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. 2024 May 2;34(13):146–160. doi: 10.1093/cercor/bhad501

Cortical dysmorphology and reduced cortico-collicular projections in an animal model of autism spectrum disorder

Kara Kosmer 1, Randy Kulesza 2,✉
PMCID: PMC11484449  PMID: 38696608

Abstract

Autism spectrum disorder is a neurodevelopmental disability that includes sensory disturbances. Hearing is frequently affected and ranges from deafness to hypersensitivity. In utero exposure to the antiepileptic valproic acid is associated with increased risk of autism spectrum disorder in humans and timed valproic acid exposure is a biologically relevant and validated animal model of autism spectrum disorder. Valproic acid-exposed rats have fewer neurons in their auditory brainstem and thalamus, fewer calbindin-positive neurons, reduced ascending projections to the midbrain and thalamus, elevated thresholds, and delayed auditory brainstem responses. Additionally, in the auditory cortex, valproic acid exposure results in abnormal responses, decreased phase-locking, elevated thresholds, and abnormal tonotopic maps. We therefore hypothesized that in utero, valproic acid exposure would result in fewer neurons in auditory cortex, neuronal dysmorphology, fewer calbindin-positive neurons, and reduced connectivity. We approached this hypothesis using morphometric analyses, immunohistochemistry, and retrograde tract tracing. We found thinner cortical layers but no changes in the density of neurons, smaller pyramidal and non-pyramidal neurons in several regions, fewer neurons immunoreactive for calbindin-positive, and fewer cortical neurons projecting to the inferior colliculus. These results support the widespread impact of the auditory system in autism spectrum disorder and valproic acid-exposed animals and emphasize the utility of simple, noninvasive auditory screening for autism spectrum disorder.

Keywords: auditory cortex, calbindin, tract tracing, valproic acid

Introduction

Autism spectrum disorder (ASD) is a neurodevelopmental disability characterized by social, communication, and behavioral challenges (Allen 1988; Wing 1997; APA 2013; CDC.gov 2022). While the prevalence of ASD varies by country and region, ~1 in 44 children are diagnosed with ASD by the age of 8 in the United States and this rate is more than 4 times higher in males compared with females (CDC.gov 2022). There are numerous signs and symptoms associated with ASD, but most subjects have some degree of hearing dysfunction (Greenspan and Wieder 1997; Tomchek and Dunn 2007; Bolton et al. 2012; reviewed in Smith et al. 2019; Mansour et al. 2021a) and this can range from deafness to hypersensitivity (Roper et al. 2003; Alcántara et al. 2004; Khalfa et al. 2004; Szelag et al. 2004; Teder-Sälejärvi et al. 2005; Gravel et al. 2006; Tharpe et al. 2006; Russo et al. 2009). Such auditory dysfunction is supported by auditory brainstem responses (ABR) in subjects with ASD. Specifically, subjects with ASD have longer latency responses (Ornitz 1969; Student and Sohmer 1978; Rosenblum et al. 1980; Sohmer 1982; Tanguay et al. 1982; Gillberg et al. 1983; Sersen et al. 1990; Thivierge et al. 1990; Wong and Wong 1991; Maziade et al. 2000; Kwon et al. 2007; Roth et al. 2012; Azouz et al. 2014; Miron et al. 2018, 2021; Ramezani et al. 2019) and smaller amplitudes in waves I through V (Ornitz et al. 1972; Gillberg et al. 1983; Martineau et al. 1987, 1992; Klin 1993). Beyond these changes in the ABR, there is an abundance of evidence for abnormal cortical responses to sound in ASD. Specifically, there is reduced cortical thickness in the temporal lobe (van Rooij et al. 2018) and reduced connectivity from auditory cortex to several other cortical regions (Wilson et al. 2022). Cortical responses to simple auditory stimuli occur at longer latency and lower amplitude in ASD (Matsuzaki et al. 2019; Williams et al. 2021) and show right–left asymmetries (Orekhova et al. 2012; Lloyd-Fox et al. 2018). There is also decreased cortical activation in response to vocal sounds but increased activation/abnormal processing of nonspeech sounds suggesting these processing streams may be impacted differently (Bidet-Caulet et al. 2017; Lloyd-Fox et al. 2018). There is evidence though that these cortical processing issues may improve with age (Alho et al. 2023). Changes in the ABR and even cortical responses to sound have been attributed to immaturity of brainstem circuits (Bidet-Caulet et al. 2017; Li et al. 2020). Taken together, these findings suggest widespread dysfunction in central auditory pathways in ASD. However, whether the cortical responses are inherited from brainstem or thalamic centers or arise de novo in the cortex is unclear. Furthermore, these hearing deficits are supported by postmortem studies demonstrating consistent auditory brainstem hypoplasia and dysmorphology in ASD (Kulesza and Mangunay 2008; Kulesza et al. 2011; Lukose et al. 2015; Mansour and Kulesza 2020). Indeed, hearing difficulties have been proposed as one of the cardinal features of ASD (Osterling and Dawson 1994).

