Autism Spectrum Disorder Model Mice Induced by Prenatal Exposure to Valproic Acid Exhibit Enhanced Empathy-Like Behavior via Oxytocinergic Signaling

Abstract

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by core symptoms, including impairments in social behavior and repetitive interests. Recent studies have revealed that individuals with ASD also display decreased empathy, ultimately leading to difficulties in social relationships; however, another report indicated that individuals with ASD have enhanced emotional empathy. Nonetheless, the neurobiological mechanisms underlying altered empathy in individuals with ASD remain unclear. In this study, we assessed empathy-like behaviors in valproic acid (VPA)-treated mice—a mouse model of ASD with observational fear learning. We then investigated the brain regions and signaling systems responsible for the altered empathy-like behaviors in VPA-treated mice. As a result, mice prenatally exposed to VPA displayed increased empathy-like behaviors, which were not attributed to altered sensitivity to auditory stimuli or enhanced memory for pain-related contexts. Immunohistochemical analysis revealed that the number of c-Fos positive oxytocinergic neurons in the paraventricular nucleus of the hypothalamus (PVN) was significantly higher in VPA-treated mice after observational fear learning. Finally, we found that pretreatment with L-368899, an antagonist of the oxytocin receptor, repressed the empathetic behavior in VPA-treated mice. These results suggest that VPA-treated ASD model animals showed increased emotional empathy-like behaviors through the hyperactivation of PVN oxytocinergic neurons for the first time. Further investigation of this hyperactivity will help to identify extrinsic stimuli and the condition which are capable of activation of PVN oxytocinergic neurons and to identify novel approach to enhance oxytocin signaling, which ultimately pave the way to development of novel therapy for ASD.

INTRODUCTION

Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by core symptoms, including impairments in social behavior and repetitive interests.^1,2) Individuals with ASD have been shown to often display other symptoms, such as decreased empathy, which leads to difficulties in social relationships.²⁾ However, a recent report indicates that individuals with ASD show enhanced emotional empathy towards other individuals with ASD compared to typically developed (TD) individuals.³⁾ Therefore, it is still under debate whether individuals with ASD have decreased or enhanced emotional empathy.

The etiology of ASD comprises a mixture of heterogeneous factors. ASD has strong genetic factors with a complex pattern of transmission^4–6) and is attributed to the involvement of approximately 1000 interacting genes^7,8); however, many “idiopathic” cases have also been reported. In addition to genetic factors, importance of environmental factors has been indicated in the pathogenesis of ASD and are reportedly linked to ASD pathophysiology.⁹⁾ The idiopathic factors affecting ASD pathogenesis include toxins, infections, air pollution, pesticides, and in utero exposure to drugs such as valproic acid (VPA).^10–16) Studies utilizing these ASD models have revealed the biological mechanisms underlying social deficits and perseverative behaviors observed in individuals with ASD. Thus, there are quite a few diagnostic tests and medical treatments available for ASD. However, the biological mechanisms underlying the apparent lack or enhancement of empathy in individuals with ASD have not yet been clarified. Therefore, further studies are needed to reveal the mechanisms underlying ASD symptoms, especially the apparent deficits in empathy.

VPA is widely used for treating epilepsy, migraines, and bipolar disorder.^17,18) However, the increased risk of malformation,¹⁹⁾ ASD, and mental retardation in children¹⁶⁾ by maternal use of VPA was reported. Notably, rodents prenatally exposed to VPA on embryonic day 12.5 (E12.5) show behavioral deficits, such as malfunction of sensory gating systems, decreased exploratory behaviors, increased repetitive behaviors, and social interaction deficits, similar to those observed in individuals with ASD.²⁰⁾ Thus, maternal exposure to VPA in mice has been utilized extensively as a mouse model of ASD for elucidation of biological mechanisms of autistic behavior and for development of therapeutic agents.^21,22) However, alterations in the empathy-like behavior of these animal models are still unknown.

