a density (puncta per 0.01 mm2). Both perisomatic and peridendritic TH-IR profiles were included in this analysis. The number of TH-IR profiles counted in each nucleus is provided in table 1.

STATISTICAL ANALYSIS 10

GraphPad Prism 6 (GraphPad Software, La Jolla, CA) was used to generate descriptive statistics, perform statistical comparisons and graph all continuous variables. Data sets that fit a normal distribution (p > 0.5, D’Agostino & Pearson omnibus normality test) were compared using a t-test or ANOVA; results are shown in the text and figures as mean ± standard deviation (SD). Additionally, data sets were screened for unequal variances (F test for variances). When variances were significantly different, Welch’s correction was applied. Data sets that failed to fit a normal distribution (p < .05, D’Agostino & Pearson omnibus normality test) were compared using non-parametric tests (Mann-Whitney U) and results are shown in the text as median and 95% confidence interval of the median (CI) and graphed as boxplots. The proportion of cell body morphologies (round/oval, fusiform, stellate) in each nucleus was compared using a Chisquare or Fisher’s exact test. Regardless of the statistical test used, differences were considered statistically significant if p values were <0.05.

RESULTS VPA EXPOSURE RESULTED IN FEWER CNIC NEURONS Consistent with previous reports (Main and Kulesza, 2017; Zimmerman et al., 2018) we found significantly lower brain weights after VPA exposure (at P50). In control animals, the brainstem weighed 0.56 ± .02 g. After VPA exposure, brainstems weighed only 0.51 ± .02 g (t10=3.37, p<.01; figure 1B).

VPA exposure resulted in a notable reduction in the size of the CNIC and reduced neuronal packing density (figure 2A, B). However, there were significantly larger neurons in the CNIC after VPA exposure (control 79 μm2; CI 77-81; VPA 116 μm2; CI 112-119; U1000, 924=195947, p < .0001; figure 2C, D, asterisk in D, 3A-C). We examined neuronal morphology across the dorsal-ventral axis of the CNIC (figure 3C). In control animals, CNIC neurons were largest in the ventral region and smallest in the center of the nucleus (figure 3C). Across the ventral (control: 84 μm2 ; VPA: 119 μm2) central (control: 72 μm2 ; VPA: 119 μm2), and dorsal regions (control: 79 μm2 ; VPA: 108 μm2) of the CNIC, neurons were larger after VPA exposure (H6,1926=215, p < .0001; figure 3C). In control animals, neurons in the CNIC had a mean 11

orientation angle of 84° (CI 77-89) and those in VPA-exposed animals had a mean orientation angel of 87° (CI 81-95; figure 3D). These populations were asymmetrically distributed (kurtosis = -1.3 and -1.25, respectively), but were statistically similar (U1000, 924=448642, p = .26). In control animals, the CNIC was composed of 82% round/oval neurons, 17% stellate neurons and 1% fusiform neurons (figure 3E). After VPA exposure, the CNIC includes 89% round/oval neurons, 10% stellate neurons and 1% fusiform neurons (figure 3E). These differences were not statistically significant (Fisher’s exact, p = .15). In control animals, the CNIC included 201,617 ± 28,331 neurons (mean ± SD) but after VPA exposure only 139,855 ± 21,886 neurons (figure 2A, B; 3F). This constitutes a decrease of nearly 31% (~61,000 neurons) and was statistically significant (t14=4.88, p < .001).

VPA EXPOSURE RESULTED IN FEWER NLL NEURONS After VPA exposure, the NLL were smaller and had markedly lower neuronal packing density and there were significantly larger neurons in the DNLL (figure 4, 5). In control animals, DNLL neurons had a cross sectional area of 183.2 μm2 (CI 175-198), while in VPA-exposed animals DNLL neurons were 215 μm2 (CI 201-227; U139, 130=6561, p = .0001; figure 4C, D; 5A). VPA exposure did not significantly impact the distribution of cell body morphologies in the DNLL (2, p > .05). Although there was a trend for fewer round/oval neurons (control: 69%; VPA: 59%) and more stellate neurons (control: 20%; VPA: 35%) after VPA exposure. Only a small number of fusiform neurons were found in the DNLL in control (11%) or VPA-exposed animals (7%). However, VPA exposure resulted in fewer neurons in the DNLL (figure 4C, D; 5C). In control animals the DNLL had 1,792 ± 310 neurons (figure 5C). After VPA exposure the DNLL had only 1,110 ± 181. This is a decrease of nearly 700 neurons (39%) and was significant (t10=4.64, p < .001; figure 5C).

VPA exposure did not significantly impact the size of neurons in the INLL (control 72.4 μm2; CI 68-79; VPA 75.9 μm2; CI 71-80; U235, 230= 26766, p = .85; figure 4A, B; 5B) or the distribution of cell body morphologies (Chi square, p = .60). In both control and VPA-exposed animals, the INLL was composed 12

of 51-52% round/oval neurons, 30-34% stellate neurons and 14-19% fusiform neurons. However, VPA exposure resulted in fewer neurons in the INLL (figure 5D). In control animals, the INLL had 5,589 ± 734 neurons. After VPA exposure the INLL had only 2,563 ± 266. This is a decrease of approximately 3,000 neurons (55%) and was significant (t10=9.47, p < .0001; figure 5D).

VPA exposure did not impact the size of VNLL neurons (control 71.7 μm2; CI 67-74; VPA 69.4 μm2; CI 66-73; U277, 264=35524, p = .56; figure 4E, F; 5B) or the distribution of cell body morphologies (Chi square, p = .07). In both control and VPA-exposed animals, the VNLL was composed of 64-65% round/oval neurons, 22-31% stellate neurons and 5-13% fusiform neurons. VPA exposure resulted in fewer neurons in the VNLL (figure 5E). In control animals the VNLL had 16,124 ± 2,008 neurons (figure 5E). After VPA exposure the VNLL had only 7,358 ± 1,401. This is a decrease of nearly 9,000 neurons (55%) and was significant (t11=8.96, p < .001; figure 5E).

VPA EXPOSURE RESULTED IN FEWER CB+ DNLL NEURONS Since VPA exposure has been shown to result in fewer CB+ neurons in the VCN and medial nucleus of the trapezoid body (MNTB), we examined CB in the NLL and CNIC. In the NLL and CNIC of both control and VPA-exposed animals, CB+ neurons were most abundant and consistently found in the DNLL (figure 6). In control animals, 59.8 ± 6.5% of DNLL neurons were CB+ (figure 6). In VPAexposed brains there was less abundant CB-immunolabeling and significantly fewer CB+ neurons (25.5 ± 16.1%; t11=5.87 [with Welch’s correction], p < .0001; figure 6A-C). There appeared to be no preferential distribution of CB+ neurons in the DNLL of control or VPA-exposed animals: CB+ neurons were dispersed throughout the nucleus in clusters and adjacent to clusters of CB-immunonegative neurons. CB+ DNLL neurons in both control and VPA-exposed animals had CB immunoreactivity in somatic, dendritic and axonal compartments (figure 6A, B). CB+ axons in the dorsal aspect of the LL (see arrowheads in figure 6A and B), had significantly smaller diameters in VPA-exposed animals (control: 1.3 ± .34µm, VPA: 1.14 ± .32 µm; t209=2.66, p < .01; figure 6D). 13

