BDNF signaling in the VTA links the drug-dependent state to drug withdrawal aversions.

Drug administration to avoid unpleasant drug withdrawal symptoms has been hypothesized to be a crucial factor that leads to compulsive drug-taking behavior. However, the neural relationship between the aversive motivational state produced by drug withdrawal and the development of the drug-dependent state still remains elusive. It has been observed that chronic exposure to drugs of abuse increases brain-derived neurotrophic factor (BDNF) levels in ventral tegmental area (VTA) neurons. In particular, BDNF expression is dramatically increased during drug withdrawal, which would suggest a direct connection between the aversive state of withdrawal and BDNF-induced neuronal plasticity. Using lentivirus-mediated gene transfer to locally knock down the expression of the BDNF receptor tropomyosin-receptor-kinase type B in rats and mice, we observed that chronic opiate administration activates BDNF-related neuronal plasticity in the VTA that is necessary for both the establishment of an opiate-dependent state and aversive withdrawal motivation. Our findings highlight the importance of a bivalent, plastic mechanism that drives the negative reinforcement underlying addiction.

Different roles of BDNF in nucleus accumbens core versus shell during the incubation of cue-induced cocaine craving and its long-term maintenance.

Brain-derived neurotrophic factor (BDNF) contributes to diverse types of plasticity, including cocaine addiction. We investigated the role of BDNF in the rat nucleus accumbens (NAc) in the incubation of cocaine craving over 3 months of withdrawal from extended access cocaine self-administration. First, we confirmed by immunoblotting that BDNF levels are elevated after this cocaine regimen on withdrawal day 45 (WD45) and showed that BDNF mRNA levels are not altered. Next, we explored the time course of elevated BDNF expression using immunohistochemistry. Elevation of BDNF in the NAc core was detected on WD45 and further increased on WD90, whereas elevation in shell was not detected until WD90. Surface expression of activated tropomyosin receptor kinase B (TrkB) was also enhanced on WD90. Next, we used viral vectors to attenuate BDNF-TrkB signaling. Virus injection into the NAc core enhanced cue-induced cocaine seeking on WD1 compared with controls, whereas no effect was observed on WD30 or WD90. Attenuating BDNF-TrkB signaling in shell did not affect cocaine seeking on WD1 or WD45 but significantly decreased cocaine seeking on WD90. These results suggest that basal levels of BDNF transmission in the NAc core exert a suppressive effect on cocaine seeking in early withdrawal (WD1), whereas the late elevation of BDNF protein in NAc shell contributes to incubation in late withdrawal (WD90). Finally, BDNF protein levels in the NAc were significantly increased after ampakine treatment, supporting the novel hypothesis that the gradual increase of BDNF levels in NAc accompanying incubation could be caused by increased AMPAR transmission during withdrawal.

Lentiviral vector-mediated dopamine d3 receptor modulation in the rat brain impairs alcohol intake and ethanol-induced conditioned place preference.

BACKGROUND: It has been reported that dopamine D3 receptor (D3R) knockout mice display similar ethanol (EtOH) consumption compared to wild types. In addition, studies with D3R pharmacological targeting were inconclusive. METHODS: In the current study, we used both gain- and loss-of-function approaches to test the effects of central D3R manipulation on voluntary alcohol intake and EtOH-induced conditioned place preference (CPP) in rats. To this aim, we developed a lentiviral-mediated gene transfer approach to examine whether D3R knockdown (LV-siD3R) or overexpression (LV-D3R) in the nucleus accumbens (NAcc) is sufficient to modulate voluntary alcohol consumption and EtOH-CPP. RESULTS: Using the standard 2-bottle choice drinking paradigm and an unbiased CPP procedure, our results indicated that, like the D3R selective antagonist SB-277011-A, LV-siD3R attenuated voluntary alcohol consumption. In contrast, LV-D3R increased EtOH intake with no effect on total fluid intake. Similarly, the D3R agonist 7-OH-DPAT also exacerbated EtOH intake. Interestingly, neither pharmacological nor genetic manipulation of D3R activity affected saccharin and quinine consumption and preference. More importantly, we report that LV-siD3R blocked, whereas LV-D3R exacerbated, EtOH-CPP. CONCLUSIONS: These results support the notion that the D3R plays an important role in alcohol reward in rats and suggest that a key threshold range of D3R levels is associated with impaired alcohol consumption. Taken together, these findings demonstrate that the D3R is an essential component of the molecular pathways underlying the reinforcing properties of alcohol. Thus, medications targeting the D3Rs may be beneficial to tackle EtOH abuse and alcoholism in humans.