Empathy is the ability to identify another person’s feelings, thoughts, and intentions and to respond appropriately to these emotions.²³⁾ This ability of empathy is vital for normal social activity and is impaired across several psychiatric disorders, resulting in secondary symptoms.^24–26) This process has been long known to be expressed by only humans; however, recent reports have indicated that rodents also display some aspects of empathy, such as rescue behavior, social modulation of distress, and socially transmitted emotion.^27–29) Observational fear learning models socially transmitted fear, which is regarded as an affective aspect of empathy, in which mice experience vicarious fear observing the distress of a conspecific.^30,31) In this system, the paraventricular nucleus of the hypothalamus (PVN), a brain region in which oxytocinergic neurons localize, plays important roles for processing socially transmitted fear,³²⁾ which is consistent with the roles of oxytocin in the enhancement of emotional empathy in humans.³³⁾ However, whether PVN is activated under the condition of observational fear learning in ASD model animals is still unclear.

In this study, we investigated the changes in empathy-like behaviors of VPA-treated mice. Moreover, we examined the necessity of oxytocinergic signaling from the PVN for alterations in the empathy-like behavior of VPA-treated mice.

MATERIALS AND METHODS

Animals

The experiments were conducted in accordance with the ethical guidelines of the Kyoto University Animal Experimentation Committee (Approval Code: 13-41-2, 19-41-1,2,3) and those of the Japanese Pharmacological Society. Male and female ICR (CD1) mice (Japan SLC, Shizuoka, Japan) were obtained at 8 weeks of age. They were kept at a constant ambient temperature of 22 ± 2 °C, under a 12 h light–dark cycle, and were tested during the light phase of this cycle. In all experiments, mice were at least 7 weeks old at the time of experimentation. The mice had free access to food and water at all times.

Vaginal cytology was performed to estimate the estrous cycle of female mice according to Hara et al.³⁴⁾ A small amount of water was used to harvest vaginal smears, and collected smears were Giemsa-stained (Nacalai Tesque Inc., Kyoto, Japan). To determine the estrous cycle stage, we analyzed the cell types in the Giemsa-stained sample according to previous reports by Caligioni³⁵⁾ and Cora et al.³⁶⁾ A male mouse was mated with a female mouse in proestrus or early estrus cycle for overnight. The next day was defined as embryonic day 0.5.

Preparation of VPA-Treated Mice

VPA (500 mg/kg; Toronto Research Chemicals Inc., ON, Canada) was dissolved in isotonic 0.9% NaCl solution (saline), at an injection volume of 10 mL/kg. Gestation day 12.5 mice were administered with VPA (500 mg/kg, intraperitoneally (i.p.)) or saline.³⁷⁾ Male offspring were selected at 3 weeks of age. Male offspring were randomly divided into cages (3–6 mice/cage) and used for behavioral and neurobiochemical analyses.

Social Interaction Test

An acrylic arena (30 × 15 × 20 cm, 350 lx) was used for analysis of social interaction. Mice were located to the arena for 60 min to habituate to environment. After habituation, a male novel conspecific mouse was placed into the arena. The behavioral interaction was digitally recorded. The sniffing (face sniffs and anogenital) behaviors of the resident mouse for 20 min were measured.

Observational Fear Test

The observational fear test was conducted according to Sakaguchi et al.³⁸⁾ with minor modifications. The test mouse and its cage mate were defined as the observer and demonstrator, respectively. Because mice in the same treatment group were housed in the same cage, we used VPA-treated mice as the demonstrator for VPA-treated observer mice, while vehicle-treated mice were used as the demonstrator for vehicle-treated mice. These mice were located into the shock chamber where these two mice were separated by a transparent plastic plate without perforation. A flat plastic plate (20 × 10 cm) was inserted just above a metal grid floor so that electric shock was not applied to the observer mouse. The chamber for the demonstrator has a metal grid floor (10 × 10 cm) connected to a shock scrambler (CBX-9M; Muromachi-Kikai, Tokyo, Japan). After 5-min habituation to the chamber, a 1.5 s electrical shock (0.6 mA) was applied to the demonstrator every 10 s for 4 min. Animals’ behavior was digitally recorded and immobility was automatically identified using a video-tracking system (ANY-MAZE version 6.00, Stoelting Co., Wood Dale, IL, U.S.A.). The threshold of freezing behavior was determined to be comparable to the detected immobility obtained manually by the experimenter. In the priming shock (PS) + fear observation (FO) group, observer mice were applied to single priming electrical shock (0.6 mA, 3 s duration). Interval between PS and FO was 2 h throughout the study.

