Introduction: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social and communication deficits, cognitive dysfunction, and stereotyped repetitive behaviors. Regional volume changes are commonly observed in individuals with ASD. To examine volumetric dysregulation across adolescence, the valproic acid (VPA) model was used to induce ASD-like phenotypes in rats. Method: Regional volumes were obtained via magnetic resonance imaging at either postnatal day 28 or postnatal day 40 (P40), which correspond to early and late adolescence, respectively. Results: Consistent with prior research, VPA animals had reduced total brain volume compared to control animals. A novel outcome was that VPA animals had overgrown right hippocampi at P40. Differences in the pattern of development of the anterior cingulate cortex were also observed in VPA animals. Differences for the posterior cingulate were only observed in males, but not females. Conclusion: These results demonstrate differences in region-specific developmental trajectories between control and VPA animals and suggest that the VPA model may capture regional volume changes consistent with human ASD.

Autism spectrum disorder (ASD) is a prevalent neurodevelopmental disorder characterized by social, communication, and repetitive behavioral issues. In order to develop better treatment options, it is critical to understand the developmental trajectory and neural underpinnings of ASD. Adolescence has been suggested as a critical period where brain changes can impact ASD symptomology [1]. For instance, in humans with ASD, there are alterations in pruning rates across adolescence [2], overgrowth of frontal cortices [3], and decreased cerebellar volumes [4]. Executive function deficits are also found in those with ASD, and these are associated with altered structural development of frontal, limbic, and cerebellar regions [1].

The hippocampus is involved in various cognitive and executive functions that appear to be aberrant in ASD. A meta-analysis examining the relationship between total hippocampal volume and memory performance across development in typically developing individuals demonstrated that larger hippocampal volumes are associated with improved memory [5]. Structural and functional alterations of the hippocampus have been found in individuals with ASD and appear to manifest at specific points in development. Several studies have found increased hippocampal volumes in adolescents with ASD [6, 7]. It has been suggested that individuals with ASD may require greater hippocampal recruitment when performing encoding tasks to compensate for prefrontal dysfunction [8].

Decreased cerebellar volume has also been associated with ASD symptomology [4], and altered cerebellar connectivity and function may contribute to cognitive and communication deficits [9‒11]. Human postmortem studies and animal models have found decreased cerebellar cell counts [12]. One magnetic resonance imaging (MRI) study has also found decreased volumes in valproic acid (VPA)-exposed animals [13]. Finally, individuals with ASD also have alterations in the amygdala [6]. However, very few studies have examined volume of the amygdala in animal models of ASD.

Animal models are useful for examining developmental differences across adolescence. Here, the VPA model was developed to induce ASD-like behaviors in rodents. Previous research has established the critical periods for VPA administration [14] and found social and repetitive behavioral deficits [15‒18] in VPA offspring. The mechanisms of VPA exposure leading to ASD symptomology are poorly understood, but hypotheses posit that mechanisms may include changes in synaptic excitation and inhibition balance, histone deacetylase inhibition, and alterations to various neurotransmitter systems as causes of ASD symptomology [19, 20]. In addition, children of females who take VPA as a prescription drug during pregnancy develop ASD at a higher rate than the general population, adding external validity to the VPA model [21‒23]. Although the VPA model is environmental, 40–50% of the variance of developing ASD is unexplained by genetic risks, which means that both genetic and environmental models are important to understand the various types of ASD [24‒26].

