Corticotropin Releasing Factor Binding Protein and CRF2 Receptors in the Ventral Tegmental Area: Modulation of Ethanol Binge Drinking in C57BL/6J Mice

Abstract

Background: Most studies with corticotropin releasing factor (CRF) and ethanol (EtOH) consumption have focused on CRF type 1 (CRF1 ) receptors; less is known about other components of the CRF system, such as the CRF type 2 (CRF2 ) receptors and the CRF binding protein (CRFBP). In humans, several nucleotide polymorphisms in the CRFBP gene have been associated with EtOH abuse.

Methods: The role of the CRFBP within the ventral tegmental area (VTA) and the central nucleus of the amygdala (CeA) was investigated in C57BL/6J mice exposed to an EtOH binge drinking paradigm (drinking in the dark [DID]), or to a dependence-producing drinking protocol (2-bottle choice, intermittent access to alcohol [IAA]) for 4 weeks. Potential interactions between VTA CRFBP and CRF2 receptors on EtOH binge drinking were also assessed. Mice were microinjected with the CRFBP antagonist CRF fragment 6-33 (CRF6-33 ) into the VTA or CeA, or with the CRF2 antagonist astressin-2B (A2B) alone or in combination with CRF6-33 into the VTA, and had access to 20% (w/v) EtOH for 4 hours (DID). Separate cohorts of mice received vehicle and doses of CRF6-33 into the VTA or CeA and had access to EtOH/water for 24 hours (IAA). Blood EtOH concentrations (BECs) were measured, and signs of withdrawal by handling-induced convulsions were determined.

Results: Intra-VTA CRF6-33 and A2B reduced EtOH intake dose dependently in mice during DID. Furthermore, a combination of a subeffective dose of CRF6-33 and a lower dose of A2B promoted additive effects in attenuating EtOH binge drinking. Intra-VTA CRF6-33 did not affect EtOH consumption in mice given IAA, and intra-CeA CRF6-33 did not change alcohol consumption in both models of drinking. DID and IAA promoted pharmacologically relevant BECs; however, only mice given IAA exhibited convulsive events during withdrawal.

Conclusions: These findings suggest that VTA CRFBP is involved in the initial stages of escalated EtOH drinking by mechanisms that may involve CRF2 receptors.

Keywords: Alcohol; CRF2 Receptor; Corticotropin Releasing Factor Binding Protein; Drinking in the Dark; Ventral Tegmental Area.

Fig. 1

Correct placements of intra-VTA and -CeA bilateral cannulae and representative photomicrographs after Nissl staining in mice exposed to the ethanol binge drinking procedure Drinking-in-the-Dark. Each figure corresponds to coronal sections of the mouse brain according to the bregma (Paxinos and Franklin, 2001). The number of points in the figures is less than the total number of animals because of overlapping injection sites.

Fig. 2

Experiment design (A) and ethanol consumption (g/kg/4h) in separate groups of mice exposed to the ethanol binge drinking procedure Drinking-in-the-Dark, 10 min after intra-VTA (B) or -CeA (C) infusions of aCSF or CRF_6–33 (0.125, 0.25 and 0.5 µg). Data are mean ± SEM. *, versus aCSF. P < 0.05, n = 6–12 mice per group.

Fig. 3

Ethanol consumption (g/kg/4h) in separate groups of mice exposed to the ethanol binge drinking procedure two-bottle choice Drinking-in-the-Dark, 10 min after intra-VTA infusions of aCSF, Astressin2-B (A2B, 0.25 and 0.5 µg) or a combination of A2B (0.25 and 0.5 µg) and CRF_6–33 (0.125 µg). Control mice (white bar) were injected with aCSF. Data are mean ± SEM. *, versus aCSF; #, versus A2B 0.25 µg. P < 0.05, n = 8–15 mice per group.

Fig. 4

(A) Experiment design and (B) correct placements of intra-VTA and -CeA bilateral cannulae in mice exposed to the dependence-producing drinking procedure intermittent access to alcohol. Each figure corresponds to coronal sections of the mouse brain according to the bregma (Paxinos and Franklin, 2001).

Fig. 5

(A) Handling-induced convulsions (HIC) ratings of severity and (B) blood ethanol concentrations (BECs) exhibited by mice exposed to the ethanol binge drinking procedure Drinking-in-the-Dark (DID) or to the dependence-producing drinking procedure intermittent access to alcohol (IAA). HIC scores were taken every 2 h after ethanol withdrawal (4 h–10 h) and are shown as median and 1^st to 3^rd quartile range (error bars); *, versus DID; #, versus other time points. BECs are plotted as a function of ethanol intake and dotted line represents the linear fit (r² = 0.60). P < 0.05, n = 8–9 mice per group.