Dysregulation of nociceptin/orphanin FQ activity in the amygdala is linked to excessive alcohol drinking in the rat

Abstract

Background: Alcoholism is a complex behavioral disorder in which interactions between stressful life events and heritable susceptibility factors contribute to the initiation and progression of disease. Neural substrates of these interactions remain largely unknown. Here, we examined the role of the nociceptin/orphanin FQ (N/OFQ) system, with an animal model in which genetic selection for high alcohol preference has led to co-segregation of elevated behavioral sensitivity to stress (Marchigian Sardinian alcohol-preferring [msP]).

Methods: The msP and Wistar rats trained to self-administer alcohol received central injections of N/OFQ. In situ hybridization and receptor binding assays were also performed to evaluate N/OFQ receptor (NOP) function in naïve msP and Wistar rats.

Results: Intracerebroventricular (ICV) injection of N/OFQ significantly inhibited alcohol self-administration in msP but not in nonselected Wistar rats. The NOP receptor messenger RNA expression and binding was upregulated across most brain regions in msP compared with Wistar rats. However, in msP rats [(35)S]GTPgammaS binding revealed a selective impairment of NOP receptor signaling in the central amygdala (CeA). Ethanol self-administration in msP rats was suppressed after N/OFQ microinjection into the CeA but not into the bed nucleus of the stria terminalis or the basolateral amygdala.

Conclusions: These findings indicate that dysregulation of N/OFQ-NOP receptor signaling in the CeA contributes to excessive alcohol intake in msP rats and that this phenotype can be rescued by local administration of pharmacological doses of exogenous N/OFQ. Data are interpreted on the basis of the anti-corticotropin releasing factor (CRF) actions of N/OFQ and the significance of the CRF system in promoting excessive alcohol drinking in msP rats.

Fig 1

Schematic representation of the sampled areas for the densitometric evaluation of mRNAs in a coronal section through the rat forebrain at Bregma levels +2 to −5 mm. CG, cingulate cortex; M1, primary motor cortex; n acc, nucleus accumbens; BNST, bed nucleus of stria terminalis; CeA, central amygdaloid nucleus; MeA, medial amygdaloid nucleus; BLA, basolateral amygdaloid nucleus; VTA, ventral tegmental area

Fig 2

Representation of the location of the tips of the injectors (filled circles) within the CeA. Numbers indicate the distances from bregma in millimeters (adapted from Paxinos and Watson 1998)

Fig 3

Self-administration for 10% ethanol under a FR-1 schedule of reinforcement in: Left Panel) msP rats treated ICV for 6 consecutive days with 0.5, 1.0 μg/rat of N/OFQ or its vehicle (Veh), and, Right Panel) Wistar rats treated ICV for 6 consecutive days with 1.0, 2.0 and 4.0 μg/rat of N/OFQ or its vehicle (Veh). Figures A) and C) correspond to the number of reinforced active lever presses, whereas, B) and D) correspond to the number of inactive lever presses. Values represent the mean (± SEM) of 6−7 subject/group. Difference from vehicle * P < 0.05; ** P< 0.01.

Fig 4

A) Left panel: Up-regulated N/OFQ mRNA expression in msP rats compared with Wistar rats (nCi/g ± SEM., n=7−8; ***P<0.001; **P<0.01; * P<0.05). Right panel : Illustrative autoradiogram showing increased N/OFQ expression in msP (Scale bars, 1 mm.). B) Left panel: Up-regulated NOP receptor mRNA expression in msP rats (black bars) compared with Wistar (white bars) rats (nCi/g ± SEM., n=7−8; ***P<0.001; ** P<0.01; * P<0.05). Right panel : Illustrative autoradiogram showing increased NOP expression in msP. (Scale bars, 1 mm.). For abbreviations see Figure 1, for details on treatment, see Material and Methods.

Fig 5

Lower panel: Elevated NOP mRNA expression in msP rats was accompanied by increased density of NOP binding sites in numerous brain regions, shown with [³H]-nociceptin using UFP-101 (N/OFQ receptor antagonist) to measure non-specific binding (rats; n = 8 per group; mean ± SEM; *** P < 0.001; ** P <0.01; * P <0.05). Upper panel: Illustrative autoradiogram showing increased total [³H]-nociceptin binding in msP rats. For abbreviations see Figure 1, for details on treatment see Material and Methods.

Fig 6

A). Illustrative autoradiogram showing [³⁵S]GTPγS binding in stimulated (upper panel) and baseline condition (lower panel). MsP rats show lower nociceptin stimulated [³⁵S]GTPγS binding in CeA (right upper panel) as compared to Wistar rats (left upper panel). B) Nociceptin stimulated [³⁵S]GTPγS binding. Percent stimulation was calculated as a difference between agonist stimulated and baseline bindings and expressed as percent of baseline value in the same region and animal (rats; n = 8 per group; mean ± SEM; *** P < 0.001; ** P <0.01; * P <0.05). Results depicted a general increase of NOP receptor functionality in msP rats except in the central amygdala where it was significantly lower. For abbreviations see Figure 1, for details on treatment see Material and Methods.

Fig 7

Self-administration for 10% ethanol under a FR-1 reinforcement schedule in msP rats treated into: Left Panel) the central amygdala (CeA) with 0.125 and 0.25 μg/site/rat of N/OFQ or its vehicle (Veh); Central Panel) the basolateral amygdala (BLA) with 0.25 and 0.5 μg/site/rat of N/OFQ or its vehicle (Veh); Right Panel) the bed nucleus of the stria terminalis (BNST) with 0.25 and 0.5 μg/site/rat of N/OFQ or its vehicle (Veh) at: A, B, C) Number of reinforced active lever presses; D, E, F) Number of inactive lever presses. Values represent the mean (± SEM) of 6−8 subject/group. Difference from controls (vehicle) * P < 0.05.