Locomotor sensitization to ethanol impairs NMDA receptor-dependent synaptic plasticity in the nucleus accumbens and increases ethanol self-administration

Abstract

Although alcoholism is a worldwide problem resulting in millions of deaths, only a small percentage of alcohol users become addicted. The specific neural substrates responsible for individual differences in vulnerability to alcohol addiction are not known. In this study, we used rodent models to study behavioral and synaptic correlates related to individual differences in the development of ethanol locomotor sensitization, a form of drug-dependent behavioral plasticity associated with addiction vulnerability. Male Swiss Webster mice were treated daily with saline or 1.8 g/kg ethanol for 21 d. Locomotor activity tests were performed once a week for 15 min immediately after saline or ethanol injections. After at least 11 d of withdrawal, cohorts of saline- or ethanol-treated mice were used to characterize the relationships between locomotor sensitization, ethanol drinking, and glutamatergic synaptic transmission in the nucleus accumbens. Ethanol-treated mice that expressed locomotor sensitization to ethanol drank significantly more ethanol than saline-treated subjects and ethanol-treated animals resilient to this form of behavioral plasticity. Moreover, ethanol-sensitized mice also had reduced accumbal NMDA receptor function and expression, as well as deficits in NMDA receptor-dependent long-term depression in the nucleus accumbens core after a protracted withdrawal. These findings suggest that disruption of accumbal core NMDA receptor-dependent plasticity may represent a synaptic correlate associated with ethanol-induced locomotor sensitization and increased propensity to consume ethanol.

Figure 1.

Development of behavioral sensitization to ethanol is associated with increased voluntary ethanol consumption. Mice were treated daily with ethanol (1.8 g/kg, i.p., n = 54) or saline (n = 28) for 21 consecutive days. A, Ambulatory distance during 15 min tests immediately after injections on days 1, 7, 14, and 21. Mice in the upper and lower tertile on day 21 were classified as sensitized (n = 27) or nonsensitized (n = 27), respectively. Sensitized mice displayed a progressive increase in activity across days (^#p < 0.05, sensitized in test 3 or 4 vs sensitized in test 1). B, Percentage increase in locomotor response on day 21 relative to day 1. Sensitized mice displayed higher locomotor activity than the other two groups. *p < 0.05. C, Daily ethanol intake for the three treatment groups during a modified Drinking in the Dark procedure (2 h, 2-bottle choice; 20% ethanol v/v, and water). Sensitized mice drank significantly more ethanol than nonsensitized and saline subjects (p < 0.05). D, Average ethanol intake for the three treatment groups. p < 0.05 for sensitized versus nonsensitized and saline groups.

Figure 2.

The development of ethanol locomotor sensitization results in decreased NAc core NMDA receptor function. For A–D, NAc core neurons were voltage clamped at −90 mV and for E–F at +40 mV and evoked EPSCs (0.05 Hz) were recorded for at least 10 min to ensure the stability of baseline recordings. In all experiments, GABA_A IPSCs were pharmacologically blocked using bicuculline methiodide (20 μm; Sigma). A, B, Average frequency (A) and amplitude of sEPSCs (B) collected during 6 min epochs at −90 mV from cells in slices from saline (n = 12 cells in 8 mice), nonsensitized (n = 6 cells in 6 mice), and sensitized (n = 8 cells in 8 mice) groups. C, Cumulative probability distributions of sEPSC amplitude obtained in recordings from saline, nonsensitized, and sensitized groups of mice. D, PPR of evoked EPSCs (25, 50, and 250 ms interpulse interval) at −90 mV from saline (n = 15 cells in 9 mice), nonsensitized (n = 12 cells in 9 mice), and sensitized (n = 12 cells in 9 mice) groups. E, Averages of 10 consecutive EPSCs evoked at +40 mV for AMPA/NMDA ratios for saline, nonsensitized, and sensitized groups. Peak AMPA receptor-mediated current was measured at +40 mV (during a 2 ms window, 13 ms after EPSC onset) and NMDA receptor-gated currents were measured in a 10 ms window, 100 ms after EPSC onset. F, AMPA/NMDA ratios in cells recorded from the saline (n = 8 cells in 8 mice), nonsensitized (n = 6 cells in 6 mice), and sensitized (n = 7 cells in 6 mice) groups. I_{AMPA at +40 mV}/I_{NMDA at +40 mv} was taken as the AMPA/NMDA ratio. *p < 0.05 for sensitized versus nonsensitized and saline groups.