In utero exposure to the antiepileptic drug valproic acid (VPA) is associated with a significant increase in the risk of an ASD diagnosis in humans (Rasalam et al. 2005; Koren et al. 2006; Bromley et al. 2013; Christensen et al. 2013). Therefore, timed in utero exposure to VPA is a biologically relevant and validated animal model of ASD (rodents: Rodier et al. 1996; Mabunga et al. 2015; primates: Zhao et al. 2019). VPA-exposed animals have significantly smaller brains and brainstems and delayed eye and ear opening (Zimmerman et al. 2018; Mansour et al. 2019). VPA-exposed rats have significantly fewer neurons in their auditory brainstem and thalamus, and this is consistent with findings in human subjects with ASD (Kulesza and Mangunay 2008; Kulesza et al. 2011; Lukose et al. 2015; Mansour and Kulesza 2021; rodent: Lukose et al. 2011; Zimmerman et al. 2018; Mansour et al. 2019; Mansour and Kulesza 2021). Furthermore, VPA-exposed animals have abnormal tonotopic maps, hyperactivation of brainstem centers in response to pure tones (Dubiel and Kulesza 2016), as well as reduced ascending projections from the superior olivary complex (SOC) and ventral cochlear nucleus (VCN) to the inferior colliculus and medial geniculate (Zimmerman et al. 2020; Mansour et al. 2021b). So, not only are there fewer neurons in the SOC and VCN in VPA-exposed animals, fewer of these surviving neurons are making the appropriate ascending projections. Immunoreactivity for the calcium binding protein calbindin (CB) is a characteristic feature of several brainstem cell groups. Specifically, the majority of neurons in the octopus cell region of the posteroventral cochlear nucleus, principal neurons in the medial nucleus of the trapezoid body (MNTB), and neurons in the dorsal nucleus of the lateral lemniscus (Friauf 1993, 1994), and nearly all cerebellar Purkinje cells (Schneeberger et al. 1985) are calbindin-positive (CB+). However, we have found that VPA exposure results in significantly fewer CB+ neurons in each of these locations (Main and Kulesza 2017; Zimmerman et al. 2018; Mansour et al. 2019). Our recent longitudinal study of ABRs in VPA-exposed rats revealed significantly higher thresholds, longer latencies, and right–left asymmetries at postnatal days (P) 22 and 28 and these differences normalize after P60 at high click intensities (Malhotra and Kulesza 2023). VPA-exposed animals also have abnormal cortical tonotopic maps (Engineer et al. 2014b; Anomal et al. 2015; Cheng et al. 2022), delayed cortical responses (Engineer et al. 2014b), and degraded temporal processing (Engineer et al. 2014a; Cheng et al. 2022). In the auditory cortex, pyramidal neurons have an increased density of dendritic spines and there is also a lower density of somatostatin immunopositive interneurons (Cheng et al. 2022). However, the impact of VPA exposure on the density of parvalbumin-immunopositive interneurons is unclear (compare Cheng et al. 2022 and Anomal et al. 2015).

Taken together, these findings provide clear evidence for disruption of the auditory brainstem and auditory cortex in ASD and VPA-exposed rats. However, we have not yet systematically examined neuronal morphology, the number of CB+ neurons or connectivity in the auditory cortex in VPA-exposed rats. Based on previous findings, we hypothesize that in utero VPA exposure will result in smaller cortical neurons across all cortical layers, fewer and smaller neurons, and fewer CB+ neurons in the auditory cortex. We examined this hypothesis by examining neuronal morphology and CB immunolabeling in P28 control and VPA-exposed animals. Furthermore, based on our findings of reduced ascending projections to central nucleus of the inferior colliculus (CNIC), we hypothesize reduced descending input from auditory cortex to the auditory midbrain. We examined this hypothesis using the injection of the retrograde tract tracer into the CNIC and counting neurons in the auditory cortex.

Materials and methods

VPA exposure

All procedures and animal handling were approved by the LECOM Institutional Animal Care and Use Committee (protocols #20-02 and 21-03) and conducted according to the National Institute of Health Guide for the Care and Use of Laboratory Animals. Sprague–Dawley rats were housed on a 12-h light/dark cycle with free access to food and water. In utero exposure to VPA was performed as previously described (Fig. 1A; Main and Kulesza 2017; Zimmerman et al. 2018, 2020; Mansour et al. 2019, 2021b, 2022; Mansour and Kulesza 2021; Malhotra and Kulesza 2023). After timed mating, dams were fed 3.1 g of peanut butter on embryonic days (E) 7 to 12. On E10 and E12, dams in the VPA group were fed peanut butter mixed with 800 mg/kg of VPA (Fig. 1A). Control animals were fed peanut butter meals according to the same schedule but without VPA. Pups were delivered without interference and litters were not culled. On P21, litters were weaned, and only male pups were included in the study since gender-specific effects of VPA exposure are established (Schneider et al. 2008). We approached each litter with the assumption that all male pups were equally afflicted by VPA exposure; previous work from our lab supports this assumption (Main and Kulesza 2017; Zimmerman et al. 2018, 2020; Mansour et al. 2019, 2021b, 2022; Mansour and Kulesza 2021; Malhotra and Kulesza 2023). Regardless, only 1 or 2 animals were randomly selected from each litter for this study.

Fig. 1.

Fig. 1

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Experimental paradigm. Animals were subjected to timed mating and exposed to vehicle (light gray bars) or VPA (dark bars) according to the timeline shown in a. Pups were weaned on P21. Animals used for cell body morphology or IHC were perfused on P28 A, B). Injections of the retrograde tracer FG were made into the IC in a separate cohort of animals between P51 and P63, and retrogradely labeled neurons were counted in the ipsilateral cerebral cortex (shown schematically in C). Cortical regions were demarcated according to Paxinos and Watson (2007; c).

Sectioning and histology

Animals used for morphology and immunohistochemistry (IHC; control, n = 9 [from 7 litters]; VPA, n = 10 [from 7 litters]; Fig. 1B) were anesthetized with an overdose of isoflurane on P28. When animals were unresponsive to toe pinch, they were perfused through the ascending aorta with normal saline followed by 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS; pH 7.4; fixative). Brains were dissected from the skull, and the right side of the brain was marked with a register pin. Brains were stored in 4% PFA-PBS (at 4°C) for at least 24 h. Brains were cryoprotected in 30% sucrose at room temperature for at least 24 h and sectioned in the coronal plane on a freezing-stage microtome at a thickness of 50 μm. Sections were collected into 3 wells—sections from well 1 were collected serially into PBS and mounted from gelatin alcohol onto glass slides in caudal-to-rostral sequence. These sections were stained for Nïssl substance with Giemsa (Sigma-Aldrich, St Louis, MO), dehydrated through ascending alcohols, cleared and coverslipped with Permount (Thermo Fisher Scientific, Waltham, MA). Sections from well 2 were used for IHC (see below) and those from well 3 were archived.

Neuronal morphology

We examined neuronal morphology in 4 adjacent cortical regions, namely, primary auditory cortex (Au1), both ventral (AuV) and dorsal (AuD) secondary auditory cortices, as well as primary somatosensory cortex (S1). Neurons in each region were studied with an Olympus BX45 microscope and were traced by an observer blinded to animal group using a 40× objective (final magnification of 680×). We followed the delineation of these cortical regions provided in Paxinos and Watson (2007). The total number of neurons traced for each group and cortical region are provided in Table 1. Neuronal profiles were only included in the quantification if they contained (i) an identifiable nucleus, (ii) a prominent nucleolus, and (iii) abundant, Giemsa-stained cytoplasm. Tracings of cell body profiles were made while focusing to ensure accurate cell body contours. Tracings of neuron cell bodies were digitized and quantified using ImageJ (1.52). An index of circularity was calculated for each cell body profile using the following equation:

Table 1.

