Rapid Temporal Changes in the Expression of a Set of Neuromodulatory Genes During Alcohol Withdrawal in the Dorsal Vagal Complex: Molecular Evidence of Homeostatic Disturbance

Abstract

BACKGROUND Chronic alcohol exposure produces neuroadaptation, which increases the risk of cellular excitotoxicity and autonomic dysfunction during withdrawal. The temporal progression and regulation of the gene expression that contributes to this physiologic and behavioral phenotype is poorly understood early in the withdrawal period. Further, it is unexplored in the dorsal vagal complex (DVC), a brainstem autonomic regulatory structure . METHODS We use a qPCR platform to precisely and simultaneously measure the expression of 145 neuromodulatory genes in more than 100 rat DVC samples from control, chronically alcohol exposed, and withdrawn rats. To gain insight into the dynamic progression and regulation of withdrawal, we focus on the expression of a subset of functionally relevant genes during the first 48 hours, when behavioral symptoms are most severe. RESULTS In the DVC, expression of this gene subset is essentially normal in chronically alcohol exposed rats. However, withdrawal results in rapid, large magnitude expression changes in this group. We observed differential regulation in 86 of the 145 genes measured (59%), some as early as 4 hours into withdrawal. Time series measurements (4, 8, 18, 32 and 48 hours after alcohol removal) revealed dynamic expression responses in immediate early genes, γ-aminobutyric acid type A, ionotropic glutamate, and G-protein coupled receptors and the Ras/Raf signaling pathway. Together, these changes elucidate a complex, temporally coordinated response that involves correlated expression of many functionally related groups. In particular, the expression patterns of Gabra1, Grin2a, Grin3a and Grik3 were tightly correlated. These receptor subunits share over-represented transcription factor binding sites for Pax-8 and other transcription factors, suggesting a common regulatory mechanism and a role for these transcription factors in the regulation of neurotransmission within the first 48h of alcohol withdrawal. CONCLUSIONS Expression in this gene set is essentially normal in the alcohol-adapted DVC, but withdrawal results in immediate, large magnitude, dynamic changes. These data support both increased research focus on the biological ramifications of alcohol withdrawal and enable novel insights into the dynamic withdrawal expression response in this understudied homeostatic control center.

Coordinated Dynamic Gene Expression Changes in the Central Nucleus of the Amygdala During Alcohol Withdrawal

“…S1). Studies using the Lieber–DeCarli liquid alcohol diet in this facility and elsewhere report 20 to 30 mM peak blood alcohol concentrations with 12 to 16 g/kg average daily alcohol intake in rats following long‐term exposure (>3 weeks), comparable to this study (Freeman et al., ,b; Lieber and DeCarli, ; Macey et al., ; Wilson et al., ).…”

“…The details of the method of alcohol exposure have been previously reported (Freeman et al., ,b). Briefly, male Sprague‐Dawley rats (>120 g; Harlan, Indianapolis, IN) were assigned to 3 treatment groups: control ( N = 33), chronic exposure ( N = 26), or withdrawal following chronic exposure ( N = 33).…”

“…Recent work in our laboratory has focused on examining withdrawal‐induced expression changes on a systems level, providing insight into regulatory dynamics in the dorsal vagal complex (DVC), a reciprocally connected portion of the medulla that supplies afferents to and receives efferents from the CeA (Freeman et al., ,b). Here, we use a similar approach to study the CeA during dependence and withdrawal.…”

Ethanol Diverts Early Neuronal Differentiation Trajectory of Embryonic Stem Cells by Disrupting the Balance of Lineage Specifiers

“…We simultaneously measured the expression of 73 genes across 7 sample groups, and 6 biological replicates using high-throughput qRT-PCR BioMark microfluidic arrays [10]–[11]. The performance of the platform across technical replicates, dilution series and primers is shown in Figure S1 ; gene annotation and primers presented in Table S1 .…”

“…Roche’s Universal Probe Library Assay Design Center (www.universalprobelibrary.com) was used to design intron spanning PCR primers and probes. cDNA samples were selectively preamplified for 14 cycles [10]. Gene expression data were obtained using Fluidigm’s high-throughput qRT-PCR BioMark microfluidic arrays (http://www.fluidigm.com; South San Francisco, CA) as described earlier [11].…”