Analysis of Auditory Sensitivity

Auditory sensitivity analysis was performed in a shock chamber using the same chamber that was used for the observational fear test. The test mouse and its cage mate were defined as the observer and demonstrator, respectively. These mice were located into the shock chamber where these two mice were separated by a transparent plastic plate without perforation, similar to what was done for the observational fear test. After 5-min habituation to the chamber, a 1.5 s auditory stimulus (4000 Hz, 85 ± 5 dB) was applied to the demonstrator every 10 s for 4 min. Similar to the observational fear test, observer mice were applied to single priming electrical shock (0.6 mA, 3 s duration) as a priming shock. The immobility time was calculated as described for the observational fear test.

Contextual Fear Conditioning

For contextual fear conditioning paradigm, mice were located to a plastic shock chamber (20 × 10 cm) with a metal grid floor. A shock scrambler (CBX-9M; Muromachi-Kikai) was connected to the metal grid to apply electrical shock. Three hundred seconds after placement in the chamber, the mice received a series of eight foot shocks (0.6 mA, 1.5 s) every 60 s. After 2 h interval, their movements in the same shock chamber were digitally recorded for 5 min. The immobility duration was calculated as described above.

Drug Treatment

L-368899 (hydrochloride) (5 mg/kg; Cayman Chemical Company, Ann Arbor, MI, U.S.A.)³²⁾ was dissolved in saline and i.p. injected into mice 30 min before fear observation, when conducting the observational fear tests. The administered dose was defined according to previous reports.^32,38) L-368899 is reported to bind preferentially with oxytocin receptors and reach the brain.³⁹⁾ For habituation purposes to drug injection, test animals received daily intraperitoneal injections of saline starting 3 d before the tests. Saline was used as the vehicle in these studies.

Immunohistochemistry

Mice were exposed to fear observation following a priming shock, as described above. As a negative control, observer and demonstrator mice were placed in the shock chamber to allow them to experience only the context of the fear observation chamber without a priming shock or foot shock. Ninety minutes after exposure to the context or fear observation test, mice were transcardially perfused with phosphate-buffered saline followed by 4% paraformaldehyde (Nacalai Tesque) diluted in 0.1 M phosphate buffer. The dissected brains were cryoprotected in 15% sucrose diluted in phosphate-buffered saline overnight at 4 °C and then frozen at −80 °C until sectioning. The brains were cryo-sectioned into 30 µm-thick coronal sections using a cryostat (Leica Biosystems, Nussloch, Germany) and stored at −20 °C until immunohistochemical processing.

For immunohistochemistry analysis of c-Fos-positive cells, the coronal sections on a slide glass were incubated with buffer containing anti-c-Fos antibody (rabbit anti-c-Fos antibody, 1 : 2000; #2250 s, Cell Signaling Technology, Danvers, MA, U.S.A.) at 4 °C overnight. After rinse with phosphate-buffered saline, the sections were incubated with buffer containing secondary antibody (Alexa Fluor 594-labeled donkey anti-rabbit immunoglobulin G (IgG), 1 : 200; A21207, Thermo Fisher Scientific, Waltham, MA, U.S.A.) at room temperature for 2 h in the dark. Fluorescence was visualized using a confocal fluorescent microscope (FluoView FV10i; Olympus, Tokyo, Japan). C-Fos-positive cells in a 0.405 mm² field of the analyzed nuclei were then enumerated.

Immunohistochemical analysis of c-Fos-and oxytocin-positive cells were performed as previously described by Kogami et al.⁴⁰⁾ First, samples were permeabilized in PBT buffer (1.0% Triton X-100 in phosphate buffered saline) for 20 min. Next, the coronal sections were incubated with primary antibodies against c-Fos (mouse anti-c-Fos antibody, 1 : 500; NBP2-50037, Novus Biologicals, CO, U.S.A.) and oxytocin (rabbit anti-oxytocin antibody, 1 : 500; The Biosignal Research Centre Antibody Supply Program, Institute for Molecular and Cellular Regulation in Gunma University, Gunma, Japan) at 4 °C for 48 h. The sections were then labeled with fluorescent-labeled secondary antibodies (Alexa Fluor 594-labeled donkey anti-rabbit IgG, 1 : 200; A21207, Thermo Fisher Scientific, and Alexa Fluor 647-labeled donkey anti-mouse IgG, 1 : 200; A31571, Thermo Fisher Scientific) at room temperature for 2 h in the dark. Images were captured using a confocal fluorescent microscope. The c-Fos-positive and oxytocin-positive cells in the PVN were enumerated.