VPA EXPOSURE HAD NO IMPACT ON THE NUMBER OF WFA PNNS Since PNNs are known to develop in an activity dependent manner and there appears to be hyperactivity in the IC after VPA exposure, we examined PNNs in the NLL and CNIC. WFA immunohistochemistry revealed PNNs throughout the pontine isthmus and midbrain, but were not found equally in auditory centers and were not uniformly distributed in the CNIC (figure 7A, B). PNNs were more abundant in the ventromedial (VM) CNIC and less numerous in the dorsolateral (DL) CNIC (figure 7A and B, arrows; C, D). In both control and VPA-exposed animals this gradient in WFA PNNs was significant (control: t test, p = .0093; VPA: t test with Welch’s correction, p = .0014). Specifically, there were more PNNs in the VL CNIC than in DL CNIC in both control and VPA brains (figure 7C, D, G). However, there was no difference in the distribution of WFA PNNs in the VM (control: 11.23 ± 4.7%, VPA: 11.72 ± 3.7%, t10=.19, p = .84) or DL IC (control: 3.76 ± 1.8%, VPA: 2.76 ± 1.3%, t10=1.09, p = .30; figure 7G) between control and VPA-exposed animals.

In both control and VPA-exposed animals, WFA PNNs were rare in the VNLL and INLL. Only 4.134.2% of neurons in the VNLL and INLL were ensheathed with WFA PNNs (VNLL: t4=.27, p = .98; INLL: t2=.01, p = .98; figure 7H). However, WFA PNNs were more abundant in the DNLL and in both control and VPA-exposed animals, PNNs surrounded cell bodies and primary dendrites (figure 7E, F). In control and VPA-exposed animals, the DNLL had 25-27% of neurons with WFA PNNs (t14=1.3, p = .21; figure 7H).

VPA EXPOSURE RESULTED IN REDUCED TH INNERVATION TO THE CNIC We examined the density of TH+ puncta, presumably representing dopaminergic inputs, in the NLL and CNIC since VPA exposure has been shown to result in fewer TH+ puncta in the VCN and SOC. In both control and VPA-exposed animals, there were more TH+ puncta in the CNIC than the NLL (figure 8). There were significantly fewer TH+ puncta in the CNIC after VPA exposure (figure 8A, B). Control 14

animals had 80.1 ± 18.7 TH+ puncta per .01 mm2 while VPA-exposed animals had only 59.6 ± 10.1 (F7, 127=20.64, p < .0001; figure 8C). Within control and VPA-exposed animals there was no difference in the density of TH+ puncta between the DNLL, INLL or VNLL (F2, 33 = .84, p = .43) and there was no difference between control and VPA-exposed animals (F5, 65 = 1.88, p = .11; figure 8C).

DISCUSSION This report provides the first evidence that in utero VPA exposure results in significantly fewer neurons, dysmorphology, decreased CB-immunolabeling and reduced dopaminergic input to the auditory midbrain. These findings are consistent with previous reports that VPA impacts structure and function of the auditory hindbrain and cortex (Lukose et al., 2011; Gandal et al., 2010; Engineer et al., 2014; Anomal et al., 2015; Dubiel and Kulesza, 2016; Zimmerman et al., 2018). Together, these studies provide evidence that in utero VPA exposure impacts nearly the entire central auditory system. The IC is a major relay station along the ascending auditory pathway and in rats, contains nearly five times more neurons than all of the subcollicular nuclei combined (Kulesza et al., 2002). Accordingly, there is a massive divergence of both excitatory and inhibitory ascending input to the CNIC contributing to complex processing networks in the IC (Chen et al., 2018; He et al., 2017). We will demonstrate below that VPA exposure has a nonuniform impact on auditory brainstem nuclei and that this differential effect appears to disrupt the normal balance of excitatory and inhibitory influence to the CNIC.

VPA HAS A HETEROGENEOUS IMPACT ON NEURON NUMBER Our neuronal estimates indicate that VPA exposure results in significantly fewer neurons in nearly all auditory brainstem nuclei, but the size of this effect varies by nucleus. Changes in neuronal number range from a 25% decrease in the LSO to decreases of 55% in the VNLL and INLL and 57% fewer neurons in the MSO. However, we have failed to find any significant loss of neurons in either the ventral or lateral nuclei of the trapezoid body (Zimmerman et al., 2018). We interpret these findings to suggest that in utero VPA does not simply cause a uniform loss of neurons throughout the brainstem or auditory pathway. 15

Further, it is important to recognize that the auditory brainstem nuclei vary considerably in the size of their neuronal populations. In control animals, the MSO includes 1,201 neurons. After VPA exposure, there is a loss of ~57% of MSO neurons and this equates to a loss of only about 684 neurons (Zimmerman et al., 2018). However, the CNIC includes over 201,000 neurons. VPA exposure results in a loss of ~31% of CNIC neurons and this equates to a loss of over 61,000 neurons. Based on previous work (Saldaña and Merchán, 1992; Merchán et al., 2005), the contralateral IC is the largest single source of input to the CNIC. How this differential loss of neurons impacts innervation patterns or neuronal response properties in the CNIC remains to be investigated.

Since we have examined all of the major auditory brainstem nuclei, we can now summarize the impact of VPA on the auditory brainstem. In figure 9A, we present the number of brainstem neurons in control and VPA-exposed animals (CN and SOC data is taken from Zimmerman et al., 2018). This figure highlights the large number of neurons lost from the CNIC, VCN and VNLL. Excluding the dorsal cochlear nucleus, the auditory brainstem includes over 266,000 neurons in control animals. After VPA exposure, this drops to 179,000. Thus, VPA exposure results in a loss of 33% of the neurons in the auditory brainstem nuclei. Of particular significance is that 71% of the neurons lost after VPA exposure were from the CNIC and that together, the NLL and CNIC account for 86% of the neurons lost after VPA exposure (figure 9B). Finally, if we normalize the number of neurons in the subcollicular nuclei to the mean number of neurons in the CNIC after VPA exposure, four nuclei (INLL, VNLL, SPON and MSO) stand out as having the most disproportionate neuronal loss (>43% of their neuronal population; figure 9C). We believe this observation provides evidence for a direct, deleterious effect of VPA on these nuclei. It is also worth noting that the VNLL projection to the CNIC is almost entirely inhibitory and that this nucleus accounts for nearly 35% of all neurons lost in the subcollicular auditory brainstem after VPA exposure (discussed further below). Based on known neurotransmitter profiles of these nuclei, it would appear that amongst these four severely affected nuclei, the balance of inhibitory neurons to excitatory neurons projecting to the CNIC is approximately 4:1 (Ito and Oliver, 2010). 16

The majority of neurons in the VCN, MSO, LSO, SPON and NLL project to the CNIC. Previous work utilizing subjects with ASD (Just et al., 2004) provides evidence for an overabundance of local axonal connections but decreased long-range projections over cortical networks. Accordingly, we hypothesize that long-range projections to the CNIC from the CN, SOC and NLL are diminished after VPA exposure and additionally that these nuclei have local aberrant axonal projections. Furthermore, both the LSO and DNLL make significant bilateral projections to the rat CNIC (Kelly et al., 1998; Kelly et al., 2009). We further hypothesize that VPA exposure results in abnormal patterns of ipsilateral and contralateral projections from the LSO and DNLL.

