Simona Perra

Endocannabinoids Mediate Presynaptic Inhibition of Glutamatergic Transmission in Rat Ventral Tegmental Area Dopamine Neurons through Activation of CB1 Receptors

The endogenous cannabinoid system has been shown to play a crucial role in controlling neuronal excitability and synaptic transmission. In this study we investigated the effects of a cannabinoid receptor (CB-R) agonist WIN 55,212-2 (WIN) on excitatory synaptic transmission in the rat ventral tegmental area (VTA). Whole-cell patch clamp recordings were performed from VTA dopamine (DA) neurons in an in vitro slice preparation. WIN reduced both NMDA and AMPA EPSCs, as well as miniature EPSCs (mEPSCs), and increased the paired-pulse ratio, indicating a presynaptic locus of its action. We also found that WIN-induced effects were dose-dependent and mimicked by the CB1-R agonist HU210. Furthermore, two CB1-R antagonists, AM281 and SR141716A, blocked WIN-induced effects, suggesting that WIN modulates excitatory synaptic transmission via activation of CB1-Rs. Our additional finding that both AM281 and SR141716A per se increased NMDA EPSCs suggests that endogenous cannabinoids, released from depolarized postsynaptic neurons, might act retrogradely on presynaptic CB1-Rs to suppress glutamate release. Hence, we report that a type of synaptic modulation, previously termed depolarization-induced suppression of excitation (DSE), is present also in the VTA as a calcium-dependent phenomenon, blocked by both AM281 and SR141716A, and occluded by WIN. Importantly, DSE was partially blocked by the D2DA antagonist eticlopride and enhanced by the D2DA agonist quinpirole without changing the presynaptic cannabinoid sensitivity. These results indicate that the two pathways work in a cooperative manner to release endocannabinoids in the VTA, where they play a role as retrograde messengers for DSE via CB1-Rs.

Prefrontal Cortex Stimulation Induces 2-Arachidonoyl-Glycerol-Mediated Suppression of Excitation in Dopamine Neurons

Endocannabinoids form a novel class of retrograde messengers that modulate short- and long-term synaptic plasticity. Depolarization-induced suppression of excitation (DSE) and inhibition (DSI) are the best characterized transient forms of endocannabinoid-mediated synaptic modulation. Stimulation protocols consisting of long-lasting voltage steps to the postsynaptic cell are routinely used to evoke DSE-DSI. Little is known, however, about more physiological conditions under which these molecules are released in vitro. Moreover, the occurrence in vivo of such forms of endocannabinoid-mediated modulation is still controversial. Here we show that physiologically relevant patterns of synaptic activity induce a transient suppression of excitatory transmission onto dopamine neurons in vitro. Accordingly, in vivo endocannabinoids depress the increase in firing and bursting activity evoked in dopamine neurons by prefrontal cortex stimulation. This phenomenon is selectively mediated by the endocannabinoid 2-arachidonoyl-glycerol (2-AG), which activates presynaptic cannabinoid type 1 receptors. 2-AG synthesis involves activation of metabotropic glutamate receptors and Ca2+ mobilization from intracellular stores. These findings indicate that dopamine neurons release 2-AG to shape afferent activity and ultimately their own firing pattern. This novel endocannabinoid-mediated self-regulatory role of dopamine neurons may bear relevance in the pathogenesis of neuropsychiatric disorders such as schizophrenia and addiction.

Adolescent exposure to cannabinoids induces long-Lasting changes in the response to drugs of abuse of rat midbrain dopamine neurons

BACKGROUND Recent studies have raised concerns about subtle long-lasting neurobiological changes that might be triggered by exposure to Cannabis derivatives, especially in a critical phase of brain maturation, such as puberty. The mesolimbic dopamine (DA) system, involved in the processing of drug-induced reward, is a locus of action of cannabinoids and endocannabinoids. Thus, we compared the effects of repeated cannabinoid administration in adolescent and adult rats on DA neuronal functions and responses to drugs of abuse. METHODS Single-unit extracellular recordings from antidromically identified mesoaccumbens DA neurons and from their target cells in the nucleus accumbens were carried out in urethane-anesthetized rats. Animals were pretreated during adolescence or adulthood, for 3 days, with the cannabinoid agonist WIN55212.2 (WIN) or vehicle and allowed a 2-week interval. RESULTS In cannabinoid-administered rats, DA neurons were significantly less responsive to the stimulating action of WIN, regardless of the age of pretreatment; however, in the adolescent group, but not in the adult, long-lasting cross-tolerance developed to morphine, cocaine, and amphetamine. CONCLUSIONS Our study suggests that an enduring form of neuronal adaptation occurs in DA neurons after subchronic cannabinoid intake at a young age, affecting subsequent responses to drugs of abuse.

Cannabinoids modulate neuronal firing in the rat basolateral amygdala: evidence for CB1- and non-CB1-mediated actions.

