Chronic ethanol exposure alters prelimbic prefrontal cortical Fast-Spiking and Martinotti interneuron function with differential sex specificity in rat brain

Abstract

Chronic ethanol exposure results in numerous neurobiological adaptations that promote deficits in medial prefrontal cortical (mPFC) function associated with blunted inhibitory control and elevated anxiety during withdrawal. Studies exploring alcohol dependence-related changes in this region have largely investigated adaptations in glutamatergic signaling, with inhibitory neurotransmission remaining relatively understudied. To address this, we used biochemical and electrophysiological methods to evaluate the effects of ethanol on the activity of deep-layer prelimbic mPFC Fast-Spiking (FS) and Martinotti interneurons after chronic ethanol exposure in male and female rats. We report that chronic alcohol exposure significantly impairs FS neuron excitability in both males and females. Interestingly, we observed a marked sex difference in the baseline activity of Martinotti cells that furthermore displayed differential sex-specific responses to alcohol exposure. In addition, alcohol effects on Martinotti neuron excitability negatively correlated with hyperpolarization-activated currents mediated by hyperpolarization-activated cyclic nucleotide gated (HCN) channels, indicative of a causal relationship. Analysis of HCN1 protein expression also revealed a substantial sex difference, although no effect of ethanol on HCN1 protein expression was observed. Taken together, these findings further elucidate the complex adaptations that occur in the mPFC after chronic ethanol exposure and reveal fundamental differences in interneuron activity between sexes. Furthermore, this disparity may reflect innate differences in intracortical microcircuit function between male and female rats, and offers a tenable circuit-level explanation for sex-dependent behavioral responses to alcohol.

Keywords: Cortex; Dependence; Electrophysiology; Ethanol; GABA; Interneurons.

FIGURE 1:. Physiological discrimination of deep-layer cortical interneurons.

A) Representative slice showing mDlx-GFP viral vector expression in the prelimbic mPFC. Co-labeling of mDlx-GFP and GAD67 demonstrates selectivity of the mDlx enhancer element for interneurons, as previously observed [20]. B) Simplified cartoon of cortical cell architecture, demonstrating approximate innervation profiles of deep-layer Fast-Spiking (FS; green) and Martinotti (MART; dark blue) interneurons. X94 cells (light blue) are somatostatin-positive cells generally located in Layer IV, and neurogliaform cells (NGF; yellow), as well as VIP-positive cells, are found largely in the superficial layers. C) For the present study, we confined our analyses to prototypical FS and Martinotti cells according to criteria outlined in the methods. Proportions of these cells were approximately equal in males and females. D-E) These neuron subtypes were readily discriminable by both spiking and input resistance. F) Single cell real-time qPCR revealed heterogeneity of Parv, Som, and Hcn1 markers in both FS and Martinotti cells, and thus did not prove especially valuable for subtype discrimination. Passive membrane properties of FS and Martinotti neurons from water-exposed animals are also shown. G) Representative traces depict fundamental differences in FS (left) and Martinotti (right) neuron functional characteristics, as well as the effect of application of the HCN channel antagonist ZD-7288 (red; 50 μM).

FIGURE 2:. Chronic ethanol exposure attenuates Fast-Spiking interneuron excitability in male and female rats.

A) Male FS cells displayed a significant reduction in current-evoked spiking with chronic ethanol treatment. B-E) Analysis of rheobase, current EC₅₀, spike threshold, and spike after-hyperpolarization (AHP) revealed no significant differences between water- and ethanol-exposed groups in males (n = 14 cells/group from 10-11 rats). F) FS neurons from female rats similarly displayed reduced current-evoked spiking after chronic ethanol exposure (n = 16 cells/group from 9-10 rats). G-J) Likewise, analysis of rheobase, current EC₅₀, spike threshold, and spike after-hyperpolarization (AHP) did not reveal any significant effects of ethanol exposure in females.

FIGURE 3:. Martinotti interneurons display sex-specific changes in excitability after alcohol exposure.

A) Male Martinotti cells displayed enhanced current-evoked spiking after chronic alcohol exposure. B-C) In males, analysis of curves revealed that the current EC₅₀ was significantly reduced, as well as significantly reduced rheobase, indicative of a general increase in excitability (n = 16-17 cells/group from 9-11 rats). D-E) Spike threshold and spike after-hyperpolarization were not significantly different between water- and ethanol-exposed groups in males. F) In contrast, female rat Martinotti interneurons displayed a modest reduction in current-evoked spiking with chronic ethanol exposure (n = 13-17 cells/group from 9-10 rats). Also of note, female rat Martinotti cells displayed greater baseline excitability relative to males. G-H) Curve analysis in female rats revealed significantly increased current EC₅₀ and rheobase values consistent with the right-ward shift in the current-event curve indicating a decrease in excitation. I-J) As with male rats, no significant effect of alcohol was observed on spike threshold or after-hyperpolarization in female rats.

FIGURE 4:. Regulation of Martinotti cell excitability by HCN channels.

A-B) Chronic alcohol exposure resulted in larger amplitude HCN-mediated currents in male rat Martinotti neurons (A), while the opposite effect was observed in alcohol-exposed female Martinotti neurons (B). In addition, female Martinotti cells displayed generally larger HCN currents relative to males consistent with observed enhanced current-evoked spiking relative to males. C-D) Representative western blots of HCN1 and the HCN-associated regulatory protein TRIP8b for males and females are shown in panel C. Analysis of normalized protein levels (D) revealed that females exhibited >5-fold more normalized HCN1 protein compared to males. However, an effect of ethanol on HCN1 or the HCN-associated regulatory protein TRIP8b was not observed. E-F) Application of the HCN antagonist ZD-7288 (50 μM; n = 4-5 cells/group) resulted in equivalent changes in input resistance to hyperpolarizing current between males and females (E), demonstrating these channels function similarly across cells from male and female rats. Representative trace shown in F illustrates ZD-7288 effect on hyperpolarizing input resistance.