Increasing Brain-Derived Neurotrophic Factor (BDNF) in medial prefrontal cortex selectively reduces excessive drinking in ethanol dependent mice

Abstract

The neurotrophin Brain-Derived Neurotrophic Factor (BDNF) has been implicated in a number of neuropsychiatric disorders, including alcohol use disorder. Studies have shown that BDNF activity in cortical regions, such as the medial prefrontal cortex (mPFC) mediates various ethanol-related behaviors. We previously reported a significant down-regulation in Bdnf mRNA in mPFC following chronic ethanol exposure compared to control mice. The present study was conducted to extend these findings by examining whether chronic ethanol treatment reduces BDNF protein expression in mPFC and whether reversing this deficit via direct injection of BDNF or viral-mediated overexpression of BDNF in mPFC alters voluntary ethanol consumption in dependent and nondependent mice. Repeated cycles of chronic intermittent ethanol (CIE) exposure was employed to model ethanol dependence, which produces robust escalation of ethanol intake. Results indicated that CIE treatment significantly increased ethanol intake and this was accompanied by a significant decrease in BDNF protein in mPFC that lasted at least 72 h after CIE exposure. In a separate study, once dependence-related increased drinking was established, bilateral infusion of BDNF (0, 0.25, 0.50 μg) into mPFC significantly decreased ethanol intake in a dose-related manner in dependent mice but did not affect moderate drinking in nondependent mice. In a third study, viral-mediated overexpression of BDNF in mPFC prevented escalation of drinking in dependent mice but did not alter intake in nondependent mice. Collectively, these results provide evidence that adaptations in cortical (mPFC) BDNF activity resulting from chronic ethanol exposure play a role in mediating excessive ethanol drinking associated with dependence.

Keywords: BDNF; Ethanol dependence; Ethanol drinking; Medial prefrontal cortex; Mice.

Figure 1:. Schematic of general study design for ethanol dependence and relapse drinking model.

Stable baseline ethanol intake was established during daily limited access (2 hr/day) sessions (filled circles). Mice were then separated into groups that received chronic intermittent ethanol (CIE) vapor or Air exposure in inhalation chambers 16 hr/day for 4 days, followed by a 72-hr forced abstinence period (stippled squares). Weekly CIE/Air exposure treatment periods alternated with 5-daily limited access Test drinking sessions for several cycles. In Study 1, mice were sacrificed at 0, 8, or 72 hr following a 5^th CIE/Air exposure cycle (denoted by red triangle). In Study 2, mice received bilateral microinjection of BDNF (0, 0.25, 0.50 μg/side) into the mPFC 24-hr prior to the first drinking session of Test cycles 4, with alternative doses given prior to Tests 5 or 6 (denoted by blue arrow). In Study 3, mice received infusions of active (AAV-BDNF) or control (AAV-GFP) virus into the mPFC 2 weeks prior to the start of study (denoted by green star). See Methods for procedural details.

Figure 2:. Repeated cycles of chronic intermittent ethanol exposure produces escalation of voluntary ethanol drinking and reduces BDNF protein content in mPFC.

(A) Data are presented as weekly average ethanol intake (g/kg) for CIE (N= 21) and CTL (N= 19) groups during the last week of baseline and four test cycles. Ethanol dependent (CIE-exposed) mice increased ethanol intake during Test 3 and Test 4 compared to their Baseline level (#; ps< 0.001), and consumed more ethanol compared to nondependent (CTL) mice during these same test cycles (*; ps< 0.001). (B) BDNF protein content in mPFC measured by ELISA at 0-hr, 8-hr, or 72-hr following the final (5^th) CIE/air exposure cycle in CIE (N= 7/time point) and CTL (N= 5– 7/time point) groups of mice sacrificed. CIE exposure produced a significant reduction in BDNF protein content in mPFC compared to controls (* p< 0.01). Values are mean ± s.e.m.

Figure 3:. BDNF infusion into mPFC selectively reduces escalated drinking in ethanol dependent (CIE-exposed) mice.

CIE and CTL mice received bilateral infusions of BDNF (0.25 or 0.50 μg/side) or vehicle (PBS) into the mPFC 48 hr following the 4^th, 5^th, or 6^th CIE/air inhalation exposure cycle in a quasi-randomized order. Data are presented as ethanol intake (g/kg) as a function of test drinking day in (A) CIE and CTL mice injected with vehicle (N= 13–15/group); (B) CIE and CTL mice injected with 0.25 μg BDNF (N= 8–9/group); and (C) CIE and CTL mice injected with 0.50 μg BDNF (N= 9–10/group). CIE-exposed mice consumed significantly more ethanol compared to CTL mice across all test days when treated with vehicle (*, p< 0.001) or 0.25 μg BDNF (*, p< 0.001), but this difference was eliminated in mice treated with 0.50 μg BDNF. (D) Average ethanol intake (g/kg) over 5-day test period for all BDNF dose groups. Ethanol intake was significantly greater in CIE compared to CTL mice that received vehicle or 0.25 μg BDNF (*, p< 0.001), and 0.50 μg BDNF significantly reduced ethanol intake in CIE mice compared to vehicle treated CIE mice (#, p< 0.001). All values are mean ± s.e.m. (E) Representative placements (black dots) of microinjection sites within the mPFC.

Figure 4:. BDNF overexpression in mPFC selectively reduces escalated drinking in ethanol dependent (CIE-exposed) mice.

Active (AAV-BDNF) and control (AAV-GFP) virus was infused into the mPFC 2 weeks prior to baseline ethanol drinking. Data are presented as average weekly ethanol intake (g/kg) during the last week of baseline and four test cycles for (A) GFP-expressing CIE (N= 10) and CTL (N= 10) mice, and (B) BDNF-expressing CIE (N= 9) and CTL (N= 11) mice. Ethanol intake during Tests 1–4 increased over baseline level of intake in CIE-GFP mice (#, p< 0.01) and intake was greater in CIE-GFP compared to CTL-GFP mice during Tests 2, 3, and 4 (*, p< 0.001). (C) Ethanol intake (g/kg) for all groups during Test 4; ethanol consumption in CIE-GFP mice was greater than intake for all other groups (*, p< 0.001). Values ate mean ± s.e.m.

Figure 5:. Verification of viral-mediated BDNF overexpression in mPFC.

(A) Representative viral expression within the mPFC visualizing the native GFP tag. (B) Schematic of tissue punch for mPFC samples analyzed for BDNF protein levels. (C) BDNF protein content (pg/mg) in mPFC samples measured by ELISA. BDNF protein expression was significantly greater in CIE-BDNF and CTL-BDNF groups of mice compared to those infused with control virus (*, p< 0.05).