Discovery of a Role for Rab3b in Habituation and Cocaine Induced Locomotor Activation in Mice Using Heterogeneous Functional Genomic Analysis

Abstract

Substance use disorders are prevalent and present a tremendous societal cost but the mechanisms underlying addiction behavior are poorly understood and few biological treatments exist. One strategy to identify novel molecular mechanisms of addiction is through functional genomic experimentation. However, results from individual experiments are often noisy. To address this problem, the convergent analysis of multiple genomic experiments can discern signal from these studies. In the present study, we examine genetic loci that modulate the locomotor response to cocaine identified in the recombinant inbred (BXD RI) genetic reference population. We then applied the GeneWeaver software system for heterogeneous functional genomic analysis to integrate and aggregate multiple studies of addiction genomics, resulting in the identification of Rab3b as a functional correlate of the locomotor response to cocaine in rodents. This gene encodes a member of the RAB family of Ras-like GTPases known to be involved in trafficking of secretory and endocytic vesicles in eukaryotic cells. The convergent evidence for a role of Rab3b includes co-occurrence in previously published genetic mapping studies of cocaine related behaviors; methamphetamine response and cocaine- and amphetamine-regulated transcript prepropeptide (Cartpt) transcript abundance; evidence related to other addictive substances; density of polymorphisms; and its expression pattern in reward pathways. To evaluate this finding, we examined the effect of RAB3 complex perturbation in cocaine response. B6;129-Rab3b^tm1Sud Rab3c^tm1sud Rab3d^tm1sud triple null mice (Rab3bcd ^-/-) exhibited significant deficits in habituation, and increased acute and repeated cocaine responses. This previously unidentified mechanism of the behavioral predisposition and response to cocaine is an example of many that can be identified and validated using aggregate genomic studies.

Keywords: QTL; cocaine sensitization; genetic; genomics; habituation.

FIGURE 1

Quantitative trait locus (QTL) mapping of cocaine response in the expanded BXD RI genetic reference population: QTL mapping of locomotor response following the second injection of cocaine in females resulted in the detection of suggestive QTLs on Chr 4 and Chr 15. (A) Green lines indicate regions where DBA/2J is associated with increased phenotypic effect the increaser allele, red lines indicate that C57BL/6J is associated with the increased phenotypic effect. (B) The two QTL additive model involving Chr 4 and Chr 15. Additive the black horizontal lines represent the 1.5 LOD support interval on Chr 4 (C) and Chr 15 (D). The LOD support interval on Chr 4 spans 101.10 (rs13477873) 114.51 (gnf04.110.583) Mb, while on Chr 15 it spans 33.43 (rs8267966) 45.11 (rs13482547) Mb. 108 and 61 positional protein coding candidate genes reside in the Chr 4 and Chr 15 intervals, respectively.

FIGURE 2

Integrative functional analysis in GeneWeaver. (A) Hierarchical Similarity tool depicting a hierarchical representation of gene sets used in the integrative analysis. Nodes at the top are highly connected genes. Pink colored nodes represent nodes containing one or more QTL candidate genes. Rab3b is revealed as the most highly connected QTL candidate gene. (B) GeneSet graph representing the underlying interconnections among the gene sets containing Rab3b.

FIGURE 3

Cocaine induced locomotor activation in Rab3bcd mutants, heterozygotes, and wild types. (A) Rab3bcd–/– mutants exhibited significantly greater cocaine sensitization (p < 0.05) relative to wild types and heterozygotes on all but the second cocaine session (B). However, Rab3bcd–/– mutants also traveled a significantly greater distance relative to wild types and heterozygotes on several sessions in the saline condition beginning with the first saline injection (C), suggesting an effect of Rab3bcd on habituation to the apparatus or injection stress. After adjusting for the effect of habituation (D–F) by including distance traveled on the first two sessions as covariates, the significant effect of Rab3bcd on cocaine sensitization persisted, whereas significant differences in distance traveled between wild types, heterozygotes, and mutants in the saline condition were no longer observed. These data indicate significant effects of Rab3bcd on both habituation and cocaine sensitization. *p < 0.05.