The Ion Channel Mechanism Of The Activate Spleen-energy Method Regulating CCK To Suppress Excitability Of VMH Neurons

Recently twenty years, with the traditional Chinese medicine theory that is Spleen transformation lying in regulation rather than nourishment guiding, The domestic scholar widely apply the activate spleen-energy law that can recover the transport mechanism of spleen and stomach and regulate the function of spleen and stomach better and this law is effective to cure child anorexia.And they systematically studied the effect of the activate spleen-energy law on digestive and absorptive function, it is confirmed that the activate spleen-energy law have significant regulation effect on the action and secretion of gastrointestinal tract. Recently, we established the juvenile rat model with anorexia by imitating the major cause of children and found that the CCK-8S concentration in hypothalamus and periphery blood of the anorexy model was obviously increased .We also found the appestat that the spontaneous discharge frequency of LHA neurons was decreased and the spontaneous discharge frequency of VMH neurons was increased. Erbao Granules was effective on decreasing CCK-8S concentration in periphery blood of the animal mode1 and regulating the spontaneous discharge frequency of LHA and VMH neurons.These results suggest that the activate spleen-energy method can achieve appetitive purpose by not only affecting the gastrointestinal tract function in periphery, but also regulating the process of ingestion in brain.The basic research about ingestion indicate: one hand, after food enters the gastro-intestinal tract, the endocrine cell of the intestinal tract is activated and secrete many kinds of brains intestines peptide, the level of the brain intestines peptide in blood as the periphery signal spreads to the central nervous system regions that control digest and feeding behavior. On the other hand, it has been shown that dorsal parabranchial neurons, containing CCK-8S, extend fibers to the VMH and are involved in the inhibition of feeding . CCK-8S is hormone and neurotransmitter, widely spread in periphery blood and the central nervous system, it plays the key role in the physiological process of ingest regulation. The VMH is the biggest nucleus in the hypothalamus basilar part. Many research suggest that VMH is satiety center and this physiological process is regulated by many neurotransmitters, like CCK-8S, 5-HT and glucose. But it's not clear that how CCK-8S affect on VMH neurons and the mechanism of its receptor.In neurons from this region, spontaneous firing and responses to perturbations of membrane potential are probably determined by currents with activation thresholds below that of the voltage-gated Na+ channel (subthreshold currents), like Ih and IA. It's more important that this subthreshold currents can control the discharge frequency and is modulated by the neurotransmitters.Some ARC neurons are also spontaneously active and subthreshold currents may shape their electrical activity. Of particular interest, Cholecystokinin (CCK-8S) can slow firing of Arcuate Nucleus neurons by activating this subthreshold currents ,such as A-type potassium channels. Therefore, using the whole-cell patch-clamp technique, we tested the hypothesis that whether endogenous CCK-8S increased VMH neuronal firing by suppressing the A-type potassium channels.According to our pass studies,we supposed it's possible that the activate spleen-energy law promote appetite through "the intestines - brain axis:Our past study confirmed that the prescription Erbao Granules not only adjusted the peptide hormone level related to ingestion in the blood ,like reducing the CCK-8S level, but also to regulated the spontaneous discharge frequency of LHA and VMH neurons. In the present study, we recorded the date under voltage and current clamp with whole cell patch-clamp on VMH brain neurons. In current clamp, we recorded the electrophysiological characteristics and the effect of CCK-8S on the firing of different types of VMH neurons. In voltage clamp, we recorded the effect of CCK-8S on the A-type potassium channels of VMH neurons. This experiment key lies in the ion channel mechanism of the activate spleen-energy method regulating neuronal excitability of VMH neurons, which is the middle link of the activate spleen-energy method adjusting neuronal excitability of VMH neurons through regulating the CCK-8S level of periphery and brain. These research can further explore the center mechanism of the activate spleen-energy method promoting ingestion through "the intestine - brain axis".Results:The first part: the electrophysiological characteristics and three distinct electrophysiological types of VMH neurons.VMH neurons are classified as three types by the spontaneous firing and electrophysiological characteristics of VMH neurons.1. TypeⅠcells : 3/31 cells are type ? cells, they had generally no spontaneous discharge with lower membrane input resistances(192±39) MΩ(n=4)and shorter membrane time constants(8±1)msec(n=4);2. TypeⅡcells : 11/31 cells are typeⅡcells , they were spontaneously active and had significantly longer membrane time constants(14±1) msec(n=11)and higher input resistances(327±2) MΩ(n=11)than typeⅠdid;3. TypeⅢcells: 17/31 cells are typeⅢcells, they was also spontaneously active and had significantly longer membrane time constants (25±2)msec(n=17)and higher input resistances(522±28) MΩ(n=17)than typeⅠand typeⅡcells did.The second part : the effect of CCK-8S on the firing of different types of VMH neurons CCK-8S (0.4μmol/L) could increase the firing of typeⅡand typeⅢneurons with spontaneous activity but had no effect on type I neurons being quiescent. The spontaneous discharge frequency is increased from (4±1) Hz to (8±1) Hz, but the spike amplitude is not change.The third part: the ion channel mechanism of CCK-8S tuning the spontaneous discharge frequency of typeⅡand typeⅢVMH neurons.1. In voltage clamp recording, Ih was not elicited by hyperpolarization in in the two types of VMH neurons ; In current clamp recording, hyperpolarizing current injected failed to elicit the typical "sag" in membrane potential characteristic of Ih activation in the two types of VMH neurons . Thus, our results suggested that Ih channels maybe are not present in the type-Ⅱand type-Ⅲneurons of VMH.2. The A-type potassium current was observed in type-ⅢVMH neurons . It was activated between -60 and -50 mv and we observed the voltage dependence of A-current inactivation.: the half-maximal inactivation occurred at -65±1mv. The current was completely eliminated by the A-current blocker 4-aminopyridine (1mM;n=4).3. CCK-8S acutely increased firing and suppressed A-type potassium currents in type-Ⅲneurons .There was a strong linear correlation between firing frequency and A-type potassium (r=0.99) .The fourth part: the receptor mechanism of CCK-8S tuning the spontaneous discharge frequency of VMH neurons.Gastrin can mimick the effects of CCK-8S on the firing by CCK-B receptor in VMH typeⅡand typeⅢneurons. The spontaneous discharge frequency is increased from (4±1) Hz to (7±1) Hz.Main conclusions:1. Our study has provided further evidence in support of neuronal heterogeneity within the ventromedial hypothalamic nucleus.2. It is suggested that CCK-8S has the selectivity of adjusting the excitability of VMH neurons and activate satiety center to suppress appetite through enhancing the excitability of majority VMH neurons.3. The inhibition of A-current clearly contributed to the potentiating effects of CCK-8S on the whole neuronal excitability of VMH.4. CCK-8S maybe tunes the spontaneous discharge frequency of VMH neurons by CCK-B receptor.5. That result confirm that the activate spleen-energy method can reduce the concentration of CCK-8S to inhibit the inactivation of IA current to selectively suppress the whole excitability of VMH neurons by the CCK-8S acceptor. This process can suppress excitability of satiety center and promote ingestive behavior.