EXCITATORY AND INHIBITORY PROJECTIONS TO THE CNIC Alterations in the excitatory:inhibitory ratio are believed to be a key contributing factor to cellular and behavioral changes in ASD (Rubenstein and Merzenich, 2003; Fujimura et al., 2016). Accordingly, previous studies of VPA-exposed animals provide evidence for alterations in the balance of excitation and inhibition (Gogolla et al., 2009; Gandal et al., 2010). In fact, when VPA-exposed animals are treated with a glutamate receptor antagonist (MPEP, an antagonists at mGluR5), there is normalization of phase locking, prepulse inhibition and startle responses (Gandal et al., 2010). We have previously reported abnormal c-Fos immunolabeling in VPA-exposed animals after exposure to pure tone stimuli consistent with hyperactive responses (Dubiel and Kulesza, 2016). We hypothesized that this pattern of hyperactivity occurs as a result of excitatory:inhibitory imbalance. Both retrograde and anterograde tract tracing experiments, combined with characterization of neurotransmitter profile will be required to fully recognize and understand VPA-induced changes to auditory brainstem circuitry.

The drastic loss of neurons in the CNIC and VCN are particularly significant as these regions comprise the largest sources of excitatory projections to the CNIC (Ito and Oliver, 2010). In fact, the input to the CNIC from the contralateral IC likely outweighs the input from the CN and SOC, combined. The 17

proportion of VCN neurons targeting the CNIC versus the SOC/NLL is not clear but would seem to favor the CNIC projection about 2:1. From the VCN, the octopus cells and both globular and spherical bushy cells make significant projections to the SOC nuclei and there is also a large projection to the contralateral CNIC from stellate/multipolar cells. VPA exposure results in a loss of ~34% of VCN neurons (Zimmerman et al., 2018) but we have not been able to determine if any neuronal populations in the VCN are impacted differently. Regardless, such a loss of VCN neurons will impact processing of sound information by both SOC and IC neurons.

When we consider known neurotransmitter profiles and proportions of neurons projecting to the rat CNIC (Gonzalez-Hernandez et al., 1996; Ito and Oliver, 2010) it appears that VPA exposure results in a more extensive loss of inhibitory neurons. Specifically, we found a drastic loss of neurons in the VNLL, SPON and DNLL and these nuclei comprise the largest sources of ascending GABAergic/glycinergic inhibition to the CNIC (Adams and Mugnaini, 1984; Riquelme et al., 2001; Kulesza and Berrebi, 2000; Kulesza et al., 2002; Ito and Oliver, 2010). Furthermore, the ascending input to the CNIC from the VNLL is almost entirely inhibitory (>91%) and, based on our neuronal estimates, outnumbers other inhibitory ascending inputs to the CNIC from the SPON, INLL, ipsilateral LSO, and DNLL nearly 10:1. This drastic loss of inhibitory neurons is consistent with our observations of hyperactivation after auditory stimulation and enlarged tonotopic maps in the CNIC (Dubiel and Kulesza, 2016). Furthermore, VPA exposure, through inhibition of HDAC and subsequent epigenetic effects (Milutinovic et al., 2007; Tou et al., 2004), interferes with axonal growth of GABAergic neurons and results in a reduction in the number of inhibitory synapses (Kumamaru et al., 2014), results in decreased glutamic acid decarboxylase (GAD) 65 and 67 expression and GAD+ neurons (Fukuchi et al., 2009). Together, these actions of VPA likely contribute to our observed long-term changes in inhibitory brainstem networks.

VPA RESULTED IN LARGER DNLL AND CNIC NEURONS

18

Neurons in the rat CNIC are, based on dendritic arborizations, classified as flat or less flat and most dendritic trees are arranged parallel to fibrodendritic lamina of the CNIC (Malmierca et al., 1993, 1995a and b). Further, there is an abundant population of large GABAergic neurons in the CNIC (Roberts and Ribak, 1987; Merchán et al., 2005; Ito et al., 2009). Of particular note, there is a significant increase in the number of GABAergic neurons throughout the IC of the genetically epilepsy-prone rat (Roberts et al., 1985; Ribak and Roberts, 1986). The increased number of GABAergic neurons is believed to result in seizure activity through disinhibition along the ascending auditory pathway (Roberts and Ribak, 1986). VPA exposure resulted in significantly larger neuronal cell bodies across the dorsal-ventral axis of the CNIC. We hypothesize that the increased size of neurons in the CNIC after VPA-exposure might be caused by an increase in the number of larger neurons which are characteristically GABAergic. Interestingly, we observed significantly more c-Fos+ neurons in the CNIC in VPA-exposed animals after both silence and sound exposures (Dubiel and Kulesza, 2016). We interpret this finding to indicate hyperactivity in the CNIC and given our recent observations of marked loss of inhibitory neurons, we hypothesis that VPA-exposed animals may be susceptible to audiogenic seizures. Recent evidence suggests that in utero VPA exposure can result in increased susceptibility to cortical seizure activity (Sakai et al., 2018). Alternatively, the larger neurons in the both the CNIC and DNLL might result from neurons with larger, more complex or more expansive dendritic branching patterns (Friede, 1963). We believe that significantly larger dendritic trees across CNIC neurons could lead to individual neurons integrating a larger repertoire of inputs, wider tuning curves and larger and less precise tonotopic maps.

CALBINDIN Expression of CB in the auditory brainstem of the adult rat is restricted to select neuronal populations. Specifically, cochlear root neurons, select populations in the dorsal cochlear nucleus, octopus cells in the PVCN, principle neurons of the MNTB, and smaller proportions in the LSO, DNLL, ECIC and DCIC (Friauf, 1994; Pór et al., 2005). Neurons in the CNIC express CB only between P2 and P20 (Friauf, 1994). Additionally, cerebellar Purkinje cells characteristically express CB (Enderlin et al., 1987). VPA 19