Morphology—number of Giemsa-stained cortical neurons analyzed.

Control
Layer Au1 AuD AuV S1 Total
II/III/IV 91 77 83 84 335
V 86 76 83 83 328
VI 82 83 90 79 334
VPA Au1 AuD AuV S1 Total
II/III/IV 98 100 111 110 419
V 109 121 110 106 446
VI 118 112 91 119 440
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Circularity = [4p*area/Perimeter2]

Neuronal profiles were classified as either pyramidal or non-pyramidal cells based on shape. Specifically, neuronal profiles were classified as pyramidal if (i) the profile was conical or triangular, (ii) the apex was directed toward the pial surface (layers III to V) or toward the deep white matter (layer VI), and (iii) an apical dendrite could be visualized. Cells that failed to meet all 3 criteria were classified as non-pyramidal. Delineation of cortical layers in control and VPA-exposed animals was based on neuronal packing density, which was statistically similar in control and VPA-exposed animals (see later). Layer I was identified at the pia surface and distinguished from layer II by its very low neuronal density. Layers II to IV were combined because of difficulty separating these layers in Giemsa-stained material and the difference in neuronal density from layer V. Layer V was distinguished by its comparatively low neuronal packing density. Layer VI was easily distinguished from layer V and the deep cortical white matter.

Immunohistochemistry

Sections from well 2 from 4 control animals (from 3 litters) and 6 VPA-exposed animals (from 5 litters) were used for CB IHC. Free-floating tissue sections were rinsed in PBS, then blocked in 1% normal horse serum (NHS; Abcam), 0.5% triton X in PBS for 1 h. Sections were incubated in rabbit anti-CB (1:1,000 with 1% NHS; Abcam, catalog #ab11426) overnight, rinsed in PBS, and incubated for 2 h in goat anti-rabbit Dylight 488 (1:100; Vector Labs). Sections were rinsed, counterstained with Neurotrace Red (NT; Thermo Fisher Scientific), mounted onto glass slides, dried and coverslipped with Entellan (Millipore Sigma). Images of NT and CB labeling were overlaid in ImageJ; in at least 2 sections per animal, we counted both the number of CB+ and NTR-labeled neurons.

Surgery and tracer injections

A total of 6 control animals (from 4 litters, between P51 and P63) and 8 VPA-exposed animals (from 4 litters, between P50 and P55) were used to study cortical projections to the IC (Fig. 1C). Animals were anesthetized with vaporized isoflurane (5% induction, 2.5% to 3% maintenance, O2: 1.2 l/min). When animals were unresponsive to toe pinch, they were fit with a custom face mask and secured in a stereotaxic frame with non-rupture ear bars (Kopf Instruments). Body temperature was maintained via a heating pad. The scalp was cleaned with 70% ethanol and washed with iodine solution. The scalp was injected with 0.25% bupivacaine and eyes were closed and covered with the face mask. A midline incision was made in the scalp to expose the dorsal aspect of the parietal and occipital bones. The IC was approached via dorsal stereotaxic craniotomy. The same stereotaxic coordinates were used for all animals: 0.2 mm rostral to lambda (as indicated by Paxinos and Watson 2007) and 1.5 mm to the right of the midline. Injections of Fluorogold (FG; 4.0% in saline; Fluorochrome) were made using a 1 μL Hamilton KH Neuros syringe (32 gage and 4 point; Fig. 1C). Two deposits of FG (100 nl each) were centered in the CNIC, −5.8 and −5.0 mm from the dural surface for a total injected volume of 200 nl. After the second injection was made, the syringe was left in place for 10 min. The syringe was then withdrawn, the bony defect was filled with dental wax and the incision closed with suture. The wound was injected with lidocaine and the animals were taken off isoflurane, returned to their home cage, and monitored until they were able to stand on all 4. From the 6 control cases and 8 VPA cases, we selected 4 injections from each group that had nearly identical injection sites centered in the CNIC with spread into both the external (EC) and dorsal cortices (DC) to control for location of the injection site and spread of FG along the coronal and parasagittal planes. Accordingly, our analysis of descending projections from the cortex to the IC is based on these 8 cases.

Quantification of neuron density

We examined the density of cortical neurons in Giemsa-stained sections, sections with CB immunolabeling and sections from animals that received FG injections. For this analysis, tissue sections were viewed live on-screen using a DP71 digital camera and neurons were counted using the object counting feature in Olympus cellSans standard software. For each view, a counting frame was drawn on-screen (see below) and placed throughout each layer to ensure counting of all neuronal populations. The number of neuronal profiles within the box was counted using stereological inclusion criteria and data collected as neurons per area. For layers II/III/IV, counting frames of ~350 × 350 μm were used (total counting area = 122,500 um2), for layer V, counting frames of ~400 × 400 μm were used (total counting area = 160,00 um2), and for layer VI, counting frames of approximately were used 350 × 350 μm were used (total counting area = 122,500 um2; see white boxes in Fig. 3A1 and B1). These values were converted to number of neurons per 0.01 mm2. Neuronal profiles were considered CB+ if they contained both NTR labeling and CB immunolabeling; neuronal profiles were considered FG+ if they contained both red NTR labeling and FG labeling.

Fig. 3.

Fig. 3

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Cortical layers are thinner, and neurons are smaller after VPA exposure. Representative Giemsa-stained sections of the cortex from control A) and VPA-exposed animals B) are shown. The regions indicated by the boxes in a and b are shown at higher magnification in A1 and B1. The thickness of the cortical layers is indicated by the lines (long = layers II to VI, top = layer I, second from top = layers II to IV, second from bottom = layer V, and bottom = layer VI). Higher magnification views of layer V from AuV are shown in A2 (control) and VPA (B2) and layer V from AuD are shown in A3 (control) and VPA (B3). Cortical neurons are significantly smaller in VPA-exposed animals. Pyramidal neurons are indicated by the black arrowhead, and non-pyramidal neurons are indicated by red/gray arrowheads. Abbreviations: Am, amygdala; D, dorsal; H, hippocampus; LV, lateral ventricle; M, medial, S1BF, primary somatosensory cortex, barrel field; St, striatum. The scale bars are equal to 500 μm b), 130 μm b1), and 40 μm b3).

