Effect Of The Central Amygdala On Gastric Motility And It’s Character Of Neuronal Activity Induced By Restraint Water-immersion Stress

“Restraint water-immersion” is a special treatment which combined physiological and psychological stressor in which conscious rats are bound and immersed into cold water(21 ±1 ℃). By integrating stimulations of immobilization and low temperature, the restraint water-immersion stress(RWIS) induces obvious gastric functional disorders and pathologies such as vagus-mediated gastric hypercontractility, gastric acid hypersecretion and gastric mucosal lesions or ulcer within a few hours. Neuronal overactivity in the dorsal vagal complex(DVC)which is the gastrointestinal primary center in medulla oblongata are the important mechanisms relative with stress-induced gastric hyperfunction and ulcer. Previous studies indicated that RWIS induced intense c-Fos expression of neurons in the extensive areas of the limbic forebrain,however, it is still unknown that what exact roles that some of these brain act in the response of RWIS.As an essential structure in the limbic stress circuits, neurons in the central nucleus of amygdala(CEA) are excited and accelerate the function of hypothalamic-pituitary-adrenocortical(HPA) axis in stress response. Excited CEA contribute to not only adaptive regulation, but also to the development of some stressful dysfunction or disorders. Previous studies have reported the specificity of neuronal activity in CEA induced by different stressors. However, as for as RWIS is concerned, the specificity of neuronal activity in CEA and the involved role in the stress-induced gastric dysfunction remain unknown. Answering these questions will further the understanding of the role of CEA in RWIS and provide necessary data for the central mechanism of the gastric dysfunction induced by RWIS.In addition, CEA may regulate gastric functions such as motility and secretion based on the connection between CEA and DVC, the primary center for controlling gastrointestinal functions.But it is inconsistent of the results of regulation effect of electrical stimulation of CEA on gastricmotility. Early researches reported excitatory effect while later ones reported mainly inhibitory effect. Abnormal gastric motility under stress is the important peripheral factor resulting in various gastrointestinal diseases. For example, excessive enhancement of gastric motility is considered to be the key factor in stress-induced gastric mucosal injury or ulcer. On the contrary,inhibition of gastric motility is considered to induce delayed gastric empty which is the character of functional dyspepsia and irritable bowel syndrome on clinic. So make sure of the regulation effect on gastric motility of CEA is the unavoidable premise to comprehend its role involved in RWIS.Based on the above-mentioned questions, we carried on the investigation from two aspects.Firstly, it is necessary to reevaluate the influence of CEA on gastric motility by distinguish the medial and lateral subdivisions of CEA(m CEA and l CEA) since they are anatomically and physiologically heterogeneous. Mainly with electrophysiological technique, we observed and compared the effects on gastric motility and neuronal spikes in DVC induced by electrical stimulation of m CEA with l CEA. Our finding here implies that stimulation of m CEA can significantly increase gastric motility, and stimulation of l CEA can significantly decrease gastric motility. Consistently, stimulation of m CEA can significantly increase the firing rate(FR) of neurons in DMV and decrease the FR of neurons in the medial NTS(m NTS), while stimulation of l CEA do exactly the opposite. The inhibitory GABAergic circuits within the CEA may be a point of such opposite effects of m CEA and l CEA. However, direct evidence has yet to be obtained by further experiments.Secondly, based on the result of above experiment and considering the discrepancy between the activity of CEA neurons induced by electrical stimulation and by RWIS, we observed the activity of CEA neurons induced by RWIS by two experiments:In the first experiment, all the rats were randomly divided into unstressed group, restraint stress(RS) group and RWIS group. Using immunohistochemical technique and neuronal spikes recording technique, we firstly compared the c-Fos expression, the firing rate(FR) of neuronal spikes and the coefficient of variation(CV) of interspike intervals of m CEA and l CEA. Our finding here implies that only RWIS induces gastric mucosal injury and the excitatory of CEA neurons. The characteristics of neuronal activity is significant different from that induced by RS:(1) although both stress induced activity in l CEA neurons, there are more Fos-positive cells in l CEA of RWIS rats than that of RS rats;(2) neuronal activity in m CEA is induced only by RWIS,and not by RS;(3) firing pattern of CEA neurons in RWIS rats are distinguished from unstressed and RS rats with lower CV.In the second experiment, all the rats were randomly divided into unstressed group, RWIS 1h group, RWIS 3h group, RS 1h group and RS 3h group. we observed the c-Fos expression of corticotrophin-releasing hormone(CRH) neurons in CEA using immunohistochemical double-labeling technique and measured adrenocorticotrophic hormone(ACHT) and corticosterone(CORT) in the blood using radioimmunoassay technique. Our finding here implies that intense c-Fos expression of CRH neurons in CEA is induced by different durations of RWIS which has not been reported in previous studies. Considering cold stress inhibits the activity of CRH neurons in CEA previously reported, it seems that the effects induced by intergraded stressors are not equal with the sum of the effects induced by each involved stressor(CRH neurons in CEA are inhibited by single cold stress by previous report). Furthermore, our finding here also implies that RWIS leads to an more increase in the activity of HPA axis than RS,judged by the concentration of ACTH and CORT in the blood. With the continuance from 1h to3 h, HPA activity in RWIS was increasing while that in RS is decreasing. The activity of CRH neurons in CEA and the HPA activity induced by RWIS show an obvious synergistic effect.Above-mentioned descriptions of the characteristics of CEA neurons induced by RWIS add necessary data for illuminating the central mechanism of the response to RWIS mediated by CEA.To summarize, we demonstrate that 1) electrical stimulation of medial and lateral CEA oppositely induce excitatory and inhibitory influence on gastric motility; 2) increased c-Fos expression of neurons in l CEA is more intense than that in m CEA induced by RWIS; 3)increased c-Fos expression of CRH neurons in CEA and HPA activity induced by RWIS are different from those induced by RS. Along with previous reports on the role of CRH and HPA activity in the development of stress ulcer, Our findings suggest that characteristic activity of CEA neurons involved in RWIS are more protective than destructive to the gastric mucosal. However, more data and further evaluation on the role of CEA in the stress-induced gastric mucosal injury areneeded. Although RWIS induce both intense activity of CEA neurons and gastric mucosal injury,it’s too easy to assure the causality existed between them. And other relative central mechanisms of the development of gastric ulcer induced by RWIS needs to be further investigated.