exposure has been shown to result in decreased expression of CB in Purkinje cells, octopus cells and the MNTB (Main and Kulesza, 2017; Zimmerman et al., 2018). In control animals, 76% of octopus cells are CB+, however in VPA-exposed animals only 33% of these neurons are CB+. Octopus cells send axonal projections to the VNLL and SPON; the main axon terminates in the ventral aspect of the VNLL as a calyx terminal (Thompson and Thompson, 1991; Thompson, 1998). In control animals, 91% of MNTB neurons were CB+, but after VPA exposure this dropped to 80% (Zimmerman et al., 2018) and CB immunelabeling was often restricted to the nucleus in octopus cells and MNTB neurons. In the current study, we found a significant reduction in the proportion of CB+ neurons in the DNLL. We observed no differences in the pattern of CB labeling within the cell body or dendritic compartments of DNLL neurons. We interpret this finding to suggest that the epigenetic effects of VPA on CB expression in the DNLL is less severe than in the MNTB or octopus cells. Additionally, we found that CB+ axons in the dorsal LL, the majority of which are likely derived from the DNLL, had significantly smaller diameters. While we found a significant reduction in the number of DNLL neurons after VPA exposure, we also found that these neurons had larger cell bodies. We interpret these findings to suggest that, after VPA exposure, DNLL neurons have larger, more complex dendritic arbors but axons with a smaller, more restricted terminal distribution in the CNIC. In a mouse model of VPA exposure, there is evidence of displacement of CB+ neurons in the striatum (Kuo and Liu, 2017). While we hypothesize the reduced CB immunolabeling in the octopus cell region, MNTB and DNLL is due to epigenetic effects of VPA and reduced expression of CB, it is possible there is some aberrant migration of CB+ neurons and ectopic displacement during development. Prenatal VPA exposure is also known to impact neurons that express the calcium binding protein, parvalbumin (PV). Specifically, there is a loss of PV+ neurons in the cerebral cortex and striatum after in utero VPA exposure (Gogolla et al., 2009; Lauber et al., 2016). In the striatum and cortex, PV+ neurons are characteristically GABAergic, commonly deficient in ASD and play roles in the temporal window of the cortical critical period and synchronization of cortical activity (Gogolla et al., 2009). However, CB is not a reliable marker for neurotransmitter in the auditory system: octopus cells are

20

glutamatergic, MNTB neurons are glycinergic and DNLL neurons are GABAergic (reviewed in Altschuler and Shore, 2010).

WFA Perineuronal nets are not present around neurons at birth, develop in an activity dependent manner over the first few postnatal weeks, have been identified throughout the auditory brainstem (Balmer, 2016; Myers et al., 2012; Foster et al., 2014) and specific binding of WFA has revealed PNNs associated with specific cell types. Specifically, in the guinea pig CNIC PNNs are preferentially associated with GABAergic neurons and are more abundant in the ventral aspect of the CNIC where neurons predominantly encode high frequency tones (Foster et al., 2014). PNNs have been associated with neurons that require fast spiking capabilities (Morris and Henderson, 2000; Balmer, 2016). Since we have observed hyperactivity in the CNIC after VPA exposure and PNNs are not uniformly distributed in the CNIC, we rationalized that VPA exposure might impact the number of neurons with PNNs. However, we found no significant changes in the number or distribution of PNNs in the DNLL or CNIC. Likewise, VPA exposure has no impact on the number of PNNs in the striatum (Lauber et al., 2016). It should be noted that WFA histochemistry reveals only a single component of PNNs and other markers reveal often different PNN patterns. Regardless, we interpret these findings to suggest that the metabolic and activitydependent events required for normal PNN development have occurred in VPA-exposed animals by P50.

TYROSINE HYDROXYLASE We recently found a significant decrease in the number of TH+ axon terminals in the VCN, LSO and MSO of VPA-exposed animals (Zimmerman et al., 2018). Herein, we report a significant reduction in the density of TH+ terminals in the CNIC, although there were no changes in the NLL. The source of TH+ terminals in the CNIC has recently been determined to be the subparafasicular nucleus (SPF) and this dopaminergic projection is mainly ipsilateral (Nevue et al., 2016a). Furthermore, the SPF also sends a dopaminergic input to the SPON and MNTB, although TH+ inputs to the LSO appear to arise from 21

another source (Nevue et al., 2016b). In both the CN and IC, TH is found in close association with both glycine and glutamate transporters and is believed to modulate neuronal responses to these neurotransmitters (Fyk-Kolodziej et al., 2015). Thus, it appears there is a robust dopaminergic innervation to the rat CNIC and that this input might be capable of complex modulation of midbrain circuits. Further, we provide evidence that this input is significantly reduced after VPA exposure. However, the functional consequences of this change remains to be investigated, but may result in abnormal modulation of IC circuits in response to behaviorally relevant stimuli.

These results, together with our previous work, indicate that prenatal VPA significantly impacts structure and function of the auditory brainstem. We have demonstrated that VPA exposure has different effects upon ascending circuits but overall, appears to preferentially impact nuclei with inhibitory projections to the CNIC. Throughout the auditory brainstem, we have also found significantly fewer CB+ neurons and diminished dopaminergic innervation after VPA exposure. Together, these findings indicate marked hypoplasia and abnormal neurochemistry of the auditory brainstem after VPA exposure. These findings led us to new questions. In particular, it is unclear how VPA impacts axonal projections of ascending glutamatergic, GABAergic and glycinergic neurons from the auditory brainstem or how VPA exposure might impact brainstem processing of sound.

ACKNOWLEDGMENTS The authors would like to thank the Lake Erie College of Osteopathic Medicine Research Collective, Dr Diana Speelman for constructive comments, Mary Petro for technical assistance and Jerome McGraw (Penn State Behrend) for technical assistance with confocal microscopy. This work was supported by the Lake Erie College of Osteopathic Medicine and the Lake Erie Consortium for Osteopathic Medical Training.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST 22

The authors declare that they have no conflicts of interest.

REFERENCES Adams JC, Mugnaini E (1984) Dorsal nucleus of the lateral lemniscus: a nucleus of GABAergic projection neurons. Brain Res Bull. Oct;13(4):585-90.

Alcántara JI, Weisblatt EJ, Moore BC, Bolton PF (2004) Speech-in-noise perception in high-functioning individuals with autism or Asperger's syndrome. J Child Psychol Psychiatry. Sep;45(6):1107-14.

Allen DA (1988) Autistic spectrum disorders: clinical presentation in preschool children. J Child Neurol. 3, 48–56 (Suppl.).

Altschuler RA, Shore SE. Central auditory neurotransmitters In: The Oxford Handbook of Auditory Science: The Auditory Brain (2010) Edited by Alan R. Palmer and Adrian Rees

Anomal RF, de Villers-Sidani E, Brandão JA, Diniz R, Costa MR, Romcy-Pereira RN (2015) Impaired Processing in the Primary Auditory Cortex of an Animal Model of Autism. Front Syst Neurosci. 2015 Nov 16;9:158.

American Psychiatric Association (2013) Diagnostic and statistical manual of mental disorders, Washington, DC.

Azouz HG, Kozou H, Khalil M, Abdou RM, Sakr M. (2014) The correlation between central auditory processing in autistic children and their language processing abilities.Int J Pediatr Otorhinolaryngol. 2014 Dec;78(12):2297-300. 23

Balmer TS. (2016) Perineuronal Nets Enhance the Excitability of Fast-Spiking Neurons. eNeuro. Jul 27;3(4).

Beebe K, Wang Y, Kulesza R (2014) Distribution of fragile X mental retardation protein in the human auditory brainstem. Neuroscience. 273:79-91.

Bennetto L, Keith JM, Allen PD, Luebke AE (2017) Children with autism spectrum disorder have reduced otoacoustic emissions at the 1 kHz mid-frequency region. Autism Res. Feb;10(2):337-345.

