Astrocytic Regulation of Endocannabinoid-Dependent Synaptic Plasticity in the Dorsolateral Striatum.

Astrocytes are pivotal for synaptic transmission and may also play a role in the induction and expression of synaptic plasticity, including endocannabinoid-mediated long-term depression (eCB-LTD). In the dorsolateral striatum (DLS), eCB signaling plays a major role in balancing excitation and inhibition and promoting habitual learning. The aim of this study was to outline the role of astrocytes in regulating eCB signaling in the DLS. To this end, we employed electrophysiological slice recordings combined with metabolic, chemogenetic and pharmacological approaches in an attempt to selectively suppress astrocyte function. High-frequency stimulation induced eCB-mediated LTD (HFS-LTD) in brain slices from both male and female rats. The metabolic uncoupler fluorocitrate (FC) reduced the probability of transmitter release and depressed synaptic output in a manner that was independent on cannabinoid 1 receptor (CB1R) activation. Fluorocitrate did not affect the LTD induced by the CB1R agonist WIN55,212-2, but enhanced CB1R-dependent HFS-LTD. Reduced neurotransmission and facilitated HFS-LTD were also observed during chemogenetic manipulation using Gi-coupled DREADDs targeting glial fibrillary acidic protein (GFAP)-expressing cells, during the pharmacological inhibition of connexins using carbenoxolone disodium, or during astrocytic glutamate uptake using TFB-TBOA. While pretreatment with the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonopentanoic acid (APV) failed to prevent synaptic depression induced by FC, it blocked the facilitation of HFS-LTD. While the lack of tools to disentangle astrocytes from neurons is a major limitation of this study, our data collectively support a role for astrocytes in modulating basal neurotransmission and eCB-mediated synaptic plasticity.

Involvement of lateral septum in alcohol's dopamine-elevating effect in the rat.

Drugs of abuse share the ability to increase extracellular dopamine (DA) levels in the mesolimbic DA system. This effect has been linked to positive and reinforcing experiences of drug consumption and is presumed to be of importance for continued use, as well as for the development of dependence and addiction. Previous rat studies from our lab have implicated a neuronal circuitry involving glycine receptors in nucleus accumbens (nAc) and, secondarily, nicotinic acetylcholine receptors in the ventral tegmental area (VTA) in ethanol's (EtOH) DA-elevating effect. The work presented here, performed in male Wistar rats, suggests that the lateral septum (LS), which has previously been associated with different aspects of EtOH-related behaviour, is involved as well. In vivo microdialysis methodology demonstrated that blocking the generation of action potentials in LS using tetrodotoxin prevented a DA increase in nAc after accumbal EtOH perfusion. Retrograde tracing and polymerase chain reaction (PCR) were used to identify and characterize cells projecting to VTA from nAc/LS and from LS to nAc. Based on the PCR results, cells projecting from both LS/nAc to anterior VTA and from LS to nAc were mainly GABAergic neurons expressing glycine receptors, and these cells are presumed to be involved in mediating the DA-elevating effect of EtOH. These results provide further evidence implicating LS in the reinforcing effects of EtOH. Additional studies are needed to investigate LS involvement in EtOH consumption behaviour and its potential role in the development of dependence and addiction.

Astrocytes modulate extracellular neurotransmitter levels and excitatory neurotransmission in dorsolateral striatum via dopamine D2 receptor signaling.

