Adolescent intermittent ethanol exposure reduces astrocyte-synaptic proximity in the adult medial prefrontal cortex in rats: Reversal by gabapentin

Abstract

Alcohol consumption in adolescence causes multiple acute negative changes in neural and behavioral function that persist well into adulthood and possibly throughout life. The medial prefrontal cortex (mPFC) and dorsal hippocampus are critical for executive function and memory and are especially vulnerable to adolescent ethanol exposure. We have reported that astrocytes, particularly in the mPFC, change both in morphology and synaptic proximity during adolescence. Moreover, adolescent intermittent ethanol (AIE) exposure produces enduring effects on both astrocyte function and synaptic proximity in the adult hippocampal formation, and the latter effect was reversed by the clinically used agent gabapentin (Neurontin), an anticonvulsant and analgesic that is an inhibitor of the VGCC α2δ1 subunit. These findings underscore the importance of investigating AIE effects on astrocytes in the mPFC, a region that undergoes marked changes in structure and connectivity during adolescence. Using astrocyte-specific viral labeling and immunohistochemistry, mPFC astrocytic morphology and colocalization with AMPA-(α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) glutamate receptor 1 (GluA1), an AMPA receptor subunit and established neuronal marker of excitatory synapses, were assessed to quantify the proximity of astrocyte processes with glutamatergic synaptic puncta. AIE exposure significantly reduced astrocyte-synaptic proximity in adulthood, an effect that was reversed by sub-chronic gabapentin treatment in adulthood. There was no effect of AIE on astrocytic glutamate homeostasis machinery or neuronal synaptic proteins in the mPFC. These findings indicate a possible glial-neuronal mechanism underlying the effects of AIE on frontal lobe-mediated behaviors and suggest a specific therapeutic approach for the amelioration of those effects.

Keywords: Adolescent; Alcohol; Astrocyte; Medial prefrontal cortex; Synaptic.

Fig. 1.. AIE + Gabapentin significantly reduced astrocyte surface area to volume.

(A) Experimental Timeline. AIW = Adolescent Intermittent Water (i.g), AIE = Adolescent Intermittent Ethanol (5 g/kg, i.g.), VEH = vehicle (0.9% sterile saline, i.p.), GBP = gabapentin (100 mg/kg, i.p.). There are four treatment groups: AIW + VEH (n = 4), AIE + VEH (n = 5), AIW + GBP (n = 5), AIE + GBP (n = 4). The number of analyzed astrocytes are as follows: AIW + VEH (n = 19), AIE + VEH (n = 20), AIW + GBP (n = 17), AIE + GBP (n = 17). (B) Pink dots represent the locations of the 73 astrocytes imaged in the mPFC. Illustration from Paxinos & Watson (1997). (C) The signal of the LCK-GFP virus (green) and the signal of the GluA1 marked synaptic puncta (red) are both strong and clear so that both the surface of the astrocyte and the colocalized synaptic puncta can be synthesized. Columns – Treatment groups. Row 1 – Raw z-series of a single slice astrocyte (green) and GluA1 synaptic puncta (red). Row 2 - Rendered 3-D structure of astrocyte (pink) using surface function of Imaris software. Scale bars are 10 μm. (D) Mean (± SEM) ratio of astrocyte surface area to volume was significantly reduced by the exposure to AIE (F _(1,14) = 4.87, p = 0.04). “#” represents main effect. Dots represent individual astrocytes while bars represent data from averaged animals.

Fig. 2.. AIE-induced reduction of astrocyte-synaptic proximity is reversed by gabapentin.

(A) Representation of colocalization methods. Left panel - rendered astrocyte surface outline (pink) and raw GluA1 puncta (red), Middle panel – GluA1 puncta (red) and a subset that are in contact (white) with astrocyte membrane (pink) (Astrocyte-Synaptic Contact). Right panel – raw z-slice of astrocyte (green) and Astrocyte-Synaptic Contact (white), GluA1 puncta (red, not colocalized with astrocyte). (B) Rows – Treatment groups: AIW = Adolescent Intermittent Water (i.g), AIE = Adolescent Intermittent Ethanol (5 g/kg, i.g.), VEH = vehicle (0.9% sterile saline, i.p.), GBP = gabapentin (100 mg/kg, i.p.). Column 1 - Rendered surface of astrocyte (pink) with colocalized synaptic puncta (white). Scale bars are 10 μm. Column 2 – Expanded magnification of inset box in Column 1. Scale bars are 5 μm. Column 3 – Expanded magnification of colocalized synaptic puncta (white). Scale bars are 5 μm. The ROI colocalized density (white) is significantly reduced in AIE exposed animals who were not treated with gabapentin compared to all other treatment groups. (C) Mean (± SEM) density of GluA1 synaptic puncta was unchanged by AIE exposure or gabapentin treatment (p > 0.05). (D) Mean (± SEM) ROI colocalization density was significantly reduced in animals exposed to AIE compared to AIW control animals (p < 0.05), and this effect was reversed by gabapentin (AIE + GBP vs. AIE, p < 0.05; AIE + GBP vs. AIW + GBP, p > 0.05). “*” represents simple main effect. Dots represent individual astrocytes while bars represent data from averaged animals.

Fig. 3.. No significant effect of either AIE on protein expression of GLT-1, GLAST, α2-δ1 or PSD-95.

(A) Mean (± SEM) protein expression of GLT-1, GLAST, α2-δ1, and PSD-95 was unchanged by AIE treatment (blue) in the synaptic fractionation. (B) Mean (± SEM) protein expression of GLT-1, GLAST, and α2-δ1 was unchanged by AIE treatment (blue) in the extra-synaptic fractionation. Images on the right include representative bands for each protein to demonstrate sufficient expression for analysis. See supplemental Figs. for full images of PVDF membranes with protein expression. AIW = Adolescent Intermittent Water (i.g), AIE = Adolescent Intermittent Ethanol (5 g/kg, i.g.).