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. 2018 Dec 27:9:66.
doi: 10.1186/s13229-018-0254-0. eCollection 2018.

Neuroglia in the autistic brain: evidence from a preclinical model

Maria Rosanna Bronzuoli #  1 , Roberta Facchinetti #  1 , Davide Ingrassia  1 , Michela Sarvadio  2 , Sara Schiavi  2 , Luca Steardo  1 , Alexei Verkhratsky  3   4   5 , Viviana Trezza  2 , Caterina Scuderi  1
Affiliations

Neuroglia in the autistic brain: evidence from a preclinical model

Maria Rosanna Bronzuoli et al. Mol Autism. .

Abstract

Background: Neuroglial cells that provide homeostatic support and form defence of the nervous system contribute to all neurological disorders. We analyzed three major types of neuroglia, astrocytes, oligodendrocytes, and microglia in the brains of an animal model of autism spectrum disorder, in which rats were exposed prenatally to antiepileptic and mood stabilizer drug valproic acid; this model being of acknowledged clinical relevance.

Methods: We tested the autistic-like behaviors of valproic acid-prenatally exposed male rats by performing isolation-induced ultrasonic vocalizations, the three-chamber test, and the hole board test. To account for human infancy, adolescence, and adulthood, such tasks were performed at postnatal day 13, postnatal day 35, and postnatal day 90, respectively. After sacrifice, we examined gene and protein expression of specific markers of neuroglia in hippocampus, prefrontal cortex, and cerebellum, these brain regions being associated with autism spectrum disorder pathogenesis.

Results: Infant offspring of VPA-exposed dams emitted less ultrasonic vocalizations when isolated from their mothers and siblings and, in adolescence and adulthood, they showed altered sociability in the three chamber test and increased stereotypic behavior in the hole board test. Molecular analyses indicate that prenatal valproic acid exposure affects all types of neuroglia, mainly causing transcriptional modifications. The most prominent changes occur in prefrontal cortex and in the hippocampus of autistic-like animals; these changes are particularly evident during infancy and adolescence, while they appear to be mitigated in adulthood.

Conclusions: Neuroglial pathological phenotype in autism spectrum disorder rat model appears to be rather mild with little signs of widespread and chronic neuroinflammation.

Keywords: Astrocyte; Autism spectrum disorder; Microglia; Oligodendrocyte; Valproic acid.

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Conflict of interest statement

