Inhibitory and excitatory alcohol-seeking cues distinct roles in behavior, neurochemistry, and mesolimbic pathway in alcohol preferring (P) rats

Abstract

Cues associated with alcohol use can readily enhance self-reported cravings for alcohol, which increases the likelihood of reusing alcohol. Understanding the neuronal mechanisms involved in alcohol-seeking behavior is important for developing strategies to treat alcohol use disorder. In all experiments, adult female alcohol-preferring (P) rats were exposed to three conditioned odor cues; CS+ associated with EtOH self-administration, CS- associated with the absence of EtOH (extinction training), and a CS^0, a neutral stimulus. The data indicated that presentation of an excitatory conditioned cue (CS+) can enhance EtOH- seeking while the CS- can inhibit EtOH-seeking under multiple test conditions. Presentation of the CS+ activates a subpopulation of dopamine neurons within the interfascicular nucleus of the posterior ventral tegmental area (posterior VTA) and basolateral amygdala (BLA). Pharmacological inactivation of the BLA with GABA agonists inhibits the ability of the CS+ to enhance EtOH-seeking but does not alter context-induced EtOH-seeking or the ability of the CS- to inhibit EtOH-seeking. Presentation of the conditioned odor cues in a non-drug-paired environment indicated that presentation of the CS+ increased dopamine levels in the BLA. In contrast, presentation of the CS- decreased both glutamate and dopamine levels in the BLA. Further analysis revealed that presentation of a CS+ EtOH-associated conditioned cue activates GABA interneurons but not glutamate projection neurons. Overall, the data indicate that excitatory and inhibitory conditioned cues can contrarily alter EtOH-seeking behaviors and that different neurocircuitries are mediating these distinct cues in critical brain regions. Pharmacotherapeutics for craving should inhibit the CS+ and enhance the CS- neurocircuits.

Keywords: Conditioned cues; Ethanol self-administration; Ethanol-seeking; Pavlovian Spontaneous Recovery.

Figure 1.

Depicts the experimental protocol employed in all experiments reported in this publication. The time points in which the conditioned cues (CS⁰, CS+, and CS−) were paired with specific events is indicated.

Figure 2.

(a) The mean (± SEM) ethanol (EtOH) lever responses during 4 PSR sessions when conditioned odor cues were present in the operant chamber during testing (n = 10–12/group). (b) The mean (± SEM) ethanol (EtOH) lever responses during 5 PSR sessions when conditioned odor cues were present in a neutral, non-drug paired environment prior to each test session (n = 10–13/group). Asterisk (*) Indicates that 1) the CS+ rats responded more on the EtOH lever than the CS⁰ or no odor control groups, 2) the CS+, CS⁰, and no odor groups responded more than baseline extinction (Ext Base), and 3) all groups were different from the CS− group, which did not respond differently from extinction levels. Plus sign (+) Indicates that: 1) CS+ is significantly greater than all other groups and 2) CS+ and no odor group significantly higher than Ext Base. Pound (#) Indicates that 1) CS+ is significantly greater than all other groups, 2) the CS+ group is significantly higher than Ext Base, and 3) CS− significantly lower than CS⁰ and no odor groups and lower than Ext Base. Asterisk (**) Indicates that CS− group is significantly higher than other groups in PSR session 5 when all groups received CS+.

Figure 3.

The mean (± SEM) number of cFos+ cells/mm2 in subregions of the nucleus accumbens (A), brain regions that contain DA neurons (B), and subregions of the amygdala (C) in P rats exposed to conditioned odor cues in a non drug-paired environment (n = 6–7/group). Plus sign (+) Indicates significant differences between the CS+ group and the CS⁰ and CS− groups. Asterisk (*) Indicates significant differences between CS− and the CS⁰ group. Abbreviations used: VTA = ventral tegmental area; SN = substantia nigra; AcbC = nucleus accumbens core; AcbSh = nucleus accumbens shell; CeA = central nucleus of the amygdala; BLA = basolateral nucleus of the amygdala.

Figure 4.

Top left panel depicts the TH (brown) and cFos (black) staining for all regions in the posterior VTA (−5.8 mm bregma). As indicated in the image, most of the cFos staining (black) occurred in the IF (dorsal, medial). The bottom left panel depicts the mean (± SEM) number of Fos+ only cells/mm2 and TH+/Fos+ cells/mm2 in the posterior ventral tegmental area (n = 10–13/group). Asterisk (*) indicates that the CS− group has significantly more Fos+/TH+ cells than the CS⁰ control group. Plus sign (+) Indicates that the CS+ group has significantly more Fos+/TH+ cells than all other groups. The right panels are representative images (40x) for rats exposed to the CS⁰ (top), CS− (middle), or CS+ (bottom).

Figure 5.

The mean (± SEM) ethanol (EtOH) lever responses during extinction baseline and PSR testing in P rats administered GABAA/B agonists or aCSF into the BLA immediately prior to EtOH-seeking testing when conditioned odor cues were present in the operant chamber during testing (n = 7–9/group). Asterisk (*) indicates that both CS⁰ groups and the CS+, GABAA/B agonists group responded more during the PSR test session than during the extinction baseline. Plus sign (+) indicates that the CS+/aCSF group responded significantly more than all other groups during the PSR test session and greater than during extinction baseline.

Figure 6.

(a) Depicts the mean (± SEM) % change in dopamine levels in the BLA in P rats exposed to conditioned cues (CS⁰, CS+, and CS−) in a non-drug-paired environment for 24 min (n = 7–10/group). Asterisk (*) indicates that DA levels in the BLA are elevated in the rats exposed to the CS+ compared to all other groups and baseline levels. Plus sign (+) indicates DA levels in the BLA are lower in the rats exposed to the CS− compared to all other groups and baseline levels. (b) Depicts the mean (± SEM) % change in glutamate levels in the BLA in P rats exposed to conditioned odor cues (CS⁰, CS+, and CS−) in a non-drug-paired environment for 24 min. Asterisk (*) indicates that glutamate levels in the BLA are lower in the rats exposed to the CS− compared to all other groups and baseline levels.

Figure 7.

Left panels depict the labeling for total GABA+ /mm3 (A), Fos+ only /mm3 (B), and GABA+/Fos+ /mm3 (C) cells in the BLA of P rats exposed to conditioned odor cues in a non drug-paired environment (n = 6–7/group). Asterisk (*) indicates that the CS+ group has significantly more Fos+ cells in the BLA than the CS⁰ or CS− group. The right panels indicate the regions of the BLA separated for analyses in Figs. 7 and 8 (top panel; 5x) and representative images (40x) for rats exposed to the CS⁰, CS−, or CS+; cFos (green), GABA (red), co-localized (yellow).

Figure 8.

Left panels depict the labeling for total GluR2/3+ /mm2 (A), Fos+ only/mm2 (B), and mGluR2/3+/Fos+ /mm2 (C) cells in the BLA of P rats exposed to conditioned odor cues in a non drug-paired environment (n = 6–7/group). Asterisk (*) indicates that the CS+ group has significantly more cFos+ cells in the BLA than the CS⁰ or CS− group. The right panels are representative images (40x) for rats exposed to the CS0 (top), CS− (middle), or CS+ (bottom).