Heterogeneous Habenular Neuronal Ensembles during Selection of Defensive Behaviors.

Optimal selection of threat-driven defensive behaviors is paramount to an animal's survival. The lateral habenula (LHb) is a key neuronal hub coordinating behavioral responses to aversive stimuli. Yet, how individual LHb neurons represent defensive behaviors in response to threats remains unknown. Here, we show that in mice, a visual threat promotes distinct defensive behaviors, namely runaway (escape) and action-locking (immobile-like). Fiber photometry of bulk LHb neuronal activity in behaving animals reveals an increase and a decrease in calcium signal time-locked with runaway and action-locking, respectively. Imaging single-cell calcium dynamics across distinct threat-driven behaviors identify independently active LHb neuronal clusters. These clusters participate during specific time epochs of defensive behaviors. Decoding analysis of this neuronal activity reveals that some LHb clusters either predict the upcoming selection of the defensive action or represent the selected action. Thus, heterogeneous neuronal clusters in LHb predict or reflect the selection of distinct threat-driven defensive behaviors.

Rationale for an adjunctive therapy with fenofibrate in pharmacoresistant nocturnal frontal lobe epilepsy.

OBJECTIVE: Nocturnal frontal lobe epilepsy (NFLE) is an idiopathic partial epilepsy with a family history in about 25% of cases, with autosomal dominant inheritance (autosomal dominant NFLE [ADNFLE]). Traditional antiepileptic drugs are effective in about 55% of patients, whereas the rest remains refractory. One of the key pathogenetic mechanisms is a gain of function of neuronal nicotinic acetylcholine receptors (nAChRs) containing the mutated α4 or ß2 subunits. Fenofibrate, a common lipid-regulating drug, is an agonist at peroxisome proliferator-activated receptor alpha (PPARα) that is a ligand-activated transcription factor, which negatively modulates the function of ß2-containing nAChR. To test clinical efficacy of adjunctive therapy with fenofibrate in pharmacoresistant ADNFLE\NFLE patients, we first demonstrated the effectiveness of fenofibrate in a mutated mouse model displaying both disease genotype and phenotype. METHODS: We first tested the efficacy of fenofibrate in transgenic mice carrying the mutation in the α4-nAChR subunit (Chrna4S252F) homologous to that found in humans. Subsequently, an add-on protocol was implemented in a clinical setting and fenofibrate was administered to pharmacoresistant NFLE patients. RESULTS: Here, we show that a chronic fenofibrate diet markedly reduced the frequency of large inhibitory postsynaptic currents (IPSCs) recorded from cortical pyramidal neurons in Chrna4S252F mice, and prevented nicotine-induced increase of IPSC frequency. Moreover, fenofibrate abolished differences between genotypes in the frequency of sleep-related movements observed under basal conditions. Patients affected by NFLE, nonresponders to traditional therapy, by means of adjunctive therapy with fenofibrate displayed a reduction of seizure frequency. Furthermore, digital video-polysomnographic recordings acquired in NFLE subjects after 6 months of adjunctive fenofibrate substantiated the significant effects on control of motor-behavioral seizures. SIGNIFICANCE: Our preclinical and clinical studies suggest PPARα as a novel disease-modifying target for antiepileptic drugs due to its ability to regulate dysfunctional nAChRs.

The neurosteroidogenic enzyme 5α-reductase modulates the role of D1 dopamine receptors in rat sensorimotor gating.