Bolton PF, Golding J, Emond A, Steer CD (2012) Autism spectrum disorder and autistic traits in the Avon Longitudinal Study of Parents and Children: precursors and early signs. J Am Acad Child Adolesc Psychiatry. Mar;51(3):249-260.e25

Bromley RL, Mawer GE, Briggs M, Cheyne C, Clayton-Smith J, García-Fiñana M, Kneen R, Lucas SB, Shallcross R, Baker GA; Liverpool and Manchester Neurodevelopment Group (2013) The prevalence of neurodevelopmental disorders in children prenatally exposed to antiepileptic drugs. J Neurol Neurosurg Psychiatry. Jun;84(6):637-43.

Brown WT, Friedman E, Jenkins EC, Brooks J, Wisniewski K, Raguthu S, French JH. (1982) Association of fragile X syndrome with autism. Lancet. Jan 9;1(8263):100.

CDC.gov. http://www.cdc.gov/ncbddd/autism/index.html. (Accessed 24 July 2018).

Chen C, Cheng M, Ito T, Song S. (2018) Neuronal Organization in the Inferior Colliculus Revisited with Cell-Type-Dependent Monosynaptic Tracing. J Neurosci. Mar 28;38(13):3318-3332. 24

Christensen J, Gronborg TK, Sorensen MJ (2013) Prenatal valproate exposure and risk of autismspectrum disorders and childhood autism. JAMA 309(16):1696–1703.

Christianson AL, Chesler N, Kromberg JG. (1994) Fetal valproate syndrome: clinical and neurodevelopmental features in two sibling pairs. Dev Med Child Neurol. Apr;36(4):361-9.

DiLiberti JH, Farndon PA, Dennis NR, Curry CJ. (1984) The fetal valproate syndrome. Am J Med Genet. Nov;19(3):473-81.

Dubiel A, Kulesza Jr RJ (2016) Prenatal valproic acid exposure disrupts tonotopic c-Fos expression in the rat brainstem. Neuroscience 324:511–523.

Enderlin S, Norman AW, Celio MR. (1987) Ontogeny of the calcium binding protein calbindin D-28k in the rat nervous system. Anat Embryol (Berl).177(1):15-28

Engineer CT, Centanni TM, Im KW, Borland MS, Moreno NA, Carraway RS, Wilson LG, Kilgard MP (2014) Degraded auditory processing in a rat model of autism limits the speech representation in nonprimary auditory cortex. Dev Neurobiol 74 (10):972–986.

Erturk O, Korkmaz B, Alev G, Demirbilek V, Kiziltan M (2016) Startle and blink reflex in high functioning autism. Neurophysiol Clin. Jun;46(3):189-92

Foran L, Blackburn K, Kulesza RJ (2017) Auditory hindbrain atrophy and anomalous calcium binding protein expression after neonatal exposure to monosodium glutamate. Neuroscience. Mar 6;344:406-417.

25

Foster NL, Mellott JG, Schofield BR. (2014). Perineuronal nets and GABAergic cells in the inferior colliculus of guinea pigs. Front Neuroanat. Jan 8;7:53.

Friauf E. (1992) Tonotopic Order in the Adult and Developing Auditory System of the Rat as Shown by c-fos Immunocytochemistry. Eur J Neurosci. 4(9):798-812.

Friauf E (1994) Distribution of calcium-binding protein calbindin-D28k in the auditory system of adult and developing rats. J Comp Neurol. Nov 8;349(2):193-211.

Friede RL (1963) The relationship of body size, nerve cell size, axon length, and glial density in the cerebellum. Proc Natl Acad Sci USA 49:187–193.

Fujimura K, Mitsuhashi T, Shibata S, Shimozato S, Takahashi T (2016) In Utero Exposure to Valproic Acid

Induces

Neocortical

Dysgenesis

via

Dysregulation

of

Neural

Progenitor

Cell

Proliferation/Differentiation. J Neurosci. Oct 19;36(42):10908-10919.

Fukuchi M, Nii T, Ishimaru N, Minamino A, Hara D, Takasaki I, Tabuchi A, Tsuda M. (2009) Valproic acid induces up- or down-regulation of gene expression responsible for the neuronal excitation and inhibition in rat cortical neurons through its epigenetic actions. Neurosci Res. Sep;65(1):35-43.

Fyk-Kolodziej BE, Shimano T, Gafoor D, Mirza N, Griffith RD, Gong TW, Holt AG (2015) Dopamine in the auditory brainstem and midbrain: co-localization with amino acid neurotransmitters and gene expression following cochlear trauma. Front Neuroanat. Jul 22;9:88.

26

Gandal MJ, Edgar JC, Ehrlichman RS, Mehta M, Roberts TP, Siegel SJ (2010) Validating γ oscillations and delayed auditory responses as translational biomarkers of autism. Biol Psychiatry. Dec 15;68(12):1100-6.

Gillberg C, Rosenhall U, Johansson E (1983) Auditory brainstem responses in childhood psychosis. J. Autism Dev. Disord. 13 (2), 181–195 (Jun).

Gogolla N, Leblanc JJ, Quast KB, Südhof TC, Fagiolini M, Hensch TK. (2009) Common circuit defect of excitatory-inhibitory balance in mouse models of autism. J Neurodev Disord. Jun;1(2):172-81.

Gomes E, Pedroso FS, Wagner MB (2008) Auditory hypersensitivity in the autistic spectrum disorder. Pro Fono. Oct-Dec;20(4):279-84.

Gonzalez-Hernandez, T., Mantolan-Sarmiento, B., Gonzalez-Gonzalez, B., Perez-Gonzalez, H (1996) Sources of GABAergic input to the inferior colliculus of the rat. J Comp Neurol 372, 30926.

Gravel JS, Dunn M, Lee WW, Ellis MA (2006) Peripheral audition of children on the autistic spectrum. Ear Hear. (3), 299–312.

Greenspan SI, Wieder S (1997) Developmental patterns and outcomes in infants and children with disorders in relating and communicating: a chart review of 200 cases of children with autistic spectrum diagnoses. J. Dev. Learn. Disord. 1, 87–141.

He N, Kong L, Lin T, Wang S, Liu X, Qi J, Yan J. (2017) Diversity of bilateral synaptic assemblies for binaural computation in midbrain single neurons. Hear Res. Nov;355:54-63. 27

Ito T, Oliver DL. (2010) Origins of Glutamatergic Terminals in the Inferior Colliculus Identified by Retrograde Transport and Expression of VGLUT1 and VGLUT2 Genes. Front Neuroanat. Sep 28;4:135.

Ito T, Bishop DC, Oliver DL. (2009) Two classes of GABAergic neurons in the inferior colliculus. J Neurosci. Nov 4;29(44):13860-9

Jebb AH, Woolsey TA. (1977) A simple stain for myelin in frozen sections: a modification of Mahon's method. Stain Technol. Nov;52(6):315-8.

Just MA, Cherkassky VL, Keller TA, Minshew NJ. (2004) Cortical activation and synchronization during sentence comprehension in high-functioning autism: evidence of underconnectivity. Brain. Aug;127(Pt 8):1811-21.