The current study was designed to examine volumetric differences at two stages of adolescent development: postnatal day 28 (P28) and postnatal day 40 (P40). These two ages correspond to early and late adolescence, respectively [27]. Comparisons between brain areas at different ages were done by normalizing volume to total brain volume. This calculation then allowed comparisons of a specific brain region at early and adolescent ages, but still accounting for overall growth (via total brain volume increases with age). The current study examined brain volumes in one cohort at P28 and another cohort at P40. The P40 rats also performed an attentional set-shifting task to assess cognitive performance and behavior published in a prior paper [28]. Brain developmental windows in rodents based on cell densities, maturation rates, and myelination are understood to correspond to developmental windows in humans [27]. For instance, P25–35 roughly corresponds to ages 4–11 in humans, in part based on the development of specialization and maturation of prefrontal neural networks [29], and P35–49 has been suggested to correspond to 12–18 years in humans. P28 and P40 fall in the middle of these developmental windows. It was hypothesized that prefrontal volumes and hippocampal volumes of VPA rats would be enlarged compared to control rats at both P28 and P40, whereas total cerebellar volume would be decreased, which aligns with human data [30]. Brain volumes were normalized to total brain volume to account for normal growth changes with age. These results would support the hypothesis that these structural brain changes are contributing to altered behaviors and could indicate that earlier interventions to boost plasticity may improve social and/or cognitive outcomes.

Subjects

Pregnant Long-Evans rat dams were shipped from Charles River on gestational day 6 to the research facility. Dams were injected intraperitoneally on gestational day 12 with a single dose of saline or VPA (sodium valproate [Sigma], 250 mg/mL, mixed in saline, administered at 600 mg/kg). Dams were briefly anesthetized with isoflurane gas to administer a less stressful injection. The isoflurane administration (required by the IACUC) was very brief, lasting only a few minutes, and should not have adversely effected the pups, as more prolonged exposure is required to see adverse effects in offspring [31]. All procedures were conducted in accordance with the Kansas State University IACUC guidelines. Rats were given free access to food except for when undergoing preparation for the set-shifting task. Lights were on from 7:00 a.m.–7:00 p.m. Rats were reared with litter mates until weaning and then were pair-housed with a same-sex littermate. One male and one female from each litter were used for each experimental condition to account for litter effects [32]. For P28, control female = 8, VPA female = 13; control male = 10, VPA male = 8. For P40, control female = 14, VPA female = 8; control male = 10, VPA male = 8.

Tissue Harvest and Preparation

At P28 or P40, animals were euthanized by pentobarbital overdose. After animals displayed complete loss of pedal withdrawal and corneal reflexes, they were transcardially perfused with 0.9% saline followed by 4% formaldehyde. After perfusion, animals were decapitated, and heads were postfixed in 4% formaldehyde prior to MRI acquisition.

MRI Volumetric Measurements and Segmentation

Anatomical images for volumetric measurements were collected via MRI using a Bruker 600 MHz dual NMR/MRI and micro 2.5/MICWB40 probe configured and 30 mm × 40 mm coil insert. Brains were placed in a flat bottom glass tube and covered with Fomblin (a safe, nontoxic, inert perfluoropolyether oil which is MRI silent and often used to increase signal-to-noise ratios and decrease magnetic susceptibility distortions [33]) and secured with a pronged glass stabilizer specially made to ensure sample position consistency. Scans were performed with rapid acquisition with refocused echo pulse sequences in ∼1 h and 45 min with one repetition, a repetition time of 2,500 ms, echo time of 30.23 ms, and a rapid acquisition with refocused echo factor of 16. The pulse sequence used a flip angle of 90°, acquired two averages, with slices acquired in an interlaced pattern. Image resolution was approximately 100 × 100 × 300 μm with matrix sizes, field of view, and number of slices varying according to brain size but balanced to maintain comparable image resolutions between image series. The utilization of adaptable parameters for imaging allows for maximal signal-to-noise and contrast-to-noise ratios dependent upon sample size.

Brain regions of interest were segmented by one trained blind-to-condition researcher. Regions were segmented with ITK-SNAP, and each region of interest was outlined based on anatomical landmarks. The volume for each region was measured by ITK-SNAP. Total brain volume included gray matter, white matter, and ventricles. The active contour segmentation tool was used to fill the entire brain including the cerebellum and brain stem, and any overfilling into the intracranial space was cleaned in coronal slice views. All region of interests followed the regions as listed below from Paxinos and Watson, 7th edition. The hippocampus was defined as the dorsal hippocampus only, and segmentation ended at the slice that matched −5.28 from bregma. Frontal regions included the anterior cingulate cortex (ACC) (areas: A24a, A24b) and the posterior cingulate cortex (PCC) (areas: A30, A29c, A29b) (Fig. 1). Other regions were amygdala central nucleus (CE) (CeM, Cel, CeC) lateral/basolateral (BMA, LaDL, BLP, LaVM, LaVL), crus I, lobule VI, and the total cerebellum all lobules, excluding the pons. Segmentations were screened for consistency through a 3D modeling in ITK-SNAP before evaluation of total volume.