Astrocytes provide structural and metabolic support of neuronal tissue, but may also be involved in shaping synaptic output. To further define the role of striatal astrocytes in modulating neurotransmission we performed in vivo microdialysis and ex vivo slice electrophysiology combined with metabolic, chemogenetic, and pharmacological approaches. Microdialysis recordings revealed that intrastriatal perfusion of the metabolic uncoupler fluorocitrate (FC) produced a robust increase in extracellular glutamate levels, with a parallel and progressive decline in glutamine. In addition, FC significantly increased the microdialysate concentrations of dopamine and taurine, but did not modulate the extracellular levels of glycine or serine. Despite the increase in glutamate levels, ex vivo electrophysiology demonstrated a reduced excitability of striatal neurons in response to FC. The decrease in evoked potentials was accompanied by an increased paired pulse ratio, and a reduced frequency of spontaneous excitatory postsynaptic currents, suggesting that FC depresses striatal output by reducing the probability of transmitter release. The effect by FC was mimicked by chemogenetic inhibition of astrocytes using Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) targeting GFAP, and by the glial glutamate transporter inhibitor TFB-TBOA. Both FC- and TFB-TBOA-mediated synaptic depression were inhibited in brain slices pre-treated with the dopamine D2 receptor antagonist sulpiride, but insensitive to agents acting on presynaptic glutamatergic autoreceptors, NMDA receptors, gap junction coupling, cannabinoid 1 receptors, µ-opioid receptors, P2 receptors or GABAA receptors. In conclusion, our data collectively support a role for astrocytes in modulating striatal neurotransmission and suggest that reduced transmission after astrocytic inhibition involves dopamine.

Differential dopamine release by psychosis-generating and non-psychosis-generating addictive substances in the nucleus accumbens and dorsomedial striatum.

Schizophrenia is associated with three main categories of symptoms; positive, negative and cognitive. Of these, only the positive symptoms respond well to treatment with antipsychotics. Due to the lack of effect of antipsychotics on negative symptoms, it has been suggested that while the positive symptoms are related to a hyperdopaminergic state in associative striatum, the negative symptoms may be a result of a reduced dopamine (DA) activity in the nucleus accumbens (nAc). Drug abuse is common in schizophrenia, supposedly alleviating negative symptomatology. Some, but not all, drugs aggravate psychosis, tentatively due to differential effects on DA activity in striatal regions. Here this hypothesis was tested in rats by using a double-probe microdialysis technique to simultaneously assess DA release in the nAc and associative striatum (dorsomedial striatum; DMS) following administration of the psychosis-generating substances amphetamine (0.5 mg/kg), cocaine (15 mg/kg) and Δ9-tetrahydrocannabinol (THC, 3 mg/kg), and the generally non-psychosis-generating substances ethanol (2.5 g/kg), nicotine (0.36 mg/kg) and morphine (5 mg/kg). The data show that amphetamine and cocaine produce identical DA elevations both in the nAc and DMS, whereas nicotine increases DA in nAc only. Ethanol and morphine both increased DMS DA, but weaker and in a qualitatively different way than in nAc, suggesting that the manner in which DA is increased might be important to the triggering of psychosis. THC elevated DA in neither region, indicating that the pro-psychotic effects of THC are not related to DA release. We conclude that psychosis-generating substances affect striatal DA release differently than non-psychosis-generating substances.

Glycine receptors involved in acamprosate's modulation of accumbal dopamine levels: an in vivo microdialysis study.

BACKGROUND: Glycine receptors (GlyRs) in the nucleus accumbens (nAc) and nicotinic acetylcholine receptors (nAChRs) in the ventral tegmental area (VTA) have been suggested to be involved in the positive reinforcing and dopamine elevating effects of ethanol. Recent studies have also shown that ethanol high-preferring rats substantially decrease their ethanol intake when treated with a glycine transporter 1 inhibitor (ORG 25935). Acamprosate, a drug used for relapse prevention in treatment of alcohol dependence, has also been demonstrated to elevate extracellular dopamine levels in the nAc. However, the underlying mechanism of action of acamprosate is not fully understood. Here we investigated whether acamprosate interferes with a neuronal circuitry that previously has been demonstrated to be involved in the dopamine elevating effects of ethanol and taurine. METHODS: In vivo microdialysis in freely moving rats was used to assess accumbal dopamine levels before and during local (nAc) or systemic administration of acamprosate. RESULTS: Perfusion of 0.5 mM acamprosate in the nAc significantly increased dopamine levels. Pretreatment either with 10 microM strychnine in the nAc or 100 microM mecamylamine in the VTA, completely antagonized the acamprosate-induced elevation of accumbal dopamine levels. Also, systemic acamprosate administration elevated accumbal dopamine output, an effect that was abolished by local (nAc) pretreatment with 10 microM strychnine. CONCLUSIONS: These results suggest that both systemic and local application of acamprosate elevate extracellular dopamine levels in the nAc by activating accumbal GlyRs, and, secondarily, tegmental nAChRs.