All procedures involving animals were performed in accordance with the guidelines of the Italian Ministry of Health (D.L. 26/2014) and with the European Parliament directive 2010/63/EU.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Effect of prenatal VPA exposure on animals’ behavior. Schematic representation of the experimental design (a). Assessment of the autistic-like phenotype in prenatally VPA-exposed rats through reduced isolation-induced USV emission at PND 13 (b), sociability in the three-chamber test at PND 35 (c), and induced stereotypic behavior in the hole-board test at PND 90 (d) (Veh n = 5, VPA n = 5). Data represent mean ± SEM. Statistical analysis was performed by t test (*p < 0.05 vs Veh group)
Fig. 2
Fig. 2
Effect of prenatal VPA exposure on astrocytes in infancy. Analysis of the neurotrophin S100B and the GFAP in the PfC, Cb, and HPC of healthy- (open bars, Veh) and autistic-like (black bars, VPA) infant rats (PND 13). Representation of the relative concentration of S100B (a) and GFAP (c) in VPA animals compared to control (Veh), normalized to both TBP and HPRT (ΔΔCq; N = 3, in triplicate). Representative western blots for S100B (b) and GFAP (d) proteins, and densitometric analyses are normalized to β-actin used as loading control. Results are expressed as percentage of control (Veh) (N = 3, in triplicate). Representative fluorescence micrographs of GFAP (green) staining in the PfCGL and MLof Cb, and CA1, CA2, CA3, and the hilus of the DG of the HPC. Nuclei were stained with Hoechst (blue) (e). The images have been analyzed by counting the number of GFAP-positive cells in 2.4 × 105 μm3 (scale bar 50 μm; N = 3, 4 times) (f). All data are presented as means ± SEM. Statistical analysis was performed by t test (*p < 0.05; ***p < 0.001 vs Veh group)
Fig. 3
Fig. 3
Effect of the prenatal VPA exposure on astrocytes in adolescence. Evaluation of the neurotrophin S100B and the cytoskeletal GFAP in the PfC, Cb, and HPC of healthy- (open bars, Veh) and autistic-like (black bars, VPA) adolescent rats (PND 35). Representation of the relative concentration of S100B (a) and GFAP (c) in VPA animals compared to control (Veh), normalized to both TBP and HPRT (ΔΔCq; N = 3, in triplicate). Representative western blots for S100B (b) and GFAP (d) proteins, and densitometric analyses normalized to β-actin used as loading control. Results are expressed as percentage of control (Veh) (N = 3, in triplicate). Representative fluorescence micrographs of GFAP (green) staining in the PfC, GL and ML of Cb, and stratum radiatum of CA1, CA2, CA3, and the hilus of the DG of the HPC. Nuclei were stained with Hoechst (blue) (e). The images have been analyzed by counting the number of GFAP-positive cells in 2.4 × 105 μm3 (scale bar 50 μm; N = 3, 4 times) (f). All data are presented as means ± SEM. Statistical analysis was performed by t test (*p < 0.05; **p < 0.01; ***p < 0.001 vs Veh group)
Fig. 4
Fig. 4
Effect of the prenatal VPA exposure on astrocytes in adulthood. Evaluation of the neurotrophin S100B and the cytoskeletal GFAP in the PfC, Cb, and HPC of healthy- (open bars, Veh) and autistic-like (black bars, VPA) adult rats (PND 90). Representation of the relative concentration of S100B (a) and GFAP (c) in VPA animals compared to control (Veh), normalized to both TBP and HPRT (ΔΔCq; N = 3, in triplicate). Representative western blots for S100B (b) and GFAP (d) proteins, and densitometric analyses normalized to β-actin used as loading control. Results are expressed as percentage of control (Veh) (N = 3, in triplicate). Representative fluorescence micrographs of GFAP (green) staining in the PfC, GL and ML of Cb, and stratum radiatum of CA1, CA2, CA3, and the hilus of the DG of the HPC. Nuclei were stained with Hoechst (blue) (e). The images have been analyzed by counting the number of GFAP-positive cells in 2.4 × 105 μm3 (scale bar 50 μm; N = 3, 4 times) (f). All data are presented as means ± SEM. Statistical analysis was performed by t test (*p < 0.05; ***p < 0.001 vs Veh group)
Fig. 5
Fig. 5
Effect of the prenatal VPA exposure on oligodendrocytes in infancy. Evaluation of the transcription factor Olig2 in the PfC, Cb, and HPC of healthy- (open bars, Veh) and autistic-like (black bars, VPA) infant rats (PND 13). Representation of the relative concentration of Olig2 (a) in VPA animals compared to control (Veh), normalized to both TBP and HPRT (ΔΔCq; N = 3, in triplicate). Representative western blots for Olig2 protein and densitometric analyses normalized to β-actin used as loading control (b). Results are expressed as percentage of control (Veh) (N = 3, in triplicate). Representative fluorescence micrographs of Olig2 (green) staining in the PfC, GL and ML of Cb, and stratum radiatum of CA1, CA2, CA3, and the hilus of the DG of the HPC. Nuclei were stained with Hoechst (blue) (c). The images have been analyzed by counting the number of Olig2-positive cells in 2.4 × 105 μm3 (scale bar 50 μm; N = 3, 4 times) (d). All data are presented as means ± SEM. Statistical analysis was performed by t test (*p < 0.05; ***p < 0.001 vs Veh group)
Fig. 6
Fig. 6