Fig. 1.

MRI scan of coronal (a), sagittal (b), horizontal (c) views of one brain. Green region in (a) represents one slice of ACC. Right and left are flipped, anterior is in the downward direction for (b) and (c). Coronal slice is ∼0.48 mm from bregma (blue horizontal line from (c) shows where coronal is located), sagittal view is barely off midline (0.4 mm lateral) to left side (blue vertical line in (c) shows location of sagittal slice), and horizontal is 2.90 mm above interaural line (see blue vertical line in (b) to see slice location).

Fig. 1.

MRI scan of coronal (a), sagittal (b), horizontal (c) views of one brain. Green region in (a) represents one slice of ACC. Right and left are flipped, anterior is in the downward direction for (b) and (c). Coronal slice is ∼0.48 mm from bregma (blue horizontal line from (c) shows where coronal is located), sagittal view is barely off midline (0.4 mm lateral) to left side (blue vertical line in (c) shows location of sagittal slice), and horizontal is 2.90 mm above interaural line (see blue vertical line in (b) to see slice location).

Close modal

Data Analysis

Brain volumes were analyzed with 3-way ANOVAs (condition × sex × age) or 2-way ANOVAs (condition × age) based on previously observed sex differences. Post hoc tests are reported for each region.

Total Body Weight

There were no significant differences between condition for male or female rats for total body weights at the time of brain collection, P28 animals: control males (90 g mean, 3.0 SD); VPA males (88 g mean, 3.8 SD); control females (74 g mean, 4.4 SD); VPA females (72 g mean, 9.4 SD); P40 animals: control males (107 g mean, 4.4 SD); VPA males (99 g mean, 4.5 SD); control females (97 g mean, 3.7 SD); VPA females (98 g mean, 5.1 SD).

Total Brain Volume

A three-way ANOVA (sex, age, condition) had significant main effects of age (F1, 75 = 11.23, p < 0.05) and condition (F1, 75 = 6.38, p < 0.05), which indicated VPA animals had smaller volumes in general, and P28 brains were smaller. Sidák post hoc tests found that female VPA P28 animals were significantly smaller compared to female control P40 animals (Fig. 2). As expected, VPA animals had smaller total brain volume compared to control animals and P28 animals had smaller brains compared to P40.

Fig. 2.

Total brain volume. P40 animals had larger volumes than P28 animals (F1, 75 = 11.23, *p < 0.05). VPA animals had smaller volumes compared to control animals (F1, 75 = 6.38, *p < 0.05).

Fig. 2.

Total brain volume. P40 animals had larger volumes than P28 animals (F1, 75 = 11.23, *p < 0.05). VPA animals had smaller volumes compared to control animals (F1, 75 = 6.38, *p < 0.05).

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Hippocampus Volumes

No sex effects were found for hippocampus volume; therefore, data were collapsed across sex. A three-way ANOVA was conducted to examine (condition, age, and hemisphere) with the Benjamini and Krieger correction for multiple comparisons. There was no significant difference between hemispheres, but there was a condition by age interaction (F1, 152 = 8.74, p < 0.05). The post hoc tests demonstrated that within the left hemisphere the proportional size of the hippocampus was larger at P40 in VPA animals compared to P28 VPA animals and P28 control animals (p < 0.05, Fig. 3), whereas the control animals did not show this excessive growth at P40. In VPA animals at P40, the right hippocampus was larger than control P28, VPA P28, and control P40 animals (p < 0.05, Fig. 3); see Table 1 for values.

Fig. 3.

