The monoamine stabilizer OSU6162 has anxiolytic-like properties and reduces voluntary alcohol intake in a genetic rat model of depression

Abstract

Alcohol use disorders (AUD) often co-occur with anxiety and depressive disorders, and anxiety often drives relapse during alcohol abstinence. Optimal AUD pharmacotherapies may thus need to target both excessive alcohol intake and elevated anxiety. (-)-OSU6162 (OSU) is a monoamine stabilizer that attenuates alcohol-mediated behaviors in both preclinical and clinical settings. However, OSU's effect on anxiety-like behavior following long-term drinking remains unknown. To this end, we utilized a genetic rat model that exhibits increased anxiety- and depression-like behaviors (Flinders Sensitive Line; FSL) and their controls (Flinders Resistant Line; FRL). Using the novelty suppressed feeding (NSF) test, we evaluated anxiety-like behaviors (1) at baseline, (2) following long-term voluntary drinking and after 24 h of alcohol deprivation, and (3) following OSU administration in the same animals. At baseline, FSL animals displayed significantly elevated anxiety-like characteristics compared to FRL. Compared to alcohol-naïve animals, long-term drinking significantly reduced anxiety-like behaviors in FSL, without any significant effects in FRL animals. Compared to vehicle, OSU administration significantly reduced anxiety-like behaviors in alcohol-naïve FSL and long-term drinking FRL animals. While there was no significant difference in alcohol intake between FSL and FRL, OSU attenuated alcohol intake in both strains. Conclusively, in addition to the compound's previously identified ability to suppress alcohol-mediated behaviors, OSU may also possess anxiolytic properties, warranting further clinical evaluation in both AUD and anxiety disorder settings.

Figure 1

Schematic experimental timeline. A total of n = 20 control-FRL (the Flinders Resistant Line) and n = 20 FSL (the Flinders Sensitive Line—a genetic rat model of depression) animals were used. Following locomotor test 1 and NSF test 1, FSL and FRL animals were divided into two groups, i.e., one group that had access to alcohol under the IA20E protocol (n = 10 animals per FSL and FRL groups) and one that had access to water only (n = 10 animals per FSL and FRL groups). The NSF test 3 was conducted during two test sessions, i.e., OSU session 1/2 and OSU session 2/2, separated by approx. 3 weeks of access to IA20E or water. For NSF test 3, all animals were given both treatments [i.e., OSU and vehicle (saline)] and were randomized to receiving either OSU or vehicle at the first test session. Single OSU or vehicle injections were given 60 min before the start of NSF test 3. To evaluate the effect of OSU on voluntary alcohol intake, rats were given access to alcohol and water immediately after locomotor tests 3.

Figure 2

FSL animals display increased anxiety-like characteristics at baseline. The Novelty Suppressed Feeding test (NSF) was used to assess anxiety-like behavior at baseline (NSF test 1). Compared to the control FRL, the FSL rats displayed (a) significantly higher latency to approach the food (n = 19–20 animals/group, n = 1 outlier) and (b) significantly lower number of approaches (n = 20 animals/group). However, (c) there was no significant difference in the latency to eat the food between the two rat strains (n = 17–18 animals/group). Graph data are presented as mean ± SEM and were analyzed by Mann Whitney tests. ***P < 0.001.

Figure 3

Alcohol intake levels in FSL and FRL animals. Animals had voluntary access to alcohol for a total of n = 33 IA20E alcohol sessions prior to the OSU experiment. Both FSL and control-FRL animals escalated significantly their alcohol intake as the result of time/drinking session. However, there was no significant difference in alcohol intake between FSL and controls at any given session (n = 10 animals per group; n = 1 outlier alcohol session). Graph data are presented as mean ± SEM and were analyzed by a mixed-effects model.

Figure 4

Long-term drinking decreases anxiety-like characteristics in FSL animals. The Novelty Suppressed Feeding (NSF) test was repeated following > 8 weeks of voluntary drinking according to the IA20E protocol to assess alcohol-associated changes in anxiety-like behaviors (NSF test 2). Analyses of the percentage change from the pre-alcohol baseline (i.e., NSF test 1), revealed anxiolytic-like effects of alcohol in the FSL rats as evidenced by (a) a significant reduction in the latency to approach the food (n = 9–10 animals/group, n = 2 outliers) and (b) a significant increase in the number of approaches (n = 9–10 animals/group, n = 1 outlier). However, (c) no significant alcohol-induced changes were found in the latency to eat (n = 6–9 animals/group, n = 1 outlier). (a–c) In the control FRL-group there were no significant alcohol-induced changes compared to pre-alcohol baseline in any of the analyzed behaviors. Graph data are presented as mean ± SEM and were analyzed by two-way ANOVA followed by Sidak’s multiple comparisons test. **P < 0.01.

Figure 5

OSU6162 has anxiolytic-like properties. The Novelty Suppressed Feeding (NSF) test was used to evaluate the effects of the monoamine stabilizer OSU6162 (OSU) on anxiety-like behaviors in long-term alcohol drinking and alcohol-naïve rats from the FSL and their controls FRL. The NSF test 3 was repeated twice to allow all animals to receive both treatments: OSU (30 mg/kg) or vehicle (saline); see also schematic experimental timeline (Fig. 1). Treatments were administered subcutaneously 60 min before the NSF test and the rats were randomized to receiving either OSU or vehicle at the first test occasion. (a) In control FRL animals, OSU had a significant main effect on latency to approach the food and interacted with alcohol exposure, and post-hoc assessments showed a significant difference in latency between OSU and vehicle in the alcohol group (n = 8–9 animal pairs/group; n = 1 outlier). In FSL animals, both alcohol exposure and OSU administration had significant main effects on the latency to approach the food, and post-hoc assessments showed a significant difference in latency between OSU and vehicle in the alcohol-naïve group, and a close-to-significant difference in the alcohol group (n = 8–9 animal pairs/group; n = 1 outlier). (b) OSU had no significant effect on the number of approaches in controls (n = 8–10 animal pairs/group) or in FSL animals (n = 9 animal pairs/group). (c) In controls, OSU had a significant main effect on the latency to eat the food, with post-hoc assessments revealing a close-to-significant difference between OSU and vehicle in the alcohol group (n = 3–6 animal pairs/group; n = 1 outlier). In addition, in FSL animals, both alcohol exposure and OSU administration had significant main effects on latency-to-eat, with post-hoc assessments showing a significant difference in latency between OSU and vehicle in the alcohol-naive group, and a trend in the alcohol group (n = 7–8 animal pairs/group; n = 1 outlier). Graph data are presented as mean ± SEM and were analyzed by mixed-effects models followed by Sidak’s multiple comparisons test. *P < 0.05, **P < 0.01.

Figure 6

The effects of OSU administration on alcohol intake in FSL and FRL animals. (a) Single OSU6162 (OSU; 30 mg/kg) administration, 60 min before the start of NSF test 3 (see also Fig. 1), significantly reduced alcohol intake both in FSL and FRL-control animals at 1 h into the alcohol session (n = 9 animal pairs/group). (b) The effect of OSU administration on reducing alcohol intake remained significant in FSL animals, but not in FRL-controls, at 24 h into the alcohol session (n = 9 animal pairs/group). Graph data are presented as mean ± SEM and were analyzed by paired t-tests. *P ≤ 0.05, **P < 0.01.