HISADQME Kazunari;TRAPP Stefan;REIMANN Frank;GRIBBLE Fiona;
<正>Glucagon-like-peptide 1(GLP-1)is a gut peptide that is released in response to ingestion of food and causes satiety.Interestingly,GLP-1 is also produced by a small population of brainstem neurons,and given its limited half-life and difficulty crossing the blood-brain-barrier,it appears feasible that GLP-1 released within brain, rather than circulating GLP-1 causes satiety.In order to enable identification of these neurons in vitro and to analyze their electrophysiological properties we have produced a transgenic mouse model where green fluorescent protein(GFP)is expressed under control of the preproglucagon promoter.Brainstem slices were obtained from adult transgenic mice after halothane anaesthesia.Slices were kept at 32℃in artificial cerebrospinal fluid(ACSF). Perforated patch-clamp recordings from 132 GFP-positive cells were performed at 32℃under visual control. From 15 cells cytoplasm was aspirated into the electrode after patch-clamp recording and used for single-cell RT-PCR. Most GFP-positive cells were detected within the caudal NTS,and few additional cells more ventral.Electrophysiological recordings were limited to the NTS.eight out of 10 GFP-positive cells,but no GFP-negative cells (n=5),tested positive for preproglucagon mRNA.Current clamp recordings revealed three subtypes of NTS GLP-1 neurons:23 cells showed TTX-resistant(n=2),large fluctuations of the membrane potential that led to burst firing.88 cells were spontaneously active with a firing rate of 1.8±0.2 Hz,a resting potential of -51±0.5 mV,and an input resistance of 1.0±0.03 GOhm.21 cells were not spontaneously active,had a resting potential of -55±1.6 mV and an input resistance of 0.7±0.1 GOhm.Injection of 14±1 pA into these cells was sufficient to elicit action potential firing(n=9).All three subpopulations exhibited a whole-cell capacitance of～30 pF.A small proportion of spontaneously-active cells hyperpolarized over time.As tested in 8 of these cells the hyperpolarisation was sensitive to tolbutamide.In contrast,tolbutamide had no effect on the spontaneously-active cells(n=4)or non-active cells (n=1).Finally,the effects of glutamate and GABA were tested on spontaneously-active cells.GLP-1 neurons depolarized under glutamate(n=12)and were inhibited by GABA(n=5).In summary,we have demonstrated that GFP-positive NTS cells are viable,produce preproglucagon and are amenable for electrophysiological recordings in vitro.We conclude that this mouse model is ideally suited for functional studies of GLP-1 neurons in vitro.
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