Neurosteroids exert diverse modulatory actions on dopamine neurotransmission and signaling. We previously documented that the enzyme 5α-reductase, which catalyzes the main rate-limiting step in neurosteroid synthesis, is required for the behavioral responses of Sprague-Dawley rats to non-selective dopaminergic agonists, such as the D1-D2 receptor agonist apomorphine. Specifically, systemic and intra-accumbal administrations of the 5α-reductase inhibitor finasteride countered apomorphine-induced deficits of sensorimotor gating, as measured by the prepulse inhibition (PPI) of the startle reflex; the classes of dopamine receptors involved in these effects, however, remain unknown. Prior rodent studies have revealed that the contributions of dopamine receptors to PPI regulation vary depending on the genetic background; thus, we analyzed the effect of finasteride on the PPI deficits induced by selective dopamine receptor agonists in Long-Evans (a strain exhibiting PPI deficits in response to both D1 and D2 receptor agonists) and Sprague-Dawley rats (which display PPI reductions following treatment with D2, and D3, but not D1 receptor agonists). In Long-Evans rats, finasteride opposed the PPI deficits induced by activation of D1, but not D2 receptors; conversely, in Sprague-Dawley rats, finasteride prevented the reductions in %PPI and accumbal dopamine extracellular levels caused by selective stimulation of D3, but not D2 receptors; however, the effects on %PPI were not confirmed by analyses on absolute PPI values. Our findings suggest that 5α-reductase modulates the effects of D1, but not D2 receptor agonists on sensorimotor gating. These data may help elucidate the role of neurosteroids in neuropsychiatric disorders featuring PPI deficits, including schizophrenia and Tourette syndrome.

Positive allosteric modulation of GABAB receptors ameliorates sensorimotor gating in rodent models.

BACKGROUND: Converging evidence points to the involvement of γ-amino-butyric acid B receptors (GABABRs) in the regulation of information processing. We previously showed that GABABR agonists exhibit antipsychotic-like properties in rodent models of sensorimotor gating deficits, as measured by the prepulse inhibition (PPI) of the acoustic startle reflex. The therapeutic potential of these agents, however, is limited by their neuromuscular side effects; thus, in this study, we analyzed whether rac-BHFF, a potent GABABR-positive allosteric modulator (PAM), could counter spontaneous and pharmacologically induced PPI deficits across various rodent models. METHODS: We tested the antipsychotic effects of rac-BHFF on the PPI deficits caused by the N-methyl-D-aspartate glutamate receptor antagonist dizocilpine, in Sprague-Dawley rats and C57BL/6 mice. Furthermore, we verified whether rac-BHFF ameliorated the spontaneous PPI impairments in DBA/2J mice. RESULTS: rac-BHFF dose-dependently countered the PPI deficits across all three models, in a fashion akin to the GABABR agonist baclofen and the atypical antipsychotic clozapine; in contrast with these compounds, however, rac-BHFF did not affect startle magnitude. CONCLUSIONS: The present data further support the implication of GABABRs in the modulation of sensorimotor gating and point to their PAMs as a novel promising tool for antipsychotic treatment, with fewer side effects than GABABR agonists.

Inhibition of 17α-hydroxylase/C17,20 lyase reduces gating deficits consequent to dopaminergic activation.

Cogent evidence points to the involvement of neurosteroids in the regulation of dopamine (DA) neurotransmission and signaling, yet the neurobiological bases of this link remain poorly understood. We previously showed that inhibition of 5α-reductase (5αR), a key neurosteroidogenic enzyme, attenuates the sensorimotor gating deficits induced by DA receptor activation, as measured by the prepulse inhibition (PPI) of the acoustic startle reflex. To extend these findings, the present study was aimed at the assessment of the role of other key neurosteroidogenic enzymes in PPI, such as 17α-hydroxylase/C17,20 lyase (CYP17A1), 3α- and 3ß-hydroxysteroid dehydrogenase (HSD), in Sprague-Dawley rats. The PPI deficits induced by the DAergic non-selective agonist apomorphine (APO, 0.25mg/kg, SC) were dose-dependently attenuated by the selective CYP17A1 inhibitor abiraterone (ABI, 10-50mg/kg, IP) in a fashion akin to that of the 5αR inhibitor finasteride (FIN, 100mg/kg, IP). These systemic effects were reproduced by intracerebroventricular injection of ABI (1 µg/1 µl), suggesting the involvement of brain CYP17A1 in PPI regulation. Conversely, the PPI disruption induced by APO was not significantly affected by the 3α- and 3ß-HSD inhibitors indomethacin and trilostane. Given that CYP17A1 catalyzes androgen synthesis, we also tested the impact on PPI of the androgen receptor (AR) antagonist flutamide (10mg/kg, IP). However, this agent failed to reverse APO-induced PPI deficits; furthermore, AR endogenous ligands testosterone and dihydrotestosterone failed to disrupt PPI. Collectively, these data highlight CYP17A1 as a novel target for antipsychotic-like action, and suggest that the DAergic regulation of PPI is modulated by androgenic neurosteroids, through AR-unrelated mechanisms.