Recent evidence indicates that the basolateral amygdala (BLA) may be involved in behavioural effects induced by cannabinoids. High levels of CB1 cannabinoid receptors have been shown in this region, where they modulate excitatory and inhibitory synaptic transmission. However, the neurophysiological effects of these opposing synaptic actions have not been investigated in vivo. To this purpose, single-unit extracellular recordings were performed in urethane anaesthetized rats in order to determine whether exogenously applied cannabinoids influenced the spontaneous or evoked electrical activity of neurons in the BLA. The effects of cannabinoids were found to be dependent on the characteristics of the neurons examined and on the properties of the agents used. We tested and compared two structurally different synthetic cannabinoid receptor agonists, the highly potent HU-210 (0.125-1.0 mg/kg, i.v.) and WIN55212-2 (WIN, 0.125-1.0 mg/kg, i.v.). With a CB1 cannabinoid receptor-dependent mechanism, HU-210 potently inhibited the firing rate of BLA interneurons whereas WIN modulated the discharge rate in a biphasic manner. By contrast, BLA projection neurons, antidromically identified from the shell of the nucleus accumbens, were significantly inhibited by WIN at all doses tested, while HU-210 administration led to less consistent effects, since only 1.0 mg/kg inhibited firing rate in the majority of recorded neurons. Additionally, WIN, but not HU-210, significantly attenuated short-latency spiking activity in BLA projection neurons evoked by electrical stimulation of the medial prefrontal cortex. In these neurons, WIN-induced effects were antagonised by the non-selective cannabinoid receptor antagonist SR141716A and by the vanilloid receptor antagonist capsazepine, but not by the selective CB1 antagonist AM-251. Taken together, our findings indicate that the overall excitability of efferent neurons in the BLA is strongly reduced by WIN in a non-CB1-dependent manner. In this effect, the contribution of a novel cannabinoid-vanilloid-sensitive putative non-CB1 receptors, the existence of which was postulated in recent reports, might play a role.

Cannabinoids modulate spontaneous neuronal activity and evoked inhibition of locus coeruleus noradrenergic neurons.

The noradrenergic pathway arising from the locus coeruleus (LC) is involved in the regulation of attention, arousal, cognitive processes and sleep. These physiological activities are affected by Cannabis exposure − both in humans and laboratory animals. In addition, exogenous cannabinoids, as well as pharmacological and genetic manipulation of the endocannabinoid system, are known to influence emotional states (e.g. anxiety) for which a contributory role of the LC‐noradrenergic system has long been postulated. However, whether cannabinoid administration would affect the LC neuronal activity in vivo is still unknown. To this end, single‐unit extracellular recordings were performed from LC noradrenergic cells in anaesthetized rats. Intravenous injection of both the synthetic cannabinoid agonist, WIN55212‐2, and the main psychoactive principle of Cannabis, Δ9‐tetrahydrocannabinol, dose‐dependently increased the firing rate of LC noradrenergic neurons, with WIN55212‐2 being the most efficacious. Similar results were obtained by the administration of these drugs into a lateral ventricle. Cannabinoid‐induced stimulation of LC noradrenergic neuronal activity was counteracted by SR141716A, a cannabinoid receptor antagonist/reverse agonist, which by itself slightly reduced LC discharge rate. Moreover, WIN55212‐2 suppressed the inhibition of noradrenergic cells produced by stimulation of the major γ‐aminobutyric acid (GABA)ergic afferent to the LC, the nucleus prepositus hypoglossi. Altogether, these findings suggest the involvement of noradrenergic pathways in some consequences of Cannabis intake (e.g. cognitive and attention deficits, anxiety reactions), as well as a role for cannabinoid receptors in basic brain activities sustaining arousal and emotional states.

Involvement of the endogenous cannabinoid system in the effects of alcohol in the mesolimbic reward circuit: electrophysiological evidence in vivo

RationaleSeveral lines of evidence indicate that the endogenous cannabinoid system is involved in the pharmacological and behavioural effects of alcohol. The mesolimbic dopaminergic (DA) system and the nucleus accumbens (NAc) process rewarding properties of drugs of abuse, including alcohol and cannabinoids, whereas endocannabinoids in these regions modulate synaptic function and mediate short- and long-term forms of synaptic plasticity.ObjectivesThe present study was designed to investigate the contribution of the endogenous cannabinoid system in alcohol electrophysiological effects in the mesolimbic reward circuit.MethodsWe utilized extracellular single cell recordings from ventral tegmental area (VTA) DA and NAc neurons in anesthetized rats. DA neurons were antidromically identified as projecting to the shell of NAc, whereas NAc putative medium spiny neurons were identified by their evoked responses to basolateral amygdala (BLA) stimulation. ResultsAlcohol stimulated firing rate of VTA DA neurons and inhibited BLA-evoked NAc neuron spiking responses. The cannabinoid type-1 receptor (CB1) antagonist rimonabant (SR141716A) fully antagonized alcohol effect in both regions. In the NAc, either inhibition of the major catabolic enzyme of the endocannabinoid anandamide, the fatty-acid amyd hydrolase, with URB597 or a pretreatment with the CB1 receptor agonist WIN55212-2 significantly depressed alcohol-induced effects in the NAc.ConclusionsThese results corroborate the notion of the involvement of endocannabinoids and their receptors in the actions of alcohol and highlight the endocannabinoid system as a valuable target in the therapy for alcoholism.