Källstrand J, Olsson O, Nehlstedt SF, Sköld ML, Nielzén S. (2010) Abnormal auditory forward masking pattern in the brainstem response of individuals with Asperger syndrome. Neuropsychiatr Dis Treat. Jun 24;6:289-96.

Kelly JB, Liscum A, van Adel B, Ito M. (1998) Projections from the superior olive and lateral lemniscus to tonotopic regions of the rat's inferior colliculus. Hear Res. Feb;116(1-2):43-54.

Kelly JB, van Adel BA, Ito M. (2009) Anatomical projections of the nuclei of the lateral lemniscus in the albino rat (Rattus norvegicus). J Comp Neurol. Feb 1;512(4):573-93.

Khalfa S, Bruneau N, Rogé B, Georgieff N, Veuillet E, Adrien JL, Barthélémy C, Collet L (2001) Peripheral auditory asymmetry in infantile autism. Eur. J. Neurosci. 13 (3), 628–632 (Feb). 28

Khalfa S, Bruneau N, Roge B, Georgieff N, Veuillet E, Adrien JL, Barthelemy C, Collet L (2004) Increased perception of loudness in autism. Hear Res. 198 (1–2), 87–92.

Klin A (1993) Auditory brainstem responses in autism: brainstem dysfunction or peripheral hearing loss? Autism Dev. Disord. 23 (1), 15–35.

Konigsmark, BW (1970) Methods for counting of neurons. In: Nauta, W.J.H., Ebbesson, S.O.E. (Eds.), Contemporary Research Methods in Neuroanatomy.Springer, Heidelberg, pp. 315–338.

Koren G, Nava-Ocampo AA, Moretti ME, Sussman R, Nulman I (2006) Major malformations with valproic acid. Can Fam Physician 52. 441-2, 444, 447.

Kulesza RJ Jr, Berrebi AS. (2000) Superior paraolivary nucleus of the rat is a GABAergic nucleus. J Assoc Res Otolaryngol. Dec;1(4):255-69.

Kulesza RJ, Viñuela A, Saldaña E, Berrebi AS. (2002) Unbiased stereological estimates of neuron number in subcortical auditory nuclei of the rat. Hear Res. Jun;168(1-2):12-24.

Kulesza RJ, Mangunay K (2008) Morphological features of the medial superior olive in autism. Brain Research 1200 (132-137)

Kulesza Jr RJ, Lukose R, Stevens LV (2011) Malformation of the human superior olive in autistic spectrum disorders. Brain Res. 1367, 360–371.

29

Kulesza RJ Jr (2007) Cytoarchitecture of the human superior olivary complex: medial and lateral superior olive. Hear Res. 225(1-2):80-90.

Kulesza RJ Jr (2008) Cytoarchitecture of the human superior olivary complex: nuclei of the trapezoid body and posterior tier. Hear Res. 241(1-2):52-63.

Kulesza RJ Jr (2014) Characterization of human auditory brainstem circuits by calcium-binding protein immunohistochemistry. Neuroscience. 258:318-31.

Kumamaru E, Egashira Y, Takenaka R, Takamori S (2014) Valproic acid selectively suppresses the formation of inhibitory synapses in cultured cortical neurons. Neurosci Lett. May 21;569:142-7.

Kuo HY, Liu FC. (2017) Valproic acid induces aberrant development of striatal compartments and corticostriatal pathways in a mouse model of autism spectrum disorder. FASEB J. Oct;31(10):4458-4471.

Kwon S, Kim J, Choe BH, Ko C, Park S (2007) Electrophysiologic assessment of central auditory processing by auditory brainstem responses in children with autismspectrumdisorders. J. Korean Med. Sci. 22 (4), 656–659.

Lauber E, Filice F, Schwaller B. (2016) Prenatal Valproate Exposure Differentially Affects ParvalbuminExpressing Neurons and Related Circuits in the Cortex and Striatum of Mice. Front Mol Neurosci. Dec 21;9:150.

Lepistö T, Kujala T, Vanhala R, Alku P, Huotilainen M, Näätänen R (2005) The discrimination of and orienting to speech and non-speech sounds in children with autism. Brain Res. 1066 (1-2), 147–157 (Dec 20, Epub 2005 Dec 1). 30

Loftus WC, Malmierca MS, Bishop DC, Oliver DL. (2008) The cytoarchitecture of the inferior colliculus revisited: a common organization of the lateral cortex in rat and cat. Neuroscience. Jun 12;154(1):196205.

Lukose R, Schmidt E, Wolski TP Jr, Murawski NJ, Kulesza RJ Jr (2011) Malformation of the superior olivary complex in an animal model of autism. Brain Res. 1398:102-12.

Lukose R, Brown K, Barber CM, Kulesza RJ Jr (2013) Quantification of the stapedial reflex reveals delayed responses in autism. Autism Res. Oct;6(5):344-53.

Lukose R, Beebe K, Kulesza RJ Jr (2015) Organization of the human superior olivary complex in 15q duplication syndromes and autism spectrum disorders. Neuroscience. 286:216-230

Main S, Kulesza RJ (2017) Repeated prenatal exposure to valproic acid results in cerebellar hypoplasia and ataxia. Neuroscience. 340:34-47

Malmierca MS, Seip KL, Osen KK. (1995a) Morphological classification and identification of neurons in the inferior colliculus: a multivariate analysis. Anat Embryol (Berl). Apr;191(4):343-50

Malmierca MS, Blackstad TW, Osen KK, Karagülle T, Molowny RL. (1995b) The central nucleus of the inferior colliculus in rat: a Golgi and computer reconstruction study of neuronal and laminar structure. J Comp Neurol. Jul 1;333(1):1-27.

Martineau J, Garreau B, Roux S, Lelord G. (1987) Auditory evoked responses and their modifications during conditioning paradigm in autistic children. J Autism Dev Disord. Dec;17(4):525-39. 31

Maziade M, Mérette C, Cayer M, Roy MA, Szatmari P, Côté R, Thivierge J (2000) Prolongation of brainstem auditory-evoked responses in autistic probands and their unaffected relatives. Arch Gen Psychiatry. Nov;57(11):1077-83.

Merchán M, Aguilar LA, Lopez-Poveda EA, Malmierca MS. (2005) The inferior colliculus of the rat: quantitative immunocytochemical study of GABA and glycine. Neuroscience. 136(3):907-25.

McClelland RJ, Eyre DG, Watson D, Calvert GJ, Sherrard E. (1992) Central conduction time in childhood autism. Br J Psychiatry. May;160:659-63.

Milutinovic S, D'Alessio AC, Detich N, Szyf M. (2007) Valproate induces widespread epigenetic reprogramming which involves demethylation of specific genes. Carcinogenesis. Mar;28(3):560-71.

Miron O, Ari-Even Roth D, Gabis LV, Henkin Y, Shefer S, Dinstein I, Geva R. (2016) Prolonged auditory brainstem responses in infants with autism. Autism Res. Jun;9(6):689-95.

Moore SJ, Turnpenny P, Quinn A, Glover S, Lloyd DJ, Montgomery T, Dean JC. (2000) A clinical study of 57 children with fetal anticonvulsant syndromes. J Med Genet. Jul;37(7):489-97.