Striatal modulation of BDNF expression using microRNA124a-expressing lentiviral vectors impairs ethanol-induced conditioned-place preference and voluntary alcohol consumption.

Alcohol abuse is a major health, economic and social concern in modern societies, but the exact molecular mechanisms underlying ethanol addiction remain elusive. Recent findings show that small non-coding microRNA (miRNA) signaling contributes to complex behavioral disorders including drug addiction. However, the role of miRNAs in ethanol-induced conditioned-place preference (CPP) and voluntary alcohol consumption has not yet been directly addressed. Here, we assessed the expression profile of miR124a in the dorsal striatum of rats upon ethanol intake. The results show that miR124a was downregulated in the dorso-lateral striatum (DLS) following alcohol drinking. Then, we identified brain-derived neurotrophic factor (BDNF) as a direct target of miR124a. In fact, BDNF mRNA was upregulated following ethanol drinking. We used lentiviral vector (LV) gene transfer technology to further address the role of miR124a and its direct target BDNF in ethanol-induced CPP and alcohol consumption. Results reveal that stereotaxic injection of LV-miR124a in the DLS enhances ethanol-induced CPP as well as voluntary alcohol consumption in a two-bottle choice drinking paradigm. Moreover, miR124a-silencer (LV-siR124a) as well as LV-BDNF infusion in the DLS attenuates ethanol-induced CPP as well as voluntary alcohol consumption. Importantly, LV-miR124a, LV-siR124a and LV-BDNF have no effect on saccharin and quinine intake. Our findings indicate that striatal miR124a and BDNF signaling have crucial roles in alcohol consumption and ethanol conditioned reward.

Infusion of brain-derived neurotrophic factor into the ventral tegmental area switches the substrates mediating ethanol motivation.

Recent work has shown that infusion of brain-derived neurotrophic factor (BDNF) into the ventral tegmental area (VTA) promotes a switch in the mechanisms mediating morphine motivation, from a dopamine-independent to a dopamine-dependent pathway. Here we showed that a single infusion of intra-VTA BDNF also promoted a switch in the mechanisms mediating ethanol motivation, from a dopamine-dependent to a dopamine-independent pathway (exactly opposite to that seen with morphine). We suggest that intra-VTA BDNF, via its actions on TrkB receptors, precipitates a switch similar to that which occurs naturally when mice transit from a drug-naive, non-deprived state to a drug-deprived state. The opposite switching of the mechanisms underlying morphine and ethanol motivation by BDNF in previously non-deprived animals is consistent with their proposed actions on VTA GABAA receptors.

Anxiety and ethanol consumption in socially defeated mice; effect of hippocampal serotonin transporter knockdown.