Statistics

Descriptive statistics were generated for all datasets using GraphPad Prism 9.5.1 (GraphPad Software, La Jolla, CA) and tested against a normal distribution using the D’Agostino and Pearson omnibus normality test. If data fit a normal distribution, comparisons were conducted using parametric tests (t test or ANOVA) and results are presented as mean ± standard deviation. If data failed to fit the normal distribution, comparisons were conducted using non-parametric tests and are presented as the median with the 95% confidence interval (CI) of the median. The proportion of CB and FG+ neurons was compared using the chi-square test. Regardless of the statistical tests used, differences were considered statistically significant if P-values were < 0.05.

Results

VPA exposure results in thinner cortical layers

The thickness of layer I was not significantly different between control and VPA-exposed animals (control: 187 ± 30 μm; VPA: 165 ± 32 μm; see red lines in Fig. 3A1 and B1). However, VPA exposure did result in a significant reduction in thickness of total cortical gray matter in Au1 (F[9, 284] = 1,101; P < 0.0001; Fig. 2, black lines in a1 and b1). In control animals, cortical layers II through VI measured 1,539 ± 140 μm and in VPA-exposed animals, these layers measured VPA 1,402 ± 115 μm (P < 0.0001). In control animals, layers II/III/IV combined to a thickness of 557 ± 74 μm and 456 ± 55 μm in VPA-exposed animals (P < 0.0001; Fig. 2, orange lines in a1 and b1). In control animals, layer V measured 481 ± 44 μm and 415 ± 47 in VPA-exposed animals (P = 0.005; Fig. 2, yellow lines in a1 and b1). Layer VI measured 534 ± 82 μm in control and 440 ± 70 μm in VPA-exposed animals (P < 0.0001; Fig. 2, green lines in a1 and b1). The difference in cortical thickness can be attributed to reductions in layers II to VI (Fig. 2B and C). A schematic of the thickness of these cortical layers is shown in Fig. 2C.

Fig. 2.

Fig. 2

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Cortical layers are thinner after VPA exposure. Shown in A are measurements of the overall (combined) cortical thickness of Au1 (layers II through VI). Shown in B are measurements of the thickness of individual cortical layers from Au1. The relative mean thickness of cortical layers is shown in C. Key to symbols: **** = P < 0.0001, ** = P < 0.01.

VPA exposure does not impact the density of cortical neurons

While there were subtle differences in neuronal density in the cortical regions analyzed, VPA exposure had no significant impact on neuronal density in Au1, AuV, AuD, or S1 (Fig. 3). In Au1, control animals had a neuronal density of 13.16 ± 3.6 neurons/0.01 mm2 in layers II/III/IV, 14.55 ± 4.8 in layer V, and 16.75 ± 10.1 in layer VI. In VPA-exposed animals, the neuronal density was 11.91 ± 3.2 in layers II/III/IV, 12.10 ± 3.6 in layer V, and 14.69 ± 8.1 in layer VI (F[5, 27] = 0.59, P = 0.70). In AuV, control animals had a neuronal density of 9.9 ± 4.8 neurons/0.01 mm2 in layers II/III/IV, 11.24 ± 1.6 in layer V, and 12.54 ± 2.8 in layer VI. In VPA-exposed animals, the neuronal density was 10.77 ± 3.6 in layers II/III/IV, 11.67 ± 4.2 in layer V, and 15.29 ± 2.3 in layer VI (F[5, 29] = 1.4, P = 0.24). In AuD, control animals had a neuronal density of 13.35 ± 3.3 neurons/0.01 mm2 in layers II/III/IV, 19.42 ± 12.1 in layer V, and 12.39 ± 0.45 in layer VI. In VPA-exposed animals, the neuronal density was 11.39 ± 2.9 in layers II/III/IV, 13.11 ± 4.2 in layer V, and 17.39 ± 8.3 in layer VI (F[5, 28] = 1.2, P = 0.32). In S1, control animals had a neuronal density of 18.4 ± 11.2 neurons/0.01 mm2 in layers II/III/IV, 22.9 ± 10.5 in layer V, and 16.93 ± 5.1 in layer VI. In VPA-exposed animals, the neuronal density was 12.83 ± 3.8 in layers II/III/IV, 13 ± 4.5 in layer V, and 21.44 ± 17.2 in layer VI (F[5, 22] = 0.77, P = 0.57).

VPA exposure results in smaller cortical neurons

VPA exposure resulted in smaller neurons across nearly all cell types and layers in Au1, AuV, AuD, and S1. However, this did not reach significance in all regions and the impact of VPA exposure was least severe in Au1 and S1 and most severe in AuV and AuD (Figs. 3 and 4; Table 2). In Au1 VPA-exposure resulted in significantly smaller pyramidal neurons in layers II/III/IV (H = 82.52, P < 0.0001, Dunn’s P < 0.0001, Fig. 4A), but there was no difference between pyramidal cells in layers V and VI or non-pyramidal cells in layers III/III/IV, V, or VI. Interestingly, in Au1, VPA-exposed animals had larger non-pyramidal cells in layer VI (Fig. 4; Table 2)—this is the only location where cell bodies were larger in VPA-exposed animals. In AuV, non-pyramidal cells were significantly smaller in layers II/III/IV (H = 170.5, P < 0.0001, Dunn’s P = 0.002). However, both pyramidal and non-pyramidal cells were significantly smaller in layer V (Dunn’s P < 0.0001 for both). Layer V in AuV had the biggest difference in cell body size between control and VPA-exposed animals. Both pyramidal and non-pyramidal cells were ~42% smaller in VPA-exposed animals (Fig. 4; Table 2). AuD was the most severely affected by VPA exposure (Figs. 3A2 and B2 and 4B) and neurons were significantly smaller in all cortical layers (H = 144.3, P < 0.0001). Pyramidal cells were significantly smaller in layers II/III/IV, V (Dunn’s, P < 0.0001) and VI (Dunn’s, P = 0.03; Fig. 3; Table 2). Non-pyramidal cells were significantly smaller in layers V (Dunn’s, P = 0.001) and VI (Dunn’s, P = 0.0009; Figs. 3 and 4; Table 2). In S1, VPA-exposed animals had significantly smaller neurons in layers V and VI (H = 123.7, P < 0.0001). Specifically, pyramidal cells in layer V were significantly smaller (Dunn’s, P = 0.009) and non-pyramidal cells in layer VI were significantly smaller (Dunn’s, P = 0.005; Figs. 3 and 4; Table 2).

