Endocannabinoids Generated by Ca2+ or by Metabotropic Glutamate Receptors Appear to Arise from Different Pools of Diacylglycerol Lipase

The identity and subcellular sources of endocannabinoids (eCBs) will shape their ability to affect synaptic transmission and, ultimately, behavior. Recent discoveries support the conclusion that 2-arachidonoyl glycerol, 2-AG, is the major signaling eCB, however, some important issues remain open. 2-AG can be synthesized by a mechanism that is strictly Ca2+-dependent, and another that is initiated by G-protein coupled receptors (GPCRs) and facilitated by Ca2+. An important question is whether or not the 2-AG in these cases is synthesized by the same pool of diacylglycerol lipase alpha (DAGLα). Using whole-cell voltage-clamp techniques in CA1 pyramidal cells in acute in vitro rat hippocampal slices, we investigated two mechanistically distinct eCB-mediated responses to address this issue. We now report that pharmacological inhibitors of DGLα have quantitatively different effects on eCB-mediated responses triggered by different stimuli, suggesting that functional, and perhaps physical, distinctions among pools of DAGLα exist

Extensive astrocyte synchronization advances neuronal coupling in slow wave activity in vivo

Slow wave activity (SWA) is a characteristic brain oscillation in sleep and quiet wakefulness. Although the cell types contributing to SWA genesis are not yet identified, the principal role of neurons in the emergence of this essential cognitive mechanism has not been questioned. To address the possibility of astrocytic involvement in SWA, we used a transgenic rat line expressing a calcium sensitive fluorescent protein in both astrocytes and interneurons and simultaneously imaged astrocytic and neuronal activity in vivo. Here we demonstrate, for the first time, that the astrocyte network display synchronized recurrent activity in vivo coupled to UP states measured by field recording and neuronal calcium imaging. Furthermore, we present evidence that extensive synchronization of the astrocytic network precedes the spatial build-up of neuronal synchronization. The earlier extensive recruitment of astrocytes in the synchronized activity is reinforced by the observation that neurons surrounded by active astrocytes are more likely to join SWA, suggesting causality. Further supporting this notion, we demonstrate that blockade of astrocytic gap junctional communication or inhibition of astrocytic Ca2+ transients reduces the ratio of both astrocytes and neurons involved in SWA. These in vivo findings conclusively suggest a causal role of the astrocytic syncytium in SWA generation

Extrasynaptic glycine receptors of rodent dorsal raphe serotonergic neurons:a sensitive target for ethanol

Alcohol abuse is a significant medical and social problem. Several neurotransmitter systems are implicated in ethanol's actions, with certain receptors and ion channels emerging as putative targets. The dorsal raphe (DR) nucleus is associated with the behavioural actions of alcohol, but ethanol actions on these neurons are not well understood. Here, using immunohistochemistry and electrophysiology we characterise DR inhibitory transmission and its sensitivity to ethanol. DR neurons exhibit inhibitory 'phasic' postsynaptic currents mediated primarily by synaptic GABAA receptors (GABAAR) and to a lesser extent by synaptic glycine receptors (GlyR). In addition to such phasic transmission mediated by the vesicular release of neurotransmitter, the activity of certain neurons may be governed by a 'tonic' conductance resulting from ambient GABA activating extrasynaptic GABAARs. However, for DR neurons extrasynaptic GABAARs exert only a limited influence. By contrast, we report that unusually the GlyR antagonist strychnine reveals a large tonic conductance mediated by extrasynaptic GlyRs, which dominates DR inhibition. In agreement, for DR neurons strychnine increases their input resistance, induces membrane depolarization and consequently augments their excitability. Importantly, this glycinergic conductance is greatly enhanced in a strychnine-sensitive fashion, by behaviourally-relevant ethanol concentrations, by drugs used for the treatment of alcohol withdrawal and by taurine, an ingredient of certain 'energy drinks' often imbibed with ethanol. These findings identify extrasynaptic GlyRs as critical regulators of DR excitability and a novel molecular target for ethanol

mGluR5 antagonism inhibits cocaine reinforcement and relapse by elevation of extracellular glutamate in the nucleus accumbens via a CB1 receptor mechanism

