ArticleChronic ethanol ingestion modulates proanxiety factors expressed in rat central amygdala
Introduction
The amygdala receives highly processed sensory and cognitive information and projects to regions regulating risk assessment (e.g., medial prefrontal cortex, bed nucleus of the stria terminalis, nucleus accumbens) as well as autonomic responses to emotionally relevant environmental stimuli. This region consequently presents as a nexus for the regulation of affective behaviors like fear and anxiety. Indeed, direct manipulation of amygdala neurotransmitter systems can modulate basal anxiety-like behavior (Moller et al., 1997, Shibata et al., 1989) and disrupt conditioned fear learning (Killcross et al., 1997, Nader et al., 2001). Recently, it has been demonstrated that various amygdala subdivisions modulate drug-seeking behaviors in rodents (Alderson et al., 2000, Di Ciano and Everitt, 2004, Fuchs and See, 2002, McLaughlin and See, 2003), drug cravings in humans (Childress et al., 1999), and the regulation of anxiety-like behaviors during withdrawal from chronic drug exposure (Menzaghi et al., 1994, Rodriguez de Fonseca et al., 1997, Watanabe et al., 2002). These findings illustrate the importance of the amygdala in the regulation of both basal affective behaviors and those associated with chronic drug exposure.
The central nucleus of the amygdala has received particular attention with regard to anxiety-like behavior during withdrawal from chronic drug exposure. For example, corticotropin releasing factor (CRF) is a neuropeptide synthesized by neurons of the central amygdala (CeN) (Cassell et al., 1986). CRF is released both locally within the CeN and at distant projection sites like the bed nucleus of the stria terminalis and the hypothalamus. CeN CRF is believed to play a principal role in the regulation of negative affect associated with drug withdrawal. There is a substantial elevation in extracellular CRF levels in the CeN following withdrawal from cocaine self-administration (Richter & Weiss, 1999), antagonist-induced withdrawal from chronic cannabinoid (Rodriguez de Fonseca et al., 1997) and morphine (Heinrichs et al., 1995) exposure, and withdrawal in ethanol-dependent animals (Menzaghi et al., 1994). Injection of CRF receptor antagonists into the CeN block anxiety-like responses to withdrawal from chronic ethanol (Rassnick et al., 1993). These findings indicate an intimate association between elevated CRF levels and withdrawal-induced anxiety.
Along with CRF, glutamate signaling in the CeN is likely to play a significant regulatory role in this region's modulation of affective responses to drug withdrawal. Chronic ethanol exposure enhances glutamate release and has been associated with adaptations in subunit-specific contributions to NMDA receptor–mediated synaptic responses (Roberto et al., 2004). Similar findings have been reported in the adjacent lateral/basolateral nuclei (Floyd et al., 2003). In addition, NMDA receptor antagonists injected directly into the CeN block the robust anxiety-like behavior precipitated by naloxone-induced withdrawal from chronic morphine (Watanabe et al., 2002). NMDA receptor activation can facilitate CRF release by CeN neurons (Cratty & Birkle, 1999); enhanced glutamate signaling in the CeN can increase CRF release at distinct sites (Gabr et al., 1995). These findings together suggest that adaptations by CeN neurotransmitter systems, particularly NMDA receptors, are likely to play a significant role in the behavioral responses to withdrawal from chronic drug exposure, including chronic ethanol.
Despite substantial evidence of functional adaptations in CRF- and NMDA-mediated signaling during or following chronic ethanol exposure, the molecular events governing such alterations remain unclear. A detailed understanding of these mechanisms would offer a clearer explanation of the potential therapies that address the long-term consequences of alcohol abuse and withdrawal. We have therefore used molecular and cellular approaches to specifically examine the impact of chronic ethanol exposure on CeN NMDA and CRF systems.
Section snippets
Animal procedures
All animal procedures were performed in accordance with the protocol approved by Wake Forest University School of Medicine Animal Care and Use Committee that was consistent with the NIH animal care and use policy. Male Sprague–Dawley rats (∼120 g; Harlan, Indianapolis, IN) were singly housed and exposed to an 8-h-on/16-h-off light/dark cycle. After 4 days of acclimation to the housing facility/conditions, rats were introduced to a commercially available Lieber–DeCarli (Lieber & DeCarli, 1989)
Chronic ethanol ingestion upregulates mRNA levels for some proanxiety factors
To assess whether neurotransmitter systems in brain regions regulating anxiety-like behavior adapt during chronic ethanol exposure, we measured the mRNA expression of several “proanxiety” factors in the CeN derived from control and chronic ethanol–exposed animals. CRF expressed in the CeN has been extensively characterized as a central regulatory component controlling anxiety-like behavior (Rassnick et al., 1993, Richter et al., 2000, Rodriguez de Fonseca et al., 1997, Sajdyk et al., 1999).
Discussion
One of the central findings of this report is the chronic ethanol–induced upregulation of prepro CRF mRNA. Chronic ethanol enhances basal levels of CRF in CeN dialysates during both the exposure and acute withdrawal (<24 h) (Merlo Pich et al., 1995). In addition, increased amygdala CRF tissue content is maintained several weeks after withdrawal (Zorrilla et al., 2001). The upregulated prepro CRF mRNA reported here could clearly provide the mechanistic basis for increased CRF release during
Acknowledgments
This work was supported by AA14445 (BAM) and the “Multi-disciplinary Training in the Biology of Alcoholism” program (T32 AA007565, KAL). We are also grateful to Dr. Dustin Dubois for his insightful comments on this manuscript.
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