Elsevier

Alcohol

Volume 36, Issue 2, June 2005, Pages 83-90
Alcohol

Article
Chronic ethanol ingestion modulates proanxiety factors expressed in rat central amygdala

https://doi.org/10.1016/j.alcohol.2005.07.004Get rights and content

Abstract

Withdrawal anxiety following chronic ethanol exposure is often associated with relapse in recovering alcoholics. It is likely that brain regions regulating anxiety-like behaviors adapt during chronic ethanol exposure to ultimately regulate such behaviors. The central amygdala contains numerous neurotransmitter systems that have been implicated in the regulation of anxiety-like behavior, including corticotropin releasing factor (CRF) and NMDA-type glutamate receptors. Chronic ethanol exposure causes functional adaptations in both CRF and NMDA receptors that are likely to regulate anxiety-like behaviors expressed during withdrawal. However, the molecular mechanisms governing these adaptations remain unexplored. We therefore evaluated these neurotransmitter systems in Sprague–Dawley rats during chronic ingestion of an ethanol-containing liquid diet. Quantitative real-time reverse transcription–PCR demonstrated that preproCRF mRNA was significantly upregulated by chronic ethanol exposure, whereas mRNA expression of CRF binding protein did not change. There were also no significant changes observed in any of the NMDA subunit mRNAs, although there was a trend toward greater NR2A mRNA expression during chronic ethanol exposure. Using Western blotting analysis we measured NMDA receptor subunit protein expression. Chronic ethanol exposure did not affect protein levels of the NR1 and NR2B subunits. Like the mRNA measures, chronic ethanol exposure did influence NR2A protein levels but the effects were modest. Our results demonstrate that NMDA receptor subunit mRNA and protein expressions are not strongly influenced by exposure to chronic ethanol. This suggests that the functional NMDA receptor adaptations identified in previous studies [Roberto, M., Schweitzer, P., Madamba, S. G., Stouffer, D. G., Parsons, L. H., & Siggins, G. R. (2004). Acute and chronic ethanol exposure alter glutamatergic transmission in rat central amygdala: an in vitro and in vivo analysis. J Neurosci 24, 1594–1603] are likely to be mediated by post-translational events. In contrast, enhanced levels of CRF during/after chronic ethanol exposure are likely to be mediated by increased levels of prepro CRF mRNA. Together, our findings suggest that adaptations to chronic ethanol exposure by proanxiety factors expressed in the central nucleus appear to be mediated by distinct cellular and molecular mechanisms.

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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