6-cyano-7-nitroquinoxaline-2-3-dione and capsazepine

Numerous studies, focused on the hypothalamus, have recently implicated endocannabinoids (EC) as orexigenic factors in the central control of food intake. However, the EC system is also highly expressed in the hindbrain autonomic integrator of food intake regulation, i.e. the dorsal vagal complex (DVC). Previous studies have shown that exogenous cannabinoids, by acting on cannabinoid 1 receptor (CB1R), suppress GABAergic and glutamatergic neuronal transmission in adult rat dorsal motor nucleus of the vagus nerve (DMNV), the principal efferent compartment of the DVC. However, no endogenous release of EC has been demonstrated in DVC to date. Using patch-clamp techniques on mouse coronal brainstem slices, we confirmed that both inhibitory and excitatory neurotransmission were depressed by WIN 55,212-2, a CB1R agonist. We demonstrated that DMNV neurons exhibited a rapid and reversible depolarization-induced suppression of electrically evoked GABAergic IPSCs (eIPSCs), classically known as DSI (depolarization-induced suppression of inhibition), while spontaneous or miniature IPSCs activity remained unaltered. Further, no depolarization-induced suppression of glutamatergic eEPSCs (DSE) occurred. Our results indicate that DSI was blocked by SR141716A (Rimonabant), a selective CB1R antagonist, and was dependent on calcium elevation in DMNV neurons, suggesting a release of EC in the DVC. Moreover, the analysis of the paired-pulse ratio, of the coefficient of variation and of the failure rate of eIPSCs support the fact that EC-mediated suppression of GABAergic inhibition takes place at the presynaptic level. These results show for the first time that DMNV neurons release EC in an activity-dependent manner, which in turn differentially regulates their inhibitory and excitatory synaptic inputs.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Arachidonic Acids; Benzoxazines; Brain Stem; Calcium Signaling; Cannabinoid Receptor Modulators; Capsaicin; Efferent Pathways; Endocannabinoids; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Inhibitory Postsynaptic Potentials; Kynurenic Acid; Male; Mice; Mice, Inbred C57BL; Morpholines; Naphthalenes; Neurons; Piperidines; Pyrazoles; Pyridazines; Receptor, Cannabinoid, CB1; Receptors, Presynaptic; Rimonabant; Tetrodotoxin; Vagus Nerve

The aim of this study was to investigate the existence of functional TRPV1 receptor by substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis (Vc), which is implicated in the processing of nociceptive information from orofacial regions. The direct membrane effects of a TRPV1 receptor agonist, capsaicin, were examined by gramicidin-perforated patch clamp recording using a trigeminal brainstem slice preparation containing Vc from immature mice. Capsaicin (1-2 microM) induced a membrane depolarization in 58 out of 71 SG neurons tested (82%). Capsaicin-induced depolarization was maintained in 20 out of 32 (63%) SG neurons in the presence of amino acid and voltage-dependent sodium channel blockers (10 microM CNQX, 20 microM AP-5, 0.5 microM TTX, 50 microM picrotoxin and 2 microM strychnine). In addition, capsaicin-induced depolarization was maintained in the presence of L-732,138 (1 microM), an NK1 receptor antagonist, in 14 out of 17 neurons (82%) tested. The capsaicin-induced depolarizing effects were blocked by a TRPV1 receptor antagonist, capsazepine (10 microM). These results indicate that a sub-population of SG neurons in the Vc express functional TRPV1 receptors, and that capsaicin can directly activate the TRPV1 receptor on the postsynaptic membrane of SG neurons.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Brain Stem; Capsaicin; Central Nervous System Stimulants; Convulsants; Excitatory Amino Acid Antagonists; In Vitro Techniques; Male; Membrane Potentials; Mice; Neurokinin-1 Receptor Antagonists; Neurons; Picrotoxin; Sensory System Agents; Sodium Channel Blockers; Strychnine; Substantia Gelatinosa; Tetrodotoxin; TRPV Cation Channels; Tryptophan

Hyperalgesia and allodynia occur as a consequence of peripheral and central sensitization that follows sustained nociceptive activation. The cellular alterations associated to this state of nociceptive network hyperexcitability represent a form of neuronal plasticity, but they are not well understood because of its complexity in situ. In this study, after treating primary spinal neuron cultures with capsaicin (0.5-1 microM) for 48 h fluorimetric recordings were performed. The activation of TRPV1 receptors with capsaicin (0.5-1.0 microM) increased the frequency of calcium transients (0.03+/-0.002 Hz vs. 0.05+/-0.006 Hz, P<0.05), mediated by AMPAergic transmission, as well as the percent of neurons with activity (37+/-3% vs. 65+/-4%, P<0.05). The effect of capsaicin was long lasting and the neurons were found to be hyperfunctional and with increased levels of phosphorylated CREB (cAMP responsive element binding) even after 72 h of treatment with capsaicin (32+/-5% vs. 52+/-5%). The effect of capsaicin was blocked by capsazepine (1 microM), TTX (100 nM) and KN-62 (1 microM), but not by K252a (200 nM) or PD98059 (50 microM) indicating the involvement of TRPV1. The results suggest the participation of Ca2+, CaMKII and CREB on the prolonged enhancement of excitability following chronic exposure to capsaicin. Thus, it is likely that chronic TRPV1 activation is capable of inducing prolonged increases in neurotransmission mediated by glutamatergic receptors.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Analysis of Variance; Animals; Calcium; Capsaicin; Cells, Cultured; CREB-Binding Protein; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Female; Humans; Mice; Mice, Inbred C57BL; Neurons; Pregnancy; Ruthenium Red; Sodium Channel Blockers; Spinal Cord; Tetrodotoxin; TRPV Cation Channels