PPAR-alpha agonists as novel antiepileptic drugs: preclinical findings.

Nicotinic acetylcholine receptors (nAChRs) are involved in seizure mechanisms. Hence, nocturnal frontal lobe epilepsy was the first idiopathic epilepsy linked with specific mutations in α4 or ß2 nAChR subunit genes. These mutations confer gain of function to nAChRs by increasing sensitivity toward acetylcholine. Consistently, nicotine elicits seizures through nAChRs and mimics the excessive nAChR activation observed in animal models of the disease. Treatments aimed at reducing nicotinic inputs are sought as therapies for epilepsies where these receptors contribute to neuronal excitation and synchronization. Previous studies demonstrated that peroxisome proliferator-activated receptors-α (PPARα), nuclear receptor transcription factors, suppress nicotine-induced behavioral and electrophysiological effects by modulating nAChRs containing ß2 subunits. On these bases, we tested whether PPARα agonists were protective against nicotine-induced seizures. To this aim we utilized behavioral and electroencephalographic (EEG) experiments in C57BL/J6 mice and in vitro patch clamp recordings from mice and rats. Convulsive doses of nicotine evoked severe seizures and bursts of spike-waves discharges in ∼100% of mice. A single dose of the synthetic PPARα agonist WY14643 (WY, 80 mg/kg, i.p.) or chronic administration of fenofibrate, clinically available for lipid metabolism disorders, in the diet (0.2%) for 14 days significantly reduced or abolished behavioral and EEG expressions of nicotine-induced seizures. Acute WY effects were reverted by the PPARα antagonist MK886 (3 mg/kg, i.p.). Since neocortical networks are crucial in the generation of ictal activity and synchrony, we performed patch clamp recordings of spontaneous inhibitory postsynaptic currents (sIPSCs) from frontal cortex layer II/III pyramidal neurons. We found that both acute and chronic treatment with PPARα agonists abolished nicotine-induced sIPSC increases. PPARα within the CNS are key regulators of neuronal activity through modulation of nAChRs. These effects might be therapeutically exploited for idiopathic or genetically determined forms of epilepsy where nAChRs play a major role.

Inhibition of 5α-reductase attenuates behavioral effects of D1-, but not D2-like receptor agonists in C57BL/6 mice.

Converging lines of evidence point to the involvement of neurosteroids in the regulation of dopamine (DA) neurotransmission and signaling, yet the neurobiological bases of this link remain poorly understood. We previously showed that inhibition of steroid 5α-reductase (5αR), the key rate-limiting enzyme in neurosteroidogenesis, attenuates the behavioral effects of non-selective DA receptor agonists in rats, including stereotyped responses and sensorimotor gating deficits, as measured by the prepulse inhibition (PPI) of the acoustic startle reflex. Since previous findings suggested that the role of DA D(1)- and D(2)-like receptor families in behavioral regulation may exhibit broad interspecies and interstrain variations, we assessed the impact of 5αR blockade on the behavioral effects of DAergic agonists in C57BL/6 mice. The prototypical 5αR inhibitor finasteride (FIN; 25-50 mg/kg, intraperitoneally, IP) dose-dependently countered the PPI deficits and the enhancement of rearing responses induced by the full D(1)-like receptor agonist SKF-82958 (0.3 mg/kg, IP); however, FIN did not significantly affect the hyperlocomotive and startle-attenuating effects of SKF-82958. Whereas the D(2)-like receptor agonist quinpirole (QUIN; 0.5 mg/kg, IP) did not induce significant changes in PPI, the combination of this agent and FIN surprisingly produced marked gating and startle deficits. In contrast with previous data on rats, FIN did not affect the reductions of startle reflex and PPI produced by the non-selective DAergic agonist apomorphine (APO; 0.5 mg/kg, IP). These findings collectively indicate that, in C57BL/6 mice, 5αR differentially modulates the effects of D(1)- and D(2)-like receptor agonists in behavioral regulation.

