Memantine/Aripiprazole Combination Alleviates Cognitive Dysfunction in Valproic Acid Rat Model of Autism: Hippocampal CREB/BDNF Signaling and Glutamate Homeostasis

Abstract

Significant efforts are increasingly directed towards identifying novel therapeutic targets for autism spectrum disorder (ASD) with a rising role of aberrant glutamatergic transmission in the pathogenesis of ASD-associated cellular and behavioral deficits. This study aimed at investigating the role of chronic memantine (20 mg/kg/day) and aripiprazole (3 mg/kg/day) combination therapy in the management of prenatal sodium valproate (VPA)-induced autistic-like/cognitive deficits in male Wistar rats. Pregnant female rats received a single intraperitoneal injection of VPA (600 mg/kg) to induce autistic-like behaviors in their offspring. Prenatal VPA induced autistic-like symptoms (decreased social interaction and the appearance of stereotyped behavior) with deficits in spatial learning (in Morris water maze) and cognitive flexibility (in the attentional set-shifting task) in addition to decreased hippocampal protein levels of phosphorylated cAMP response element-binding protein (p-CREB), brain-derived neurotrophic factor (BDNF), and gene expression of glutamate transporter-1 (Glt-1) with a decline in GABA/glutamate ratio (both measured by HPLC). These were accompanied by the appearance of numerous neurofibrillary tangles (NFTs) with enhanced apoptosis in hippocampal sections. Memantine/aripiprazole combination increased the protein levels of p-CREB, BDNF, and Glt-1 gene expression with restoration of GABA/glutamate balance, attenuation of VPA-induced neurodegenerative changes and autistic-like symptoms, and improvement of cognitive performance. This study draws attention to the favorable cognitive effects of memantine/aripiprazole combination in autistic subjects which could be mediated via enhancing CREB/BDNF signaling with increased expression of astrocytic Glt-1 and restoration of GABA/glutamate balance, leading to inhibition of hippocampal NFTs formation and neuronal apoptosis.

Keywords: Aripiprazole; Astrocyte; Autism spectrum disorder; Cognitive dysfunction; Glutamate; Memantine.

Fig. 1

A diagram showing the experimental exposures of the study. VPA Sodium valproate, MEM Memantine, ARI Aripiprazole, G Gestational day, PND Postnatal day, OFT Open field test, 3-CST Three-chamber sociability test, MBT Marble burying test, TNT Tail-immersion nociceptive test, MWM Morris water maze, ASST Attentional set-shifting task

Fig. 2

Effects of MEM and ARI on A body weight, B sociability index, C social novelty preference index, and D number of buried marbles in prenatal VPA rat model of autism. Data are presented as mean ± S.E.M (n = 10). ^**P < 0.01; ^****P < 0.0001 vs. Control group; ^#P < 0.05; ^##P < 0.01; ^####P < 0.0001 vs. VPA/VEH group; ^$P < 0.05; ^$$P < 0.01; ^$$$$P < 0.0001 vs. VPA/MEM group. Treatments were compared by one-way ANOVA followed by Tukey's post-hoc test. For body weight, treatments were compared by repeated-measures ANOVA followed by Tukey's post-hoc test

Fig. 3

Effects of MEM and ARI on open field behavioral changes in prenatal VPA rat model of autism. Data are presented as mean ± S.E.M (n = 10). ^*P < 0.05 vs. Control group; ^#P < 0.05; ^##P < 0.01; ^###P < 0.001 vs. VPA/VEH group. Treatments were compared by one-way ANOVA followed by Tukey's post-hoc test

Fig. 4

Effects of MEM and ARI on A latency to reach MWM hidden platform, B % time spent in the target quadrant in MWM, and C TNT tail withdrawal latency in prenatal VPA rat model of autism. Data are presented as mean ± S.E.M (n = 10). ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001 vs. Control group; ^#P < 0.05; ^###P < 0.001; ^####P < 0.0001 vs. VPA/VEH group; ^$P < 0.05 vs. VPA/MEM group; ^@@P < 0.01 vs. VPA/ARI group. Treatments were compared by one-way ANOVA followed by Tukey's post-hoc test. For the latency to reach MWM hidden platform, treatments were compared by repeated-measures ANOVA followed by Tukey's post-hoc test

Fig. 5

Effects of MEM and ARI on A Glutamate level, B GABA level, C GABA/glutamate ratio, and D Glt-1 relative gene expression in prenatal VPA rat model of autism. Data are presented as mean ± S.E.M (n = 6). ^****P < 0.0001 vs. Control group; ^#P < 0.05; ^##P < 0.01; ^###P < 0.001; ^####P < 0.0001 vs. VPA/VEH group; ^$$P < 0.01; ^$$$$P < 0.0001 vs. VPA/MEM group; ^@@@@P < 0.0001 vs. VPA/ARI group. Treatments were compared by one-way ANOVA followed by Tukey's post-hoc test. E Example of HPLC chromatogram showing Glutamate (Glu) and GABA peaks