The comorbidity of generalized anxiety disorders (GAD) with alcohol use disorders (AUD) is common and there is an association between the serotonin transporter (SERT) genetic variation and the comorbid conditions of GAD and AUD. However, few mechanistic studies have systematically explored the role of direct SERT manipulation in stress-elicited mood disorders. Therefore, the aim of this study was to determine whether reductions in SERT expression in the hippocampus were sufficient to ameliorate anxiety- and ethanol-related behaviors in socially defeated mice. Following stress exposure, and using stereotaxic surgery, SERT was knocked down using specific shRNA-expressing lentiviral vectors and anxiety-like behavior was evaluated by open-field, elevated plus maze, and marbles burying test. The two-bottle choice (TBC) drinking paradigm was used to assess stress-induced voluntary ethanol intake and preference. Results showed that hippocampal SERT loss-of-function prevented stress-elicited anxiogenic-like effects with no differences in spontaneous locomotor activity. Moreover, in the TBC paradigm, SERT shRNA-injected mice consistently showed a significantly decreased consumption and preference for ethanol when compared to Mock-injected controls. In contrast to ethanol, SERT shRNA-injected mice exhibited similar consumption and preference for saccharin and quinine. Interestingly, we confirmed that SERT hippocampal mRNA expression correlated with measures of anxiety- and ethanol-related behaviors by Pearson correlation analysis. Our findings show that social defeat recruits hippocampal serotoninergic system and that these neuroadaptations mediate the heightened anxiety-like behavior and voluntary alcohol intake observed following stress exposure, suggesting that this system represents a major brain stress element responsible for the negative reinforcement associated with the "dark side" of alcohol addiction.

Effects of chronic psychosocial stress on 'binge-like' sucrose intake in mice.

Binge eating episodes are persistent and are essential features of numerous eating disorders (EDs). Susceptibility to EDs is largely presumed to be associated with early life stress. In fact, converging evidence from preclinical animal studies have implicated stress as a driver of binge eating. Still, literature examination indicates that vulnerability to EDs may depend on factors such as severity, time, and the type of stressor. Therefore, we aimed at exploring the link between chronic psychosocial stress and 'binge-like' sucrose intake in adolescent mice. To this aim, intruders' experimental mice were exposed to the chronic subordinate colony (CSC) housing, in the presence of a resident aggressive mouse for 2 weeks. At the end of the stress period, mice were tested for anxiety-like behavior then assessed for 'binge-like' intake of sucrose using a long-term drinking in the dark (DID) method that successfully replicates binge eating in humans. As expected, and compared to single housed colony controls (SHC), CSC exposure elicited an anxiogenic-like response in the open field (OF) and elevated-plus maze (EPM) tests and reduced weight gain. Most importantly, we report here for the first time, that mice exposed to chronic psychosocial stress displayed a 'binge-like' consumption of sucrose. However, neither quinine (bitter) nor saccharin (sweet) intakes were affected by CSC exposure. Finally, using Pearson's correlation, results showed a strong correlation between anxiety-like behavior parameters and sucrose intake. Overall these findings support the validity of our chronic psychosocial stress to model binge EDs and establish the long-term consequences of stress on 'binge-like' eating in male mice. These data suggest that chronic psychosocial stress is a risk factor for developing anxiety-associated EDs.

Chronic knockdown of the tetraspanin gene CD81 in the mouse nucleus accumbens modulates anxiety and ethanol-related behaviors.

CD81, a member of the tetraspanin family, plays important roles in many physiological processes, such as cell motility, attachment, and entry. Yet, CD81 functions in the brain remain unclear. In this study, we investigated the effects of CD81 knockdown, using lentiviral vectors (LV), on anxiety- and ethanol-related behaviors. For this purpose, mice were stereotaxically injected with CD81 shRNA-expressing LV into the nucleus accumbens (Nacc) and were assessed for anxiety-like behavior using the elevated plus maze (EPM) and open field (OF) tests. Alcohol's sedative effects were studied using loss-of-righting-reflex (LORR) and voluntary ethanol intake was assessed using a two-bottle choice (TBC) procedure. Results showed that mice depleted of CD81 exhibited an anxiolytic-like response in the EPM and OF tests with no effect on locomotor activity. In addition, genetic reduction of CD81 in the Nacc increased mice' sensitivity to alcohol's sedative effects in the LORR test, although plasma alcohol concentrations were unaffected. Interestingly, CD81 loss-of-function-induced anxiolysis was accompanied by a significant decrease in ethanol, but not saccharin nor quinine, intake in the TBC procedure. Finally, and following CD81 mRNA quantification, Pearson's correlations showed a significant positive relationship between accumbal CD81 mRNA with anxiety and ethanol-related behaviors. Our data indicate that CD81 is implicated in the pathogenesis of anxiety and alcoholism. Indeed the targeted disruption of CD81, with the resultant decrease in CD81 mRNA in the Nacc, converted ethanol-"preferring" mice into ethanol "non-preferring" mice. Collectively, these findings demonstrate that future CD81-targeted pharmacotherapies may be beneficial for the treatment of anxiety and alcoholism.