Hippocampus volume: within the left hippocampus, the proportional size of the hippocampus was larger at P40 in VPA animals compared to P28 VPA animals and P28 control animals (p < 0.05). In VPA animals at P40, the right hippocampus was larger than control P28, VPA P28, and control P40 animals (*p < 0.05, significant interaction [F1, 152 = 8.74, *p < 0.05]).

Fig. 3.

Hippocampus volume: within the left hippocampus, the proportional size of the hippocampus was larger at P40 in VPA animals compared to P28 VPA animals and P28 control animals (p < 0.05). In VPA animals at P40, the right hippocampus was larger than control P28, VPA P28, and control P40 animals (*p < 0.05, significant interaction [F1, 152 = 8.74, *p < 0.05]).

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Table 1.

Normalized hippocampus volumes with standard error (st err)

ControlVPAControlVPA
L HippL HippR HippR Hipp
meansst errmeansst errmeansst errmeansst err
P28 
 Female 0.009809 0.000405 0.010076 0.000314 0.010017 0.000557 0.00965 0.000266 
 Male 0.010417 0.00046 0.010025 0.000271 0.01022 0.000364 0.010035 0.000207 
P40 
 Female 0.010572 0.000259 0.011682 0.000519 0.010275 0.000322 0.011464 0.00042 
 Male 0.010806 0.000291 0.011096 0.000151 0.010663 0.000234 0.011219 0.000337 
ControlVPAControlVPA
L HippL HippR HippR Hipp
meansst errmeansst errmeansst errmeansst err
P28 
 Female 0.009809 0.000405 0.010076 0.000314 0.010017 0.000557 0.00965 0.000266 
 Male 0.010417 0.00046 0.010025 0.000271 0.01022 0.000364 0.010035 0.000207 
P40 
 Female 0.010572 0.000259 0.011682 0.000519 0.010275 0.000322 0.011464 0.00042 
 Male 0.010806 0.000291 0.011096 0.000151 0.010663 0.000234 0.011219 0.000337 

Cortex Volumes

Frontal cortex volumes were separated by sex based on past behavioral findings [28] and analyzed with 2-way ANOVAs (condition and age). For the ACC, females showed a significant interaction (F1, 39 = 6.70, p < 0.05) and main effect of age (F1, 39 = 5.47, p < 0.05) (Fig. 4a). LSD post hoc tests found P28 control animals had larger ACC volumes compared to P40 control animals (Fig. 4a). For the PCC, there were no significant differences in the females (Fig. 5a). For the ACC of males, there were no significant findings (Fig. 4a). For the male PCC, there was a significant main effect of age (F1, 36 = 12.47, p < 0.05), where for normalized volume the PCC was smaller at P40 compared to P28 (regardless of condition) (Fig. 5b).

Fig. 4.

ACC. a In females, the normalized ACC volume was larger at P28 compared to P40 for control animals but not for VPA animals (F1, 39 = 6.70, *p < 0.05). b No significant differences were found in the male ACC.

Fig. 4.

ACC. a In females, the normalized ACC volume was larger at P28 compared to P40 for control animals but not for VPA animals (F1, 39 = 6.70, *p < 0.05). b No significant differences were found in the male ACC.

Close modal
Fig. 5.

PCC. a No significant differences were found in the female PCC. b Both control and VPA males demonstrated a relative decrease in PCC volume from P28 to P40 (F1, 36 = 12.47, *p < 0.05).

Fig. 5.

PCC. a No significant differences were found in the female PCC. b Both control and VPA males demonstrated a relative decrease in PCC volume from P28 to P40 (F1, 36 = 12.47, *p < 0.05).

Close modal

Amygdala Volumes

The amygdala was segmented into the CE (left and right) as well as lateral (lateral and basolateral nuclei) and combined left or right (central and lateral combined for each hemisphere). For the CE, a 3-way ANOVA with (sex, hemisphere, and age) found a significant interaction between age and sex (F1, 156 = 7.34, p < 0.05). LSD post hoc tests found that P40 males had larger left CE proportionally compared to both P28 males and P40 females (Fig. 6, p < 0.05). This was not found for the right CE; see Table 2 for values.