The smoking cessation medication varenicline attenuates alcohol and nicotine interactions in the rat mesolimbic dopamine system.

Varenicline was recently approved as an aid for smoking cessation. Patients treated with varenicline have reported a concomitant reduction in their alcohol consumption. This compound has also been demonstrated to reduce alcohol seeking and consumption in alcohol high-preferring rats. Based on the extensive coabuse of nicotine and alcohol, the aim of the present study was to explore whether interactions among varenicline, nicotine, and ethanol in the brain reward system could indicate the use of varenicline also for alcohol dependence. Using the in vivo microdialysis method, we investigated the effects of systemic injections of varenicline on the extracellular accumbal dopamine levels in response to a systemic challenge of ethanol, nicotine, or the combination of nicotine and ethanol in the experimental rat. Acute systemic coadministration of varenicline and ethanol counteracted each others' respective enhancing effect on dopamine levels in the nucleus accumbens. However, after 5 days of varenicline pretreatment, acute combined varenicline and ethanol administration raised dopamine levels to the same extent as either drug alone. Furthermore, after varenicline pretreatment an acute injection of varenicline antagonized the dopamine stimulatory effect of acute nicotine as well as that of systemic coadministration of ethanol and nicotine. In contrast, a pronounced additive dopamine increase was observed when nicotine and ethanol were coadministered in vehicle-pretreated rats. The antismoking agent varenicline exhibits properties with respect to its interaction with ethanol and nicotine in the brain reward system that may be beneficial for treating patients with alcohol dependence with (and possibly also without) concomitant nicotine dependence.

The glycine reuptake inhibitor org 25935 interacts with basal and ethanol-induced dopamine release in rat nucleus accumbens.

BACKGROUND: The mesolimbic dopamine (DA) projection from the ventral tegmental area to nucleus accumbens (nAc), a central part of the reward system, is activated by ethanol (EtOH) and other drugs of abuse. We have previously demonstrated that the glycine receptor in the nAc and its amino acid agonists may be implicated in the DA activation and reinforcing properties of EtOH. We have also reported that the glycine transporter 1 inhibitor, Org 25935, produces a robust and dose-dependent decrease in EtOH consumption in Wistar rats. The present study explores the interaction between EtOH and Org 25935 with respect to DA levels in the rat nAc. METHODS: The effects of Org 25935 (6 mg/kg, i.p.) and/or EtOH (2.5 g/kg, i.p.) on accumbal DA levels were examined by means of in vivo microdialysis (coupled to HPLC-ED) in freely moving male Wistar rats. The effect of Org 25935 on accumbal glycine output was also investigated. RESULTS: Systemic Org 25935 increased DA output in a subpopulation of rats (52% in Experiment 1 and 38% in Experiment 2). In Experiment 2, EtOH produced a significant increase in DA levels in vehicles (35%) and in Org 25935 nonresponders (19%), whereas EtOH did not further increase the DA level in rats responding to Org 25935 (2%). The same dose of Org 25935 increased glycine levels by 87% in nAc. CONCLUSIONS: This study demonstrates that Org 25935, probably via increased glycine levels, (i) counteracts EtOH-induced increases of accumbal DA levels and (ii) increases basal DA levels in a subpopulation of rats. The results are in line with previous findings and it is suggested that the effects observed involve interference with accumbal GlyRs and are related to the alcohol consumption modulating effect of Org 25935.

Nicotinic acetylcholine receptors in the anterior, but not posterior, ventral tegmental area mediate ethanol-induced elevation of accumbal dopamine levels.