Morris NP, Henderson Z. (2000) Perineuronal nets ensheath fast spiking, parvalbumin-immunoreactive neurons in the medial septum/diagonal band complex. Eur J Neurosci. Mar;12(3):828-38.

Myers AK, Ray J, Kulesza RJ Jr. (2012) Neonatal conductive hearing loss disrupts the development of the Cat-315 epitope on perineuronal nets in the rat superior olivary complex. Brain Res. Jul 17;1465:3447. 32

Nevue AA, Elde CJ, Perkel DJ, Portfors CV (2016a) Dopaminergic Input to the Inferior Colliculus in Mice. Front Neuroanat. Jan 21;9:168.

Nevue AA, Felix RA 2nd, Portfors CV (2016b) Dopaminergic projections of the subparafascicular thalamic nucleus to the auditory brainstem. Hear Res. Nov;341:202-209.

Nicolini C, Fahnestock M. (2018) The valproic acid-induced rodent model of autism. Exp Neurol. Jan;299(Pt A):217-227

Ornitz EM, Atwell CW, Kaplan AR, Westlake JR (1985) Brain-stem dysfunction in autism. Results of vestibular stimulation. Arch Gen Psychiatry. Oct;42(10):1018-25.

Pór A, Pocsai K, Rusznák Z, Szucs G. (2005) Presence and distribution of three calcium binding proteins in projection neurons of the adult rat cochlear nucleus. Brain Res. Mar 28;1039(1-2):63-74.

Rasalam AD, Hailey H, Williams JH, Moore SJ, Turnpenny PD, Lloyd DJ, Dean JC (2005) Characteristics of fetal anticonvulsant syndrome associated autistic disorder. Dev Med Child Neurol. Aug;47(8):551-5.

Riquelme R, Saldaña E, Osen KK, Ottersen OP, Merchán MA. (2001) Colocalization of GABA and glycine in the ventral nucleus of the lateral lemniscus in rat: an in situ hybridization and semiquantitative immunocytochemical study. J Comp Neurol. Apr 16;432(4):409-24.

Roberts RC, Ribak CE, Oertel WH. (1985) Increased numbers of GABAergic neurons occur in the inferior colliculus of an audiogenic model of genetic epilepsy. Brain Res. Dec 30;361(1-2):324-38. 33

Ribak CE, Roberts RC. (1986) The ultrastructure of the central nucleus of the inferior colliculus of the Sprague-Dawley rat. J Neurocytol. Aug;15(4):421-38.

Roberts RC, Ribak CE. (1987) GABAergic neurons and axon terminals in the brainstem auditory nuclei of the gerbil. J Comp Neurol. Apr 8;258(2):267-80.

Rodier PM, Ingram JL, Tisdale B, Nelson S, Romano J (1996) Embryological origin for autism: developmental anomalies of the cranial nerve motor nuclei. J Comp Neurol 370(2):247–261.

Roper L, Arnold P, Monteiro B (2003) Co-occurrence of autism and deafness: diagnostic considerations. Autism. Sep;7(3):245-53.

Rosenblum SM, Arick JR, Krug DA, Stubbs EG, Young NB, Pelson RO. (1980) Auditory brainstem evoked responses in autistic children. J Autism Dev Disord. Jun;10(2):215-25.

Rosenhall U, Nordin V, Brantberg K, Gillberg C (2003) Autism and auditory brain stemresponses. Ear Hear. 24 (3), 206–214 (Jun).

Roth DA, Muchnik C, Shabtai E, Hildesheimer M, Henkin Y (2012) Evidence for atypical auditory brainstem responses in young children with suspected autism spectrum disorders. Dev Med Child Neurol. Jan;54(1):23-9.

Rubenstein JL, Merzenich MM. (2003) Model of autism: increased ratio of excitation/inhibition in key neural systems. Genes Brain Behav. Oct;2(5):255-67.

34

Ruby K, Falvey K, Kulesza RJ (2015) Abnormal neuronal morphology and neurochemistry in the auditory brainstem of Fmr1 knockout rats. Neuroscience. 303:285-98.

Rumsey JM, Grimes AM, Pikus AM, Duara R, Ismond DR (1984) Auditory brainstem responses in pervasive developmental disorders. Biol. Psychiatry 19 (10), 1403–1418.

Russo N, Nicol T, Trommer B, Zecker S, Kraus N (2009) Brainstem transcription of speech is disrupted in children with autism spectrum disorders. Dev. Sci. 12 (4), 557–567

Sakai A, Matsuda T, Doi H, Nagaishi Y, Kato K, Nakashima K. (2018) Ectopic neurogenesis induced by prenatal antiepileptic drug exposure augments seizure susceptibility in adult mice. Proc Natl Acad Sci U S A. Apr 17;115(16):4270-4275

Saldaña E, Merchán MA. (1992) Intrinsic and commissural connections of the rat inferior colliculus. J Comp Neurol. May 15;319(3):417-37.

Santos M, Marques C, Nóbrega Pinto A, Fernandes R, Coutinho MB, Almeida E Sousa C (2017) Autism spectrum disorders and the amplitude of auditory brainstem response wave I. Autism Res. Jul;10(7):13001305.

Schneider T, Roman A, Basta-Kaim A, Kubera M, Budziszewska B, Schneider K, Przewłocki R (2008) Gender-specific behavioral and immunological alterations in an animal model of autism induced by prenatal exposure to valproic acid. Psychoneuroendocrinology. Jul;33(6):728-40.

Skoff BF, Mirsky AF, Turner D (1980) Prolonged brainstem transmission time in autism. Psychiatry Res. 2 (2), 157–166. 35

Szelag E, Kowalska J, Galkowski T, Poppel E (2004) Temporal processing deficits in high-functioning children with autism. Br. J. Psychol. 95 (Pt 3), 269–282 (Aug).

Tas A, Yagiz R, Tas M, Esme M, Uzun C, Karasalihoglu AR (2007) Evaluation of hearing in children with autism by using TEOAE and ABR. Autism 11 (1), 73–79.

Teder-Salejarvi WA, Pierce KL, Courchesne E, Hillyard SA (2005) Auditory spatial localization and attention deficits in autistic adults. Brain Res. Cogn. Brain Res. (2-3), 221–234.

Tharpe AM, Bess FH, Sladen DP, Schissel H, Couch S, Schery T (2006) Auditory characteristics of children with autism. Ear Hear. (4), 430–441 (3).

Thompson CK, Brenowitz EA (2005) Seasonal change in neuron size and spacing but not neuronal recruitment in a basal ganglia nucleus in the avian song control system. J. Comp. Neurol. 481 (3), 276– 283.

Thompson AM, Thompson GC. (1991) Posteroventral cochlear nucleus projections to olivocochlear neurons. J Comp Neurol. Jan 8;303(2):267-85.

Thompson AM. (1998) Heterogeneous projections of the cat posteroventral cochlear nucleus. J Comp Neurol. Jan 19;390(3):439-53

Tomchek SD, Dunn W (2007) Sensory processing in children with and without autism: a comparative study using the short sensory profile. Am. J. Occup. Ther. 61 (2), 190–200 (Mar-Apr, (3)).

