Deep Brain Stimulation Modified Autism-Like Deficits via the Serotonin System in a Valproic Acid-Induced Rat Model

Abstract

Deep brain stimulation (DBS) is known to be a promising treatment for resistant depression, which acts via the serotonin (5-hydroxytryptamine, 5-HT) system in the infralimbic prefrontal cortex (ILPFC). Previous study revealed that dysfunction of brain 5-HT homeostasis is related to a valproate (VPA)-induced rat autism spectrum disorder (ASD) model. Whether ILPFC DBS rescues deficits in VPA-induced offspring through the 5-HT system is not known. Using VPA-induced offspring, we therefore explored the effect of DBS in autistic phenotypes and further investigated the underlying mechanism. Using combined behavioral and molecular approaches, we observed that applying DBS and 5-HT_1A receptor agonist treatment with 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) reversed sociability deficits, anxiety and hyperactivity in the VPA-exposed offspring. We then administered the selective 5-HT_1A receptor antagonist N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate (WAY 100635), following which the effect of DBS in terms of improving autistic behaviors was blocked in the VPA-exposed offspring. Furthermore, we found that both 8-OH-DPAT and DBS treatment rescued autistic behaviors by decreasing the expressions of NR2B subunit of N-methyl-D-aspartate receptors (NMDARs) and the β₃ subunit of γ-aminobutyric acid type A receptors (GABA_AR) in the PFC region. These results provided the first evidence of characteristic behavioral changes in VPA-induced offspring caused by DBS via the 5-HT system in the ILPFC.

Keywords: autism spectrum disorder; deep brain stimulation; serotonin system; valproic acid.

Figure 1

ILPFC DBS improved social interaction, anxiety and hyperlocomotion in the VPA-exposed offspring. (A) Time spent by the VPA-exposed offspring in chamber E, the center chamber, and chamber S1 during the three-chamber social interaction test following seven days of DBS treatment; (B) preference indices of the saline- and VPA-exposed offspring after DBS treatment; (C) bar chart showing the percentage of time spent in the open arms after DBS treatment; (D) bar chart revealing the total distance traveled in the open field test. * p < 0.05 vs. saline/sham; *** p < 0.001 vs. saline/sham; ^# p < 0.05 vs. VPA/sham; ^## p < 0.01 vs. VPA/sham; ^### p < 0.001 vs. VPA/sham. Sample sizes (n): saline/sham n = 7, saline/DBS n = 5, VPA/sham n = 9, VPA/DBS n = 7.

Figure 2

WAY 100635 blocked the improvements in social interaction, anxiety and hyperlocomotion instigated by ILPFCDBS in the VPA-exposed offspring. (A) Duration of latency on entering chamber E, the center chamber, and chamber S1 during the three-chamber social interaction test; (B) preference index in the social interaction test; (C) percentage of time spent in the open arms; (D) total distance traveled in the open field test. * p < 0.05 vs. VPA/sham; *** p < 0.05 vs. VPA/sham; ^# p < 0.05 vs. VPA/DBS; ^## p < 0.01 vs. VPA/DBS; ^### p < 0.001 vs. VPA/DBS. Sample sizes (n): saline/sham n = 7, VPA/sham n = 9, VPA/DBS n = 5, VPA/DBS/WAY 100635 n = 5.

Figure 3

8-OH-DPAT treatment increased social interaction and ameliorated anxiety and hyperlocomotion in the VPA-exposed offspring. (A) Time spent by the VPA-exposed offspring in chamber E, the center chamber, and chamber S1 during the three-chamber social interaction test following local infusion of 8-OH-DPAT (10 μg/μL) into the ILPFC; (B) preference index in the social interaction test; (C) the percentage in the open arms after 8-OH-DPAT treatments; (D) bar chart revealing the total distance traveled in the open field test. * p < 0.05 vs. saline/vehicle; ** p < 0.01 vs. saline/vehicle; *** p < 0.001 vs. saline/vehicle; ^# p < 0.05 vs. VPA/vehicle; ^## p < 0.01 vs. VPA/vehicle; ^### p < 0.001 vs. VPA/vehicle. Sample sizes (n): saline/vehicle n = 6, saline/8-OH-DPAT n = 7, VPA/vehicle n = 8, VPA/8-OH-DPAT n = 5.

Figure 4

One-day ILPFC DBS combined with 8-OH-DPAT treatment facilitated improvement of social interaction and social preference in the VPA-exposed offspring. (A) Duration of latency on entering chamber E, the center chamber, and chamber S1 during the three-chamber social interaction test; (B) preference index in the social interaction test. *** p < 0.001 vs. saline-; ^### p < 0.001 vs. VPA. Sample sizes (n): saline n = 5, VPA n = 8, VPA/DBS 1 day/8-OH-DPAT n = 4, saline/DBS 7 days n = 5.

Figure 5

DBS and 8-OH-DPAT treatment reversed the increased expressions of NMDAR subunit NR2B and the GABA_AR β3 subunit in the VPA-exposed offspring. (A) Representative blots and quantification showing the synapse NMDAR subunit NR2B expression after 8-OH-DPAT treatment in the VPA-exposed offspring. Sample sizes (n): Saline n = 12, VPA n = 9, VPA/8-OH-DPAT n = 5; (B) representative blots and quantification showing the synapse GABA_AR β3 subunit expression after 8-OH-DPAT treatment in the VPA-exposed offspring. Sample sizes (n): Saline n = 4, VPA n = 4, VPA/8-OH-DPAT n = 4; (C) immunoblots and quantification showing the synapse NMDAR subunit NR2B expression after seven days of DBS and after three days of DBS combined with 8-OH-DPAT treatments in the VPA-exposed offspring. Sample sizes (n): VPA n = 14, VPA/DBS n = 12, VPA/DBS/8-OH-DPAT n = 12; (D) western blotting showing the synapse GABA_AR β3 subunit expression after seven days of DBS and after three days of DBS combined with 8-OH-DPAT treatment in the VPA-exposed offspring. Sample sizes (n): VPA n = 8, VPA/DBS n = 6, VPA/DBS/8-OH-DPAT n = 6. * p < 0.05 vs. saline/vehicle; ** p < 0.01 vs. saline/vehicle; * p < 0.05 vs. saline; ** p < 0.01 vs. saline; ** p < 0.01 vs. VPA; *** p < 0.001 vs. VPA; ^## p < 0.01 vs. VPA; ^### p < 0.001 vs. VPA.

Figure 6

Experimental design of ILPFC DBS applied to the VPA-exposed offspring. (A) Schematic representation flow chart of the experimental design; (B) verification of unilateral DBS electrode placement on the right-hand side by Nissl staining of the ILPFC. Scale bar, 1 mm.