Statistical Analysis

Statistical analysis was performed using Prism software, version 8 (GraphPad Software, San Diego, CA, U.S.A.). A one-way ANOVA with Sidak’s post hoc test or two-way ANOVA with Sidak’s post hoc test was used for comparisons between more than two experimental groups. An unpaired t-test was used to compare two experimental groups. In all cases, differences were considered statistically significant at p < 0.05. Data are presented as mean ± standard error of the mean (S.E.M.). The number of animals used in each experiment is indicated in the figure legend.

RESULTS

Mice Prenatally Exposed to VPA Display Lower Social Behavior

Impairments in social behavior are one of the characteristic symptoms of ASD.¹⁾ We first assessed the alterations in VPA-treated mice in social interaction tests. In this test, the mice were placed in a novel cage with a novel conspecific. We analyzed social interaction behaviors with the novel conspecific, such as sniffing behavior. Mice prenatally exposed to VPA showed decreased social interaction behaviors compared to control mice prenatally exposed to saline (Fig. 1B), suggesting that VPA-treated animals showed lower social behavior, which is consistent with previous reports.²⁰⁾

Fig. 1. Mice Prenatally Exposed to VPA Displayed Lower Social Behavior

(A) Schematic of the social interaction test. In this test, we analyzed social interaction behavior from subject to stranger without prior contact between them. (B) Social interaction time of saline/VPA-treated mice is displayed. VPA; valproic acid. * p < 0.05 by unpaired t-test; n = 7–8.

Mice Prenatally Exposed to VPA Manifest Increased Empathy-Like Behavior

To clarify the alterations in empathy-like behavior in an ASD mouse model, we assessed the socially transmitted fear in VPA-treated mice using observational fear systems. In this test, an “observer” mouse observed a “demonstrator” mouse that received repetitive foot shocks (fear observation, FO). We assessed the freezing behavior of observer mice during this observation as an index of empathy.^30,31) The observer received a single foot shock before observing the other mouse being shocked, to promote the social transmission of fear (priming shock, PS). Mice prenatally exposed to VPA showed increased freezing behavior compared to mice prenatally exposed to saline (Fig. 2B), suggesting that VPA-treated animals showed increased empathy. To exclude the possibility that auditory sensitivity contributed to the apparent increase in vicarious freezing in VPA-treated mice, we investigated the sensitivity to auditory stimuli in model animals. In this analysis, the demonstrator and observer mice were placed in a shock chamber, similar to the chamber that used for observational fear tests. Notably, we delivered no foot shocks but applied a sound stimulus of constant frequency, which has a similar intensity to a mouse scream in this setup (85 ± 5 dB). Mice prenatally exposed to VPA showed similar immobility duration to the auditory stimulus to mice prenatally exposed to saline (Fig. 3B). To exclude the possibility that enhanced memory of pain-related contexts contributed to an apparent increase in vicarious freezing in VPA-treated mice, we investigated alterations in contextual fear conditioning in model animals. In this analysis, the animals first received electric shocks to associate the shock chamber with the foot shocks. After 2 h, which was the same interval between PS and FO in observational fear learning, mice were placed in a shock chamber, and freezing behaviors were measured. Mice prenatally exposed to VPA showed similar freezing behaviors in this context compared to mice prenatally exposed to saline (Fig. 4B).

Fig. 2. Mice Prenatally Exposed to VPA Manifest Increased Empathy-Like Behavior

(A) Time course and the schematic of the observational fear test. A single 0.6 mA foot shock was applied to the observer as a priming shock (PS), and fear observation (FO) was performed with delivery of 24 0.6 mA foot shocks to the demonstrator. (B) The percentage of immobility time in saline/VPA-treated mice. VPA; valproic acid. * p < 0.05 by unpaired t-test; n = 8–10.

Fig. 3. Assessing Sensitivity to Auditory Stimulus in VPA-Treated Mice

(A) Time course and the schematic of measurement of sensitivity to sound under the conditions of the fear observation test. Priming shock (PS) was applied to the observer. No foot shock was applied to the demonstrator, however, white noise was delivered during the test session. (B) The percentage of immobility time of saline/VPA-treated mice in the test session. ns; not significant. n = 5.

Fig. 4. Assessing Memory in Pain-Related Contexts in VPA-Treated Mice

(A) Time course and the schematic of the contextual fear conditioning test. Conditioning was performed by applying eight 0.6 mA foot shocks. (B) The percentage of immobility time in saline/VPA-treated mice. VPA; valproic acid. ns; not significant. n = 5–6.

