Dopamine Restores Limbic Memory Loss, Dendritic Spine Structure, and NMDAR-Dependent LTD in the Nucleus Accumbens of Alcohol-Withdrawn Rats

Abstract

Alcohol abuse leads to aberrant forms of emotionally salient memory, i.e., limbic memory, that promote escalated alcohol consumption and relapse. Accordingly, activity-dependent structural abnormalities are likely to contribute to synaptic dysfunctions that occur from suddenly ceasing chronic alcohol consumption. Here we show that alcohol-dependent male rats fail to perform an emotional-learning task during abstinence but recover their functioning by l-3,4-dihydroxyphenylalanin (l-DOPA) administration during early withdrawal. l-DOPA also reverses the selective loss of dendritic "long thin" spines observed in medium spiny neurons of the nucleus accumbens (NAc) shell of alcohol-dependent rats during abstinence, as well as the reduction in tyrosine hydroxylase immunostaining and postsynaptic density-95-positive elements. Patch-clamp experiments in NAc slices reveal that both in vivo systemic l-DOPA administration and in vitro exposure to dopamine can restore the loss of long-term depression (LTD) formation, counteract the reduction in NMDAR-mediated synaptic currents and rectify the altered NMDAR/AMPAR ratio observed in alcohol-withdrawn rats. Further, in vivo microdialysis experiments show that blunted dopaminergic signaling is revived after l-DOPA treatment during early withdrawal. These results suggest a key role of an efficient dopamine signaling for maintaining, and restore, neural trophism, NMDA-dependent LTD, and ultimately optimal learning.SIGNIFICANCE STATEMENT Blunted dopamine signaling and altered glutamate connectivity in the nucleus accumbens represent the neuroanatomical basis for the impairment in aversive limbic memory observed during withdrawal in alcohol dependence. Supplying l-DOPA during withdrawal re-establishes synaptic morphology and functional neuroadaptations, suggesting a complete recovery of nucleus accumbens glutamatergic synaptic plasticity when dopamine is revived. Importantly, restoring dopamine transmission allows those synapses to encode emotionally relevant information and rescue flexibility in the neuronal circuits that process limbic memory formation. Under these conditions, drugs capable of selectively boosting the dopaminergic function during the "fluid" and still responsive state of the early withdrawn maladaptive synapses may help in the treatment of alcohol addiction.

Keywords: LTD; alcohol abuse; confocal microscopy; dopamine; glutamate; memory.

Figure 1.

Alcohol withdrawal disrupts limbic memory formation. Limbic memory was assessed in rats by (a) the EOR test, here schematically represented. Four hours after the cued fear-conditioned learning, rats were put into the central zone of Context A chamber and tested for individual zone preference in epoch BSL. Afterward emotional-object avoidance and target-zone aversion were assessed in epoch ON-1 (objects in the arena), OFF (objects removed from the arena), ON-2 (objects in the arena). Twelve hour alcohol-withdrawn rats (EtOH-WDL) displayed reduced (b) emotional object avoidance and (c) target-zone aversion with respect to alcohol-naive control (CTRL) and chronically-EtOH-exposed (EtOH-CHR) rats. No significant differences between CTRL and EtOH-CHR were recorded. EtOH-WDL did not show sensory-motor impairment, in terms of (d) TDT when video-tracked during the BSL epoch in context A chamber, and (e) tail-flick latency following the EOR test. Each bar represents the mean ± SEM; n = 8 rats. Each box-and-whisker plot represents the median (horizontal line in the box), 25–75% (box), and min-to-max (whiskers) values of n = 8 rats. ***p < 0.001, *p < 0.05.

Figure 2.

Limbic memory disruption was rescued by l-DOPA/benserazide (l -DOPA) after 12 h withdrawal. Twelve hour alcohol-withdrawn rats administered with l-DOPA (EtOH-WDL + l -DOPA) displayed increased (a) emotional object avoidance and (b) target-zone aversion score with respect to 12 h alcohol-withdrawn rats receiving vehicle (EtOH-WDL + vehicle), up to alcohol-naive control (CTRL) rats' level. Limbic memory disruption was long lasting: alcohol-withdrawn rats showed decreased (c) emotional object avoidance and (d) target-zone aversion score following 48 h [EtOH-WDL (48 h)] and 14 d [EtOH-WDL (14 d)] of withdrawal. Long lasting limbic memory disruption in EtOH-withdrawn rats was not rescued by late stimulation of dopamine transmission. Preconditioning l-DOPA is not effective at significantly increasing (c) emotional object avoidance and (d) target-zone aversion score in EtOH-WDL (48 h) and EtOH-WDL (14 d) rats. In contrast, the rescuing effect of early (12 h withdrawal) l-DOPA administration was dose-dependent on both (e) emotional object avoidance and (f) target-zone aversion score. Each bar represents the mean ± SEM; n = 8 rats. ***p < 0.001; **p < 0.01; *p < 0.05.

Figure 3.

DA increase reverts aberrant structural plasticity in the NAc of alcohol-withdrawn rats. a, Representative 3D reconstruction of the simultaneous visualization of Golgi-Cox stained MSNs. b, 3D reconstruction of the simultaneous visualization of Golgi-Cox stained MSNs (red), DA projections (TH+; green), PSD-95 (yellow), and long-thin spines (blue) in alcohol-naive control (CTRL), 12 h alcohol-withdrawn rats (EtOH-WDL), and 12 h alcohol-withdrawn rats administered with l-DOPA (EtOH-WDL + l-DOPA) Scale bar 20 μm. l-DOPA administration restores the aberrant structural plasticity of the synaptic triad in the NAc of withdrawn-rats. Indeed, l-DOPA (c) selectively expanded the density of long-thin spines to similar values as controls; (d) increased TH levels and (e) produced a complete restoring of the immunolabeling for PSD-95 in the NAc. Each bar represents the mean ± SEM; n = 6–8 rats. ***p < 0.001, *p < 0.05.

