The BDNF Valine 68 to Methionine Polymorphism Increases Compulsive Alcohol Drinking in Mice That Is Reversed by Tropomyosin Receptor Kinase B Activation

Abstract

Background: The valine 66 to methionine (Met) polymorphism within the brain-derived neurotrophic factor (BDNF) sequence reduces activity-dependent BDNF release and is associated with psychiatric disorders in humans. Alcoholism is one of the most prevalent psychiatric diseases. Here, we tested the hypothesis that this polymorphism increases the severity of alcohol abuse disorders.

Methods: We generated transgenic mice carrying the mouse homolog of the human Met66BDNF allele (Met68BDNF) and used alcohol-drinking paradigms in combination with viral-mediated gene delivery and pharmacology.

Results: We found that Met68BDNF mice consumed excessive amounts of alcohol and continued to drink despite negative consequences, a hallmark of addiction. Importantly, compulsive alcohol intake was reversed by overexpression of the wild-type valine68BDNF allele in the ventromedial prefrontal cortex of the Met68BDNF mice or by systemic administration of the tropomyosin receptor kinase B agonist, LM22A-4.

Conclusions: Our findings suggest that carrying this BDNF allele increases the risk of developing uncontrolled and excessive alcohol drinking that can be reversed by directly activating the BDNF receptor, tropomyosin receptor kinase B. Importantly, this work identifies a potential therapeutic strategy for the treatment of compulsive alcohol drinking in humans carrying the Met66BDNF allele.

Keywords: Addiction; Alcohol; BDNF; Ethanol; Growth factor; Polymorphism.

Figure 1. Excessive, but not moderate, alcohol intake is exacerbated in Met68BDNF mice

Met68BDNF and Val68BDNF mice underwent intermittent access to 10% (A–B) or 20% (C–D) alcohol 2-bottle choice paradigm for 24 hrs starting at noon on Mondays, Wednesdays, and Fridays for 4 weeks. Intake (g/kg/24 hrs) of a 10% (A) or 20% (C) alcohol solution. Preference for a 10% (B) or 20% (D) alcohol solution. Alcohol preference was calculated as a ratio of alcohol consumed relative to total fluid intake (alcohol + water). Results are expressed as mean ± SEM; *P < 0.05, **P < 0.01 and ***P < 0.001 compared to Val68BDNF mice during the same session of alcohol access, LSD post hoc test; (A–B) n=19 per genotype, (C–D) n=18–19 per genotype.

Figure 2. Met68BDNF mice consume high levels of alcohol despite the addition of quinine

After 4 weeks of intermittent access to 10% (A–B) or 20% (C–D) alcohol, quinine was added to the alcohol solution in increasing concentrations (0.0–0.30 g/l; each concentration was maintained for 3 weeks before being increased). Intake (g/kg/24 hrs) of a 10% (A) or 20% (C) alcohol solution in the absence and presence of quinine. Preference for a 10% (B) or 20% (D) alcohol solution in the absence or presence of quinine was calculated as described in Figure 1. See also Supplementary Figure S7. Results are expressed as mean ± SEM, ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001 compared to 0.0 g/l quinine in the same genotype; *P < 0.05 Met68BDNF mice compared to Val68BDNF at the same dose of quinine, method of contrasts; n=9–11 per genotype.

Figure 3. The Met68BDNF polymorphism does not alter quinine or quinine-adulterated saccharin intake

(A–B) Met68BDNF and Val68BDNF mice underwent an intermittent access to quinine paradigm (0.10 and 0.15 g/kg, 2 bottle choice) for 24 hrs starting at 12:00 PM (Mondays, Wednesdays, and Fridays). Each quinine concentration was maintained for 2 weeks. Intake of (A) and preference for (B) for quinine. Results are expressed as mean ± SEM, ^##P < 0.01 compared to 0.10 g/l quinine, LSD post hoc test; n=7 per genotype. (C–D) Mice had intermittent access to 0.03% saccharin (2 bottle choice) for 24 hrs starting at 12:00 PM (Mondays, Wednesdays, and Fridays). After 2 weeks, the saccharin solution was adulterated with quinine (0.10, 0.15, 0.20 and 0.30 g/l; concentrations were increased every 2 weeks). Intake of (A), and preference for (B) saccharin in the absence and presence of quinine. Results are expressed as mean ± SEM, ^##P < 0.01 and ^###P < 0.001 compared to 0.0 g/l quinine, LSD post hoc test; n=7 per genotype.