Fig. 4.

Fig. 4

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Quantification of soma size. Graphs show box plots of soma size in Au1 A), AuV B), AuD C), and S1 D) split by cell body shape (St, stellate; R, round) and layer. The whiskers represent 5 to 95th percentile. Key to symbols: ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001.

Table 2.

Cell body size (median, 95% CI of median).

Au1 Cell type Control VPA
II/III/IV Pyramidal 226.5 μm2 (209 to 251 μm2) 171.5 μm2 (123, 187 μm2)b
Non-pyramidal 259.3 μm2 (217 to 282 μm2) 219.1 μm2 (175 to 250 μm2)
V Pyramidal 227.8 μm2 (206 to 248 μm2) 202 μm2 (176 to 224 μm2)
Non-pyramidal 267.3 μm2 (244 to 287 μm2) 254 μm2 (215 to 292 μm2)
VI Pyramidal 208.7 μm2 (155 to 255 μm2) 170 μm2 (133 to 193 μm2)
Non-pyramidal 240.2 μm2 (216 to 255 μm2) 245 μm2 (210 to 274 μm2)
AuV
II/III/IV Pyramidal 247.3 μm2 (217 to 297 μm2) 187.4 μm2 (164 to 221 μm2)
Non-pyramidal 252.8 μm2 (232 to 263 μm2) 208.1 μm2 (177 to 231 μm2)b
V Pyramidal 302.9 μm2 (268 to 335 μm2) 177.8 μm2 (150 to 199 μm2)d
Non-pyramidal 362.1 μm2 (312 to 399 μm2) 210.4 μm2 (179 to 239 μm2)d
VI Pyramidal 222.4 μm2 (175 to 268 μm2) 174.4 μm2 (150 to 200 μm2)
Non-pyramidal 271.3 μm2 (242 to 289 μm2) 223 μm2 (197 to 247 μm2)
AuD
II/III/IV Pyramidal 227.4 μm2 (202 to 276 μm2) 141.3 μm2 (131 to 174 μm2)d
Non-pyramidal 271.8 μm2 (232 to 296 μm2) 217.7 μm2 (206 to 265 μm2)
V Pyramidal 267.8 μm2 (228 to 319 μm2) 169.7 μm2 (140 to 189 μm2)d
Non-pyramidal 297.2 μm2 (253 to 319 μm2) 226.9 μm2 (201 to 245 μm2)b
VI Pyramidal 206.7 μm2 (175 to 248 μm2) 130.6 μm2 (112 to 149 μm2)a
Non-pyramidal 230.3 μm2 (210 to 262 μm2) 176.4 μm2 (155 to 201 μm2)c
S1
II/III/IV Pyramidal 213.5 μm2 (191 to 243 μm2) 184.7 μm2 (159 to 200 μm2)
Non-pyramidal 275.4 μm2 (237 to 317 μm2) 226 μm2 (202 to 278 μm2)
V Pyramidal 246.4 μm2 (199 to 290 μm2) 192.5 μm2 (160 to 234 μm2)b
Non-pyramidal 291.7 μm2 (249 to 317 μm2) 247.5 μm2 (225 to 277 μm2)
VI Pyramidal 171.8 μm2 (136 to 227 μm2) 149 μm2 (139 to 166 μm2)
Non-pyramidal 242.3 μm2 (218 to 287 μm2) 195.3 μm2 (172 to 230 μm2)b
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a = P < .05, b = P < .01, c = P < .001, d = P < .0001.

VPA exposure results in fewer CB+ neurons

VPA exposure resulted in a lower density of CB+ neurons in all regions of the cerebral cortex examined; however, significant differences were limited to Au1 and AuD (Figs. 5 and 6; Table 3). In Au1, there was no difference in the density of CB+ neurons in layers II/III/IV. But in layer V, control animals had 2.6 ± 1.2 CB+ neurons/0.01 mm2 and VPA-exposed animals had only 1.25 ± 0.46 (F[5, 35] = 5.73, P = 0.0006, Šídák’s, P = 0.04, Fig. 5). Likewise, in layer VI control animals had 3.09 ± 2.1 CB+ neurons and VPA-exposed animals had only 0.66 ± 0.23 (Šídák’s, P = 0.0008). Overall, in control animals, 18.95 ± 3.7% of neurons in Au1 layer V were CB+, but in VPA-exposed animals, only 12.26 ± 3.8% were CB+. In Au1 layer VI, 15.65 ± 10.2% of neurons were CB+ in control, but only 6.06 ± 2.5% were CB+ (Fig. 5). There were no significant differences in the density of CB+ neurons in AuV or S1. In AuV, there was a significantly lower density of CB+ neurons in layer VI only (F[5, 28] = 3.46, P = 0.01, Šídák’s, P = 0.01; Table 3). Specifically, control animals had 3.6 ± 1.9 CB+ neurons/0.01 mm2, but VPA-exposed animals had only 1.5 ± 0.60. In control animals, 32.22 ± 8.8% of neurons in AuD layer VI were CB+, but in VPA-exposed animals, only 7.7 ± 1.3% were CB+.

Fig. 5.

Fig. 5

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CB+ neurons in Au1. The images show CB immunolabeling and NT in control A) and VPA-exposed animals B). CB+ neurons are indicated by the arrowheads. There is a higher density of CB+ neurons in layers V and VI in control animals. Abbreviations: D, dorsal; M, medial. The scale bar in b is equal to 200 μm.

Fig. 6.

Fig. 6

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VPA exposure results in fewer CB+ neurons in Au1. The graphs show the density of CB+ neurons in Au1 A), AuD B), AuV C), and S1 D) split by cortical layer. The whiskers represent the mean and SD. Key to symbols: * = P < 0.05, *** = P < 0.001.

Table 3.

Density of CB+ neurons (neurons/0.01 mm2).

Au1 Control VPA
II/III/IV 2.5 ± 0.76 1.3 ± 0.69
V 2.6 ± 1.2 1.25 ± 0.46a
VI 3.09 ± 2.1 0.66 ± 0.23b
AuV
II/III/IV 2.3 ± 1.5 1.28 ± 0.67
V 2.4 ± 0.99 1.54 ± 0.63
VI 2.02 ± 0.69 1.27 ± 0.35
AuD
II/III/IV 2.5 ± 1.2 1.2 ± 0.99
V 1.98 ± 0.74 1.34 ± 0.74
VI 3.6 ± 1.9 1.5 ± 0.60a
S1
II/III/IV 2.0 ± 0.72 1.27 ± 1.0
V 4.136 ± 2.3 1.69 ± 1.83
VI 3.0 ± 2.1 1.02 ± 0.43
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a = P < .05, b = P < .01, c = P < .001, d = P < .0001.