Abstract Metabotropic glutamate receptor 5 (mGluR5) antagonism inhibits cocaine self-administration and reinstatement of drug-seeking behavior. However, the cellular and molecular mechanisms underlying this action are poorly understood. Here we report a presynaptic glutamate/cannabinoid mechanism that may underlie this action. Systemic or intra-nucleus accumbens (NAc) administration of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) dose-dependently reduced cocaine (and sucrose) self-administration and cocaine-induced reinstatement of drug-seeking behavior. The reduction in cocaine-taking and cocaine-seeking was associated with a reduction in cocaine-enhanced extracellular glutamate, but not cocaine-enhanced extracellular dopamine (DA) in the NAc. MPEP alone, when administered systemically or locally into the NAc, elevated extracellular glutamate, but not DA. Similarly, the cannabinoid CB1 receptor antagonist, rimonabant, elevated NAc glutamate, not DA. mGluR5s were found mainly in striatal medium-spiny neurons, not in astrocytes, and MPEP-enhanced extracellular glutamate was blocked by a NAc CB1 receptor antagonist or N-type Ca++ channel blocker, suggesting that a retrograde endocannabinoid-signaling mechanism underlies MPEP-induced glutamate release. This interpretation was further supported by our findings that genetic deletion of CB1 receptors in CB1-knockout mice blocked both MPEP-enhanced extracellular glutamate and MPEP-induced reductions in cocaine self-administration. Together, these results indicate that the therapeutic anti-cocaine effects of mGluR5 antagonists are mediated by elevation of extracellular glutamate in the NAc via an endocannabinoid-CB1 receptor disinhibition mechanism

Rat Nucleus Accumbens Core Astrocytes Modulate Reward and the Motivation to Self-Administer Ethanol after Abstinence

Our understanding of the active role that astrocytes play in modulating neuronal function and behavior is rapidly expanding, but little is known about the role that astrocytes may play in drug-seeking behavior for commonly abused substances. Given that the nucleus accumbens is critically involved in substance abuse and motivation, we sought to determine whether nucleus accumbens astrocytes influence the motivation to self-administer ethanol following abstinence. We found that the packing density of astrocytes that were expressing glial fibrillary acidic protein increased in the nucleus accumbens core (NAcore) during abstinence from EtOH self-administration. No change was observed in the nucleus accumbens shell. This increased NAcore astrocyte density positively correlated with the motivation for ethanol. Astrocytes can communicate with one another and influence neuronal activity through gap-junction hemichannels. Because of this, the effect of blocking gap-junction hemichannels on the motivation for ethanol was examined. The motivation to self-administer ethanol after 3 weeks abstinence was increased following microinjection of gap-junction hemichannel blockers into the NAcore at doses that block both neuronal and astrocytic channels. In contrast, no effect was observed following microinjection of doses that are not thought to block astrocytic channels or following microinjection of either dose into the nucleus accumbens shell. Additionally, the motivation for sucrose after 3 weeks abstinence was unaffected by NAcore gap-junction hemichannel blockers. Next, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) were selectively expressed in NAcore astrocytes to test the effect of astrocyte stimulation. DREADD activation increased cytosolic calcium in primary astrocytes, facilitated responding for rewarding brain stimulation, and reduced the motivation for ethanol after 3 weeks abstinence. This is the first work to modulate drug-seeking behavior with astrocyte-specific DREADDs. Taken together, our findings demonstrate that NAcore astrocytes can shape the motivation to self-administer ethanol; suggesting that the development of ligands which selectively stimulate astrocytes may be a successful strategy to abate ethanol-seeking behavior

Cannabinoids Occlude the HIV-1 Tat-Induced Decrease in GABAergic Neurotransmission in Prefrontal Cortex Slices

In the era of combined antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) is now considered a chronic disease that specifically targets the brain and causes HIV-1-associated neurocognitive disorders (HAND). Endocannabinoids exhibit neuroprotective and anti-inflammatory properties in several central nervous system (CNS) disease models, but their effects in HAND are poorly understood. To address this issue, whole-cell recordings were performed on young (14 – 21 day old) C57BL/6J mice. We investigated the actions of the synthetic cannabinoid WIN55,212-2 (1 μM) and the endocannabinoid N-arachidonoyl ethanolamine (anandamide; AEA, 1 μM) in the presence of HIV-1 Tat on GABAergic neurotransmission in mouse prefrontal cortex (PFC) slices. We found a Tat concentration dependent (5 – 50 nM) decrease in the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs). The cannabinoid 1 receptor (CB(1)R) antagonist rimonabant (1 μM) and zero extracellular calcium prevented the significant Tat-induced decrease in mIPSCs. Further, bath-applied WIN55,212-2 or AEA by itself, significantly decreased the frequency, but not amplitude of mIPSCs and/or spontaneous IPSCs (sIPSCs), and occluded a further down-regulation of IPSCs by Tat. Pretreatment with rimonabant but not the CB(2)R antagonist AM630 (1 μM) prevented the WIN55,212-2- and AEA-induced decrease in IPSCs frequency without any further Tat effect. Results indicated a Tat-induced decrease in GABAergic neurotransmission, which was occluded by cannabinoids via a CB(1)R-related mechanism. Understanding the relationship between Tat toxicity and endocannabinoid signaling has the potential to identify novel therapeutic interventions to benefit individuals suffering from HAND and other cognitive impairments