Effect of acute administration of Pistacia lentiscus L. essential oil on rat cerebral cortex following transient bilateral common carotid artery occlusion.

BACKGROUND: Ischemia/reperfusion leads to inflammation and oxidative stress which damages membrane highly polyunsaturated fatty acids (HPUFAs) and eventually induces neuronal death. This study evaluates the effect of the administration of Pistacia lentiscus L. essential oil (E.O.), a mixture of terpenes and sesquiterpenes, on modifications of fatty acid profile and endocannabinoid (eCB) congener concentrations induced by transient bilateral common carotid artery occlusion (BCCAO) in the rat frontal cortex and plasma. METHODS: Adult Wistar rats underwent BCCAO for 20 min followed by 30 min reperfusion (BCCAO/R). 6 hours before surgery, rats, randomly assigned to four groups, were gavaged either with E.O. (200 mg/0.45 ml of sunflower oil as vehicle) or with the vehicle alone. RESULTS: BCCAO/R triggered in frontal cortex a decrease of docosahexaenoic acid (DHA), the membrane highly polyunsaturated fatty acid most susceptible to oxidation. Pre-treatment with E.O. prevented this change and led further to decreased levels of the enzyme cyclooxygenase-2 (COX-2), as assessed by Western Blot. In plasma, only after BCCAO/R, E.O. administration increased both the ratio of DHA-to-its precursor, eicosapentaenoic acid (EPA), and levels of palmytoylethanolamide (PEA) and oleoylethanolamide (OEA). CONCLUSIONS: Acute treatment with E.O. before BCCAO/R elicits changes both in the frontal cortex, where the BCCAO/R-induced decrease of DHA is apparently prevented and COX-2 expression decreases, and in plasma, where PEA and OEA levels and DHA biosynthesis increase. It is suggested that the increase of PEA and OEA plasma levels may induce DHA biosynthesis via peroxisome proliferator-activated receptor (PPAR) alpha activation, protecting brain tissue from ischemia/reperfusion injury.

Inhibition of 5α-reductase in the nucleus accumbens counters sensorimotor gating deficits induced by dopaminergic activation.

Cogent evidence highlights a key role of neurosteroids and androgens in schizophrenia. We recently reported that inhibition of steroid 5α-reductase (5αR), the rate-limiting enzyme in neurosteroid synthesis and androgen metabolism, elicits antipsychotic-like effects in humans and animal models, without inducing extrapyramidal side effects. To elucidate the anatomical substrates mediating these effects, we investigated the contribution of peripheral and neural structures to the behavioral effects of the 5αR inhibitor finasteride (FIN) on the prepulse inhibition (PPI) of the acoustic startle reflex (ASR), a rat paradigm that dependably simulates the sensorimotor gating impairments observed in schizophrenia and other neuropsychiatric disorders. The potential effect of drug-induced ASR modifications on PPI was excluded by measuring this index both as percent (%PPI) and absolute values (ΔPPI). In both orchidectomized and sham-operated rats, FIN prevented the %PPI deficits induced by the dopamine (DA) receptor agonists apomorphine (APO, 0.25mg/kg, SC) and d-amphetamine (AMPH, 2.5mg/kg, SC), although the latter effect was not corroborated by ΔPPI analysis. Conversely, APO-induced PPI deficits were countered by FIN infusions in the brain ventricles (10µg/1µl) and in the nucleus accumbens (NAc) shell and core (0.5µg/0.5µl/side). No significant PPI-ameliorating effect was observed following FIN injections in other brain regions, including dorsal caudate, basolateral amygdala, ventral hippocampus and medial prefrontal cortex, although a statistical trend was observed for the latter region. The efflux of DA in NAc was increased by systemic, but not intracerebral FIN administration. Taken together, these findings suggest that the role of 5αR in gating regulation is based on post-synaptic mechanisms in the NAc, and is not directly related to alterations in DA efflux in this region.