VPA exposure results in fewer neurons projecting to the IC

Descending projections from cortical regions were investigated in 4 control and 4 VPA-exposed animals with matched FG injection sites (Fig. 7). These injection sites were largely centered in the CNIC but had tracer spread into the adjacent DC and EC. Additionally, these injection sites were localized along the caudal and central aspect of the IC; we had no matched injection sites in the rostral IC (Fig. 7). In VPA-exposed animals, there were significantly fewer retrogradely labeled neurons in Au1, AuV, AuD but not S1 (Figs. 8 and 9). In Au1, there were 3.5 ± 0.72 FG+ neurons/0.01 mm2 in layer V in control animals but only 2.5 ± 0.81 in VPA-exposed animals, but this difference was not significant. In layer VI of Au1, control animals had 3.56 ± 1.4 FG+ neurons/0.01 mm2 and VPA-exposed animals had 2.0 ± 0.96 (F[3, 34] = 5.13, P = 0.004, Šídák’s, P = 0.003; Fig. 9A). More specifically, in control animals, 16.8 ± 2.4% of neurons layer VI of Au1 were FG+ after IC injections, but in VPA-exposed animals, only 8.46 ± 1.12% were FG+. This equates to a 50% decrease in the descending projection from layer VI in Au1. In layers V and VI of AuD in control animals, there were 3.39 ± 0.81 and 3.5 ± 0.86 FG+ neurons/mm2, respectively (F[3, 32] = 18.17, P < 0.0001; Fig. 9B). In VPA exposed animals, this was reduced to 2.4 ± 0.5 in layer V (Šídák’s P = 0.008) and 1.3 ± 0.48 in layer VI (P < 0.0001). In control animals, 26.9 ± 5.3% of neurons in layer V of AuD were FG+ after IC injections, but in VPA-exposed animals, only 18.1 ± 3.1% were FG+. In control animals, 17.6 ± 8.6% of neurons in layer VI of AuD were FG+ after IC injections, but in VPA-exposed animals, this dropped to only 6.3 ± 1.6%. This amounts to a 64% decrease in the descending projection to the IC from layer VI in AuD. In layers V and VI of AuV in control animals, there were 3.8 ± 0.83 and 3.7 ± 1.4 FG+ neurons/mm2, respectively (F[3, 33] = 5.98, P = 0.002; Fig. 9C). In VPA exposed animals, this was reduced to 2.6 ± 0.47 in layer V and 2.3 ± 0.8 in layer VI (Šídák’s P = 0.01, 0.007, respectively). In control animals, 27.5 ± 1.17% of neurons in layer V of AuV were FG+ after IC injections, but in VPA-exposed animals, only 15.7 ± 1.9% were FG+. In control animals, 17 ± 4.6% of neurons in layer VI of AuV were FG+ after IC injections, but in VPA-exposed animals, this dropped to only 9.1 ± 1.4%. There was a decrease in the density of FG+ neurons in S1 after injections in the CNIC, but this difference was not significant (F[3, 34] = 3.34, P = 0.03; Fig. 9D). In layer V, control animals had 2.0 ± 0.61 FG+ neurons/mm2 and VPA-exposed animals had 1.74 ± 0.50 (P = 0.49). In layer VI, control animals had 1.8 ± 0.87 FG+ neurons/mm2 and there were 1.14 ± 0.59 in VPA-exposed animals (P = 0.06). Even though there was no difference in the density of FG+ neurons in S1, there was a notable drop in the % of FG+ neurons. Specifically, in control animals, 20 ± 4.8% of neurons in layer V of S1 were FG+ after CNIC injections, but in VPA-exposed animals, only 12.8 ± 3.1% were FG+. In layer VI of S1, 9.4 ± 3% of neurons were FG+ in control, but in VPA-exposed animals, only 4.2 ± 1.7% were FG+.

Fig. 7.

Fig. 7

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Location of matched injections sites. To control as much as possible for location and spread of FG deposits, we selected matched injection sites from 4 control A) and 4 VPA-exposed animals B). These injections sites were biased toward the caudal half of the IC and included tracer spread into the EC and DC of the inferior colliculus. The scale bar in b is equal to 500 μm.

Fig. 8.

Fig. 8

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VPA exposure results in a lower density of cortical neurons retrogradely labeled from the IC. Shown in a (control) and B (VPA) is FG labeling after injection in the ipsilateral IC. Shown in C (control) and D (VPA) are representative sections through Au1 showing FG and NT. The whiskers in C and D indicate the thickness of layer V; the arrowheads indicate FG+ neurons in layer VI. The scale bar in B represents 720 μm and the scale bar in D represents 250 μm.

Fig. 9.

Fig. 9

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VPA exposure results in reduced projections from primary and secondary auditory cortices to the IC. The graphs show the density of FG+ neurons after injection in the ipsilateral CNIC in Au1 A), AuD B), AuV C), and S1 D) split by layers cortical layer. The whiskers represent the mean and SD. Key to symbols: * = P < 0.05, ** = P < 0.01, **** = P < 0.0001.

Discussion

This report provides the first evidence that in utero exposure to VPA results in thinner cortical layers, significant neuronal dysmorphology in auditory and somatosensory cortices, reduced CB-immunolabeling in Au1 and AuD and reduced corticocollicular projections from Au1 and AuV. We have previously found that in utero, VPA exposure results in fewer neurons in the auditory and vestibular brainstem (Lukose et al. 2011; Zimmerman et al. 2018; Mansour et al. 2019, 2022) and thalamus (Mansour et al. 2021b), fewer CB+ neurons in the brainstem (Zimmerman et al. 2018; Mansour et al. 2019, 2022) and cerebellum (Main and Kulesza 2017), imbalance of glutamatergic/GABAergic inputs (Alhelo and Kulesza 2022), reduced ascending projections to the CNIC and MG (Mansour et al. 2020), elevated hearing thresholds and longer latency ABR (Malhotra and Kulesza 2023). We have also found that VPA exposure results in delayed eye and ear opening, and lower body and brain weights after weening (Zimmerman et al. 2018; Mansour et al. 2019). These findings, together with the literature (Mabunga et al. 2015; Zhao et al. 2019), indicated that in utero VPA exposure impacts a number of brain regions but impacts the auditory pathway from the cochlear nucleus to the auditory cortex.