Associative learning and CA3-CA1 synaptic plasticity are impaired in D1R null, Drd1a-/- mice and in hippocampal siRNA silenced Drd1a mice.

Associative learning depends on multiple cortical and subcortical structures, including striatum, hippocampus, and amygdala. Both glutamatergic and dopaminergic neurotransmitter systems have been implicated in learning and memory consolidation. While the role of glutamate is well established, the role of dopamine and its receptors in these processes is less clear. In this study, we used two models of dopamine D(1) receptor (D(1)R, Drd1a) loss, D(1)R knock-out mice (Drd1a(-/-)) and mice with intrahippocampal injections of Drd1a-siRNA (small interfering RNA), to study the role of D(1)R in different models of learning, hippocampal long-term potentiation (LTP) and associated gene expression. D(1)R loss markedly reduced spatial learning, fear learning, and classical conditioning of the eyelid response, as well as the associated activity-dependent synaptic plasticity in the hippocampal CA1-CA3 synapse. These results provide the first experimental demonstration that D(1)R is required for trace eyeblink conditioning and associated changes in synaptic strength in hippocampus of behaving mice. Drd1a-siRNA mice were indistinguishable from Drd1a(-/-) mice in all experiments, indicating that hippocampal knockdown was as effective as global inactivation and that the observed effects are caused by loss of D(1)R and not by indirect developmental effects of Drd1a(-/-). Finally, in vivo LTP and LTP-induced expression of Egr1 in the hippocampus were significantly reduced in Drd1a(-/-) and Drd1a-siRNA, indicating an important role for D(1)R in these processes. Our data reveal a functional relationship between acquisition of associative learning, increase in synaptic strength at the CA3-CA1 synapse, and Egr1 induction in the hippocampus by demonstrating that all three are dramatically impaired when D(1)R is eliminated or reduced.

A decrease in gamma-synuclein expression within the nucleus accumbens increases cocaine intravenous self-administration in the rat.

Except as a marker of cancer progression, gamma-synuclein (GSyn) had received little attention. Recent data showed however that GSyn modulates cocaine-induced locomotor effects, suggesting that it could also play a role in cocaine reinforcing effects. In the rat, siRNAs targeting GSyn expression were injected in the nucleus accumbens and cocaine reinforcing effects were evaluated by means of intravenous self-administration. A dose-response curve was followed by procedures of progressive ratio, extinction, cocaine- and cue-induced reinstatements. Decrease of GSyn expression increased self-administration over a large range of doses. This effect was associated with an increase in cocaine-induced reinstatement. The present data reveal that GSyn exert a specific negative control on cocaine-induced reinforcing and incentive effects.

The brain-specific Neural Zinc Finger transcription factor 2b (NZF-2b/7ZFMyt1) causes suppression of cocaine-induced locomotor activity.

Chronic cocaine induces high expression of the brain-specific Neural-Zinc-Finger transcription factor-2b (NZF-2b/7ZFMyt1), particularly in the mesolimbic dopaminergic pathway, resulting in a 11-fold increase in NZF-2b/7ZFMyt1 expression in the Nucleus Accumbens (NAc). Overexpression of this gene in the NAc with a NZF-2b/7ZFMyt1-expressing lentivirus resulted in >55% decrease in locomotor activity upon chronic cocaine administration, compared to control animals. In contrast knocking-down the gene in the NAc with lentiviruses expressing shRNAs against NZF-2b/7ZFMyt1 induced strong hyperlocomotor activity upon cocaine. Strong inhibition of BDNF is observed upon NZF-2b/7ZFMyt1 expression, concomitant with strong induction of transcription factors REST1 (RE silencing transcription factor-1) and NAC1, probably leading to regulation of gene expression by interaction with histone deacetylases. These changes lead to decreased responsiveness of the animal to the locomotor-activating effects of cocaine, indicating that NZF-2b/7ZFMyt1 expression plays an important role in phenotypic changes induced by the drug.