Figure 3.

The development of ethanol locomotor sensitization results in decreased NAc NMDA receptor surface expression. Two weeks after the end of ethanol or saline treatment, NAc tissue was isolated from brain slices prepared using a protocol identical to that described for the electrophysiological studies. The slices were divided and incubated in either ACSF (total expression) or BS3-ACSF (internal expression). Graphs on the left are representative of total expression in arbitrary units; graphs on the right are representative of percentage surface expression. A, Average total accumbal GluN1 expression in saline (n = 6), nonsensitized (n = 6), and sensitized (n = 6) tissue isolates. *p < 0.05 for sensitized versus nonsensitized and saline groups. B, Average percentage of GluN1 subunit surface expression in NAc tissue isolates from saline (n = 6), nonsensitized (n = 6), and sensitized (n = 6) groups. *p < 0.05 for sensitized versus nonsensitized and saline groups. C, Average total accumbal GluN2A expression in saline (n = 6), nonsensitized (n = 6), and sensitized (n = 6) tissue isolates. *p < 0.05 for sensitized versus nonsensitized and saline groups. D, Average percentage GluN2A subunit surface expression in NAc tissue isolates from saline (n = 6), nonsensitized (n = 6), and sensitized (n = 6) groups. E, Average total accumbal GluN2B expression in saline (n = 6), nonsensitized (n = 6), and sensitized (n = 6) tissue isolates *p < 0.05 for sensitized versus nonsensitized and saline groups; ^#p < 0.05 for nonsensitized versus saline groups. F, Average percentage GluN2B subunit surface expression in NAc tissue isolates from saline (n = 6), nonsensitized (n = 6), and sensitized (n = 6) groups. G, Average percentage GluA2/3 subunit surface expression in NAc tissue isolates from saline (n = 6), nonsensitized (n = 6), and sensitized (n = 6) groups.

Figure 4.

Impairment of NAc core NMDA-dependent LTD in ethanol-sensitized mice. NAc neurons were voltage clamped at −90 mV. LTD was induced using a pairing protocol: 3 × 5 Hz for 3 min, paired with a depolarization to −50 mV with a 5 min intertrain interval. EPSC amplitude (mean ± SEM) was measured as the maximal response 15–30 min after LTD induction. A, Time course of NAc core LTD in cells recorded from saline (n = 9 cells in 7 mice), nonsensitized (n = 7 cells in 6 mice), and sensitized (n = 6 cells in 6 mice) groups. B, Representative EPSCs before (1) and after (2) the NAc core LTD protocol (A) for the three treatment groups. C, EPSC amplitude (% baseline) after NAc core LTD induction. *p < 0.05 for sensitized versus nonsensitized and saline groups. The magnitude of LTD was significantly lower in cells recorded from ethanol-sensitized mice compared with cells from nonsensitized and saline groups. D, Ambulatory distance during a 15 min test immediately after intraperitoneal administration of saline (n = 6) or ethanol 1.8 g/kg. Based on their locomotor response to a single ethanol injection, ethanol-treated mice were classified as low responders (lower tertile; n = 6) or high responders (upper tertile; n = 6). *p < 0.05 for high versus low responders and saline groups. E, EPSC amplitude (% baseline) after NAc core LTD induction. No difference in the magnitude of LTD was observed in cells recorded from ethanol high responders, ethanol low responders, or saline-treated mice. F, EPSC amplitude (% baseline) after NAc core NMDA-independent LTD induction (recorded in the presence of the NMDA receptor antagonist APV 50 μm). No difference in the magnitude of NMDA-independent LTD was observed in cells recorded from the saline (n = 7), ethanol nonsensitized (n = 7), and sensitized (n = 6) groups. G, EPSC amplitude (% baseline) after NAc shell LTD induction. No difference in the magnitude of LTD was observed in cells recorded from the saline (n = 5), nonsensitized (n = 5), and sensitized (n = 8) groups.