Fig. 6.

CE of amygdala: there was a significant interaction between age and sex (F1, 156 = 7.34, *p < 0.05) where P40 males had larger left CE proportionally compared to both P28 males and P40 females (*p < 0.05), regardless of condition.

Fig. 6.

CE of amygdala: there was a significant interaction between age and sex (F1, 156 = 7.34, *p < 0.05) where P40 males had larger left CE proportionally compared to both P28 males and P40 females (*p < 0.05), regardless of condition.

Close modal
Table 2.

Normalized amygdala volumes with standard error (left and right amygdala, CE, BLA)

ControlVPAControlVPAControlVPA
left amygdalaleft amygdalaleft BLAleft BLAleft CEleft CE
meansst errmeansst errmeansst errmeansst errmeansst errmeansst err
P28 
 Female 0.0034156 0.000206 0.0032828 0.0001717 0.0024863 0.0001629 0.0023453 0.0001458 0.0009518 0.000104 0.0009375 0.000051 
 Male 0.0033728 0.00018 0.0030075 0.0001797 0.0023808 0.0001673 0.002213 0.000137 0.000992 0.0001034 0.0008033 0.000048 
P40 
 Female 0.0032435 0.00013 0.0030009 0.0003388 0.0023399 0.000104 0.0021186 0.0003041 0.0008931 0.000037 0.0008559 0.000061 
 Male 0.003477 0.000199 0.0036214 0.0001668 0.00258 0.0001964 0.002572 0.0001888 0.0010282 0.000043 0.000996 0.000065 
ControlVPAControlVPAControlVPA
left amygdalaleft amygdalaleft BLAleft BLAleft CEleft CE
meansst errmeansst errmeansst errmeansst errmeansst errmeansst err
P28 
 Female 0.0034156 0.000206 0.0032828 0.0001717 0.0024863 0.0001629 0.0023453 0.0001458 0.0009518 0.000104 0.0009375 0.000051 
 Male 0.0033728 0.00018 0.0030075 0.0001797 0.0023808 0.0001673 0.002213 0.000137 0.000992 0.0001034 0.0008033 0.000048 
P40 
 Female 0.0032435 0.00013 0.0030009 0.0003388 0.0023399 0.000104 0.0021186 0.0003041 0.0008931 0.000037 0.0008559 0.000061 
 Male 0.003477 0.000199 0.0036214 0.0001668 0.00258 0.0001964 0.002572 0.0001888 0.0010282 0.000043 0.000996 0.000065 
ControlVPAControlVPAControlVPA
right amygdalaright amygdalaright BLAright BLAright CEright CE
meansst errmeansst errmeansst errmeansst errmeansst errmeansst err
P28 
 Female 0.003262 0.000179 0.0033804 0.000177 0.0023777 0.0001344 0.0024321 0.0001543 0.0008953 0.000060 0.0009465 0.000050 
 Male 0.0031202 0.000246 0.0030042 0.0001566 0.0022437 0.0002343 0.0021617 0.0001261 0.0008896 0.000054 0.0008151 0.000057 
P40 
 Female 0.0031611 0.000108 0.0034382 0.0003197 0.0023008 0.000085 0.0025022 0.0002773 0.000860 0.000032 0.0009361 0.000077 
 Male 0.0033632 0.000241 0.0034294 0.0002294 0.002449 0.0002156 0.0024753 0.0001735 0.0009142 0.000034 0.0009541 0.000077 
ControlVPAControlVPAControlVPA
right amygdalaright amygdalaright BLAright BLAright CEright CE
meansst errmeansst errmeansst errmeansst errmeansst errmeansst err
P28 
 Female 0.003262 0.000179 0.0033804 0.000177 0.0023777 0.0001344 0.0024321 0.0001543 0.0008953 0.000060 0.0009465 0.000050 
 Male 0.0031202 0.000246 0.0030042 0.0001566 0.0022437 0.0002343 0.0021617 0.0001261 0.0008896 0.000054 0.0008151 0.000057 
P40 
 Female 0.0031611 0.000108 0.0034382 0.0003197 0.0023008 0.000085 0.0025022 0.0002773 0.000860 0.000032 0.0009361 0.000077 
 Male 0.0033632 0.000241 0.0034294 0.0002294 0.002449 0.0002156 0.0024753 0.0001735 0.0009142 0.000034 0.0009541 0.000077 

CE, central nucleus; BLA, basolateral and lateral.