Social withdrawal and gambling-like profile after lentiviral manipulation of DAT expression in the rat accumbens.

Dysfunction of brain dopamine transporter (DAT) has been associated with sensation seeking and impulse-control disorders. We recently generated a new animal model by stereotaxical inoculation of lentiviral vectors, which allowed localized intra-accumbal delivery of modulators for DAT gene: GFP (green fluorescent protein) control, silencers (Sil), a regulatable enhancer (DAT+), or both (DAT+Sil). Wistar male rats were followed both for socio-emotional profiles and for propensity to seek risky, uncertain rewards. Elevated anxiety and affiliation towards an unfamiliar partner emerged in Sil rats. Interestingly, in DAT+Sil rats (and Sil rats to a lesser extent) levels of playful social interaction were markedly reduced compared to controls. These DAT+Sil rats displayed a marked 'gambling-like' profile (i.e. preference for a large/uncertain over a small/sure reward), which disappeared upon doxycycline-induced switch-off onto DAT enhancer, but consistently reappeared with doxycycline removal. MRI-guided 1H-MRS (at 4.7 T) examinations in vivo (under anaesthesia) revealed changes in the bioenergetic metabolites (phosphocreatine and total creatine) for DAT+Sil rats, indicating a functional up-regulation of dorsal striatum (Str) and conversely a down-regulation of ventral striatum (i.e. nucleus accumbens, NAc). A combined profile of (1) enhanced proneness to gambling and (2) strong social withdrawal is thus associated with altered DAT-induced balance within forebrain dopamine systems. In fact, risk of developing a gambling-prone, social-avoidant psychopathology might be associated with (1) dominant semi-automatic strategies and/or habits, developed within Str circuits, and (2) reduced NAc function, with poorer feedback adjustment on decisions by aversive experiences.

microRNAs miR-124, let-7d and miR-181a regulate cocaine-induced plasticity.

MicroRNAs play key regulatory roles in cellular processes including neurogenesis, synapse development and plasticity in the brain. Psychostimulants induce strong neuroadaptive changes through a surfeit of gene regulatory mechanisms leading to addiction. MicroRNA profiling for identifying miRNAs regulating cocaine-induced, plasticity-related genes revealed significant regulation of a set of miRNAs upon cocaine administration, especially let-7d, miR-181a and the brain-specific miR-124. These miRNAs target many genes involved in cocaine addiction. Precursor and mature miRNA quantification by qRT-PCR showed that miR-124 and let-7d are significantly downregulated, whereas miR-181a is induced in the mesolimbic dopaminergic system under chronic cocaine administration. Results were confirmed by in situ hybridization, Northern blots, FISH analysis and RNase protection assay. Using lentiviral-mediated miRNA expression, we show a significant downregulation of BDNF and D3R both at mRNA and protein levels by miR-124 and let-7d, respectively. Our data suggest that miR-124, let-7d and miR-181a may be involved in a complex feedback loop with cocaine-responsive plasticity genes, highlighting the possibility that some miRNAs are key regulators of the reward circuit and may be implicated in addiction.

Environmental enrichment decreases chronic psychosocial stress-impaired extinction and reinstatement of ethanol conditioned place preference in C57BL/6 male mice.

