Hampered long-term depression and thin spine loss in the nucleus accumbens of ethanol-dependent rats

Abstract

Alcoholism involves long-term cognitive deficits, including memory impairment, resulting in substantial cost to society. Neuronal refinement and stabilization are hypothesized to confer resilience to poor decision making and addictive-like behaviors, such as excessive ethanol drinking and dependence. Accordingly, structural abnormalities are likely to contribute to synaptic dysfunctions that occur from suddenly ceasing the use of alcohol after chronic ingestion. Here we show that ethanol-dependent rats display a loss of dendritic spines in medium spiny neurons of the nucleus accumbens (Nacc) shell, accompanied by a reduction of tyrosine hydroxylase immunostaining and postsynaptic density 95-positive elements. Further analysis indicates that "long thin" but not "mushroom" spines are selectively affected. In addition, patch-clamp experiments from Nacc slices reveal that long-term depression (LTD) formation is hampered, with parallel changes in field potential recordings and reductions in NMDA-mediated synaptic currents. These changes are restricted to the withdrawal phase of ethanol dependence, suggesting their relevance in the genesis of signs and/or symptoms affecting ethanol withdrawal and thus the whole addictive cycle. Overall, these results highlight the key role of dynamic alterations in dendritic spines and their presynaptic afferents in the evolution of alcohol dependence. Furthermore, they suggest that the selective loss of long thin spines together with a reduced NMDA receptor function may affect learning. Disruption of this LTD could contribute to the rigid emotional and motivational state observed in alcohol dependence.

Keywords: Golgi; dopamine; glutamate; synaptic plasticity.

Fig. 1.

Morphological and morphometrical characteristics used for spine classification. (Left) Four different morphological (shape) types were identified and categorized based on morphometrical features (criteria), as indicated. hd, head diameter; nd, neck diameter; nL, neck length; TL, total length. (Right) Example of a typical observation made on the dendritic tree of an MSN belonging to a rat of the control group. The image is color-coded. Colored bands on dendrites indicate the origin of spines.

Fig. 2.

Representative MSN dendritic branch (orange) showing anatomical differences among CTRL, EtOH-CHR, and EtOH-W groups in TH-positive fiber density (green) (A) and colocalized PSD-95 (white) (B). (C) Selective loss of long thin (blue) spines in the EtOH-W group. Colored bands on dendrites indicate the origin of spines. Software used was Imaris 7.4. (Scale bars, 5 μm.)

Fig. 3.

Histograms represent the mean ± SEM of dendritic spine densities of second-order dendrites of NAcc shell MSNs in the three experimental conditions. Various spine typologies are represented within each group. *P < 0.0001 vs. CTRL.

Fig. 4.

Depiction of changes affecting presynaptic (Upper) and postsynaptic indices (Lower) observed in the three experimental conditions. (Upper) TH volume (Left) is halved by EtOH-W and unaltered by EtOH-CHR, whereas DAT (Right) is unmodified by treatments. (Lower) Long thin spine density (Left) is reduced by about 40%, and this effect is paralleled by a reduction in PSD-95 (Right) immunolabeling of similar proportions (∼60%). *P < 0.0001 vs. CTRL. Error bars represent SEM in all figures.

Fig. 5.

Effects of chronic ethanol treatment and withdrawal on GLUergic mEPSCs and paired-pulse ratio measured in MSNs of the Nacc. (A) Representative tracings of mEPSCs from single voltage-clamped MSNs of the different experimental groups. (B) Averaged mEPSCs recorded during periods of 3 min from which analysis of the kinetic properties has been conducted. (C–E) The bar graphs summarize changes in mEPSC amplitude, fast and slow decay time constants, and frequency, and are expressed as the mean of absolute values ± SEM. (F) Representative traces of averaged synaptically evoked EPSCs obtained with an interstimulus of 50 ms. (G) Paired-pulse ratio in the different experimental groups. The number of cells analyzed is indicated in each bar. *P < 0.05, **P < 0.001 vs. CTRL.

Fig. 6.

Changes in NMDA:AMPA ratio in Nacc MSNs induced by chronic ethanol exposure and withdrawal. I–O curves of AMPAr- (A) and NMDAr-mediated (C) eEPSCs. The values represent current amplitude and are normalized to the maximal response. (B and D) The graphs summarize the values of stimulus intensity that evoked a half-maximal response in the different experimental groups. Data are expressed as averaged absolute values ± SEM. (E) Representative eEPSCs mediated by NMDA and AMPA receptors recorded in single MSNs. (F) The graph summarizes the NMDA:AMPA ratio obtained from MSNs of the different experimental groups. The number of cells analyzed is indicated in each bar. *P < 0.05, **P < 0.001 vs. CTRL.

Fig. 7.

Effect of chronic ethanol treatment and withdrawal on LTD induced in Nacc MSNs. (A) AMPAr-mediated EPSCs were recorded in single voltage-clamped (−65 mV) MSNs of the Nacc shell obtained from the different groups of animals. Representative EPSCs recorded before (1) and after (2) conditioning are shown above the graph. (B) The graph illustrates the degree of LTD, calculated by averaging the percent change in EPSC amplitude from baseline 70–80 min after LFS. The number of cells analyzed is indicated in each bar. **P < 0.01 vs. CTRL. (C) Field EPSPs were recorded in Nacc shell slices obtained from the different groups of rats. LTD was elicited by LFS (500 stimuli at 1 Hz), and representative traces are shown above the graph. Data are expressed as mean percent change in fEPSP slope ± SEM from baseline. (D) The graph summarizes the degree of LTD, calculated by averaging the percentage change in fEPSP slope from baseline 70–80 min after LFS. The number of recordings analyzed is indicated in each bar. *P < 0.05 vs. CTRL.

Fig. 8.

Effects of chronic ethanol treatment and withdrawal on passive membrane properties and excitability of Nacc MSNs. (A) The graph summarizes changes in membrane capacitance observed in different experimental groups. Data are expressed as absolute values (pF) and are means ± SEM from the number of cells indicated in each bar. *P < 0.05 vs. CTRL. (B) Representative voltage responses to negative and subthreshold positive current pulses applied to MSNs in current-clamp mode. (C and D) V/I relationship (C) and comparison of membrane input resistance (D) (MΩ). *P < 0.05 vs. CTRL. (E) Representative membrane voltage responses to negative (−20 pA) and positive (160 pA) pulses. (F–I) Effects of chronic ethanol treatment and withdrawal on action potential threshold (F), minimum current intensity required for inducing the first AP (G), AP latency (H), and AP frequency (I). Data are expressed as means ± SEM from the number of cells indicated in each bar. *P < 0.05 vs. CTRL.