Effect of the prenatal VPA exposure on oligodendrocytes in adolescence. Evaluation of the transcription factor Olig2 in the PfC, Cb, and HPC of healthy- (open bars, Veh) and autistic-like (black bars, VPA) adolescent rats (PND 35). Representation of the relative concentration of Olig2 (a) in VPA animals compared to control (Veh), normalized to both TBP and HPRT (ΔΔCq; N = 3, in triplicate). Representative western blots for Olig2 protein and densitometric analyses normalized to β-actin used as loading control (b). Results are expressed as percentage of control (Veh) (N = 3, in triplicate). Representative fluorescence micrographs of Olig2 (green) staining in the PfC, GL and ML of Cb, and stratum radiatum of CA1, CA2, CA3, and the hilus of the DG of the HPC. Nuclei were stained with Hoechst (blue) (c). The images have been analyzed by counting the number of Olig2-positive cells in 2.4 × 105 μm3 (scale bar 50 μm; N = 3, 4 times) (d). All data are presented as means ± SEM. Statistical analysis was performed by t test (*p < 0.05; **p < 0.01; ***p < 0.001 vs Veh group)
Fig. 7
Fig. 7
Effect of the prenatal VPA exposure on oligodendrocytes in adulthood. Evaluation of the transcription factor Olig2 in the PfC, Cb, and HPC of healthy- (open bars, Veh) and autistic-like (black bars, VPA) adult rats (PND 90). Representation of the relative concentration of Olig2 (a) in VPA animals compared to control (Veh), normalized to both TBP and HPRT (ΔΔCq; N = 3, in triplicate). Representative western blots for Olig2 protein and densitometric analyses normalized to β-actin used as loading control (b). Results are expressed as percentage of control (Veh) (N = 3, in triplicate). Representative fluorescence micrographs of Olig2 (green) staining in the PfC, GL and ML of Cb, and stratum radiatum of CA1, CA2, CA3, and the hilus of the DG of the HPC. Nuclei were stained with Hoechst (blue) (c). The images have been analyzed by counting the number of Olig2-positive cells in 2.4 × 105 μm3(scale bar 50 μm; N = 3, 4 times) (d). All data are presented as means ± SEM. Statistical analysis was performed by t test (*p < 0.05; **p < 0.01; ***p < 0.001 vs Veh group)
Fig. 8
Fig. 8
Effect of the prenatal VPA exposure on microglia in infancy. Evaluation of the cluster of differentiation 11b (CD11b) and Iba1 in the PfC, Cb, and HPC of healthy- (open bars, Veh) and autistic-like (black bars, VPA) infant rats (PND 13). Representation of the relative concentration of CD11b (a) and Iba1 (c) in VPA animals compared to control (Veh), normalized to both TBP and HPRT (ΔΔCq; N = 3, in triplicate). Representative western blots for CD11b (b) and Iba1 (d) proteins, and densitometric analyses normalized to β-actin used as loading control. Results are expressed as percentage of control (Veh) (N = 3, in triplicate). Representative fluorescence micrographs of Iba1 (green) staining in the PfC, GL and ML of Cb, and stratum radiatum of CA1, CA2, CA3, and the hilus of the DG of the HPC. Nuclei were stained with Hoechst (blue) (e). The images have been analyzed by counting the number of Iba1-positive cells in 2.4 × 105 μm3 (scale bar 50 μm; N = 3, 4 times) (f). All data are presented as means ± SEM. Statistical analysis was performed by t test (*p < 0.05; **p < 0.01; ***p < 0.001 vs Veh group)
Fig. 9
Fig. 9
Effect of the prenatal VPA exposure on microglia in adolescence. Evaluation of the cluster of differentiation 11b (CD11b) and Iba1 in the PfC, Cb, and HPC of healthy- (open bars, Veh) and autistic-like (black bars, VPA) adolescent rats (PND 35). Representation of the relative concentration of CD11b (a) and Iba1 (c) in VPA animals compared to control (Veh), normalized to both TBP and HPRT (ΔΔCq; N = 3, in triplicate). Representative western blots for CD11b (b) and Iba1 (d) proteins, and densitometric analyses normalized to β-actin used as loading control. Results are expressed as percentage of control (Veh) (N = 3, in triplicate). Representative fluorescence micrographs of Iba1 (green) staining in the PfC, GL and ML of Cb, and stratum radiatum of CA1, CA2, CA3, and the hilus of the DG of the HPC. Nuclei were stained with Hoechst (blue) (e). The images have been analyzed by counting the number of Iba1-positive cells in 2.4 × 105 μm3 (scale bar 50 μm; N = 3, 4 times) (f). All data are presented as means ± SEM. Statistical analysis was performed by t test (*p < 0.05; **p < 0.01; ***p < 0.001 vs Veh group)
Fig. 10
Fig. 10
Effect of the prenatal exposure to VPA on microglia in adulthood. Evaluation of the cluster of differentiation 11b (CD11b) and Iba1 in the PfC, Cb, and HPC of healthy- (open bars, Veh) and autistic-like (black bars, VPA) adult rats (PND 90). Representation of the relative concentration of CD11b (a) and Iba1 (c) in VPA animals compared to control (Veh), normalized to both TBP and HPRT (ΔΔCq; N = 3, in triplicate). Representative western blots for CD11b (b) and Iba1 (d) proteins, and densitometric analyses normalized to β-actin used as loading control. Results are expressed as percentage of control (Veh) (N = 3, in triplicate). Representative fluorescence micrographs of Iba1 (green) staining in the PfC, GL and ML of Cb, and stratum radiatum of CA1, CA2, CA3, and the hilus of the DG of the HPC. Nuclei were stained with Hoechst (blue) (e). The images have been analyzed by counting the number of Iba1-positive cells in 2.4 × 105 μm3 (scale bar 50 μm; N = 3, 4 times) (f). All data are presented as means ± SEM. Statistical analysis was performed by t test (*p < 0.05; **p < 0.01; ***p < 0.001 vs Veh group)
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