Chronic ethanol exposure differentially alters neuronal function in the medial prefrontal cortex and dentate gyrus

Abstract

Alterations in the function of prefrontal cortex (PFC) and hippocampus have been implicated in underlying the relapse to alcohol seeking behaviors in humans and animal models of moderate to severe alcohol use disorders (AUD). Here we used chronic intermittent ethanol vapor exposure (CIE), 21d protracted abstinence following CIE (21d AB), and re-exposure to one vapor session during protracted abstinence (re-exposure) to evaluate the effects of chronic ethanol exposure on basal synaptic function, neuronal excitability and expression of key synaptic proteins that play a role in neuronal excitability in the medial PFC (mPFC) and dentate gyrus (DG). CIE consistently enhanced excitability of layer 2/3 pyramidal neurons in the mPFC and granule cell neurons in the DG. In the DG, this effect persisted during 21d AB. Re-exposure did not enhance excitability, suggesting resistance to vapor-induced effects. Analysis of action potential kinetics revealed that altered afterhyperpolarization, rise time and decay time constants are associated with the altered excitability during CIE, 21d AB and re-exposure. Molecular adaptations that may underlie increases in neuronal excitability under these different conditions were identified. Quantitative polymerase chain reaction of large-conductance potassium (BK) channel subunit mRNA in PFC and DG tissue homogenates did not show altered expression patterns of BK subunits. Western blotting demonstrates enhanced phosphorylation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), and reduced phosphorylation of glutamate receptor GluN2A/2B subunits. These results suggest a novel relationship between activity of CaMKII and GluN receptors in the mPFC and DG, and neuronal excitability in these brain regions in the context of moderate to severe AUD.

Keywords: BK channel; CaMKII; Ethanol vapor exposure; GluN; Hippocampus; qPCR.

Figure 1.. Intrinsic firing properties are differentially affected during CIE, abstinence and re-exposure.

(a) Schematic illustrating the coronal section through the rat brain at 2.7 mm from bregma and the location of patch clamp recordings restricted to the prelimbic cortex. (b) Recording pipette patching a pyramidal neuron at layer 2/3, and pyramidal neurons were identified by their triangular shape with IR-DIC optics at 40x magnification. (c) Representative traces of action potentials elicited by depolarizing current injection from pyramidal cells from control (black), CIE (red), 21dAB (blue) and re-exposure (green) groups. The depicted sweeps resulted from injection of 120 pA into all cells. (d-e) Resting membrane potential and membrane resistance of cells from all groups. (f) Graphical relationship between the number of spikes elicited by increasing current injections in current-clamp recording from pyramidal cells from all groups. Action potential threshold (g), fAHP (h), rise tau (i) and decay tau (j) were calculated for each group. (k) Schematic illustrating the coronal section through the rat brain at −3.1 mm from bregma and the location of patch clamp recordings restricted to the granule cell layer of the dentate gyrus. (l) Recording pipette patching a granule cell neuron (GCN), and GCNs were identified by their oval shape with IR-DIC optics at 40x magnification. (m) Representative traces of action potentials elicited by depolarizing current injection from GCNs from control (black), CIE (red), 21d AB (blue) and re-exposure (green) groups. The depicted sweeps resulted from injection of 60 pA into all cells. (n-o) Resting membrane potential and membrane resistance of cells from all groups. (p) Graphical relationship between the number of spikes elicited by increasing current injections in current-clamp recording from GCNs from all groups. Group analysis is indicated as interaction (^$p<0.05) and main effect of current injection (^&p<0.05). Posthoc analysis is indicated as ^p<0.05 vs. control, ^#p<0.05 vs. CIE, +p<0.05 vs. re-exposure. Data are expressed as mean ± S.E.M.. Pyramidal neurons (animals/cells): n=3/9–12 controls, n=3/8–10 CIE, n=3/8–11 21d AB, n= 3/9–14 re-exposure. GCNs (animals/cells): n=5–7/21–27 controls, n=3/7–10 CIE, n=5/21–27 21d AB, n= 5/14–18 re-exposure.

Figure 2.. Baseline spontaneous excitatory postsynaptic currents (sEPSCs) are not significantly different between naïve, CIE, 21d AB and re-exposure groups.

(a) Representative sEPSC trace from one pyramidal neuron from a naïve (control) rat. (b) Group comparisons in mPFC pyramidal neurons demonstrated no significant change in sEPSC frequency and amplitude. (c) Representative sEPSC trace from one GCN from a control rat. (d) Group comparisons in GCNs demonstrated no significant change in sEPSC frequency or amplitude. Data are expressed as mean ± S.E.M.. mPFC pyramidal neurons (animals/cells): n=3/11 controls, n=3/6–7 CIE, n=3/11–15 21d AB, n=3/11–12 re-exposure. GCNs (animals/cells): n=7/29–31 controls, n=3/5–7 CIE, n=5/23–29 21d AB, n= 5/16–35 re-exposure.

Figure 3.. Expression of BK channel subunits are differentially affected during CIE, abstinence and re-exposure.

(a) Schematic illustrating the coronal section through the rat brain at 2.7 mm from bregma and the location of tissue punches used for qPCR assay. (b) Releative mRNA expression of BK channel subunits in the PFC by qPCR. (c) Schematic illustrating the coronal section through the rat brain at −3.1 to −4.3 mm from bregma and the location of tissue punches used for qPCR assay. (d) Relative mRNA expression of BK channel subunits in the PFC (b) and DG (c) by qPCR. (e-i) Group analysis of BK channel subunits in the PFC expressed as percent change from control. (j-n) Group analysis of BK channel subunits in the DG expressed as percent change from control. Data is expressed as mean ± S.E.M.. n=8 controls, n=9–10 CIE, n=9–11 21d AB, n= 7–10 re-exposure.

Figure 4.. Expression of plasticity related proteins are differentially affected during CIE, abstinence and re-exposure.

(a) Schematic illustrating the coronal section through the rat brain at 2.7 mm from bregma and the location of tissue punches used for Western blotting. (b) Group analysis of the density of proteins in the PFC. Below each set of bar graphs, a representative immunoblot is provided for each protein that was quantified. (c) Schematic illustrating the coronal section through the rat brain at −3.1 to −4.3 mm from bregma and the location of tissue punches used for qPCR assay. (d) Group analysis of the density of proteins in the DG. Below each set of bar graphs, a representative immunoblot is provided for each protein that was quantified. Significance is indicated based on one-way ANOVA. Data is expressed as mean ± S.E.M.. n=8 controls, n=9–10 CIE, n=9–11 21d AB, n= 9–11 re-exposure.

Figure 5.. Expression of potassium channels are differentially affected during CIE, abstinence and re-exposure.

Quantitative analysis of the potassium channel KCNK10 in the PFC (a), and KCNK1 in the DG (b). Representative immunoblots are indicated below each set of bar graphs. Significance is indicated based on one-way ANOVA. Data is expressed as mean ± S.E.M.. Data is expressed as mean ± S.E.M.. n=8 controls, n=9–10 CIE, n=9–11 21d AB, n= 9–11 re-exposure.