Ethanol-induced elevations of accumbal dopamine levels have been linked to the reinforcing properties of the drug. However, it has not yet been demonstrated where the primary point of action of ethanol is in the mesolimbic dopamine system, and there appear to be conflicting findings depending on methodology (electrophysiology, microdialysis, or intracranial self-administration). We have suggested that ethanol acts in the nucleus accumbens (nAc), where it activates a neuronal loop involving ventral tegmental nicotinic acetylcholine receptors (nAChRs) to elevate dopamine levels in the nAc. Application of ethanol in the nAc results in elevated dopamine levels in the same brain region, whereas administration in the anterior ventral tegmental area (VTA) fails to influence dopamine output. In the present study, we were able to repeat these findings. In addition, application of ethanol in the posterior VTA also failed to influence nAc dopamine levels. Perfusion of the nAChR antagonist mecamylamine in the anterior VTA completely blocked the elevation of accumbal dopamine levels observed after ethanol perfusion in nAc, whereas mecamylamine in the posterior VTA had no effect. To detect a possible influence on phasic dopamine release, the dopamine transporter inhibitor nomifensine was included in the accumbal perfusate. In addition, under these conditions, ethanol in the anterior or posterior VTA failed to influence dopamine release in the nAc. These results support previous suggestions of distinct functions of the anterior and posterior VTA and give further evidence for our hypothesis of a nAc-anterior VTA-nAc neuronal circuitry involved in the dopamine-activating effects of ethanol.

Nicotinic acetylcholine receptors in the ventral tegmental area mediate the dopamine activating and reinforcing properties of ethanol cues.

RATIONALE: Cues associated with alcohol can elicit craving, support drug-seeking and precipitate relapse. OBJECTIVES: We investigated the possible involvement of nicotinic acetylcholine receptors (nAChRs) in the ventral tegmental area (VTA) in the conditioned reinforcing properties of ethanol-associated stimuli in the rat. MATERIALS AND METHODS: First, using in vivo microdialysis, we analyzed the effect of VTA perfusion of the nonselective nAChR antagonist mecamylamine (MEC) or the selective alpha4beta2* nAChR antagonist dihydro-beta-erythroidine (DHbetaE) on the nucleus accumbens (nAc) dopaminergic response to the presentation of an ethanol-associated conditioned stimulus (CS). Second, rats were trained to associate a tone+light CS with the presentation of 10% ethanol and were subsequently tested on the acquisition of a new instrumental response with conditioned reinforcement (CR) after local VTA infusion of MEC, DHbetaE, or alpha-Conotoxin MII (alpha-CtxMII, a selective alpha3beta2* and alpha6* nAChR antagonist). RESULTS: The ethanol-associated CS elevated nAc dopamine, an effect that was blocked by VTA perfusion of MEC but not DHbetaE. Systemic administration of MEC or local VTA infusion of MEC or alpha-CtxMII selectively blocked ethanol-associated CR, whereas systemic DHbetaE had no effect. CONCLUSIONS: We hypothesize a novel mechanism by which alcohol-associated cues promote drug-seeking behavior via activation of dopamine-stimulating alpha-CtxMII-sensitive nAChRs in the VTA. Pharmacological manipulations of selective nAChRs may thus be possible treatment strategies to prevent cue-induced relapse.

Characterization of ethanol-induced dopamine elevation in the rat nucleus accumbens.

Ethanol-induced accumbal dopamine elevations have been linked to ethanol consumption. It is unclear, however, where along the mesolimbic dopamine system this effect is initiated and why the ethanol-induced dopamine elevations are transient, returning to pre-drug baseline before brain and blood ethanol levels decline. Using in vivo microdialysis, Experiment 1 investigated the effect of local ethanol application in the nucleus accumbens, the ventral tegmental area and the nucleus accumbens+the ventral tegmental area, on accumbal dopamine. Experiment 2 examined whether the rapid withdrawal of dopamine response to ethanol involves activation of GABA(A)-receptors, by analyzing the effect of accumbal co-perfusion of picrotoxin and ethanol. In Experiment 1, ethanol perfusion into the ventral tegmental area alone did not affect accumbal dopamine. Ethanol co-perfusion of one of the tested doses into the ventral tegmental+the nucleus accumbens produced higher dopamine levels than ethanol perfusion into the nucleus accumbens alone during 120-160 min following perfusion onset. In Experiment 2, accumbal ethanol perfusion caused a transient increase in nucleus accumbens dopamine. Co-perfusion of ethanol and picrotoxin produced a sustained dopamine elevation. These data support the hypothesis that the primary effect of ethanol on accumbal dopamine is in the nucleus accumbens, but that a secondary effect of nucleus accumbens ethanol perfusion, such as release of acetylcholine in the ventral tegmental area, enables ethanol to act as a nicotinic acetylcholine receptor co-agonist in this area. Moreover, recruitment of GABA(A)-receptor activity appears responsible for the second, declining phase with respect to dopamine levels following ethanol administration.