Increased Neuronal Activity in the PVN and Decreased Activity in the Basolateral Nucleus of the Amygdala (BLA) Are Associated with Increased Socially Transmitted Fear in VPA-Treated Mice

Previous studies have shown that modulating specific nuclei in observational fear systems, such as the anterior cingulate cortex (ACC), PVN, and BLA, play significant roles in regulating vicarious fear.^30,32,41) We analyzed activity changes induced by socially transmitted fear in these nuclei by counting the number of c-Fos positive cells (a neuronal activity marker in VPA/saline-treated mice). Statistical analysis of c-Fos-positive cells in the ACC revealed a significant effect in the PS + FO group (F_1,8 = 40.5, p = 0.0002), but no prenatal drug effect nor interaction between prenatal drug and PS + FO was observed (for all comparisons, p > 0.05 – Figs. 5B, C). Statistical analysis of c-Fos-positive cells in the PVN revealed a significant effect of prenatal drug and PS + FO (PS + FO, F_1,8 = 12.94, p = 0.0048; prenatal drug, F_1,8 = 7.621, p = 0.0247) but no interaction between prenatal drug and PS + FO (p > 0.05). The post hoc Sidak’s multiple comparisons test indicated that the number of c-Fos-positive cells in the PVN of the VPA-treated PS + FO group was larger than that in the PS + FO group of saline-exposed mice (Figs. 5B, D). Statistical analysis of c-Fos-positive cells in the BLA revealed a significant interaction between prenatal drug and PS + FO (F_1,8 = 6.87, p = 0.0306), but neither prenatal drug nor PS + FO alone effected the number of c-Fos positive cells (for all comparisons, p > 0.05). Contrary to the results for PVN, the post hoc Sidak’s multiple comparisons tests indicated that the number of BLA c-Fos-positive cells in the PS + FO group of VPA-treated animals was lower than that of the PS + FO group of saline-exposed mice (Figs. 5B, E). These results suggest that neuronal activity in the PVN and BLA are positively and negatively associated with the expression of empathy-like behaviors, respectively.

Fig. 5. Increased Neuronal Activity in the PVN and Decreased Activity in the BLA Are Associated with Increased Socially Transmitted Fear in VPA-Treated Mice

(A) Time course of (left) and illustration of brain regions examined in this figure (right). Representative images (B) and quantitative analyses (C–E) for c-Fos positive cells in each region. VPA; valproic acid. ACC; anterior cingulate cortex. PVN; paraventricular nucleus of the hypothalamus. BLA; basolateral amygdala. * p < 0.05, ** p < 0.01 by two-way ANOVA with Sidak’s multiple comparison test; n = 3. Scale bar, 20 µm.

The PVN is a nucleus in the hypothalamus in which neuronal cell bodies produce various hormones such as corticotropin-releasing hormone, vasopressin, and oxytocin (OT).⁴²⁾ They release the hormones to peripheral organs as well as to the central nervous system as neurotransmitters. Notably, OT modulates various emotional processes, such as fear, social behaviors, and empathy,^32,43,44) as well as reproductive functions. In addition, a study showed that intranasal administration of OT ameliorated social deficits in ASD individuals.⁴⁵⁾ Together with the increased PVN activity observed in VPA-treated mice experiencing vicarious fear (Figs. 5B, D), there is a possibility that vicarious fear is associated with the activity of PVN OT neurons. To test this hypothesis, we analyzed changes in the activity of the PVN OT neurons and the association with altered empathy-like behaviors. In this study, after exposure to observational fear tests, we analyzed the ratio of c-Fos-positive OT cells to only OT positive cells. The activity of PVN OT neurons in the PS + FO group of VPA-treated animals was higher than that in the PS + FO group of saline-exposed mice (Figs. 6B, C), suggesting that the activity of PVN OT neurons is associated with enhanced socially transmitted fear in ASD model mice.

Fig. 6. Increased Activity of PVN Oxytocinergic Neurons Is Associated with Increased Vicarious Fear in VPA-Treated Mice

(A) Time course of experiments (left) and the brain region examined in this figure (right). Representative images (B) and quantitative analysis (C) of the percentage of c-Fos and oxytocin double positive cells compared to oxytocin positive cells. VPA; valproic acid. PVN; paraventricular nucleus of the hypothalamus. *** p < 0.005 by two-way ANOVA with Sidak’s multiple comparison test; n = 3. Scale bar, 20 µm.