Cerebellar Volumes

The volume of the total cerebellum was normalized to total brain volume, whereas cerebellar lobules were normalized to total cerebellar volume. For total cerebellar volume, there were no significant effects. Based on past sex differences [34], cerebellar lobules were separated by sex and analyzed by condition and age. For females, there were no main effects in left crus I volume, but simple comparisons found that VPA animals at P40 had enlarged left crus I compared to P28, whereas this was not the case for controls. For female right crus I, there was a main effect age (F1, 34 = 5.31, p < 0.05; Fig. 7), and LSD post hoc tests demonstrated that for VPA animals right crus I was enlarged at P40 compared to P28; see Table 3 for values.

Fig. 7.

Right cerebellum crus I. a No significant differences were found in male right crus I volume. b Female right crus I was enlarged at P40 in VPA animals relative to P28, but not in control animals (F1, 34 = 5.31, *p < 0.05).

Fig. 7.

Right cerebellum crus I. a No significant differences were found in male right crus I volume. b Female right crus I was enlarged at P40 in VPA animals relative to P28, but not in control animals (F1, 34 = 5.31, *p < 0.05).

Close modal
Table 3.

Normalized cerebellum volumes with standard error (total cerebellum, left crus I, right crus I, lobule VI)

ControlVPAControlVPA
left crus Ileft crus Iright crus Iright crus I
meansst errmeansst errmeansst errmeansst err
P28 
 Female 0.039454 0.002535 0.040153 0.002233 0.037117 0.003352 0.038755 0.002047 
 Male 0.036304 0.002082 0.03776 0.001672 0.035206 0.001848 0.037558 0.001919 
P40 
 Female 0.041002 0.001542 0.047799 0.003223 0.050025 0.002202 0.048575 0.005819 
 Male 0.038111 0.00117 0.040605 0.003708 0.039385 0.001552 0.039443 0.003604 
ControlVPAControlVPA
left crus Ileft crus Iright crus Iright crus I
meansst errmeansst errmeansst errmeansst err
P28 
 Female 0.039454 0.002535 0.040153 0.002233 0.037117 0.003352 0.038755 0.002047 
 Male 0.036304 0.002082 0.03776 0.001672 0.035206 0.001848 0.037558 0.001919 
P40 
 Female 0.041002 0.001542 0.047799 0.003223 0.050025 0.002202 0.048575 0.005819 
 Male 0.038111 0.00117 0.040605 0.003708 0.039385 0.001552 0.039443 0.003604 
ControlVPAControlVPA
total cerebellumtotal cerebellumlobule VIlobule VI
meansst errmeansst errmeansst errmeansst err
P28 
 Female 0.150407 0.005901 0.145477 0.002658 0.045443 0.012267 0.050763 0.002327 
 Male 0.154223 0.00392 0.145433 0.002147 0.045427 0.007909 0.051569 0.002768 
P40 
 Female 0.153795 0.003276 0.145917 0.004246 0.043667 0.002231 0.049437 0.002377 
 Male 0.154223 0.002816 0.156163 0.004859 0.045586 0.001734 0.046493 0.002053 
ControlVPAControlVPA
total cerebellumtotal cerebellumlobule VIlobule VI
meansst errmeansst errmeansst errmeansst err
P28 
 Female 0.150407 0.005901 0.145477 0.002658 0.045443 0.012267 0.050763 0.002327 
 Male 0.154223 0.00392 0.145433 0.002147 0.045427 0.007909 0.051569 0.002768 
P40 
 Female 0.153795 0.003276 0.145917 0.004246 0.043667 0.002231 0.049437 0.002377 
 Male 0.154223 0.002816 0.156163 0.004859 0.045586 0.001734 0.046493 0.002053 