RATIONALE: During the last few decades, alcohol use disorders (AUD) have reached an epidemic prevalence, yet social influences on alcoholism have not been fully addressed. Several factors can modulate alcohol intake. On one hand, stress can reinforce ethanol-induced behaviors and be an important component in AUD and alcoholism. On the other hand, environmental enrichment (EE) has a neuroprotective role and prevents the development of excessive ethanol intake in rodents. However, studies showing the role of EE in chronic psychosocial stress-impaired ethanol-conditioned rewards are nonexistent. AIM: The purpose of the current study is to explore the potential protective role of EE on extinction and reinstatement of ethanol-conditioned place preference (EtOH-CPP) following chronic psychosocial stress. METHODS: In the first experiment and after the EtOH-CPP test, the mice were subjected to 15 days of chronic stress, then housed in a standard (SE) or enriched environment (EE) while EtOH-CPP extinction was achieved by repeated exposure to the CPP chambers without ethanol injection. In the second experiment and after the EtOH-CPP test, extinction was achieved as described above. Mice were then exposed to chronic stress for 2 weeks before being housed in a SE or EE. EtOH-CPP reinstatement was induced by a single exposure to the conditioning chambers. RESULTS: As expected, stress exposure increased anxiety-like behavior and reduced weight gain. More importantly, we found that EE significantly shortened chronic stress-delayed extinction and decreased the reinstatement of EtOH-CPP. CONCLUSION: These results support the hypothesis that EE reduces the impact of alcohol-associated environmental stimuli, and hence it may be a general intervention for reducing cue-elicited craving and relapse in humans.

Lentiviral-mediated up-regulation of let-7d microRNA decreases alcohol intake through down-regulating the dopamine D3 receptor.

Recent studies have shown that Lethal-7 (let-7) microRNA (miRNA) is involved in a wide range of psychiatric disorders such as anxiety, depression, schizophrenia, and cocaine addiction. However, the exact role of let-7d miRNA in regulating ethanol intake and preference remains to be elucidated. The aim of the present study was to clarify the role of accumbal let-7d in controlling ethanol-related behaviors in adult rats. For this purpose, stereotaxic injections of let-7d-overexpressing lentiviral vectors (LV) were administered bilaterally into the nucleus accumbens (Nacc) of Wistar rats. The ethanol-related behaviors were investigated using the two-bottle choice (TBC) access paradigm, in which the rats had access to 2.5, 5, and 10% ethanol solutions, the grid hanging test (GHT) and ethanol-induced loss-of-righting-reflex (LORR) test. The results showed that intra-accumbally administered let-7d-overexpressing LV significantly decreased ethanol intake and preference without having significant effects on body weight, consumption or preference for tastants (saccharin and quinine) or ethanol metabolism. Furthermore, accumbal let-7d increased resistance to ethanol-induced sedation in the GHT and LORR test. Most importantly, the data showed that the dopamine D3 receptor (D3R) was a candidate target of let-7d In fact, and using real time PCR, let-7d was found to directly target D3R mRNA to decrease its expression. Further analyses proved that D3R expression was negatively correlated with the levels of let-7d and ethanol-related behaviors parameters. Taken together, the data indicating that let-7d impaired ethanol-related behaviors by targeting D3R will open up new exciting possibilities and might provide potential therapeutic evidence for alcoholism.

Dopamine transporter (DAT) knockdown in the nucleus accumbens improves anxiety- and depression-related behaviors in adult mice.

Many epidemiological and clinical studies have demonstrated a strong comorbidity between anxiety and depression, and a number of experimental studies indicates that the dopamine transporter (DAT) is involved in the pathophysiology of anxiety and depression. However, studies using laboratory animals have yielded inconclusive results. The aim of the present study was to examine the effects of DAT manipulation on anxiety- and depression-like behaviors in mice. For this purpose, animals were stereotaxically injected with DAT siRNA-expressing lentiviral vectors (siDAT) in the caudate putamen (CPu) or in the nucleus accumbens (Nacc) and the behavioral outcomes were assessed using the open-field (OF), elevated-plus maze (EPM), light-dark box (LDB), sucrose preference (SPT), novelty suppressed feeding (NSF), and forced-swim (FST) tests. The results showed that in the Nacc, but not in the CPu, siDAT increased the time spent at the center of the arena and decreased the number of fecal boli in the OF test. In the EPM and LDB tests, Nacc siDAT injection increased the entries and time spent on open arms, and increased the time spent in the light side of the box, respectively, suggesting an anxiolytic-like activity. In addition, siDAT, in the Nacc, induced significant antidepressant-like effects, evidenced by increased sucrose preference, shorter latency to feed in the NSF test, and decreased immobility time in the FST. Most importantly, Pearson's test clearly showed significant correlations between DAT mRNA in the Nacc with anxiety and depression parameters. Overall, these results suggest that low DAT levels, in the Nacc, might act as protective factors against anxiety and depression. Therefore, targeting DAT activity might be a very attractive approach to tackle affective disorders.

