在体膜片钳方法的建立及其在神经痛研究中的应用

The local circuits, which were involved in nociceptive transmission and antinociceptive modulation, and the related neurotransmitters, neuromodulators, receptors and intracellular signal transduction mechanisms in the spinal dorsal horn were studied in the present project supported national natural science fundation of China (No.39970239) by using combined modern neuroscience research methods. The main results are as follows:(1) The g-aminobutyric acid receptor B subtype (GABABR) immunoreactive (GABABR-ir) neurons were observed densely in the brainstem raphe nuclei, periaqueductal gray (PAG) and alpha part of the gigantocellular reticular nucleus (GiA) of the rat by using immunohistochemical staining technique. Most of these GABABR-ir neurons also contained 5-hydroxytryptamine (5-HT). The GABA-ir terminals contacted closely with the somata and dendrites of the GABABR/5-HT double-labeled neurons in the brainstem raphe nuclei, PAG and GiA. These results suggested that GABA might affect the activities of the 5-HT-sergic neurons through binding to GABABR.(2) The electrophysiological and morphological properties of the neurons in laminae I and III of the rat medullary dorsal horn (MDH) were studied by using intracellular recording and biocytin-injections combined with histochemical and immunohisto- chemical staining methods. According to their electrophysiological and morphological properties, neurons in lamina I were divided into two kind projection neurons, while neurons in the suoperficial part of lamina III were grouped into projection neurons and intrinsic interneurons, these two kind neurons were subdivided into two kind projection neurons and two kind interneurons. A new green fluorescence protein (GFP) gene recombinant virus labeling method was established and employed in investigating the morphological features of the neurons in the deeper part of laminae III. According to the morphological features of the neurons in the deeper layer of laminae III, they were also divided into rojection neurons and interneurons.(3) There were some enkephaline or protein kinase C g subunit (PKCg) and three main calcium-binding proteins: calbindin-D28k, calretinin and parvalbumin co-existed neurons in the medullary dorsal horn of the rat confimed by the mmunohistochemical double-staining method. Of the projection neurons which send their axons to the thalamus, over 90% of them showed immunoreactivity for PKCg. These results suggested that PKCg and enkephalin might play important roles of in the ransmission and modulation of nociceptive information processing. (4) 5-HT-immunoreactive terminals made close contacts with the somata and dendrites of the 5-HT2A receptor (5-HT2AR)/GABA, 5-HT2AR/glycine (Gly) double-labeled neurons in the spinal dorsal horn were observed by using immunofluorescence triple-labeling technique. By using whole cell voltage-clamp recording technique, it was found that 5-HT potentiated the inhibitory effects of GABA and Gly to neurons in the spinal dorsal horn, which were mediated by 5-HT2AR and through Ca2+-indepentent PKC intracellular signal transduction pathway. Those results provid evidence for the antinociceptive effect of 5-HT through intereaction with inhibitory interneurons..(5) Using whole cell voltage-clamp recording technique, we found that noradrenaline (NE) inhibited the miniature excitatory postsynaptic currents (mEPSCs) which were induced by capsaicin application and mediated by glutamate release in substantia gelatinosa of the spinal dorsal horn. The results suggest that NE might play significant roles in inhibiting nociceptive inputs at the presynaptic sites. (6) Topical capsaicin application to the sciatic nerve inducing the Fos protein expression in the lumbar dorsal horn were observed by using immunohistochemical staining technique, while topical applications of capsazepin, lidocaine and colchicine to the sciatic nerve reduced the number of Fos-like immunoreactive neurons. The results suggest that capsaicin receptors are involved in nociceptive transmitsion..(7) Intrathecal pretreat

首先建立在体膜片钳方法。用在体膜片钳全细胞记录正常和神经损伤大鼠经皮给予触、压、痛刺激在脊髓II层神经元诱发的突触应答，根据潜伏期计算纤维传导速度推论传入纤维的种类并用药理学方法证实；中枢刺激和脊髓表面施予神经递质受体阻断剂对突触应答的影响；最后用形态手段确认机能学研究的结果。为神经损伤后顽固性疼痛的治疗提供线索和依据。