Neuronal morphology

In utero, VPA exposure results in a significant reduction in the total number of neurons in the VCN, SOC, NLL, CNIC, and MG (Lukose et al. 2011; Zimmerman et al. 2018; Mansour et al. 2019, 2021b). Notably, in human subjects with ASD, there are significantly fewer neurons in the SOC (Kulesza et al. 2011; Lukose et al. 2015). However, not all nuclei in the SOC are equally impacted. VPA exposure does not significantly impact the total number of neurons in the ventral nucleus of the trapezoid body in VPA-exposed rats or humans (rat: Mansour and Kulesza 2021; human: Kulesza et al. 2011; Lukose et al. 2015). VPA exposure does not impact the number of neurons in the medial vestibular nucleus (Mansour et al. 2022) or oropharyngeal lower motor neurons (Alhelo and Kulesza 2022). We did not estimate total neuron number in Au1 or surrounding cortical regions because of challenges ensuring clear rostro-caudal boundaries of physiologically defined regions. Nonetheless, we did find that VPA exposure results in a significant reduction in the thickness of layers II to IV, V, and VI in Au1, but there was no difference in the density of neurons. This reduced thickness is consistent with findings in humans with ASD (van Rooij et al. 2018). We interpret these findings to indicate there are fewer neurons in Au1 and surrounding cortical regions after VPA exposure. While we did find significantly fewer CB+ (likely GABAergic, discussed further below) neurons here, it is unclear what cell types are predominantly impacted.

Since VPA exposure results in 50% fewer neurons in the vMG (Mansour et al. 2021b) and this region of the thalamus is the main source of ascending input to Au1 (Lee and Winer 2008), it was not surprising to find fewer neurons in layer V of Au1. Layer V of auditory cortex projects to secondary cortical areas, the contralateral auditory cortex, and provides descending projections to the medial geniculate and brainstem (Games and Winer 1988; Moriizumi and Hattori 1991; Romanski and LeDoux 1993; Saldaña et al. 1996; Weedman and Ryugo 1996). As such, we propose that the decrease in neuron number we report is consistent with physiological changes in the auditory cortex in VPA-exposed animals (Engineer et al. 2014a, 2014b; Anomal et al. 2015; Cheng et al. 2022). In rats, layer V of Au1 includes mainly glutamatergic pyramidal neurons (Feliciano and Potashner 1995) that project to the contralateral cortex and send a descending projection to the thalamus (Games and Winer 1988). The non-pyramidal cells are most likely all GABAergic (Ottersen and Storm-Mathisen 1984; Winer and Larue 1989). In the auditory brainstem, VPA exposure appears to have a more severe impact on inhibitory neurons. Specifically, nuclei composed of predominantly GABAergic neurons have the largest loss of neurons and VPA exposure. The ventral nucleus of the lateral lemniscus provides the largest sub-collicular GABAergic projection to the CNIC and VPA exposure results in a 55% decrease in the number of neurons in this nucleus (Mansour et al. 2019). While there is some evidence from immunolabeling for calcium binding proteins that VPA exposure may result in fewer GABAergic neurons in the cerebral cortex (Watanabe et al. 2017; Cheng et al. 2022; Santos-Terra et al. 2022; current study), further work is needed to clarify what specific neuronal populations and circuits are impacted after VPA exposure, what degree brainstem hyperexcitability extends to auditory cortex (Dubiel and Kulesza 2016) and how this loss of GABAergic and/or glutamatergic neurons in layer V impacts hearing.

Throughout the auditory brainstem, cerebellum, and thalamus, we have found smaller neurons with only a few exceptions. The vast majority of neurons in the ventral cochlear nuclei, SOC, NLL, and MG are significantly smaller than in control animals (Lukose et al. 2011; Zimmerman et al. 2018; Mansour et al. 2019, 2021b). Interestingly, VPA exposure results in significantly larger neurons in the CNIC and dorsal nucleus of the lateral lemniscus (Mansour et al. 2019), the spinal vestibular nucleus, and lateral vestibular nucleus (Mansour et al. 2022). VPA exposure also results in significantly smaller Purkinje cells across all 10 lobules of the cerebellar vermis (Main and Kulesza 2017). In Au1, we found that stellate neurons in layer V tended to be larger in VPA-exposed animals. The reason for this finding is unclear but might be because there are fewer stellate neurons in this layer, and surviving ones have larger, more complex dendrites and/or more extensive axons and ectopic projections. On the contrary, in both AuD and AuV, we found more drastic changes in neuron size. In AuD, there were significantly smaller stellate and round/oval neurons in layers V and VI. In AuV, only the round/oval neurons were significantly smaller. Our morphological analysis was performed in animals at P28. So, the finding of smaller cell bodies might be attributed to delayed maturation/immature neurons with underdeveloped dendrites.

Calbindin

We have previously shown that in utero VPA exposure results in fewer CB+ neurons in the VCN, MNTB (Zimmerman et al. 2018), the NLL (Mansour et al. 2019), and cerebellum (Main and Kulesza 2017). Axons from the MNTB and DNLL traverse the lateral lemniscus en route to the midbrain and/or thalamus and are significantly thinner in VPA-exposed animals (Mansour et al. 2019). In the vestibular nuclei, there are fewer CB+ perisomatic puncta after VPA-exposure (Mansour et al. 2022). In rat auditory cortex, the highest density of CB+ neurons is found in layers II/III with fewer in layers IV to V (Friauf 1994) and in cortical regions, CB colocalizes with GABAergic neurons. While there was a trend for fewer CB+ neurons in all regions we examined, there were significantly fewer CB+ neurons in layers V and VI in Au1 and layer VI in AuD. Currently, it is unclear if the reduction in CB immunolabeling is from death of CB+ neurons or if surviving neurons are simply no longer producing this calcium binding protein at detectable levels. Alternatively, the loss of CB/GABAergic neurons in the auditory cortex could result in hyperexcitability and seizure activity. We believe that reduced GABAergic inputs could explain some of the physiological observations in auditory cortex. Specifically, reduced speech discrimination (Engineer et al. 2014a), increased amplitudes in evoked potentials, and reduced vector strengths (Engineer et al. 2014b). However, increased latencies and decreased firing rates (Engineer et al. 2014b) might be attributed to disinhibition pathways. Therefore, it seems that neuron number, connectivity, and excitatory/inhibitory balance collectively contribute to these abnormal brainstem and cortical responses in VPA-exposed animals (Engineer et al. 2014a, 2014b; Anomal et al. 2015; Cheng et al. 2022; Malhotra and Kulesza 2023).