Effects of fatty acid amide hydrolase inhibition on neuronal responses to nicotine, cocaine and morphine in the nucleus accumbens shell and ventral tegmental area: involvement of PPAR-alpha nuclear receptors.

The endocannabinoid system regulates neurotransmission in brain regions relevant to neurobiological and behavioral actions of addicting drugs. We recently demonstrated that inhibition by URB597 of fatty acid amide hydrolase (FAAH), the main enzyme that degrades the endogenous cannabinoid N-acylethanolamine (NAE) anandamide and the endogenous non-cannabinoid NAEs oleoylethanolamide and palmitoylethanolamide, blocks nicotine-induced excitation of ventral tegmental area (VTA) dopamine (DA) neurons and DA release in the shell of the nucleus accumbens (ShNAc), as well as nicotine-induced drug self-administration, conditioned place preference and relapse in rats. Here, we studied whether effects of FAAH inhibition on nicotine-induced changes in activity of VTA DA neurons were specific for nicotine or extended to two drugs of abuse acting through different mechanisms, cocaine and morphine. We also evaluated whether FAAH inhibition affects nicotine-, cocaine- or morphine-induced actions in the ShNAc. Experiments involved single-unit electrophysiological recordings from DA neurons in the VTA and medium spiny neurons in the ShNAc in anesthetized rats. We found that URB597 blocked effects of nicotine and cocaine in the ShNAc through activation of both surface cannabinoid CB1-receptors and alpha-type peroxisome proliferator-activated nuclear receptor. URB597 did not alter the effects of either cocaine or morphine on VTA DA neurons. These results show that the blockade of nicotine-induced excitation of VTA DA neurons, which we previously described, is selective for nicotine and indicate novel mechanisms recruited to regulate the effects of addicting drugs within the ShNAc of the brain reward system.

GABAB receptor activation exacerbates spontaneous spike-and-wave discharges in DBA/2J mice.

Rich evidence has highlighted that stimulation of gamma-amino-butyric acid (GABA)(B) receptors increases the occurrence of spike-and-wave discharges (SWDs), the electroencephalographic (EEG) landmark of absence epilepsy (AE). Recent findings suggest that the outcomes of GABA(B) activation in vivo are contingent on the chemical characteristics of the agonist. In particular, the endogenous ligand gamma-hydroxybutyrate (GHB) and its precursor gamma-butyro-lactone (GBL) have been shown to elicit different effects than the prototypical GABA(B) agonist baclofen. In view of these premises, the present study was aimed at the characterization of the effects of baclofen (0.5-10 mg/kg, i.p.) and GBL (5-100 mg/kg, i.p.) on the spontaneous SWDs and locomotor activity of DBA/2J mice. While both baclofen and GBL dose-dependently increased SWDs episodes, high doses of the latter (100 mg/kg, i.p.) reduced the occurrence of these phenomena and increased the number of isolated spikes. Interestingly, both compounds elicited a dose-dependent reduction of locomotor activity, in comparison with their vehicle-treated controls. The GABA(B) selective antagonist, SCH50911 (50 mg/kg, i.p.), reversed the changes in SWD occurrence and locomotion induced by baclofen and GBL, but failed to elicit intrinsic effects on either paradigm. These results indicate that GABA(B) receptor signaling might exert differential effects on SWDs in DBA/2J mice.

Electrophysiological properties of dopamine neurons in the ventral tegmental area of Sardinian alcohol-preferring rats.