Ethanol-induced dopamine elevation in the rat--modulatory effects by subchronic treatment with nicotinic drugs.

Chronic nicotine administration is associated with increased ethanol consumption in laboratory animals and in humans. Some smokers report less sedation during acute ethanol intoxication after nicotine administration and the sedative effects from ethanol are mediated by inhibitory GABA(A)-receptors. In a series of in vivo microdialysis experiments we investigated whether subchronic pre-treatment with nicotinic drugs known to enhance ethanol consumption in the rat (nicotine or the peripheral nicotinic antagonist hexamethonium) could modulate the alterations in extracellular dopamine observed in response to administration of ethanol or the sedative GABA(A)-agonist diazepam. In the nucleus accumbens and the dorsal striatum, systemic and/or local ethanol administration resulted in transient increases in extracellular dopamine levels that returned to baseline before the local levels of ethanol started to decline. In hexamethonium pre-treated rats, however, the nucleus accumbens dopamine levels were time-locked to the ethanol levels in the same area after systemic or local ethanol administration. Perfusion of diazepam into the nucleus accumbens produced a significant reduction in nucleus accumbens dopamine in controls. Prior subchronic treatment with nicotine or hexamethonium abolished this effect. The present results suggest that subchronic treatment with the nicotinic acetylcholine receptor antagonist hexamethonium reduces a GABA(A)-R mediated counteraction of the nucleus accumbens dopamine response to ethanol. Additionally, we demonstrate that modulation of nicotinic receptors may reduce the sensitivity of GABA(A) receptors to benzodiazepines. These phenomena may offer a novel explanation to why nicotine and alcohol are often co-abused.

Nicotinic acetylcholine receptors are required for the conditioned reinforcing properties of sucrose-associated cues.

RATIONALE: We recently demonstrated that blocking specific nicotinic acetylcholine receptors (nAChRs) abolishes the conditioned reinforcing properties of ethanol-associated cues in rat, suggesting nAChRs as promising pharmacological targets for prevention of cue-induced relapse. OBJECTIVES: The present study investigated the involvement of nAChR subtypes in the conditioned reinforcing properties of stimuli associated with a natural reward (sucrose). METHODS: Water-deprived rats were trained to associate a tone + light stimulus (CS) with the presentation of a 0.1 M sucrose solution for 10 consecutive days. On the subsequent day, the animals were tested on the stringent acquisition of a new instrumental response with conditioned reinforcement, following a systemic injection of the nonselective nAChR antagonist mecamylamine (MEC) or the selective α7 and α6/α3ß2ß3* nAChR antagonist methyllycaconitine (MLA). At testing, the rats were presented with two novel levers. Responding on the lever assigned as active (CR lever) resulted in a presentation of the CS alone, while pressing the inactive lever (NCR lever) had no programmed consequences. RESULTS: Control animals pressed the CR lever significantly more than the NCR lever, demonstrating that the CR had acquired conditioned reinforcing properties. Systemic MEC as well as MLA reduced the CR lever responses to the same level as for the NCR lever. CONCLUSIONS: These results demonstrate a role for the α7 and/or α6/α3ß2ß3* nAChRs in conditioned reinforcement to a natural reward and suggest neuronal nAChRs as common mediators of the impact of cues on incentive processes.

Taurine elevates dopamine levels in the rat nucleus accumbens; antagonism by strychnine.