36

Tou L, Liu Q, Shivdasani RA (2004) Regulation of mammalian epithelial differentiation and intestine development by class I histone deacetylases. Mol Cell Biol. Apr;24(8):3132-9.

Wagoner JL, Kulesza RJ Jr (2009) Topographical and cellular distribution of perineuronal nets in the human cochlear nucleus. Hear Res. 254(1-2):42-53.

Wang CC, Chen PS, Hsu CW, Wu SJ, Lin CT, Gean PW. (2012) Valproic acid mediates the synaptic excitatory/inhibitory balance through astrocytes--a preliminary study. Prog Neuropsychopharmacol Biol Psychiatry. Apr 27;37(1):111-20.

Williams G, King J, Cunningham M, Stephan M, Kerr B, Hersh JH (2001) Fetal valproate syndrome and autism: additional evidence of an association. Dev. Med. Child Neurol. 43 (3), 202–206 (Mar).

Wing L (1997) The autistic spectrum. Lancet 350, 1761–1766.

Zimmerman R, Patel R, Smith A, Pasos J, Kulesza RJ Jr. (2018) Repeated Prenatal Exposure to Valproic Acid Results in Auditory Brainstem Hypoplasia and Reduced Calcium Binding Protein Immunolabeling. Neuroscience. May 1;377:53-68.

37

FIGURE LEGENDS Figure 1. VPA exposure. Shown in A is a schematic of the VPA exposure model. All pregnant females were administered vehicle on E7-12 (gray bars). Females in the experimental groups were administered vehicle and VPA (blue bars) on E10 and E12. Pups were weaned on P21 and animals were perfused between P50 and P64. Shown in B are brainstem weights from control and VPA-exposed animals (P50 only; horizontal line represents the median, whiskers represent the 95% percentile). Key to symbols: * = p < .05.

Figure 2. VPA exposure impacted the CNIC. Shown in A (control) and B (VPA) are low magnification views of the IC. The CNIC is highlighted in yellow. The CNIC is smaller and has a lower neuronal packing density after VPA exposure. The angle arrows in A (top right) indicate the direction of angles for the orientation of the long axis of neuronal cell bodies in the CNIC. Shown in C (control) and D (VPA) are higher magnification views of the CNIC. The black arrows indicate stellate neurons and the arrowheads indicate round/oval neurons. The asterisk in D indicates a very large oval neuron. D = dorsal, M = medial.

Figure 3. VPA exposure results in larger but fewer CNIC neurons. Shown in A is a box and whisker plot of the all CNIC neurons measured in control and VPA animals. CNIC neurons are significantly larger after VPA exposure. Shown in B is a histogram of the proportion of neurons according to cell body size. In VPA animals, there are more neurons with larger cell bodies. Shown in C are CNIC neurons split by dorsoventral region of the CNIC. In all regions, CNIC neurons are larger after VPA exposure. Shown in D are the orientation angles of cell bodies from the CNIC. Shown in E is a histogram of the proportion of cell body morphologies (fusiform cells were rare and were not plotted). Shown in F is the number of 38

CNIC neurons in control and VPA animals (mean + standard deviation). There were significantly fewer CNIC neurons after VPA exposure. Key to symbols: ***p <.001, ****p <.0001.

Figure 4. VPA exposure impacted the NLL. Shown in A (control) and B (VPA) are low magnification views of the NLL. The DNLL is highlighted in yellow, the INLL in green and the VNLL in blue. Shown in C and D are higher magnification views of the DNLL. DNLL neurons were larger after VPA. The black arrows indicate stellate neurons and the arrowheads represent round/oval neurons. Shown in E and F are higher magnification views of the VNLL. The neuronal packing density was markedly lower in the VNLL after VPA exposure.

Figure 5. VPA exposure results in fewer NLL neurons. Shown in A and B are boxplots of soma size in the NLL. Neurons in the DNLL were significantly larger after VPA exposure but there was no change in the INLL or VNLL. Shown in C-E are estimates of neuronal number in the NLL. In all three nuclei, there were significantly fewer neurons after VPA exposure. Key to symbols: ***p <.001, ****p <.0001.

Figure 6. VPA exposure resulted in fewer CB+ neurons in the DNLL. Shown in A (control) and B (VPA) are images of the DNLL showing CB immunolabeling and Nissl staining. The majority of DNLL neurons were CB+ in control animals. The arrowheads indicate CB+ neurons and the black arrows indicate CBimmunonegative neurons. Shown in C is the number of CB+ neurons. After VPA-exposure, there were significantly fewer CB+ neurons in the DNLL. CB+ axons were found in the LL dorsal to the DNLL (white arrowheads). The diameters of these axons are shown in D. LPN – lateral parabrachial nucleus. Key to symbols: ** p < .01, ***p <.001.

Figure 7. VPA had no impact on WFA PNNs. Shown in A (control) and B (VPA) are low magnification views of the IC with PNN histochemistry. There is a higher density of PNN in the VM aspect of the CNIC. The arrowheads in A and B indicate areas of high PNN density. Shown in C and D are higher 39

magnification views of the VM CNIC and E and F show views of the DNLL. Neurons surrounded by PNNs are indicated by arrowheads; neurons lacking a PNN are indicated by white arrows. Shown in G are the proportion of PNNs in the VM and DL CNIC and shown in H are the proportions of PNNs in the NLL. There were no significant differences in these comparisons.

Figure 8. VPA exposure resulted in fewer TH+ boutons in the CNIC. Shown in A (control) and B (VPA) is TH-immunolabeling in the CNIC. Both TH+ axonal profiles (black arrowheads) and TH+ puncta (white arrowheads) are observed. The density of TH+ puncta in the CNIC and NLL is shown in C. Key to symbols: ****p <.0001.

Figure 9. The impact of VPA on the auditory brainstem. Based on the current report and Zimmerman et al (2018), the number of auditory brainstem neurons in control and VPA-exposed auditory nuclei is summarized in A. The pie chart in B shows the location and proportion of neuronal loss after VPA exposure. Together, the NLL and CNIC account for ~86% of the neuronal loss after VPA exposure. However, if the neuronal loss in each nucleus is normalized to the number of neurons in the CNIC, a disproportionate impact of VPA is observed. The MSO, VNLL, INLL and SPON are the most severely affected by VPA exposure.

40

41

42

43

44

45

Table 1. Number of Neurons included in Morphology. Neurons VNLL INLL Control 135 119 VPA 117 98 Screened for PNNS Control 495 229 VPA 466 241 CB Somata Control VPA TH puncta Control 656 613 VPA 605 580 CB Axons LL Control 105 VPA 106

DNLL 139 98

CNIC 1000 924

810 1063

1789 2018

840 855 435 357

2562 1909

Highlights

   

In utero exposure to VPA results in an increased risk of ASD in humans VPA exposure resulted in fewer neurons in the NLL and CNIC VPA exposure resulted in fewer CB+ neurons in the DNLL and smaller CB+ axons VPA exposure resulted in decreased dopaminergic innervation to the CNIC

46