Oxytocin Signaling Is Responsible for Increased Empathy-Like Behaviors in VPA-Treated Mice

Given the positive association between PVN OT neuronal activity and empathy-like behaviors in VPA-treated mice, we examined the effects of an OT receptor antagonist on socially transmitted fear in model mice. In this study, L-368899, an OT receptor antagonist, was administered systemically (5 mg/kg, i.p.) to mice prenatally exposed to VPA, before fear observation during observational fear learning. Systemic injection of L-368899 significantly decreased the duration of immobility (Fig. 7B), suggesting that OT signaling is necessary for enhanced socially transmitted fear in VPA-treated mice.

Fig. 7. Oxytocin Signaling Is Responsible for Increased Empathy-Like Behaviors in VPA-Treated Mice

(A) Time course and the schematic of the observational fear test after injection of the oxytocin receptor antagonist, L-368899 (5 mg/kg, i.p.). (B) The percentage of immobility time in VPA-treated mice after injection of Saline/L-368, 899. VPA; valproic acid. n = 7. * p < 0.05 by unpaired t-test.

DISCUSSION

In the present study, we found mice exposed to VPA prenatally (ASD mice) exhibited enhanced empathy-like behaviors, in which OT neurons in the PVN played critical roles.

In this study, mice prenatally exposed to VPA exhibited enhanced empathy-like behavior. To the best of our knowledge, no study has yet reported enhanced affective empathy-like behaviors in ASD mice. While previous reports have focused primarily on lack of social behavior and perseverative behaviors as disease-related aberrant behaviors,⁴⁶⁾ apparent lack of empathy is often observed in individuals with ASD.²⁵⁾ One hypothesis explaining this discrepancy is that the ASD animal model shows increased psychological apprehension to others. Empathy is classified into prosocial behaviors that regard helping others in trouble and experiencing the emotion of others, which play a role in motivating prosocial behaviors.⁴⁷⁾ Komeda et al. investigated the difference between prosocial behaviors and the experience of others’ emotions in individuals with ASD.³⁾ In this study, participants read stories of TD characters or characters with ASD that were having trouble in human relationships. Then, the participants rated how they empathized with the character (empathic response) and how they were motivated to help the character (motivation to help). They found that individuals with ASD show a greater empathic response to other individuals with ASD than TD individuals, while showing similar motivation to help individuals with ASD and TD individuals who were in trouble. Taken together with this report, it is possible that the increased socially transmitted fear observed in our study is comparable to a strong empathic response between individuals with ASD. Future studies investigating the motivation to help others in VPA-treated mice by utilizing measures evaluating rescue behavior are necessary for a better understanding of the neural basis of ASD.²⁹⁾ Moreover, from the perspective of psychology, dividing symptoms of mental diseases, including ASD (e.g., social deficit, perseveration, empathic response, and motivation to help others), will be important to clarify the pathology and neurobiology of illnesses and pave the way for drug development.

We explored the brain regions responsible for enhanced socially transmitted fear in VPA-treated mice and found that PVN neuronal activity is associated with enhanced vicarious fear in VPA-treated mice. In addition, we found that ACC activity did not differ between VPA- and saline-exposed mice. This may be due to enhanced neurotransmission from the PVN to other regions, which contributes to enhanced empathy in VPA-treated animals. Another possible explanation for this phenomenon is the ceiling effect, as many c-Fos-positive cells were found in the ACC of both groups of animals after observational fear tests, which occlude further increases by the PVN OT neuronal innervation to the ACC.