RATIONALE: Sardinian alcohol-preferring (sP) or -nonpreferring (sNP) rats are one of the few pairs of lines of rats selectively bred for their voluntary alcohol preference or aversion, respectively. Ventral tegmental area (VTA) dopamine (DA) neurons have long been implicated in many drug-related behaviors, including alcohol self-administration. However, the electrophysiological properties of these cells in sP and sNP rats remain unknown. OBJECTIVES: This study was designed to examine the properties of posterior VTA DA neurons and to unveil functional differences between sP and sNP rats. MATERIALS AND METHODS: The electrophysiological properties of DA cells were examined performing either single-cell extracellular recordings in anesthetized rats or whole-cell patch-clamp recordings in slices. RESULTS: Extracellular single-unit recordings revealed an increased spontaneous activity in sP rats. However, a corresponding difference was not found in vitro. Moreover, DA cells of sP and sNP rats showed similar intrinsic properties, suggesting changes at synaptic level. Therefore, inhibitory- and excitatory-mediated currents were studied. A decreased probability of GABA release was found in sP rats. Additionally, sP rats showed a reduced depolarization-induced suppression of inhibition, which is an endocannabinoid-mediated form of short-term plasticity. Additionally, the effect of cannabinoid-type 1 (CB1) receptor agonist WIN55,212-2 on GABAA IPSCs was smaller in sP rats, suggesting either a reduced number or functionality of CB1 receptors in the VTA. CONCLUSIONS: Our findings suggest that both decreased GABA release and endocannabinoid transmission in the VTA play a role in the increased impulse activity of DA cells and, ultimately, in alcohol preference displayed by sP rats.

Endogenous fatty acid ethanolamides suppress nicotine-induced activation of mesolimbic dopamine neurons through nuclear receptors.

Nicotine stimulates the activity of mesolimbic dopamine neurons, which is believed to mediate the rewarding and addictive properties of tobacco use. Accumulating evidence suggests that the endocannabinoid system might play a major role in neuronal mechanisms underlying the rewarding properties of drugs of abuse, including nicotine. Here, we investigated the modulation of nicotine effects by the endocannabinoid system on dopamine neurons in the ventral tegmental area with electrophysiological techniques in vivo and in vitro. We discovered that pharmacological inhibition of fatty acid amide hydrolase (FAAH), the enzyme that catabolizes fatty acid ethanolamides, among which the endocannabinoid anandamide (AEA) is the best known, suppressed nicotine-induced excitation of dopamine cells. Importantly, this effect was mimicked by the administration of the FAAH substrates oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), but not methanandamide, the hydrolysis resistant analog of AEA. OEA and PEA are naturally occurring lipid signaling molecules structurally related to AEA, but devoid of affinity for cannabinoid receptors. They blocked the effects of nicotine by activation of the peroxisome proliferator-activated receptor-alpha (PPAR-alpha), a nuclear receptor transcription factor involved in several aspects of lipid metabolism and energy balance. Activation of PPAR-alpha triggered a nongenomic stimulation of tyrosine kinases, which might lead to phosphorylation and negative regulation of neuronal nicotinic acetylcholine receptors. These data indicate for the first time that the anorexic lipids OEA and PEA possess neuromodulatory properties as endogenous ligands of PPAR-alpha in the brain and provide a potential new target for the treatment of nicotine addiction.

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.

Oxytocin injected into the ventral tegmental area induces penile erection and increases extracellular dopamine in the nucleus accumbens and paraventricular nucleus of the hypothalamus of male rats.

The neuropeptide oxytocin (20-100 ng), induces penile erection when injected unilaterally into the caudal but not rostral mesencephalic ventral tegmental area (VTA) of male Sprague-Dawley rats. Such pro-erectile effect started 30 min after treatment and was abolished by the prior injection of d(CH2)5Tyr(Me)(2)-Orn(8)-vasotocin (1 microg), an oxytocin receptor antagonist injected into the same caudal ventral tegmental area or of haloperidol (1 microg), a dopamine receptor antagonist, injected either into the nucleus accumbens shell (NAs) or into the paraventricular nucleus of the hypothalamus (PVN) ipsilateral to the injected ventral tegmental area. Penile erection was seen 15 min after the occurrence of, or concomitantly to, an increase in extracellular dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the dialysate obtained from the nucleus accumbens or the paraventricular nucleus, which was also abolished by d(CH2)5Tyr(Me)(2)-Orn(8)-vasotocin (1 microg), injected into the ventral tegmental area before oxytocin. In the caudal ventral tegmental area oxytocin-containing axons/fibres (originating from the paraventricular nucleus) appeared to closely contact cell bodies of mesolimbic dopaminergic neurons retrogradely labelled with Fluorogold injected into the nucleus accumbens shell, suggesting that oxytocin effects are mediated by the activation of mesolimbic dopaminergic neurons, followed in turn by that of incerto-hypothalamic dopaminergic neurons impinging on oxytocinergic neurons mediating penile erection. As the stimulation of paraventricular dopamine receptors not only induces penile erection, but also increases mesolimbic dopamine neurotransmission by activating oxytocinergic neurons, these results provide further support for the existence of a neural circuit in which dopamine and oxytocin influence both the consummatory and motivational/rewarding aspects of sexual behaviour.