Figure 4. The Met68BDNF polymorphism does not alter general compulsive-like behavior and anxiety levels in mice

To assess compulsive-like behavior, the inclination of mice to make an alternative arm-entry choice in a T-Maze was measured (32, 33). In this procedure, animals learn to associate each goal arm with access to a reward (chocolate milk solution). A persistent entry into the same arm across several consecutive trials reflects an inflexible strategy to obtain the reward and represents a behavioral demonstration of compulsivity. During the test, the alternation between the two goal arms and the latency to reach the reward for each trial were measured. (A) Number of repeated arm entries before alternation designated as the first entry into the opposite goal arm). (B) Latency to reach the reward. (C–F) Anxiety-like behaviors were measured in the elevated plus maze (EPM). (C) Percentage of time spent in the open arms and (D) number of visits to the open arms. (E–F) Locomotor activity was measured by the number of entries to closed arm (E) and the total arm entries (F). Results are expressed as mean ± SEM; (A–B) n = 9 per genotype, (C–F) n=8–12 per genotype.

Figure 5. Met68BDNF polymorphism does not affect the sensitivity to ataxic effects of alcohol

Mice were trained for 2 consecutive days to perform the rotarod task (Supplementary Figure S3C), and on the third day mice were given a systematic administration of 1.5 g/kg alcohol and placed on the rod every 15 minutes for 2 hrs. Arrow indicates the time point of the alcohol injection. (A) Latency to fall. (B) Number of trials necessary to reach the criterion of 180 seconds on the rod without falling. Results are expressed as mean ± SEM; n = 11–17 per genotype.

Figure 6. Overexpression of Val68BDNF in the vmPFC reduces consumption of quinine-adulterated alcohol by Met68BDNF mice

(A–B) Infection of vmPFC of the Met68BDNF mice with AAV overexpressing the wildtype Val68BDNF gene (AAV-BDNF). (A) AAV-BDNF infected neurons. AAV-BDNF (1 × 10¹² TU/ml) was bilaterally infused and the brains were fixed for the immunochemistry 3 weeks after the viral infusion. The left-hand panel depicts the specific, regionally constrained injection site. Slices were co-stained with anti-GFP (green) and anti-NeuN (red) antibodies. Scale bar: 500µm. Right-hand panels are representative images of GFP (green) co-stained with either GFAP (red; top panels) or NeuN (red; bottom panels), showing localization of viral infection within neurons but not GFAP-positive astrocytes. Scale bar: 50µm. (B) AAV-BDNF increases BDNF expression in the vmPFC of Met68BDNF mice. Western blot analysis was conducted 3 weeks after the viral infusion. The levels of BDNF were assessed using anti-BDNF antibody. Actin immunoreactivity was used as an internal loading control. (C–D) Met68BDNF mice undergoing an intermittent access to 10% alcohol containing 0.10 g/l quinine were bilaterally infused into the vmPFC with either AAV-BDNF or the AAV-GFP control. After 5 days of recovery, access to the alcohol+quinine solution was resumed. Arrows indicates the time point of the intra-vmPFC infusion of the viruses. Alcohol intake (g/kg/24 hrs) (C), and preference for the alcohol solution (D) were recorded for 6 weeks. Results are expressed as mean ± SEM; *P < 0.05, **P < 0.01 and ***P < 0.001 compared to AAV-GFP, LSD post hoc test; n=10–11 per group.

Figure 7. Activating the TrkB signaling pathway reduces consumption of quinine-adulterated alcohol by Met68BDNF mice

Mice undergoing an intermittent access (2-bottle choice) to 10% alcohol/0.10 g/l quinine solution (see Supplementary Figure S12 A–B for basal level of alcohol drinking with or without quinine and Supplementary Fig. S12 C–D for basal level of saccharin drinking with our without quinine) received i.p. administration of 100 mg/kg LM22A-4 or saline immediately before the beginning of the drinking session. Intake of alcohol (g/kg/24 hrs) (A) or saccharin (ml/kg/24 hrs) (C). Preference for the alcohol+quinine (B) or saccharin+quinine (D) solution. Results are expressed as mean ± SEM; ^###P < 0.001 compared to Met68BDNF mice receiving saline, *P < 0.05 and ***P < 0.001; LSD post hoc test; (A–B) n=8 per genotype, (C) n=7 per genotype.