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

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.Alcohol addiction is a major public health problem in the Western world. In the United States alone, about 15% of adults have an alcohol-related disorder at some point in their life, and alcohol abuse costs the economy over $220 billion per y in medical care and productivity loss (1). A general consensus has emerged on drug addiction as a substance-induced, aberrant form of neural plasticity (2, 3). The nucleus accumbens (Nacc) plays a central role in the neural circuits that are responsible for goal-directed behaviors (4, 5) and in addictive states. Its activity is heavily modulated by glutamate- (GLU) and dopamine- (DA) containing projections that originate in cortical and limbic regions and converge on a common postsynaptic target: the medium spiny neuron (MSN). Furthermore, DA modulates GLU inputs to Nacc neurons (6, 7), both by directly influencing synaptic transmission and by modulating voltage-dependent conductances (8). Accordingly, interactions between DA and GLU are involved in drug-induced locomotor stimulation and addiction (9, 10) and may represent useful potential therapeutic targets (11, 12). In the distal portion of the dendrites of MSNs a significant subpopulation of spines shows a particular synaptic architecture, called the “striatal microcircuit” or “synaptic triad” (13, 14), which is characterized by a double, discrete, and reciprocal interaction between DA and GLU afferents: The former establishes synaptic contact on the spine neck, whereas the latter reaches the head (13). This classical, widely accepted picture has been integrated with the coexistence of DA and GLU on the same neurons (15), but because this phenomenon appears to regress with growth in vitro (16) and its role is unclear at present (17), in the present study GLU and DA will be considered as originating from cortex and ventral tegmental area (VTA), respectively.At present, little information is available concerning the effects produced by ethanol withdrawal in dependent rats (18), although a selective increase in the density of mushroom-type spines following chronic intermittent ethanol intake has recently been reported on the basal dendrites of layer V neurons of the rodent prefrontal cortex (19). In addition, reduced expression of tyrosine hydroxylase (TH) has been demonstrated in the ventral striatum of rats maintained on a chronic ethanol-containing diet (20), and a decrease of neurofilament protein immunoreactivity in the VTA (21) has been reported. Thus, in the present work, we sought to investigate possible alterations produced by ethanol withdrawal on mesocorticolimbic transmission by exploring critical elements whose presence is strictly correlated with DAergic and GLUergic function, respectively: TH- and dopamine transporter (DAT)-positive fibers and postsynaptic density 95 (PSD-95). Spine density, morphology, and morphometry of MSNs in the Nacc shell were also investigated to obtain structural insights into pre- and postsynaptic elements of the triad simultaneously. Although considered impossible until recently (22), we have developed a new method (23) that allows visualizing the finest morphological details of spinous neurons (Golgi-Cox staining) together with the immunofluorescent neuronal elements under study. By exploiting this novel approach, we are able to visualize (in the same slice) spine morphology, TH- and DAT-positive fibers, and PSD-95–positive elements to gather information on DA and GLU transmission. Notably, because recent work suggests (24, 25) a potential relationship between spine shape, synaptic function, and morphological rearrangements of the spines as forms of developmental or experience-dependent plasticity (26), we performed patch-clamp experiments in Nacc shell slices obtained from ethanol-withdrawn rats to evaluate whether long-term depression (LTD) formation and its underlying synaptic currents are modified by experimental conditions.