Involvement of the Limbic Basal Ganglia in Ethanol Withdrawal Convulsivity in Mice Is Influenced by a Chromosome 4 Locus

Abstract

Physiological dependence and associated withdrawal episodes are thought to constitute a motivational force that sustains ethanol (alcohol) use/abuse and may contribute to relapse in alcoholics. Although no animal model duplicates alcoholism, models for specific factors, like the withdrawal syndrome, are useful for identifying potential genetic and neural determinants of liability in humans. We generated congenic mice that confirm a quantitative trait locus (QTL) on chromosome 4 with a large effect on predisposition to alcohol withdrawal. Using c-Fos expression as a high-resolution marker of neuronal activation, congenic mice demonstrated significantly less neuronal activity associated with ethanol withdrawal than background strain mice in the substantia nigra pars reticulata (SNr), subthalamic nucleus (STN), rostromedial lateral globus pallidus, and ventral pallidum. Notably, neuronal activation in subregions of the basal ganglia associated with limbic function was more intense than in subregions associated with sensorimotor function. Bilateral lesions of caudolateral SNr attenuated withdrawal severity following acute and repeated ethanol exposures, whereas rostrolateral SNr and STN lesions did not reduce ethanol withdrawal severity. Caudolateral SNr lesions did not affect pentylenetetrazol-enhanced convulsions. Our results suggest that this QTL impacts ethanol withdrawal via basal ganglia circuitry associated with limbic function, and that the caudolateral SNr plays a critical role. These are the first analyses to elucidate circuitry by which a confirmed addiction-relevant QTL influences behavior. This mouse QTL is syntenic with human chromosome 9p. Given the growing body of evidence that a gene(s) on chromosome 9p influences alcoholism, our results can facilitate human research on alcohol dependence and withdrawal.

Limbic circuitry activation in ethanol withdrawal is regulated by a chromosome 1 locus

“…Results using mean densities across a brain region and representative sections are comparable (Chen et al, 2008), so representative sections were analyzed for each brain region as follows (from Paxinos & Franklin, 2001): cingulate and prelimbic cortices (Cg1 and PrL, plate 18), ectorhinal-perirhinal cortex (EcP, plate 49), the central and basolateral nuclei of the amygdala (CeA and BLA, plate 42), the bed nucleus of the stria terminalis (BNST, plate 30), rostral substantia nigra pars reticulata (SNr, plate 55), caudal SNr (plate 61), subthalamic nucleus (STN, plate 48), lateral globus pallidus (LGP, plate 35) and rostromedial LGP (plate 33), medial globus pallidus (MGP, plate 42), ventral pallidum (VP, plate 30), discrete regions (dorsomedial dorsolateral and rostroventral) of the striatum (STR) (plate 23), ventral tegmental area (VTA, plate 56), nucleus accumbens (NAc) shell and core (plate 23), intermediate layers of superior colliculus (SCi, plate 61), lateral and medial septum (plate 27), and hippocampal formation CA1, CA2, CA3, stratum radiatum, dentate gyrus (DG), and DG molecular cell layer (plate 47). All images were taken at 10X and signals were quantified using Image Pro Plus (Media Cybernetics).…”

“…c-Fos immunostaining was performed as described in our previous work (Chen et al, 2008; Kozell et al, 2005). Notably, the mice used were not tested for convulsions in order to avoid potential confounds of evoked convulsions on c-Fos immunoreactivity.…”

“…First because previous results using congenic and background strain mice demonstrate that withdrawal-associated handling-induced convulsions begin approximately 4–5 h post-ethanol exposure and peak in severity approximately 6–7 h post-ethanol exposure (Kozell et al, 2008). Second because c-Fos protein induction typically occurs within 1 h of such stimuli (Chang, Kenigs, Moldow, & Zadina, 1995; Morgan et al, 1987), and lastly, to facilitate comparison of analyses using standard inbred strains (Chen et al, 2009; Kozell et al, 2005) and other congenic models for different withdrawal QTLs (Chen & Buck, 2010; Chen et al, 2008). …”

“…c-Fos is a high-resolution histological marker of neuronal stimulation (Herdegen & Leah, 1998; Morgan, Cohen, Hempstead, & Curran, 1987), which identifies a distinct activation pattern associated with alcohol withdrawal (Borlikova, Le Merrer, & Stephens, 2006; Chen & Buck, 2010; Chen, Kozell, Hitzemann, & Buck, 2008; Dave, Tabakoff, & Hoffman, 1990; Knapp, Duncan, Crews, & Breese, 1998; Kozell, Hitzemann, & Buck, 2005; Morgan, Nadi, Karanian, & Linnoila, 1992; Wilce, Beckmann, Shanley, & Matsumoto, 1994). Previous analyses have identified brain regions that differ between standard inbred strains in c-Fos expression associated with withdrawal after chronic and acute ethanol exposure (Chen, Reilly, Kozell, Hitzemann, & Buck, 2009; Kozell et al, 2005).…”

“…Significant strain differences in withdrawal-associated activation of limbic basal ganglia brain regions were found using acute alcohol exposure models, with an extended limbic circuit (i.e., hippocampus, amygdala, and prefrontal cortex) apparently recruited following chronic alcohol exposure. Because of the near elimination of genetic “noise” from loci elsewhere in the genome, comparisons between congenic and background strain animals are invaluable to address a QTL’s influence on neural activity and identify brain regions potentially involved in mediating its impact on behavior (Chen et al, 2008, 2009). …”