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. 2020 May 18;21(10):3576.
doi: 10.3390/ijms21103576.

Prenatal Exposure to Valproic Acid Affects Microglia and Synaptic Ultrastructure in a Brain-Region-Specific Manner in Young-Adult Male Rats: Relevance to Autism Spectrum Disorders

Magdalena Gąssowska-Dobrowolska  1 , Magdalena Cieślik  1 , Grzegorz Arkadiusz Czapski  1 , Henryk Jęśko  1 , Małgorzata Frontczak-Baniewicz  2 , Magdalena Gewartowska  2 , Agnieszka Dominiak  3 , Rafał Polowy  4 , Robert Kuba Filipkowski  4 , Lidia Babiec  1 , Agata Adamczyk  1
Affiliations

Prenatal Exposure to Valproic Acid Affects Microglia and Synaptic Ultrastructure in a Brain-Region-Specific Manner in Young-Adult Male Rats: Relevance to Autism Spectrum Disorders

Magdalena Gąssowska-Dobrowolska et al. Int J Mol Sci. .

Abstract

Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental conditions categorized as synaptopathies. Environmental risk factors contribute to ASD aetiology. In particular, prenatal exposure to the anti-epileptic drug valproic acid (VPA) may increase the risk of autism. In the present study, we investigated the effect of prenatal exposure to VPA on the synaptic morphology and expression of key synaptic proteins in the hippocampus and cerebral cortex of young-adult male offspring. To characterize the VPA-induced autism model, behavioural outcomes, microglia-related neuroinflammation, and oxidative stress were analysed. Our data showed that prenatal exposure to VPA impaired communication in neonatal rats, reduced their exploratory activity, and led to anxiety-like and repetitive behaviours in the young-adult animals. VPA-induced pathological alterations in the ultrastructures of synapses accompanied by deregulation of key pre- and postsynaptic structural and functional proteins. Moreover, VPA exposure altered the redox status and expression of proinflammatory genes in a brain region-specific manner. The disruption of synaptic structure and plasticity may be the primary insult responsible for autism-related behaviour in the offspring. The vulnerability of specific synaptic proteins to the epigenetic effects of VPA may highlight the potential mechanisms by which prenatal VPA exposure generates behavioural changes.

Keywords: autism spectrum disorders (ASD); microglia; neuroinflammation; oxidative stress; pre- and postsynaptic proteins; synaptic ultrastructure; synaptopathology; valproic acid (VPA).