No effect of sex on ethanol intake and preference after dopamine transporter (DAT) knockdown in adult mice.

RATIONALE: Dopamine levels are controlled in part by transport across the cell membrane by the dopamine transporter (DAT), and recent evidence showed that a polymorphism in the gene encoding DAT is associated with alcoholism. However, research in animal models using DAT knockout mice has yielded conflicting results. OBJECTIVES: The present study was planned to evaluate the effects of DAT knockdown in the nucleus accumbens (Nacc) on voluntary ethanol consumption and preference in male and female C57BL/6J mice. METHODS: For this purpose, animals were stereotaxically injected with DAT siRNA-expressing lentiviral vectors in the Nacc, and using a voluntary, continuous access two-bottle choice model of alcohol, we investigated the importance of accumbal DAT expression in voluntary alcohol intake and preference. We also investigated the effects of DAT knockdown on saccharin and quinine consumption and ethanol metabolism. RESULTS: We show that females consumed more alcohol than males. Interestingly, DAT knockdown in the Nacc significantly decreased alcohol intake and preference in both groups, but no significant sex by group interaction was observed. Also, DAT knockdown did not alter total fluid consumption, saccharin or quinine consumption, or blood ethanol concentrations. Using Pearson correlation, results indicated a strong positive relationship between DAT mRNA expression and ethanol consumption and preference. CONCLUSIONS: Taken together, these data provide further evidence that DAT plays an important role in controlling ethanol intake and that accumbal DAT contributes in the modulation of the reinforcing effects of ethanol. Overall, the results suggest that DAT inhibitors may be valuable in the pharmacotherapy of alcoholism.

The role of gamma-synuclein in cocaine-induced behaviour in rats.

The aim of this study was to investigate the role of gamma-synuclein in the rewarding effects of chronic cocaine administration and its putative interaction with the dopamine transporter (DAT). For this purpose, regulatable lentiviruses driving overexpression of the rat gamma-synuclein or DAT have been prepared, as well as lentiviruses expressing siRNAs, aimed at silencing either DAT or gamma-synuclein mRNA expression. Overexpression of DAT in the nucleus accumbens (NAc) induced a 35% decrease in locomotor activity, which could be abolished when the same animal was fed doxycycline. Furthermore, local inhibition of DAT in the NAc, using lentiviruses expressing siRNAs targeted against DAT, resulted in significant hyperlocomotion activity (72% increase over controls). By contrast, overexpression of gamma-synuclein in the NAc alone had no effect, while local silencing lead to a significant decrease in cocaine-induced locomotor activity (47% decrease compared with controls). Surprisingly, coinjection lentiviruses expressing DAT and gamma-synuclein - leading to overexpression of both proteins in the NAc - resulted in a strong increase in cocaine-induced rat locomotor activity (52% increase compared with controls), which was abolished upon locally silencing these genes, suggesting a synergetic role of both proteins, possibly mediated through a direct interaction.