This novel study examined brain volume at two different developmental ages during adolescence in the VPA model. Normalized volumetric measurements allowed direct comparisons between ages. Consistent with past findings, VPA animals had reduced total brain volume compared to controls, and P28 animals had smaller brains than P40 animals [35]. Despite reduced overall brain volumes, the right and left hippocampi were overgrown in VPA animals at P40 but not at P28. In addition, there were volumetric differences in VPA animals. The pattern of brain development between controls and VPA animals was observed to be different in the ACC and PCC. For the ACC, control animals had smaller volumes at P40 compared to P28. This was only observed in females and was not the case for the VPA animals. This pattern across groups suggests that control animals may increase synaptic pruning from P28 to P40, thereby reducing volume, whereas this did not happen in VPA animals. These data highlight differences in region-specific developmental trajectories between control and VPA animals and suggest that the VPA model may capture region-specific changes in volume consistent with human ASD. Within the PCC, both control and VPA males had smaller volumes at P40 compared to P28 animals, which could be related to regional developmental sex differences.

Hippocampus

The VPA animals had overgrowth of the dorsal right and left hippocampi proportionally compared to P28 animals. Additionally, the right hippocampus was enlarged compared to aged-matched controls at P40. This was not observed at P28. Studies in adolescent humans with ASD have found enlargements of the hippocampus at this age, suggesting that this may be another region-specific change captured by the VPA model [6, 7, 36]. The increase in volume observed here is probably not linked to microglia as these were not elevated at the same time point [37] but could be related to other cell types. In humans, these increased volumes have been linked to worse social interactions [38], and it has also been suggested that enlarged volumes are being utilized to compensate for impaired posterior medial network function [8, 39]. This paper was focused on structural changes, but future studies should use tasks known to recruit hippocampal function (e.g., novel object, Morris water maze) to examine how behaviors and function differ in adolescent VPA and control animals. The hippocampus is also important for pairing cues across time, such as in trace conditioning. Even though the hippocampus has been extensively studied for its role in learning and memory, it may also interact with the cerebellum for timing tasks [40]. Additionally, it interacts with limbic structures that play a role in social interactions and interpreting non-verbal social cues [41]. Future studies should include social memory tasks in ASD models to examine how hippocampal function impacts behavioral phenotypes. This study suggests the VPA model is inducing hippocampal overgrowth which is also observed in humans with ASD, and future studies examining how the hippocampus is recruited during tasks in vivo may provide additional insight into how it impacts this wide array of functions within ASD.

Prefrontal and Cortical Differences

VPA females had increased ACC volumes compared to age-matched controls at P40. However, for the control group, normalized volumes at P40 were smaller than at P28. This may indicate that control animals are pruning from P28 to P40, whereas this process may not occur in VPA animals, which may result in VPA animals having overgrowth in later adolescence. Excessive gray matter during adolescence has been observed in humans with ASD [42]. Furthermore, adolescents with ASD have demonstrated greater activation of anterior cingulate regions during cognitive attention tasks [43]. The increases in frontal volumes observed in this study support that the VPA model is capturing another component of ASD, with the increases in ACC volume being greater in the VPA females compared to control females. This may be driven by synaptic pruning in controls that is not happening in the VPA group, leading to the observed overgrowth later in adolescence.

Human longitudinal studies suggest that the frontal cortex and other cortical regions with overgrowth are then followed by periods where gray matter declines at a faster rate in those with ASD [30, 44, 45]. The data suggest that synaptic pruning may be happening in control animals but not in VPA animals. Pruning rates have been found to be altered across different animal models of ASD [46] and support this hypothesis. Longitudinal within-subject MRI studies in the animal model would be beneficial to examine changes in volume across age. Additionally, finer-grain histological analysis could examine if changes in cells are linked to neurons or glial cells, which have been found in human postmortem studies [47, 48]. Some research suggests that differences in volume may be related to delayed pruning mechanisms [49]. This could be happening in the ACC of the VPA females.