How in utero VPA exposure impacts CB expression at P28 is unclear. We proposed that this is mediated through VPAs effect on histone deacetylases (Göttlicher et al. 2001; Ornoy 2009) resulting in reduced expression of CB in surviving neurons. CB is also known to provide protection from apoptosis (Sun et al. 2011). Therefore, it is possible that CB-expressing neurons may die during the early postnatal period when CB is first detectable (Friauf 1994). Since CB expression and GABAergic lineages are linked in the cerebral cortex, we hypothesize the loss of CB/GABAergic neurons in cortical regions occurs from early cell death. VPA also increases levels of GABA and blocks voltage-gated ion channels (Zona and Avoli 1990; Owens and Nemeroff 2003). But it is unclear to what degree this mechanism is utilized during embryonic exposures from E10 to 12. It seems possible that VPA could increase levels of GABA in the embryonic brain and this might serve as a negative feedback for development of GABAergic neuronal populations.

Corticollicular projections

VPA exposure results in reduced ascending projections from the VCN, SOC, and NLL to the CNIC and from the VCN and SOC to the MG (Mansour et al. 2020). While we did find significantly fewer neurons throughout the auditory brainstem, proportionally fewer neurons were labeled after tracer injections in the CNIC and vMG. So, not only are there fewer neurons in VPA-exposed animals, but fewer surviving neurons make the appropriate projections. We did not adjust the volume of the tracer injection for animal’s body weight. Since VPA-exposed animals have smaller brains, the injected volume was relatively larger than reported in these animals. Therefore, we believe the difference in these projections is larger between control and VPA-exposed animals. However, alteration in axonal projection patterns is not ubiquitous. In the ventral nucleus of the trapezoid, we found that VPA exposure did not impact descending projections to the cochlea or ascending projections to the IC of vMG (Mansour et al. 2021b). Furthermore, there was no difference in the projection from the DNLL or MSO to the vMG, although these are relatively minor projections (Mansour et al. 2020).

Descending cortical projections to the IC are well established (Beyerl 1978; Games and Winer 1988; Saldaña et al. 1996) and target the entire ipsilateral IC and the dorsal regions of the contralateral medial IC (Saldaña et al. 1996). This descending projection is glutamatergic and contacts distal dendrites of IC neurons (Saldaña et al. 1996). Large injections of retrograde tracers in the IC results in many retrogradely labeled neurons in layer V with a smaller population in the deepest part of layer VI (Games and Winer 1988; Doucet et al. 2003; Bajo and Moore 2005). This is consistent with our injection sites (Fig. 7). Projections from ipsilateral Au1/AuD/AuV to the IC outweigh those from the contralateral side nearly 4:1 (Olthof et al. 2019). However, this is not the case for S1 where the ipsi:contra projection to CNIC is 1.2:1 (Olthof et al. 2019). These contralateral projections, along with projections from prefrontal, motor, and visual cortical regions, will be the subject of further study. Importantly, significant alterations in cortical connectivity have been found in humans with ASD (Wilson et al. 2022; Alho et al. 2023). We found fewer FG+ neurons in layer V in AuD and AuV and layer VI in Au1, AuD, and AuV. Besides there very likely being fewer neurons in these cortical regions projecting to the auditory midbrain, we suspect there are also significant changes in how these cortical axons are distributed in the IC—anterograde tracing studies will be required to examine terminal fields. Finally, we found no difference in the number of FG+ neurons in S1. The reason for this is unclear but it may be that development and maintenance of such descending somatosensory projections to the auditory midbrain are less dependent on activity in the ascending auditory pathway, more specifically activity in the MG and Au1.

Mechanism and implications

In VPA-exposed animals, the reduction in neuron number extending from the cochlear nucleus to the auditory cortex and reduced connectivity is most likely attributed to deficits in neuronal proliferation and migration in utero or in the early postnatal period. Regardless, the drastic loss of neurons throughout the auditory pathway likely has a significant impact in processing of auditory information. It is unclear how in utero VPA exposure is impacting the number of CB+ neurons, but this may arise from altered expression and/or developmental loss of CB/GABAergic neurons. Regardless, in the cortex this likely results in hyperexcitability, abnormal tonotopic maps, and loss of timing information (decreased vector strengths, poor spectral coding) that is essential for interpretation of and production of vocalizations. Indeed, there is evidence for impaired vocalization in VPA-exposed animals (Gzielo et al. 2020; Potasiewicz et al. 2020). We have shown that VPA-exposed animals have higher ABR thresholds and increased latencies at P22. While latencies normalize by P60, there are significant differences in latency and interpeak latencies that persist near threshold up to P360 (Malhotra and Kulesza 2023). Beyond these differences in timing of evoked potentials, we believe our findings provide evidence that in utero VPA exposure results in impaired temporal and spectral coding extending all the way to association auditory cortex. Our results also suggest that VPA exposure impacts descending projections to the IC and likely cortical influence on the SOC and CN. Together, these findings highlight the widescale disruption of the auditory system in this animal model of ASD, the utility of simple, noninvasive auditory tests for early screening, and the importance of speech and sound therapy in ASD.

Author contributions

Kara Kosmer (Formal analysis, Investigation, Data curation, Writing—original draft, review & editing, visualization) and Randy Kulesza (Conceptualization, Methodology, Formal analysis, Investigation, Resources, Writing—original draft, review& editing, Visualization, Supervision, Project administration, Funding acquisition)

Funding

A training grant from the Lake Erie Consortium for Osteopathic Medical Training.

 

Conflict of interest statement: None declared.

Contributor Information

Kara Kosmer, RWJBH Monmouth Medical CenterLong Branch, NJ 07740, United States.

Randy Kulesza, Department of Anatomy, Lake Erie College of Osteopathic Medicine, Erie, PA 16509, United States.

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