Activation of melanocortin 4 receptors (MC4-Rs) in brain nuclei associated with food intake profoundly influences consummatory behavior. Of these nuclei, the dorsal motor vagal nucleus (DMV), which has a dense concentration of MC4-Rs, is an important regulator of gastric tone and motility. Hence, the present study sought to examine the role of MC4-Rs in this nucleus on these activities. Using an in vivo approach, MC4-R agonists, melanotan-II (MT-II) or ␣-melanocyte stimulating hormone (␣-MSH), were unilaterally microinjected into the DMV of rats, and their effects were noted on gastric activity. MT-II decreased phasic contractions, whereas ␣-MSH increased their amplitude. Both effects were blocked by the MC4-R antagonist SHU9119 or by ipsilateral vagotomy. Microinjection of the agonists (MT-II and ␣-MSH) into the overlying nucleus of the solitary tract (NTS), an important component of "vago-vagal" gastric circuitry, decreased phasic contractions. In addition, ␣-MSH reduced gastric tone and mean arterial blood pressure. To study the underlying mechanisms of the effect of MC4-R stimulation on gastric activity, electrophysiological recordings were made from labeled DMV antrum neurons in rat pups and MC4-R Ϫ/Ϫ mice. Bath application of MT-II or ␣-MSH significantly reduced spontaneous action potentials (but not in MC4-R Ϫ/Ϫ mice). However, in low-calcium ACSF, MT-II decreased neuronal firing, whereas ␣-MSH increased it. These effects mirror those of our in vivo DMV studies. Altogether, our novel findings show that activation of MC4-Rs in the brainstem, particularly in the medial NTS by the endogenous peptide ␣-MSH, modulates gastric activity, which may have physiological relevance for food intake and gastric function.
We have previously shown that local GABA signalling in the brainstem is an important determinant of vagally-mediated gastric activity. However, the neural identity of this GABA source is currently unknown. To determine this, we focused on the somatostatin positive GABA (Sst-GABA) interneuron in the dorsal motor nucleus of the vagus (DMV), a nucleus that is intimately involved in regulating gastric activity. Also of particular interest was the effect of melanocortin and μ-opioid agonists on neural activity of Sst-GABA DMV neurons because their in vivo administration in the DMV mimics GABA blockade in the nucleus. Experiments were conducted in brain slice preparation of transgenic adult Sst-IRES-Cre mice expressing tdTomato fluorescence, channelrhodopsin-2, archaerhodopsin or GCaMP3. Electrophysiological recordings were obtained from Sst-GABA DMV neurons or DiI labelled gastric-antrum projecting DMV neurons. Our results show that optogenetic stimulation of Sst-GABA neurons results in a robust inhibition of action potentials of labelled premotor DMV neurons to the gastric-antrum through an increase in inhibitory post-synaptic currents. The activity of the Sst-GABA neurons in the DMV is inhibited by both melanocortin and μ-opioid agonists. These agonists counteract the pronounced inhibitory effect of Sst-GABA neurons on vagal pre-motor neurons in the DMV that control gastric motility. These observations demonstrate that Sst-GABA neurons in the brainstem are crucial for regulating the activity of gastric output neurons in the DMV. Additionally, they suggest that these neurons serve as targets for converging CNS signals to regulate parasympathetic gastric function.
Key pointsr Pharmacological activation of striatal Group I metabotropic glutamate receptors (mGluRs) increases the occurrence of GABA A -mediated currents in striatal spiny projection neurones (SPNs).r Genetically identified striatal interneurones are depolarized by Group I mGluR activation. r Group I mGluR activation elevates intracellular calcium in genetically identified striatal interneurones expressing a genetically encoded calcium indicator.r Group I mGluR activation results in increased intracellular calcium in SPNs only after priming with calcium influx.r Combined electrophysiology and calcium imaging reveals that mGluR activation is not accompanied by depolarization in SPNs.Abstract In mouse striatum, metabotropic glutamate receptor (mGluR) activation leads to several modulatory effects in synaptic transmission. These effects range from dampening of glutamate release from excitatory terminals to depolarization of divergent classes of interneurones. We compared the action of group I mGluR activation on several populations of striatal neurones using a combination of genetic identification, electrophysiology, and Ca 2+ imaging techniques. Patch-clamp recordings from spiny projection neurones (SPNs) and various interneurone populations demonstrated that the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) robustly depolarizes several interneurone classes that form GABAergic synapses onto SPNs. We further utilized the genetic reporter mouse strain Ai38, which expresses the calcium indicator protein GCaMP3 in a Cre-dependent manner. Breeding Ai38 mice with various neurone selective, promoter-driven Cre recombinase mice resulted in GCaMP3 expression in defined cell populations in striatum. Consistent with our electrophysiological findings, group I agonist applications increased intracellular levels of calcium ([Ca 2+ ] i ) in all interneurone populations tested. We also found that acute DHPG application evoked a transient, rapid increase in [Ca 2+ ] i from only a small percentage of identifiable SPNs. Surprisingly, this fast [Ca 2+ ] i response exhibited a robust enhancement or sensitization, in a calcium-dependent fashion. Following several procedures to increase [Ca 2+ ] i , the vast majority of SPNs responded with rapid changes in [Ca 2+ ] i to mGluR agonists in a time-dependent fashion. These findings extend our understanding on group I mGluR influence of striatal output via powerful, local GABAergic connections in addition to [Ca 2+ ] i dynamics that impact on activity or spike-timing-dependent forms of synaptic plasticity.
AT-1001 [N-(2-bromophenyl)-9-methyl-9-azabicyclo[3.3.1] nonan-3-amine] is a high-affinity and highly selective ligand at a3b4 nicotinic cholinergic receptors (nAChRs) that was reported to decrease nicotine self-administration in rats. It was initially reported to be an antagonist at rat a3b4 nAChRs heterologously expressed in HEK293 cells. Here we compared AT-1001 actions at rat and human a3b4 and a4b2 nAChRs similarly expressed in HEK 293 cells. We found that, as originally reported, AT-1001 is highly selective for a3b4 receptors over a4b2 receptors, but its binding selectivity is much greater at human than at rat receptors, because of a higher affinity at human than at rat a3b4 nAChRs. Binding studies in human and rat brain and pineal gland confirmed the selectivity of AT-1001 for a3b4 nAChRs and its higher affinity for human compared with rat receptors. In patch-clamp electrophysiology studies, AT-1001 was a potent partial agonist with 65-70% efficacy at both human and rat a3b4 nAChRs. It was also a less potent and weaker (18%) partial agonist at a4b2 nAChRs. Both a3b4 and a4b2 nAChRs are upregulated by exposure of cells to AT-1001 for 3 days. Similarly, AT-1001 desensitized both receptor subtypes in a concentration-dependent manner, but it was 10 and 30 times more potent to desensitize human a3b4 receptors than rat a3b4 and human a4b2 receptors, respectively. After exposure to AT-1001, the time to recovery from desensitization was longest for the human a3b4 nAChR and shortest for the human a4b2 receptor, suggesting that recovery from desensitization is primarily related to the dissociation of the ligand from the receptor.
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