Alterations in PCC structure [50], activation [51], and connectivity have also been found in those with ASD [52‒54]. Here, we found both control and VPA males had decreased PCC volumes at P40 compared to P28, a finding that was not observed in females. Altered connectivity between the PCC and frontal regions and decreased regional homogeneity have been observed in males with ASD [55]. This project observed changes occurring across development in males and not females within the PCC; it is possible that this trajectory then accelerates through adolescence. Studying the different trajectories of specific brain areas through development is an ongoing endeavor, and future studies could examine this longitudinally to better understand sex differences. Examining if these structures in adult animal models are further differentiated between male control and male VPA animals could be a future direction.

Amygdala

The left amygdala and left CE were smaller in female rats than males, regardless of condition. Volumetric studies examining the amygdala of individuals with ASD have found that young children with ASD have enlarged amygdala volumes [36], while adults with ASD have decreased amygdala volumes [6]. These age-related differences indicate volumetric dysregulation of the amygdala across development. The fact that no impact of condition was found aligns with human studies where overgrowth in those with ASD occurs at an earlier age than in adolescence [36, 56]. Amygdala differences in individuals with ASD have been linked to different behavioral deficits [57]. Specifically, abnormalities of the amygdala are thought to be linked to impaired social behavior and emotional regulation [7]. VPA models have found increased levels of anxiety-like behavior linked to changes in the GABA system [58]. It is likely that volumetric dysregulation of the amygdala across development contributes to some of the core symptoms of ASD.

Cerebellar Regions

VPA animals had decreased total cerebellar volumes, but it did not reach statistical significance. This relationship is consistent with human studies [4, 9, 59, 60]. Right crus I was enlarged at P40 in VPA females only compared with P28, a relationship that was not observed in control or male animals. Previous research has found loss of Purkinje cells in animal models of ASD in crus I [13, 61‒63]. Prior scans in adult VPA rats found overgrowth of lobule VI in male VPA rats and decreased cerebellar volumes for female VPA rats [34]. In the human literature, bilateral crus I volume is decreased in those with ASD [4, 10]. However, most of these studies were completed at older ages and were not balanced for sex. The developmental trajectories of these regions may be different in females with ASD, but this requires more research [64]. However, these prior results examined brains in adulthood. Perhaps, this decrease occurs after adolescence; future longitudinal studies could address this hypothesis.

Mechanisms of Change

As mentioned previously, the VPA model has been a useful tool to create a rodent phenotype overlapping in biological, structural, functional, and behavioral changes that encapsulate some facets of ASD [65]. VPA exposure may cause changes in brain volume throughout development by altering pruning mechanisms [46], changing inflammation responses [66], altering astrocyte and microglia function [67]. Combining methodologies and examining multiple animal models may assist with the elucidation of complex interplay between these systems that impact brain development and which lead to behavioral symptomology.

Taken together, the results of this study suggest that VPA animals have structural changes during adolescence that are consistent with human results in adolescents with ASD. Future studies could manipulate specific structures such as the hippocampus and record from another region in the network to better understand the functional relationship across brain areas and how they are impacted within this model. The fact that volumetric changes are similar during the same ages in rodents and humans indicates that the model is capturing some of the components observed in ASD, meaning that, despite recent advances in genetic models of ASD, environmental models like the VPA model are still invaluable in examining the development of ASD.

This study protocol 4024 was reviewed and approved by the IACUC at Kansas State University.

There are no conflicts of interest.

This work was supported by start-up funds to Dr. Plakke from KSU and the National Institutes of Health NIGMS-GM113109.

Cole King and Hunter Strating: investigation and writing. Ivina Mali: methodology, data analysis, and software. Elizabeth Fangman and Jenna Neyhard: investigation. Macy Payne: methodology and data analysis. Stefan H. Bossmann: project administration, methodology, and funding acquisition. Bethany Plakke: conceptualization, formal analysis, resources, writing – original draft and editing, visualization, supervision, and funding acquisition.

Additional Information

Cole King and Ivina Mali should be considered as the co-first authors.

The data that support the findings of this study can be made available from the corresponding author B.P.

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