Effect Of Paeoniflorin On Electric Activity In The Rat Ventromedial Hypothalamus In Vitro

Hypothalamus plays a key role in food intaking as the primary integration system in controlling food intake. The ventromedial hypothalamic nucleus (VMH),as an important nuclear group in the circuit loop of feeding control, is named as satiety center for outstanding inhibitive effect on feeding. And glucose-receptive neurons (GRNs) in this nuclear group take action in controlling feeding. Recently, the physiological study about feeding showed that a variety of signals related with feeding affected the lateral hypothalamic area and evoked the feeding behavior via the corresponding pathways and neurotransmitters. The factors, including endocrine, humour changed after food,. affect VMH via the corresponding pathways so that the feeding behavior stopped. The vagus nerve in the gastrointestinal tract is one of the pathways in which the afferent fibers in the stomach vagus nerve conducts negative feedback signal into VMN and then VMH suppressed the gastrointestinal motility via efferent fibers in the stomach vagus nerve so that feeding behavior is terminated.The Chinese peony, with character of bitter, acid and property of slightly cold, plays roles of"enriching blood and softening liver"and"relieving spasm and pain"so that it is commonly used in enriching YING and softening liver. Recently, some studies have confirmed that the Chinese peony could play significant roles in the digestive tract movement and inhibiting activity of nerve centre. It is recoded in Compendium of Materia Medica that white peony can play role of YIPI and flow swiftly the wood in the earth. It is concluded that white peony can regulate the function of digestive tract to some extent. However, most of the domestic and international studies were restricted at the complex prescription or at the observation of whole effect so that the studies generally focus on the effects of gastrointestinal tract functional status itself on appetite and took less attention in the regulative effects of nerve centre on feeding behavior.Some studies demonstrated that both Chinese peony and PF could play roles in regulating immunological function of organism, inhibitting excitement of parasympathetic nerve, extenuating the tension of smooth muscle and modulating the movement of digestive tract [1, 2]. However there were few studies that focus on the effects of PF on the feeding center of central nervous system. Our provious studies aimming directly at the patent medicine (ErBao Granule and Baobaole oral liquid) in which white peony was taken as principal agent confirmed that ErBao Granule could not only regulative many fators related with feeding in periphlevel, such as promoting gastro-intestinal tract creeping motion, enhancing digestive enzyme activity, promoting small intestine absorptive function, adjusting gastro-intestinal tract hormone level, but also affect the control of feeding behavior in level of central nerve. We further explore the action mechanisms of PF on feeding behavior in VMH neurons in juvenile rat with use of Infrared visibility whole-cell patch-clamp technique in this study. To begin with, the electrophysiological characteristics of different types of VMH neurons and the effects of PF on discharge in different types of VMH neurons were observed with current clamp. Moreover, the effects of PF on the electric current change and the spontaneous discharge in VMH neurons were detected with method of voltage clamp. This study lied point to expore the effects of PF on electric activity in hypothalamus feeding center VMH neurons so as to investigate and analyze the feeding regulatory mechanism of PF on nerve centre. Our main observations were reported as following.Main conclusions:ExperimentⅠExploration of electrophysiological properties of rat VMH neurons with infrared visual patch clampObjective To observe the electrophysiological characteristics of rat VMH neurons using the visual patch clamp technique. Methods With infrared-visualized patch clamp technique, the morphosis and the electrophysiological characteristics of VMH neurons from the selected hypothalamic slices of healthy juvenile Sprague-Dawley rats were recorded in whole-cell mode. Results The morphosis of somas and axon was observed non-invasively by patch clamp with infrared videomicroscopy. 81 VMH neurons cells were observed and classified into three types, among which, 15 cells were type ? cells that had generally no spontaneous discharge with lower membrane input resistances(193.0±31.0) M? and shorter membrane time constants (7.5±2.3) msec (n=15); 45 cells were typeⅡcells that got spontaneously active and had significantly longer membrane time constants (14.1±2.2) msec and higher input resistances (338.8±42.6) M? (n=45) than typeⅠdid; 21 cells were typeⅢcells that got also spontaneously active and had significantly longer membrane time constants (23.8±2.5) msec and higher input resistances (511.8±70.5) M?(n=21)than typeⅠand typeⅡcells did.ExperimentⅡEffects of PF on electric activity in the rat VMH in vitroObjective To investigate the effects of PF on neuronal excitability in the juvenile rat VMH. Methods Whole-cell configuration of the patch clamp technique was used to record the spontaneous discharge of 35 normal neurons in the VMH slice before and after the application of PF. Results when slices were continuously perfused with ACSF containing 300μmol/L PF, the firing rate of 62.8% neurons were significantly reduced(n=22), the average firing rates of VMH neurons decreased from(7.1±2.2)Hz to (3.2±0.7)Hz(P <0.01), the number of discharges also decreased significantly ,which were dose- dependent decreases. when giving a kind of depolarizing square-wave,. the firing rate recovered after washing out the PF. 22.9% neurons didn't responsed(n=8), and the firing rate of 14.3% VMH neurons were obviously increased(n=5) from(4.7±0.8)Hz to(5.2±0.4)Hz.ExperimentⅢEffect of Paeoniflorin on the glucose-responsive neurons in hypothalamus of ratsObjective In order to explore the mechanism of PF-induced feeding behavior on glucoresponsive neurons in hypothalamus were observed. Methods by using the brain slices whole-cell patch-clamp technique, the electrical activity of the LHA and VMH neurons,on the basis of their responsiveness by adding in 100μmol/L glucose solution, glucose-sensitive neurons(GSNs) in LHA and glucoreceptor (GRNs) in VMH were recognized, Effects of PF on neural activities of glucoresponsive neurons and non-glucoresponsive neurons were examined. The mixture of PF and glucose were used to GSNs and GRNs which were sensitive of PF. Results In LHA, PF increased activity of GSNs significantly(P<0.05 vs non-glucose-sensitive neurons group). Whereas in VMH, PF significantly decresed the activities of GRNs (P<0.05 vs NGRNs group). The mixture of PF and glucose had no effect on GRNs.Main conclusions:1. VMH neurons are classified as three types according to the morphology and electrophysiological characteristics of VMH neurons. Our study has provided further evidence in support of neuronal heterogeneity within the ventromedial hypothalamic nucleus.2. PF has the selectivity of adjusting the excitability of VMH neurons.3. PF can increase the excitability of LHA neurons and decrease the excitability of majority VMH neurons, further in VMH, PF can significantly descend the activity of satiety center.4. PF can stimulate GSNs in LHA and suppress GRNs in VMH and this may contribute to central motilin's effect on feeding behavior.