Dept. of Behavioral Neuroscience
Oregon Health & Science University
Alcohol use disorders (AUDs) are a leading cause of preventable death and illness that contribute significant social and economic costs to society. Genetic factors contribute substantially to AUD development but identifying and characterizing their phenotypic expression at the cellular level has been incomplete. One of the greatest challenges in treating problem alcohol (ethanol; EtOH) use is our poor understanding of the diversity of factors that promote sensitivity to EtOH reward and intoxication. The cerebellum has long been ignored as a brain region that could influence EtOH intake. However, recent functional, anatomical, and behavioral studies have established a cerebellar link to cognitive function, elements of reward processing, and risk for developing AUDs in humans and enhanced EtOH consumption in rodents. Low sensitivity to EtOH-induced cerebellar ataxia is a genetically-regulated factor associated with increased EtOH consumption and elevated risk for developing AUDs, but the cellular mechanisms that promote variation in sensitivity to EtOH impairment are unknown. In this dissertation I sought to identify the cellular mechanisms that underlie sensitivity to EtOH-induced cerebellar impairment in rodents with opposite sensitivity to EtOH-induced cerebellar ataxia and EtOH consumption phenotypes. Previous reports have identified that EtOH enhancement of cerebellar GABAA receptor (GABAAR) signaling induces ataxia, and GABAAR inhibition of granule cells (GCs) is a particularly sensitive target for modulation by EtOH. I therefore hypothesized that sensitivity to EtOH enhancement of GC GABAAR inhibition is a cellular substrate linking genetic risk for high EtOH consumption to a low sensitivity to EtOH-induced cerebellar impairment. Using patch-clamp slice electrophysiology, immunohistochemistry, and behavioral techniques, I confirmed this hypothesis by revealing that two genetically-regulated factors, GC layer neuronal nitric oxide synthase (nNOS) expression and GC protein kinase C (PKC) determine sensitivity to EtOH-induced enhancement of GC GABAAR inhibition and modulate glutamatergic input to Purkinje cells, the sole output of the cerebellar cortex. Lastly, I demonstrate that EtOH’s action on GC tonic GABAAR inhibition contributes to EtOH intake. In Chapter 2, I tested whether EtOH differentially affected GC GABAARs in rats and mice with opposite sensitivities to EtOH-induced cerebellar ataxia and consumption phenotypes. I specifically predicted that EtOH would strongly enhance GABAAR inhibition in Sprague-Dawley rats (SDRs) and DBA/2J (D2) mice with high sensitivity to EtOH-induced cerebellar impairment and low EtOH consumption phenotypes, and induce little enhancement in behaviorally insensitive, high EtOH consuming, C57BL/6J (B6) mice. Instead, I found that EtOH’s effect on GC tonic GABAAR inhibition ranged across a spectrum from strong enhancement in behaviorally sensitive, low EtOH consuming, SDRs and D2 mice to suppression in behaviorally insensitive, high EtOH consuming B6 mice. The net effect of EtOH on GC tonic GABAAR inhibition was determined by the relative expression of two genetically regulated factors: nNOS expression in the GC layer determined the magnitude of enhancement in GABAergic transmission to GCs by EtOH, and low PKC activity enabled EtOH to directly inhibit the GABAARs that mediate the tonic inhibitory current. Thus, Chapter 2 revealed that low nNOS expression and low GC PKC activity are genetic factors, common in high EtOH consuming rodents, which may perhaps underlie cerebellar-mediated genetic AUD risk in humans. Based on the findings from Chapter 2, Chapter 3 tested the predictive validity of the hypothesis that EtOH-induced suppression of GC tonic GABAAR inhibition is a neural substrate common in high EtOH consuming genotypes. I tested and confirmed this hypothesis in the high-EtOH consuming prairie vole (PV) genotype. Consistent with the results from Chapter 2, EtOH suppression of tonic GABAAR inhibition in the majority of GCs was associated with relatively low nNOS expression in comparison to age-matched low EtOH consuming rat and mice genotypes. Together with the genotypes investigated in Chapter 2, as well as our lab’s previously collected data from non-human primate GCs, I confirmed in seven different genotypes that the effect of EtOH on GC tonic GABAAR inhibition varied as a function of EtOH-consumption phenotype, ranging from strong enhancement in low EtOH consuming genotypes to suppression in high EtOH consuming genotypes. These results suggest an important contribution of EtOH’s actions on GC tonic GABAAR inhibition in behavioral phenotypes associated with elevated EtOH intake in rodents and AUD risk in humans. Despite EtOH’s differential effects on GC tonic GABAAR inhibition, it was unknown if EtOH differentially regulated excitatory transmission through the cerebellar cortex. Chapter 4 tested the hypothesis that EtOH-induced suppression of GC tonic GABAAR inhibition would be less disruptive of spontaneous and mossy fiber-evoked transmission to PCs. This hypothesis was tested in B6 and D2 mice that have high- and low-EtOH consumption phenotypes, respectively. In support of my hypothesis, a low 9mM EtOH concentration reduced spontaneous glutamatergic input to PCs in D2s, but not B6s, through effects on GC GABAAR inhibition. However, 9mM EtOH paradoxically caused slight enhancement of mossy fiber-evoked glutamatergic input to PCs in D2 mice and a slight reduction in B6 mice. GABAergic molecular layer interneurons were found to be relatively insensitive to low-moderate EtOH concentrations, further supporting the importance of EtOH’s opposite actions on GC tonic GABAAR inhibition in the diversity of cerebellar-mediated EtOH-related behavioral phenotypes. Chapters 2-4 suggested that EtOH suppression of GC tonic GABAAR inhibition is common in high-EtOH consuming genotypes and differentially regulates glutamatergic input to PCs. So in Chapter 5, I tested the hypothesis that EtOH-induced suppression of GC tonic GABAAR inhibition contributes to EtOH intake. To test this, B6 mice were stereotaxically implanted with unliteral cannula into lobes IV/V/VI of the cerebellar cortex and given limited access to 10% EtOH in a two-bottle choice drinking paradigm. GC tonic GABAAR inhibition was then selectively enhanced by microinjection of the selective δ subunit-containing GABAAR agonist, THIP, which significantly reduced EtOH intake compared to vehicle infusion in two separate experimental passes. THIP reduced EtOH intake without impairing the ability to consume fluids. These results are the first to show an isolated cerebellar contribution to EtOH intake by revealing that enhancing GC tonic GABAAR inhibition suppresses EtOH consumption. Together, the findings presented in this dissertation provide important insight into the cellular mechanism underlying the link between heritable cerebellar-related behavioral phenotypes and EtOH consumption. GC tonic GABAAR inhibition may be a promising target for directed pharmacological intervention for treatment and prevention of AUDs. Future research should be directed towards gaining a more thorough understanding of the cerebellum in reward processing and EtOH intake.
School of Medicine
Kaplan, Joshua Steven, "The Cerebellar GABAa receptor contribution to alcohol intake and intoxication" (2015). Scholar Archive. 3646.