We found that PVN OT neurons were responsible for increased empathy-like behavior in VPA-treated mice. Anatomical studies have revealed that the PVN OT neurons are innervated by most of the forebrain and part of the brain stem.⁴⁸⁾ Afferents from the superficial layer of the superior colliculus (sSC) (a nucleus responsible for visual sensation, such as saccade eye movement)⁴⁹⁾ are one of the best candidates for upstream regulators. Optogenetic stimulation of sSC fibers projecting to PVN OT neurons enhance social transmission of maternal behaviors.⁵⁰⁾ This is similar to the observational fear system in that both systems analyze socially transmitted emotions from the other animal, which is the core concept of empathy. Given that visual inputs are required for vicarious fear,³⁰⁾ sSC may play a role in regulating the PVN to govern empathy-like behaviors in VPA-treated mice. PVN OT neurons innervate most of the forebrain and the brainstem. Among these nuclei, PVN OT neurons densely innervate the central nucleus of the amygdala (CeA), a nucleus governing emotions such as fear⁵¹⁾ which is socially transmitted in observational fear systems. Chemogenetic inhibition of PVN OT fibers projecting to the CeA inhibits discrimination between distress and neutral emotion.⁵²⁾ This is an important cognitive process for empathy because discrimination of emotion enables social transmission of emotion from other individuals. Therefore, it is possible that the PVN regulates CeA to enhance empathy in VPA-treated mice. In addition, we found that neuronal activity in the BLA was decreased in the PS + FO groups of VPA-treated mice compared to saline-exposed mice. The CeA is innervated by the BLA directly and indirectly via an intercalated cell mass lying between the CeA and BLA.⁵³⁾ Therefore, it is possible that simultaneous alteration of PVN OT neurons and BLA neurons cooperatively enhance discrimination of emotion via activity changes in the CeA, which ultimately leads to enhanced emotion-like behaviors in VPA-treated mice.

Previous reports have shown that Fmr1-KO mice and Cntnap2-KO mice, ASD model mice, show impaired oxytocinergic signaling due to decreased number of OT-positive cells in the PVN.^54,55) Another report suggests that Nlgn3-KO mice show impaired oxytocinergic signaling due to decreased response to OT in postsynaptic neurons without affecting the number of OT-positive cells in the PVN.⁵⁶⁾ Moreover, we previously reported that the number of OT-positive cells in the PVN of POGZ-Q1038R knock-in mice, an ASD model mice,⁵⁷⁾ was unaltered whereas expression of OTR is decreased in the knock-in mice.⁵⁸⁾ On the other hand, patients with Williams Syndrome, a genetic disease with enhanced sociality, show increased serum OT level.⁵⁹⁾ Collectively these reports indicate that oxytocinergic signaling bidirectionally controls the level of sociality. We have previously reported that impaired sociality in VPA-treated mice was improved by OT treatment.⁶⁰⁾ Therefore, it is possible that oxytocinergic signaling was decreased in VPA-treated mice, which may underlie impaired sociality in these mice. In this study, we have found that VPA-treated mice showed enhanced activity in OT-positive cells in the PVN after fear observation, although it is unclear whether this enhanced activity was observed after social interaction test. Further investigation of the molecular mechanisms underlying the enhanced activity in OT-positive cells in VPA-treated mice will be necessary to extend our understanding of neural mechanisms of empathy and sociality.

Although our data indicate a critical role of PVN OT neurons, we cannot rule out the possibility that OT neurons in other brain nuclei such as supraoptic nucleus (SON) may mediate enhanced affective empathy in VPA-treated mice. Previous reports have demonstrated the critical role of SON OT neurons in lactation.^61,62) However, Takayanagi et al. have shown that activation of SON OT neurons enhances sociality in mice through activation of OTR.⁶³⁾ Moreover, histological analyses indicate that SON OT neurons innervate the amygdala,⁶⁴⁾ a key brain region for sociality. Collectively, it is possible that activity of SON OT neurons may contribute to enhanced affective empathy in VPA-treated mice.

In summary, we showed that ASD mice, a mouse model induced by prenatal exposure to VPA, showed increased affective empathy for the first time, which is consistent with a previous report in individuals with ASD. In addition, our findings that hyperactivity of PVN OT neurons and the subsequent increase in OT signaling underlie this increased affective empathy in VPA-treated mice. Further investigation of the mechanisms of this hyperactivity in PVN OT neurons in VPA-treated mice will help to identify extrinsic stimuli and the condition which are capable of activation of PVN OT neurons in ASD model mice and ultimately in individuals with ASD. Therefore, these findings may help researchers to understand the neurobiological mechanisms underlying the affective empathy and to identify novel approach to enhance OT signaling by extrinsic stimuli, which ultimately pave the way to development of novel therapy for ASD.

Acknowledgments

We thank Hisae Kobayashi and Ken Sato (Gunma University) for generously providing anti-oxytocin antibodies under the joint/usage research program of the Institute for Molecular and Cellular Regulation, Gunma University (antibody ID: HAC-HM06-03RBP90).

Conflict of Interest

The authors declare no conflict of interest.

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