Figure 4.

Effects of EtOH withdrawal on LTD formation in rat NAc MSNs. AMPAR-mediated eEPSCs were recorded in single voltage-clamped (−65 mV) MSNs of the NAc shell obtained from the different groups of animals [n of animals: CTRL = 5; EtOH-WDL (12 h) = 5; EtOH-WDL (48 h) = 4; EtOH-WDL (14 d) = 3]. a, Representative eEPSCs recorded before (black trace) and 60 min after (blue trace) LFS paired with depolarization (−50 mV). b, Scatter plot graph of the changes in eEPSC amplitude in CTRL and EtOH-WDL (12 h, 48 h, and 14 d) with data expressed as percentage of baseline. c, The graph illustrates the degree of LTD, calculated by averaging the eEPSC amplitude values measured 50–60 min after LFS and expressed as percentage of baseline. The number of cells analyzed is indicated for each group. ***p = 0.0004. d, The scatter graph illustrates the distribution of individual values, averaged in c. Color code is the same as in c. ***p = 0.0006.

Figure 5.

Single acute administration of l-DOPA and bath perfusion of NAc slices with DA restores the hampered LTD formation in NAc shell MSNs from EtOH-withdrawn rats. a, Representative EPSCs recorded before (black trace) and 60 min after (blue trace) LFS paired with depolarization (−50 mV) obtained in single MSNs from CTRL and EtOH-dependent rats that were tested after 12 h withdrawal. EtOH-WDL rats were subjected to an acute administration of l-DOPA (6 mg/kg, s.c.) and benserazide (6 mg/kg, s.c.), or vehicle, 1 h before their kill. b, Scatter plot graph of the changes in EPSC amplitude with data expressed as percentage of baseline. c, Scatter graph illustrating the distribution of individual data, averaged in b, calculated by averaging the EPSC amplitude values measured 50–60 min after LFS and expressed as percentage of baseline. The number of cells analyzed is indicated in each group and were obtained from five animals per group. ***p = 0.0002. d, Representative EPSCs recorded before (black trace) and 60 min after (blue trace) LFS paired with depolarization (−50 mV) obtained in single MSNs from EtOH-dependent rats that were tested after 12 h of EtOH-WDL. Slices from EtOH-WDL rats were acutely perfused with dopamine (10 μm) 5 min before application of LFS (indicated in green in graph e). e, Scatter plot graph of the changes in EPSC amplitude with data expressed as percentage of baseline. f, Scatter graph illustrating the distribution of individual data averaged in e. The number of cells analyzed is indicated in each group and were obtained from five animals per group. ***p = 0.0002. A single intraperitoneal injection of l-DOPA or DA perfusion in slice did not alter LTD formation in NAc shell MSNs from CTRL (g) or EtOH-CHR (h) rats. The bar graphs illustrate the degree of LTD, calculated by averaging the EPSC amplitude values measured 50–60 min after LFS and expressed as percentage of baseline. The number of cells analyzed is indicated in each group and were obtained from five animals per group.

Figure 6.

Single acute administration of l-DOPA and bath perfusion of NAc slices with DA restore the decrease in NMDA/AMPA ratio in NAc shell MSNs from EtOH-withdrawn rats. a, Representative EPSCs mediated by NMDA and AMPA receptors recorded in single MSNs clamped at −70 mV (for AMPA) and +40 mV (for NMDAR in the presence of NBQX 5 μm) from the different experimental groups. b, The graph summarizes the NMDAR/AMPAR ratio obtained from MSNs of the different groups. c, Scatter graph illustrating the distribution of individual data averaged in a and b. The number of cells analyzed is indicated in each graph bar and were obtained from five animals per group. ***p = 0.0001.

Figure 7.

The selective antagonist of D1 but not D2 receptors prevents the restoring effect of DA on LTD levels in EtOH-WDL (12 h) rats. NAc slices of EtOH-WDL rats were bath-perfused with DA (10 μm), in the absence or presence of SCH23390 (10 μm) or sulpiride (10 μm), for 5 min prior LFS paired with depolarization (−50 mV). a, b, Scatter plot graph of the changes in EPSC amplitude with data expressed as percentage of baseline. c, The scatter graph illustrates the degree of LTD, as in graphs a and b, and were obtained from five animals. The number of cells analyzed is indicated for each experimental group. ***p = 0.0006.

Figure 8.

Single acute administration of l-DOPA restores extracellular DA levels in NAc shell of EtOH-withdrawn rats. a, EtOH-WDL (12 h) rats displayed lower levels of dialysate DA in the NAc shell, with respect to CTRL rats; l-DOPA/BENSERAZIDE (6/6 mg/kg, s.c.) administration significantly increased DA levels in the NAc shell of EtOH-WDL (12 h) rats, up to CTRL's levels. b, Notably, l-DOPA induced a larger increase of dialysate DA in the NAc shell of EtOH-WDRL (12 h) compared with basal (filled symbol) and to dialysate DA in the NAc shell of CTRL rats expressed as percentage of baseline. Each value represents the mean ± SEM; n = 4 rats. **p < 0.01, ***p < 0.01.