Role of accumbens BDNF and TrkB in cocaine-induced psychomotor sensitization, conditioned-place preference, and reinstatement in rats.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is involved in the survival and function of midbrain DA neurons. BDNF action is mediated by the TrkB receptor-tyrosine kinase, and both BDNF and TrkB transcripts are widely expressed in the rat mesolimbic pathway, including the nucleus accumbens (NAc) and the ventral tegmentum area (VTA). OBJECTIVE: BDNF was previously shown to be involved in cocaine reward and relapse, as assessed in rat models. The goal of this study is to explore the role of BDNF and TrkB in the rat nucleus accumbens (NAc) in cocaine-induced psychomotor sensitization and in conditioned-place preference acquisition, expression, and reinstatement. MATERIALS AND METHODS: In vivo genetic manipulations of BDNF and TrkB were performed using a lentiviral gene delivery approach to over-express these genes in the NAc and siRNA-based technology to locally knockdown gene expression. Behavioral experiments consisted of locomotor activity monitoring or cocaine-induced conditioned-place preference (CPP). RESULTS: BDNF and/or its receptor TrkB in the NAc enhance drug-induced locomotor activity and induce sensitization in rats. Furthermore, LV-BDNF- and LV-TrkB-treated rats display enhanced cocaine-induced CPP, delayed CPP-extinction upon repeated measurements, and increased CPP reinstatement. In contrast, expression of TrkT1 (truncated form of TrkB, acting as a dominant negative) inhibits these behavioral changes. This inhibition is also observed when rats are fed doxycycline (to block lentivirus-mediated gene expression) or when injected with siRNAs-expressing lentiviruses against TrkB. In addition, we investigate the establishment, maintenance, extinction, and reinstatement of cocaine-induced CPP. We show that BDNF and TrkB-induced CPP takes place during the learning period (conditioning), whereas extinction leads to the loss of CPP. Extinction is delayed when rats are injected LV-BDNF or LV-TrkB, and in turn, priming injections of 2 mg/kg of cocaine reinstates it. CONCLUSIONS: These results demonstrate the crucial function of BDNF-through its receptor TrkB-in the enhancement of locomotor activity, sensitization, conditioned-place preference, CPP-reinstatement, and rewarding effects of cocaine in the mesolimbic dopaminergic pathway.

Effects of urokinase-type plasminogen activator in the acquisition, expression and reinstatement of cocaine-induced conditioned-place preference.

Cocaine and many other psychostimulants strongly induce urokinase-type plasminogen activator (uPA) expression in the mesolimbic dopaminergic pathway, which plays a major role in drug-mediated behavioral plasticity [Bahi A, Boyer F, Gumy C, Kafri T, Dreyer JL. In vivo gene delivery of urokinase-type plasminogen activator with regulatable lentivirus induces behavioral changes in chronic cocaine administration. Eur J Neurosci 2004;20:3473-88; Bahi A, Boyer F, Kafri T, Dreyer JL. Silencing urokinase in the ventral tegmental area in vivo induces changes in cocaine-induced hyperlocomotion. J Neurochem 2006;98:1619-31; Bahi A, Dreyer JL. Overexpression of plasminogen activators in the nucleus accumbens enhances cocaine-, amphetamine- and morphine-induced reward and behavioral sensitization. Genes Brain Behav 2007]. In this study, the role of mesolimbic dopamine (DA) pathways in the development of cocaine reward was examined by conditioned-place preference in rats with bilateral intra-accumbens injections of uPA-expressing lentiviral vectors. We show that overexpression of uPA in the Nac significantly augments cocaine-induced place preference. Furthermore, while this did not affect the ability of preference to be extinguished, reinstatement with a low dose of cocaine produced significantly greater preference to the cocaine-associated context. Once CPP had been established, and the preference extinguished, reinstatement induced by a priming dose of cocaine was facilitated by uPA. Inhibition of this serine protease expression using doxycycline abolished the augmented acquisition produced by overexpression of uPA but not the expression of the cocaine-induced CPP. When uPA is inhibited during the acquisition phase, animals no longer demonstrate place preference for the environment previously paired with cocaine. B428, a specific uPA inhibitor does not affect drug reinstatement after extinction if uPA has been activated during acquisition, a clear indication that uPA is involved in the acquisition phase of conditioned-place preference. Our results suggest that that increased uPA expression with repeated drug exposure produces conditions for enhanced acquisition of cocaine-induced CPP, indicating that cocaine-induced CPP and reinstatement may be dependent on active extracellular uPA.