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
The effect of prenatal exposure to valproic acid (VPA) on offspring behaviour. (I). The impact of prenatal exposition to VPA on offspring–mother communication. The behaviour of control rats and rats exposed to VPA was analysed at postnatal day 11 in the juvenile isolation test. (A) The total number of ultrasonic vocalization events during the trial, n = 90 (CTRL), n = 88 (VPA); (B) the duration of average ultrasonic vocalization event, n = 90 (CTRL), n = 91 (VPA). (II). The impact of prenatal exposition to VPA on exploratory activity and anxiety. The behaviour of control rats and rats exposed to VPA was analysed at postnatal day 55 in the open-field test. (A) The total distance travelled by animals, n = 58 (CTRL), n = 48 (VPA); (B) the time spent in the central zone, n = 50 (CTRL), n = 39 (VPA); (C) the number of entries to the central zone, n = 45 (CTRL), n = 33 (VPA). (III). The impact of prenatal exposition to VPA on anxiety and repetitive behaviour. The behaviour of control rats and rats exposed to VPA was analysed at postnatal day 55 in the open-field test. (A) The number of rearing events, n = 10 (CTRL), n = 7 (VPA); (B) the number of climbing events, n = 10 (CTRL), n = 7 (VPA); (C) the total time spent on self-grooming, n = 9 (CTRL), n = 5 (VPA). Data represent the mean values ± SEM. (IV,V). The impact of prenatal exposition to VPA on social behaviour. The behaviour of control rats and rats exposed to VPA was analysed at postnatal day 57 in a three-chamber sociability and social novelty test (Crawley’s test). (IV). (A) The time spent on the exploration of each chamber during the social preference test, (B) the time spent on exploration of each cage during social preference test, and (C) the time spent on direct interaction with the other animal during social preference test. (V). (A) the time spent on the exploration of each chamber during a social novelty test, (B) the time spent on exploration of each cage during a social novelty test, and (C) the time spent on direct interaction with the other animal during a social novelty test. Data represent the mean values ± SEM from n = (5–8). * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 2
Figure 2
(I). The effect of prenatal exposure to VPA on the ultrastructure of neuronal cells in the CA1 region of the hippocampus of the offspring. (A–B’) Control group. Ultrastructurally unchanged neuronal cells, unaltered structure of neuropil with normal appearance of the synaptic cleft (black arrowheads), well-defined structure of the synapses with accurate postsynaptic density (PSD), the correct distribution of synaptic vesicles (SVs) (long arrows), and ultrastructurally unchanged mitochondria (M). (C–G) VPA-exposed group. Reduced packing density of SVs in the presynaptic area (release of SVs from the presynaptic area accompanied by disruption of the synaptic membranes) (long arrow), nerve ending swelling (S), blurred and thickened structure of the synaptic cleft without clearly marked pre- and postsynaptic membranes (black arrowheads). Ultrastructurally changed mitochondria with a blurred cristae structure (M). Astrocytes with features of swelling (SA) and swollen perivascular astrocyte processes (SPAP) were observed. Microglial cell activation (Mi); (VL) Vascular lumen; (G) Golgi apparatus; (ER) Endoplasmatic reticulum; (N) Neuron. Representative pictures from n = 6 independent experiments for the control and experimental animals are presented. (II). The effect of prenatal exposure to VPA on the ultrastructure of neuronal cells in the cerebral cortex of the offspring. (A–B’) Control group. Ultrastructurally unchanged neuronal cells, unaltered structure of neuropil with normal appearance of the synaptic cleft (black arrowheads), well-defined structure of synapses with accurate postsynaptic density (PSD), correct distribution of SVs (long arrows), and well preserved mitochondria (M). (C–F’) VPA-exposed group. Reduced packing density of SVs in the presynaptic area (release of SVs from the presynaptic area accompanied by the disruption of the synaptic membranes) (long arrows), nerve ending swelling (S), and blurred and thickened structure of the synaptic cleft, without clearly marked pre- and postsynaptic membranes (black arrowheads). Ultrastructurally changed mitochondria with a blurred cristae structure (M). Changed myelin structure (CHM); (DN) Degenerating neuron; (N) Neuron. Representative pictures from n = 6 independent experiments for the control and experimental animals are presented. (III). The effect of prenatal exposure to VPA on the synaptic vesicles (SVs) number. The effect of VPA on the number of synaptic vesicles (SVs) in the CA1 region of the hippocampus (A) and the cerebral cortex (B) was analysed at postnatal day 58 (PND58). The number of SVs was counted in 30 nerve endings of each animal from the control and experimental groups. Data represent the mean values ± SEM from n = 4 independent experiments. * p ˂ 0.05, vs. control.
Figure 2
Figure 2