Fig. 6

Effects of MEM and ARI on protein levels of total, p-CREB, and BDNF in prenatal VPA rat model of autism. A A representative image of quantitative protein levels of total and p-CREB normalized to β-actin, B Total CREB, C p-CREB, and D BDNF. Data are presented as mean ± S.E.M (n = 6). ^****P < 0.0001 vs. Control group; ^###P < 0.001; ^####P < 0.0001 vs. VPA/VEH group; ^$$$$P < 0.0001 vs. VPA/MEM group; ^@@@P < 0.001; ^@@@@P < 0.0001 vs. VPA/ARI group. Treatments were compared by one-way ANOVA followed by Tukey's post-hoc test

Fig. 7

A Photomicrographs representing H&E, toluidine blue, Marsland, Glees and Erikson's Silver, GFAP, caspase-3, BAX, and Bcl-2-stained sections of CA3 hippocampal area from the study groups. The control group showed many intact well-organized neurons with distinct nuclear and subcellular details (black arrow) and minimal reactive glial cell infiltrates (black arrowhead) and normal neuronal argyrophilia (blue arrow) alongside an increased optical density of Nissl’s granules, a high immunoreactivity of Bcl-2, and a low immunoreactivity of GFAP, caspase-3, and BAX. VPA/VEH group showed abundant degenerated hypereosinophilic neurons without distinct subcellular details (red arrow) alternated with few dispersed intact cells (black arrow) with marked higher reactive glial cell infiltrates (black arrowhead) and several NFTs (blue arrowhead) alongside a low optical density of Nissl’s granules, low immunoreactivity of Bcl-2 and a high immunoreactivity of GFAP, caspase-3, and BAX. Treated groups showed improvement of all the histological changes that was obvious in the VPA/MEM/ARI group which exhibited many well-organized intact neurons and minimal reactive glial cell infiltrates with minimal NFTs besides a high optical density of Nissl’s granules, a high immunoreactivity of Bcl-2 and a low immunoreactivity of GFAP, caspase-3, and BAX. B Effects of MEM and ARI on the number of intact neurons, mean optical density of Nissl’s granules, number of NFTs, and area percentage of immunohistochemical expression of GFAP, caspase-3, BAX, and Bcl-2 in CA3 hippocampal area in prenatal VPA rat model of autism. Data are presented as mean ± S.E.M. (n = 6). ^****P < 0.0001 vs. Control group; ^###P < 0.001; ^####P < 0.0001 vs. VPA/VEH group; ^$$P < 0.01; ^$$$$P < 0.0001 vs. VPA/MEM group; ^@P < 0.05; ^@@@@P < 0.0001 vs. VPA/ARI group. Treatments were compared by one-way ANOVA followed by Tukey's post-hoc test

Fig. 8

A Photomicrographs representing H&E, toluidine blue, Marsland, Glees and Erikson's Silver, GFAP, caspase-3, BAX, and Bcl-2-stained sections of dentate gyrus from the study groups. The control group exhibited granule cells with intact subcellular details (black arrow). VPA/VEH group showed marked degenerated pyknotic granule neurons (red arrow) with moderate edema and vacuolation of brain matrix (star) accompanied by mild higher reactive glial cell infiltrates (arrowhead) with multiple NFTs (blue arrowhead). Treated groups showed improvement of all the histological changes, especially in the VPA/MEM/ARI group which showed preserved morphological features with abundant intact neurons (black arrow) and minimal sporadic degenerated granule cells (red arrow). B Effects of MEM and ARI on the number of intact neurons, mean optical density of Nissl’s granules, number of NFTs, and area percentage of immunohistochemical expression of GFAP, caspase-3, BAX, and Bcl-2 in the dentate gyrus in prenatal VPA rat model of autism. Data are presented as mean ± S.E.M. (n = 6). ^***P < 0.001; ^****P < 0.0001 vs. Control group; ^#P < 0.05; ^##P < 0.01; ^####P < 0.0001 vs. VPA/VEH group; ^$P < 0.05; ^$$P < 0.01 vs. VPA/MEM group; ^@P < 0.05; ^@@P < 0.01; ^@@@P < 0.001; ^@@@@P < 0.0001 vs. VPA/ARI group. Treatments were compared by one-way ANOVA followed by Tukey's post-hoc test