Characterizing neuroadaptation processes in chronic alcohol dependence and acute alcohol withdrawal in rats

Chronic alcohol dependence and acute alcohol withdrawal affect homeostatic and emotional regulation through processes of neuroadaptation that are currently poorly characterized. In this dissertation, we employ experimental, analytical, and computational techniques to improve our understanding of the underlying molecular dynamics producing this neuroadaptation in two interconnected rat brain regions: the nucleus tractus solitarius (NTS), an integrative center for homeostatic regulation, and the central nucleus of the amygdala (CeA), the nexus of emotional regulation.The NTS and the CeA were collected from alcohol-naive control rats, chronic alcohol dependent rats, and rats undergoing acute alcohol withdrawal. Rats in the third category were subjected to one of five different durations of acute alcohol withdrawal enabling the measurement of the dynamic progression of alcohol withdrawal. As a functional readout of cellular activity in the NTS and the CeA, precise, high-throughput gene expression measurements were obtained with the BioMarkTM dynamic array platform, a new microfluidic technology that enables simultaneous, high-throughput, quantitative reverse transcription polymerase chain reactions (qRT-PCR). This is the first in vivo investigation of neuronal gene expression dynamics during alcohol withdrawal, as well as the first study that enables the deconvolution of alcohol withdrawal-induced changes from those caused by chronic alcohol dependence.The gene expression data were analyzed using a variety of analytical and statistical techniques including principal component analysis, analysis of variance, and temporal pattern classification. These analyses show that: (i) the NTS and the CeA have distinguishable transcriptomic profiles that are more distinct than any experimentally-induced perturbations (ii) there are diurnal rhythms in gene expression in the majority of genes studied, and these patterns are disrupted in chronic alcohol dependent rats and (iii) alcohol withdrawal induces dynamic changes in gene expression in both the NTS and the CeA. While these results have led to a better understanding of the dynamics of alcohol withdrawal-induced neuroadaptation, they also have broad implications in neuroscience research. In particular, the characterization of distinct transcriptomic phenotypes in functionally related brain regions highlights another level of heterogeneity in the brain, and the identification of transcriptional diurnal rhythms that are modulated by chronic alcohol consumption introduces a novel regulatory mechanism for perturbations influencing the central nervous system.The standard method of quantifying qRT-PCR fluorescence data involves the arbitrary determination of a fluorescence threshold. The validity of this quantification method in high-throughput applications is questionable. Therefore, an automated framework was developed to characterize each qRT-PCR reaction individually without the use of an arbitrary threshold. The framework was tested and validated with more than 10,000 qRT-PCR reactions.A hallmark of alcohol withdrawal is excessive neuronal excitation, which suggests an overactive control response. Therefore, using computational modeling, we tested possible control system hypotheses that involve the primary means of excitatory neurotransmission in the brain. Simulation results suggest that, individually, neither of the two regulatory mechanisms proposed from experiments can produce the dynamics observed clinically. However, a synergistic combination of the two mechanisms produces the desired dynamics, and the resulting computational model may serve as an in silico framework for preliminary testing of alcohol withdrawal treatment strategies.Finally, we discovered a subpopulation of rats that perished inexplicably during the adaptation to chronic alcohol consumption. A subsequent characterization of this subpopulation revealed that: (i) these rats had similar intake patterns prior to their deaths, (ii) deaths occurred primarily within a particular window of susceptibility, and (iii) there is no evidence in our data to support a genetic heritability for death during alcohol adaptation. Our results suggest that the dynamic processes involved in the adaptation to chronic alcohol intake are complex and variable, a result that correlates well with our gene expression results during alcohol withdrawal.