(I). The effect of prenatal exposure to VPA on the ultrastructure of neuronal cells in the CA1 region of the hippocampus of the offspring. (A–B’) Control group. Ultrastructurally unchanged neuronal cells, unaltered structure of neuropil with normal appearance of the synaptic cleft (black arrowheads), well-defined structure of the synapses with accurate postsynaptic density (PSD), the correct distribution of synaptic vesicles (SVs) (long arrows), and ultrastructurally unchanged mitochondria (M). (C–G) VPA-exposed group. Reduced packing density of SVs in the presynaptic area (release of SVs from the presynaptic area accompanied by disruption of the synaptic membranes) (long arrow), nerve ending swelling (S), blurred and thickened structure of the synaptic cleft without clearly marked pre- and postsynaptic membranes (black arrowheads). Ultrastructurally changed mitochondria with a blurred cristae structure (M). Astrocytes with features of swelling (SA) and swollen perivascular astrocyte processes (SPAP) were observed. Microglial cell activation (Mi); (VL) Vascular lumen; (G) Golgi apparatus; (ER) Endoplasmatic reticulum; (N) Neuron. Representative pictures from n = 6 independent experiments for the control and experimental animals are presented. (II). The effect of prenatal exposure to VPA on the ultrastructure of neuronal cells in the cerebral cortex of the offspring. (A–B’) Control group. Ultrastructurally unchanged neuronal cells, unaltered structure of neuropil with normal appearance of the synaptic cleft (black arrowheads), well-defined structure of synapses with accurate postsynaptic density (PSD), correct distribution of SVs (long arrows), and well preserved mitochondria (M). (C–F’) VPA-exposed group. Reduced packing density of SVs in the presynaptic area (release of SVs from the presynaptic area accompanied by the disruption of the synaptic membranes) (long arrows), nerve ending swelling (S), and blurred and thickened structure of the synaptic cleft, without clearly marked pre- and postsynaptic membranes (black arrowheads). Ultrastructurally changed mitochondria with a blurred cristae structure (M). Changed myelin structure (CHM); (DN) Degenerating neuron; (N) Neuron. Representative pictures from n = 6 independent experiments for the control and experimental animals are presented. (III). The effect of prenatal exposure to VPA on the synaptic vesicles (SVs) number. The effect of VPA on the number of synaptic vesicles (SVs) in the CA1 region of the hippocampus (A) and the cerebral cortex (B) was analysed at postnatal day 58 (PND58). The number of SVs was counted in 30 nerve endings of each animal from the control and experimental groups. Data represent the mean values ± SEM from n = 4 independent experiments. * p ˂ 0.05, vs. control.
Figure 3
Figure 3
The effect of prenatal exposure to VPA on the expression of v-SNARE proteins: VAMP1/2, Syp, Syn1, p-Syn1(Ser62/Ser67), and Syt1. The level of mRNA of Vamp1, Vamp2, Syp, Syn1, and Syt1 in the hippocampus (A) and cerebral cortex (C) of the control and VPA-exposed rats is presented. The level of mRNA was measured by real-time PCR and calculated by the ΔΔCt method with Actb (β-actin) as a reference gene. The data represent the mean values ± SEM from n = (4–5) independent experiments in the hippocampus and n = (4–6) in the cerebral cortex. The immunoreactivity of the v-SNARE proteins in the control and the VPA-exposed rats was monitored using a Western blot analysis. Densitometric analysis and representative pictures of VAMP1/2, Syp, Syn1, p-Syn1, and Syt1 in the hippocampus (B) and cerebral cortex (D) are shown. Results were normalized to GAPDH levels. Data represent the mean values ± SEM from n = (3–9) independent experiments in both the hippocampus and cerebral cortex. * p < 0.5, ** p < 0.01, *** p < 0.001 vs. control.
Figure 4
Figure 4
The effect of prenatal exposure to VPA on the expression of the t-SNARE proteins: SNAP25 and Stx1. The gene expression of Snap25, Stx1a, and Stx1b in the hippocampus (A) and cerebral cortex (C) of the control and VPA-exposed rats was measured via quantitative RT-PCR and calculated by the ΔΔCt method with Actb (β-actin) as a reference gene. Data represent the mean values ± SEM from n = (4–6) independent experiments in both the hippocampus and cerebral cortex. The immunoreactivity of the t-SNARE proteins in the control and the VPA-exposed rats was monitored using a Western blot analysis. Representative pictures and a densitometric analysis of SNAP25 and syntaxin 1 (Stx1) in the hippocampus (B) and cerebral cortex (D) are presented. The results were normalized to GAPDH levels. The data represent the mean values ± SEM from n = 5 independent experiments in both the hippocampus and cerebral cortex. ** p < 0.01, vs. control.
Figure 5
Figure 5
The effect of prenatal exposure to VPA on the expression of the postsynaptic density proteins: PSD95, Shank2, and Shank3. The gene expression of Dlg4, Shank2, and Shank3 in the hippocampus (A) and cerebral cortex (C) of the control and VPA-exposed rats was measured by quantitative RT-PCR and calculated by the ΔΔCt method with Actb (β-actin) as a reference gene. Data represent the mean values ± SEM from n = (4–5) independent experiments in the hippocampus and n = (4–8) in cortex. The immunoreactivity of the postsynaptic density proteins in the control and VPA-exposed rats was monitored using Western blot analysis. Densitometric analysis and representative pictures for PSD95, Shank2, and Shank3 in the hippocampus (B) and cerebral cortex (D) are presented. The results were normalized to GAPDH or vinculin levels. Data represent the mean values ± SEM from n = (4–9) independent experiments in both the hippocampus and cerebral cortex. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.
Figure 6
Figure 6
