| OBJECTIVEThe purpose of this experiment is to record and identify GnRH neurons in hypothalamus and to confirm the particular acupoints and/or parts of body that activate GnRH neurons effectively by acupuncture stimulation, Besides, we observed the background discharges of GnRH neurons and their response to simulated sex stimulation, pinch and acupuncture at the different stages of the estrous of female rats.METHODSThe experiment was performed on 49 Sprague-Dawley virgin adult female rats weighing 250 ± 20g and 11 virgin adult male rats weighing 300 ± 60g. Each female rat's estrous stage was evaluated at beginning of the experiment by vaginal smears. The rats was anesthetized by urethane (10%, 1.0-1.2g/kg,i.p).A catheter was inserted into one of the jugular veins to allow an infusion of reagents (|3-Estradiol, Testosterone Propionate and Hydrocortisonethe) arousing a feedback regulation mechanism. Body temperature was monitored and kept around 37 ℃ by a feedback-controlled heating blanket.The head of rat was fixed in a stereotaxic apparatus. Incisions were made in the skin along the midline over the head. For the medial preoptic area, the arcuate nucleus and the paraventricular nucleus of hypothalamus extracellular recording the discharges of neurons with glass microelectrodes, a craniotomy was performed to expose the area of cortex above the hypothalamus. The dura mater was cut and brain tissue was exposed and then covered with warm paraffin oil.Simulated sex stimulation was generate by electrical stimulating (duration 0.5ms) the dorsal clitoris nerve, tampon rubbing clitoris , vibrator (12Hz) vibrating vagina and cervix in order to activate the neurons related with neuroendocrine of procreatione inthe hypothalamus.Noxious mechanical cutaneous stimulation was produced by pinching various skin fields and needing 26 acupoints which last for 30 seconds, and observed if there was any change in discharge activity of the hypothalamic neurons related with neuroendocrine of procreation and the magnitude of change to identify the most effective parts and acupoints of body.RESULTS1 The activity for the neurons related with neuroendocrine of procreatione in the hypothalamus of female rats by acupuncture and identification of effective acupointsThere were 354 neurons recorded in the hypothalamus. 120 neurons were excited by three kinds of stimulated sex stimulation, 65 neurons were inhibited and 169 neurons were not response. Both the excitory neurons and the inhibitory neurons had evident response to simulated sex stimulation (P<0.01~0.001).132 neurons were recorded in 18 estrous female rats, accounting for 37% of all neurons recorded, 72 neurons were excited, 21. neurons were inhibited. 222 neurons were recorded in 31 anestrous female rats, accounting for 63% of all neurons recorded, 48 neurons were excited, 44 were inhibited. The results showed that the excitory neurons at the stage of estrous were more than at the stage of anestrous.Means of the background discharges of the excitory neurons at the stage of estrus (1.75 ± 0.25) was greater than at the stage of anestrous (1.02 ± 0.14). Response after stimulation at the stage of estrus was higher than at the stage of anestrous (P<0.01). However, Means of the background discharges of the inhibitory neurons at the stage of estrus (1.64 ± 0.35) was smaller than at the stage of anestrous (4.22 ± 0.95). Response after stimulation at the stage of estrus and at the stage of anestrous did not change (P>0.05).The background discharge of 33 excitory neurons selected randomly reduced from 2.05 ± 0.36 spikes/s to 0.97 ± 0.22 spikes/s after the female rats being injected p-Estradiol (P<0.001), with an reducing rate of 45.81% ,indicating that (3-Estradiol acts negative feedback regulation on the excitory neurons in hypothalamus. The background discharge of 2 excitory neurons injected Hydrocortisonethe did not respond. However, the background discharge of 14 inhibitory neurons injected (3-Estradiol did not distinctlychange (reducing rate 11.42%), hinting P-Estradiol could not act negative feedback regulation on the inhibitory neurons in hypothalamus. The background discharge of 3 inhibitory neurons injected Hydrocortisonethe also did not respond.Pinching of various skin fields on or near "zigong (Ex-CAl)", "zusanli (S36)", "guanyuan (CV4)", "neiguan (P6)" acupoints, the discharges of the excitory neurons obviously increased (P<0.05~0.01). Comparing with acupuncture these acupoints, their response almost was identical (P>0.05).We compare the response of the excitory neurons after acupuncture 26 acupoints to ascertain the most effective acupoints activating the neurons related with neuroendocrine of procreatione in the hypothalamus. The result showed that stimulation of acupoints located in low abdominal, low back and hind limb area can induced the most significant effect (P<0.01~0.001) and stimulation of acupoints located in face and ear can induced the very visible effect (P<0.01). Because stimulating "Ex-CAl" acupoint in low abdominal produced the most effective response, we compared the response stimulated "Ex-CAl" acupoint with "sanyinjiao (Sp6)", "quchi (LIU)", "tanzhong (CV17)" acupoints and ear area. Between "Ex-CAl" and "Sp6", "ear" had not remarkable difference (P>0.05), indicating that their acupuncture effect was identical. However, between "Ex-CAl" and "LIU", "CV 17" had remarkable difference (P<0.01), indicating that their acupuncture effect was distinct. To the inhibitory neurons, its result was similar to the excitory neurons.2 The activity for the neurons related with neuroendocrine of procreatione in the hypothalamus of female rats by acupuncture and identification of effective acupointsThere were 95 neurons recorded in the hypothalamus. 33 neurons were excited by stimulated sex stimulation, 27 neurons were inhibited. Compared with female rats, the rate of the inhibitory neurons was greater. Both the excitory neurons and the inhibitory neurons had evident response to simulated sex stimulation (P<0.01~0.001).The background discharge of 14 excitory neurons selected randomly reduced from 2.23 ± 0.44 spikes/s to 0.69 ± 0.25 spikes/s after the male rats were injected Testosterone Propionate (P<0.0/),with an reducing rate of 71.18%, indicating that Testosterone Propionate acted negative feedback regulation on the excitory neurons in hypothalamus. The background discharge of 3 inhibitory neurons injected Testosterone Propionate did not distinctly changed.The result was similar to female rats, compared the response of the neurons related... |
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