Medial forebrain bundle stimulation evokes endocannabinoid-mediated modulation of ventral tegmental area dopamine neuron firing in vivo.

RATIONALE: Endocannabinoid-mediated forms of transient synaptic depression have been described in several brain structures, including the dopaminergic ventral tegmental area (VTA). However, their functional and/or behavioural correlates are yet to be determined. OBJECTIVES: The present study was designed to investigate whether back-propagating action potentials in dopamine (DA) neurons, evoked by the stimulation of the medial forebrain bundle (MFB), could trigger endocannabinoid-mediated forms of synaptic modulation. The MFB contains axons ascending from DA neurons to the nucleus accumbens and other forebrain structures, and its stimulation is rewarding because it elicits intra-cranial self-stimulation. MATERIALS AND METHODS: Single cell extracellular recordings were carried out from anti-dromically identified VTA DA neurons in chloral hydrate anesthetized rats. RESULTS: DA neurons responded to MFB stimulation (1 s, 20-80 Hz) with a frequency-dependent increase in spontaneous firing rate, which was enhanced by the cannabinoid type-1 receptor antagonist SR141716A (1 mg/kg) and depressed by the agonist WIN55212-2 (0.125 mg/kg). Increasing brain levels of the endocannabinoid anandamide by blocking its major hydrolysing enzyme, fatty-acid amide hydrolase, with URB597 (0.1 mg/kg) was ineffective, whereas blockade of the endocannabinoid membrane transporter with UCM707 (1 mg/kg) enhanced post-stimulus firing rate. CONCLUSIONS: Our study indicates that stimulation of the MFB evokes an endocannabinoid-mediated short-term modulation of DA neuron activity. Thus, endocannabinoids might play an important role in the mechanisms underlying the rewarding properties of MFB stimulation.

Protective activation of the endocannabinoid system during ischemia in dopamine neurons.

Endocannabinoids act as neuroprotective molecules promptly released in response to pathological stimuli. Hence, they may represent one component of protection and/or repair mechanisms mobilized by dopamine (DA) neurons under ischemia. Here, we show that the endocannabinoid 2-arachidonoyl-glycerol (2-AG) plays a key role in protecting DA neurons from ischemia-induced altered spontaneous activity both in vitro and in vivo. Accordingly, neuroprotection can be elicited through moderate cannabinoid receptor type-1 (CB1) activation. Conversely, blockade of endocannabinoid actions through CB1 receptor antagonism worsens the outcome of transient ischemia on DA neuronal activity. These findings indicate that 2-AG mediates neuroprotective actions by delaying damage and/or restoring function of DA cells through activation of presynaptic CB1 receptors. Lastly, they point to CB1 receptors as valuable targets in protection of DA neurons against ischemic injury and emphasize the need for a better understanding of endocannabinoid actions in the fine control of DA transmission.

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, Delta9-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 gamma-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.

RATIONALE: Several 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. OBJECTIVES: The present study was designed to investigate the contribution of the endogenous cannabinoid system in alcohol electrophysiological effects in the mesolimbic reward circuit. METHODS: We 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. RESULTS: Alcohol 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. CONCLUSIONS: These 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.

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.