Alcohol Inhibits Spontaneous Activity of Basolateral Amygdala Projection Neurons in the Rat: Involvement of the Endocannabinoid System

BACKGROUND A large body of evidence indicates that the limbic system is involved in the neural processing underlying drug addiction. Among limbic regions, the basolateral nucleus of amygdala (BLA) is implicated in some aspects of the neurobiological mechanisms of drugs of abuse, including alcohol and cannabinoids. It is recently emerging that the endocannabinoid system is involved in many pharmacological and behavioral effects of alcohol. The BLA possesses a very high density of CB1 cannabinoid receptors, and endocannabinoids modulate forms of synaptic plasticity in this region. The aims of our study were first to investigate in vivo the sensitivity of BLA pyramidal neurons to alcohol and second to determine the role of the endocannabinoid system in the acute effects of alcohol. METHODS We utilized extracellular single cell recordings in urethane anesthetized rats from BLA principal neurons, antidromically identified from their projection site in the nucleus accumbens. RESULTS Alcohol (0.25 to 2.0 g/kg i.v.) induced a marked decrease in the spontaneous firing rate of BLA projecting neurons (51.1 +/- 16% of baseline at 0.5 g/kg alcohol, p < 0.0001). The involvement of the endogenous cannabinoid system was investigated by administering the CB1 receptor antagonist SR141716A (rimonabant, SR) (1.0 mg/kg i.v.) before alcohol. SR per se did not significantly affect firing rate of BLA neurons, but it prevented the inhibition produced by alcohol (98 +/- 18% of baseline firing at 0.5 g/kg alcohol, p < 0.01). Then, we studied the actions of alcohol following a chronic treatment with the CB1 agonist WIN55212-2 (WIN). Animals were administered WIN for 6.5 days (2.0 mg/kg, i.p. twice daily) and alcohol dose-response curves were carried out on firing rate of BLA neurons 24 hours following the last injection of the cannabinoid agonist. In WIN-treated animals the inhibitory effect of alcohol was significantly reduced as compared with controls (95 +/- 16% of baseline firing at 0.5 g/kg, p < 0.05). CONCLUSIONS Our results provide evidence of the involvement of the endocannabinoid system in the effects of alcohol on BLA projection neurons. They also further point to the endocannabinoid system as a possible molecular target in the treatment of alcoholism.

γ-Hydroxybutyric acid (GHB) and the mesoaccumbens reward circuit: Evidence for GABAB receptor-mediated effects

γ-Hydroxybutyric acid (GHB) is a short-chain fatty acid naturally occurring in the mammalian brain, which recently emerged as a major recreational drug of abuse. GHB has multiple neuronal mechanisms including activation of both the GABAB receptor, and a distinct GHB-specific receptor. This complex GHB-GABAB receptor interaction is probably responsible for the multifaceted pharmacological, behavioral and toxicological profile of GHB. Drugs of abuse exert remarkably similar effects upon reward-related circuits, in particular the mesolimbic dopaminergic system and the nucleus accumbens (NAc). We used single unit recordings in vivo from urethane-anesthetized rats to characterize the effects of GHB on evoked firing in NAc “shell” neurons and on spontaneous activity of antidromically identified dopamine (DA) cells located in the ventral tegmental area. GHB was studied in comparison with the GABAB receptor agonist baclofen and antagonist (2S)(+)-5,5-dimethyl-2-morpholineacetic acid (SCH50911). Additionally, we utilized a GHB analog, γ-(p-methoxybenzil)-γ-hydroxybutyric acid (NCS-435), devoid of GABAB binding properties, but with high affinity for specific GHB binding sites. In common with other drugs of abuse, GHB depressed firing in NAc neurons evoked by the stimulation of the basolateral amygdala. On DA neurons, GHB exerted heterogeneous effects, which were correlated to the baseline firing rate of the cells but led to a moderate stimulation of the DA system. All GHB actions were mediated by GABAB receptors, since they were blocked by SCH50911 and were not mimicked by NCS-435. Our study indicates that the electrophysiological profile of GHB is close to typical drugs of abuse: both inhibition of NAc neurons and moderate to strong stimulation of DA transmission are distinctive features of diverse classes of abused drugs. Moreover, it is concluded that addictive and rewarding properties of GHB do not necessarily involve a putative high affinity GHB receptor.