| ObjectiveBombesin is a 14 amino acid-containing peptide first isolated from the skin of the frog Bombina bombina. Since its isolation, two related mammalian neuropeptides, neuromedin B (NMB) and gastrin-releasing peptide (GRP), have been isolated and shown to have a widespread distribution. Bombesin, as a brain-gut peptide, plays an important role in food intake and behavior regulations. Hypothalamic arcuate nucleus (ARC) is an area in brain that regulates energy balance in adult mammalian animals. The arcuate nucleus NPY neurons are thought to play an important anabolic role in energy homeostasis.As a brain-gut peptide, bombesin have profound effects on glioblastoma, ischemia and memory processing. Bombesin concentration increased in Parkinson's disease patients, and it can act as anti-epileptic peptide in the hippocampus. There are increasing evidences suggesting that bombesin have neuroprotective effects.In this study, we look at the possible effects of bombesin on NPY neurons in hypothalamic arcuate nucleus, using NPY-GFP transgenetic mice. Further more, we investigate the effect of bombesin on ischemia-induced memory deficts rats and on rat hippocampal CA1 neruonal channel activity, trying to provide another insight for neuroprotective effect of bombesin.MethodsUse NPY-GFP transgenetic mice to study the possible effect of bombesin on NPY neurons. The expression of green fluorescent protein (GFP) is confirmed by immunohistochemistry and single-cell reverse transcription-PCR. The effects of bombesin on NPY neurons were studied using patch-clamp techniques in the hypothalamic brain slice. The input-resistance and the reverse potential were measured. Various artificial cerebrospinal fluid (ACSF) and pipette solutions were used to access the mechanism of the effects of bombesin. Besides, for comparision purpose, we looked at the effects of bombesin-like peptide (GRP and NMB) and other food regulating peptides on NPY neurons. Finally, we examined the effect of bombesin on proopiomelanocortin (POMC) neurons.Twenty-four male Wistar rats were randomly divided into three groups: sham-operation group, ischemia group and ischemia plus bombesin group. Sustained global cerebral ischemia was induced by bilateral occlusion of the carotid arteries. Morris water maze test and long-term potentiation (LTP) recording were performed to access the effect of bombesin on cognitive deficits with chronic cerebral ischemia.Besides, Young male Wistar rats on postnatal days 21-28 were decapitated and cut using an automatic oscillating tissue slicer. Using whole-cell patch clamp recording, the sodium currents (INa), were tested before and after applying bombesin in 350μm thick hippocampal slices.Results1) Single GFP-positive neurons harvested from ARC were proved to have NPY mRNS using single-cell reverse transcription-PCR.2) Bombesin (250nM) increased the spike frequency by 61.5±14.2%(n=15, P <0.05) with a mean depolarization of 5.0±0.5 mV (n=15,P<0.05)3) Neuromedin B, NMB (250nM) increased the spike frequency by 66.5±10.6%(n=16, P<0.05) with a mean depolarization of 5.6±0.4 mV (n=16,P< 0.05. Gastrin-releasing peptide,GRP (250nM) increased the spike frequency by 9.2±10.5%(n=13, P>0.05) with a mean depolarization of 2.6±0.7 mV(n=13, P< 0.05)4) Bombesin (250nM) decreased whole-cell input resistance by 22.4±5.7%(n =8, P<0.05) and the induced inward-current reversed～-18.3±3.4 mV.5) With BAPTA (10mM) in the pipette solution the bombesin (250 nM)-mediated depolarization of the NPY neuron was reduced to 2.0 mV±0.6 mV, (n=5, P<0.05). In contrast, when a Ca2+-free extracellular buffer was used, the bombesin-mediated depolarization was increased to 9.0±1.4 mV, (n=5, P<0.05). With SKF96365 (30μM) in the bath the bombesin-mediated depolarization of the NPY neuron was reduced to 2.7 mV±0.6 mV, (n=5, P<0.05). With NiCl2 in the bath, the depolarization by bombesin was 2.4±0.7 mV (n=5, P<0.05). With Na+/Ca2+ exchanger blocker KB-R7943 in the bath, the depolarization by bombesin was reduced to 2.3±0.9 mV (n=6, P<0.05)6) In Morris water maze test, after five days of training, On day 6, animals in the ischemia group spent less time in the target quadrant (ischemia:24.7±3.8%; sham: 37.8±6.1%; ischemia+bombesin:35.5±3.6%,), whereas the sham-operated (P< 0.05, ischemia vs sham) and the ischemia+bombesin-treated (P<0.05, ischemia vs ischemia+bombesin) animals spent significantly longer times, respectively.7) In LTP recording, bombesin protects against LTP changes in the hippocampus induced by chronic cerebral ischemia. In the ischemia group chronic ischemia significantly reduced the normalized slope of the fEPSP (ischemia:114.0±3.1%; sham:129.8±4.5%, P<0.05) but in the bombesin treated group there was no significant impairment of the fEPSP slope this being similar to sham levels (ischemia+bombesin:125.8±4.3%, P>0.05 vs sham; P<0.05 vs ischemia).8) Bombesin decreased whole-cell sodium currents by 83.32±5.98%(n=6, P< 0.05) and after wash-out, the currents recovered to 97.55±2.19%(n=6,P>0.05) of its original amplitude.Conclusion1) Bombesin reversibly excites NPY and POMC neurons in a dose-dependent manner.2) The excitatory effects of bombesin are most consistent with two mechanisms, activation of both a nonselective cation channel and of the sodium/calcium transporter.3) Bombesin improved the performance of learning and memory of chronic cerebral ischemic rats, reversed the LTP deficit induced by ischemia4) Bombesin decreased whole-cell sodium currents, which could be the underlying mechanism for its neuroprotective effects.
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