The effect of prenatal exposure to VPA on the expression of the synaptic cell adhesion molecules: Nlgn1 and Nlgn3. The gene expression of Nlgn1 and Nlgn3 in the hippocampus (A) and cerebral cortex (C) of the control and VPA-exposed rats was measured with quantitative RT-PCR and calculated by the ΔΔCt method with Actb (β-actin) as a reference gene. The data represent the mean values ± SEM from n = (5–6) independent experiments in both the hippocampus and cerebral cortex. The immunoreactivity of Nlgn1 and Nlgn3 in the control and VPA-exposed brains was monitored using a Western blot analysis. Densitometric analysis and representative pictures for Nlgn1 and Nlgn3 in the hippocampus (B) and cerebral cortex (D) are shown. Results were normalized to vinculin levels. Data represent the mean values ± SEM from n = (11–14) independent experiments in both the hippocampus and cerebral cortex. * p < 0.5, ** p < 0.01 vs. control.
Figure 7
Figure 7
The effect of prenatal exposure to VPA on the glutathione level. The glutathione redox imbalance was determined by spectrophotometric assays. We determined the level of total glutathione, oxidized glutathione (GSSG), reduced glutathione (GSH), and the ratio of GSH/GSSG in the hippocampus ((A–D), respectively), as well as in the cerebral cortex ((E–H), respectively), of the control and VPA-exposed animals. Data represent the mean values ± SEM from n = (10–12) independent experiments in the hippocampus and n = (3–5) in the cerebral cortex. * p < 0.05, ** p < 0.01 vs. control.
Figure 8
Figure 8
The effect of prenatal exposure to VPA on free oxygen radicals (ROS) generation. The level of ROS was determined using a fluorescent probe (H2DCF-DA) on the hippocampus and cerebral cortex of the control and VPA-exposed animals for 30 min ((A,C), respectively) and after 30 min ((B,D), respectively). The results are presented as the relative increase in fluorescence. Data represent the mean values ± SEM from n = 8 independent experiments in both the hippocampus and cerebral cortex. ** p < 0.01, *** p < 0.001 vs. control.
Figure 9
Figure 9
The effect of prenatal exposure to VPA on the status of microglia in the hippocampus. (A) The immunoreactivity of Iba-1 was monitored using a Western blot analysis. Densitometric analysis and representative pictures are shown. Results were normalized to GAPDH levels. Data represent the mean values ± SEM from n = (6–8). independent experiments. The gene expression of Ptgs2, Alox12, (B); Il1b, Il6, and Tnf (C); and the markers of the M2 phenotype’s microglia activation: Arg1, Chi3l1, Mrc1, Cd86, Fcgr1a, Tgfb1, and Sphk1 (D), in the hippocampus of the control and VPA-exposed rats were measured with quantitative RT-PCR and calculated by the ΔΔCt method using Actb (β-actin) as a reference gene. Data represent the mean values ± SEM from n = (4–6) independent experiments. * p < 0.5, ** p < 0.01 vs. control.
Figure 10
Figure 10
The effect of prenatal exposure to VPA on the status of microglia in the cerebral cortex. (A) The immunoreactivity of Iba-1 was monitored using a Western blot analysis. Densitometric analysis and representative pictures are shown. Results were normalized to GAPDH levels. Data represent the mean values ± SEM from n = (8–11) independent experiments. The gene expression of Ptgs2, Alox12 (B); Il1b, Il6, and Tnf (C); and the markers of the M2 phenotype’s microglia activation: Arg1, Chi3l1, Mrc1, Cd86, Fcgr1α, Tgfb1, and Sphk1 (D) in the cerebral cortex of the control and VPA-exposed rats were measured with quantitative RT-PCR and calculated by the ΔΔCt method with Actb (β-actin) as a reference gene. Data represent the mean values ± SEM for n = (3–6) independent experiments. * p < 0.5, ** p < 0.01 vs. control.
Figure 11
Figure 11
Experimental design. (A) Pregnant Wistar rats were injected at gestation day 12.5 (GD 12.5) with NaCl or VPA (450 mg/kg b.w.). One group of offspring rats (males and females) was subjected to an ISO test at postnatal day 11 (PND 11) and then to an Open-field test (males only) at PND 55. Other groups of offspring rats (males) was subjected to Open-field test at PND 55 and then to a 3-chamber test at PND 57. All offspring rats were euthanized at PND 58; tissue samples from the hippocampus and cerebral cortex were collected and used for biochemical, immunochemical, genetic, and microscopic analysis. (B) Design of the 3-chamber test. A detailed description is included in the behavioural analysis section.
Figure 12
Figure 12
Schematic diagram showing the effect of prenatal exposure to VPA on the pathological changes in brains of young-adult male offspring. A single i.p. injection of VPA at 12.5 days of pregnancy-induced brain structure-specific defects in the expression of key pre- and postsynaptic proteins, consequently leading to pathological alterations in the structure of the synaptic endings (the hippocampus and cerebral cortex). In the hippocampus, the importance of oxidative stress and ROS generation in synaptic pathology is suggested. In the cerebral cortex, in addition to oxidative stress and ROS generation, neuroinflammation associated with microglia mobilization and M1 activation have also been proposed as potential triggers of a molecular cascade leading to synaptopathology. VPA induced long-lasting changes in microglia status in a brain structure-specific manner. In the cerebral cortex, both the pro-inflammatory M1 and the potentially beneficial recovery-promoting M2 phenotype were activated, while in the hippocampus, only M2 was stimulated.
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