The mesolimbic dopamine (DA) system, projecting from the ventral tegmental area (VTA) to the nucleus accumbens (nAcc), is involved in reward-related behaviours and addictive processes, such as alcoholism and drug addiction. It was recently suggested that strychnine-sensitive glycine receptors (GlyR) in the nAcc regulate both basal and ethanol-induced mesolimbic DA activity via a neuronal loop involving endogenous activation of nicotinic acetylcholine receptors (nAChR) in the VTA. However, as the nAcc appears to contain few glycine-immunoreactive cell bodies or fibres, the question as to what may be the endogenous ligand for GlyRs in this brain region remains open. Here we have investigated whether the amino acid taurine could serve this purpose using in vivo microdialysis in awake, freely moving male Wistar rats. Local perfusion of taurine (1, 10 or 100 mm in the perfusate) increased DA levels in the nAcc. The taurine (10 mm)-induced DA increase was, similarly to that previously observed after ethanol, completely blocked by (i) perfusion of the competitive GlyR antagonist strychnine in the nAcc, (ii) perfusion of the nAChR antagonist mecamylamine (100 microm) in the VTA, and (iii) systemic administration of the acetylcholine-depleting drug vesamicol (0.4 mg/kg, i.p). The present results suggest that taurine may be an endogenous ligand for GlyRs in the nAcc and that the taurine-induced elevation of DA levels in this area, similarly to that observed after local ethanol, is mediated via a neuronal loop involving endogenous activation of nAChRs in the VTA.

Involvement of accumbal glycine receptors in the regulation of voluntary ethanol intake in the rat.

BACKGROUND: Extracellular dopamine (DA) levels in the nucleus accumbens (nAc) increase after ethanol (EtOH) administration in the rat, a response that may be involved in the positive reinforcing effects of EtOH. The mechanisms underlying this DA activation and how they relate to EtOH reinforcement remain to be elucidated, but recent data indicate that glycine receptors (GlyRs) in the nAc may be involved. Here this hypothesis was further challenged by examining the influence of bilateral accumbal application of glycine (a GlyR agonist), strychnine (a GlyR competitive antagonist), or Ringer on EtOH intake and preference, as well as on the concomitant DA output in the nAc, in EtOH high-preferring male Wistar rats. METHODS: EtOH high-preferring male Wistar rats [EtOH preference >60% during continuous access to a bottle of EtOH (6% v/v) and a bottle of water] were limited to drink 1 hr/day (limited access drinking). Thereafter, the animals were equipped bilaterally with microdialysis probes aimed at the mAc, and were subjected to in vivo microdialysis (coupled to high-pressure liquid chromatography with electrochemical detection) and reversed microdialysis (for drug application) during two experimental days (balanced study), during which the animals were allowed a choice between EtOH and water. RESULTS: The EtOH consumption in rats that were perfused with Ringer in the nAc was approximately 0.9 g/kg/hr and associated with a significant increase in extracellular accumbal DA levels. In a subpopulation of rats, bilateral accumbal glycine (100 microM) perfusion produced a significant increase in accumbal DA output and a decrease in EtOH preference and intake. In these glycine responders, the EtOH consumed (approximately 0.7 g/kg/hr) did not produce a further increase of DA levels. In other rats, bilateral glycine perfusion did not change the accumbal DA output, and voluntary EtOH intake was not altered. In these glycine nonresponders, EtOH tended to increase accumbal DA levels. Bilateral accumbal strychnine (20 microM) perfusion significantly decreased DA output in the nAc, and the DA levels remained decreased despite a statistically significant increase of EtOH intake. Finally, the increase in accumbal DA levels observed after EtOH consumption in Ringer-treated rats was significantly larger in glycine responders than in glycine nonresponders. CONCLUSIONS: The present findings suggest that glycine and strychnine alter extracellular DA levels in the nAc, probably via GlyR stimulation and blockade, respectively, and concomitantly glycine and strychnine reciprocally alter also EtOH consumption in EtOH high-preferring male Wistar rats. The possibility of developing selective GlyR agonists and/or antagonists should be explored. Such agents could prove of value in the treatment of alcoholism.