河蟹眼柄神经分泌细胞离子通道的膜片钳研究

采用全细胞膜片钳技术对培养12－24小时不同形态河蟹眼柄视节端髓X器官（MTXO）神经分泌细胞离子通道进行了研究。结果表明,河蟹眼柄MTXO中分布的A、B、C三种类型神经分泌细胞均可记录到由向电流和外向电流组成的正常全细胞电流。内向电流由高电压激活钙离子通道电流（Lca)和对TTX敏感钠离子通道电流（INa）组成。ICa的激活电压为－30mV,在0－ 20mV电压下达到峰值,在－40mV和－70mV保持电压下记录的ICa激活阈值、初始峰值及I－V曲线无明显差别。外向电流明显,幅值较大,包括对4－AP敏感的快速激活、快速失活钾离子通道电流（IA）和对TEA敏感的缓慢激活、缓慢失活钾离子通道电流（IK）。正常蟹种、二龄成蟹和早熟蟹种MTXO神经分泌细胞均表达电压门控钠、钾、钙离子通道,通道电流和电压特征无明显区别.     

Neurosecretion in the external and internal zone of the median eminence of the cat and dog

Summary The immunoglobulin-enzyme bridge technique, in association with rabbit antiporcine neurophysin-II has been applied to the median eminence of the dog and cat in order to study the distribution of neurophysin-like antigens throughout this area of the brain and correlate the findings with the corresponding distribution of neurosecretory material (NSM) as revealed by the crotonaldehyde fuchsin stain.Neurophysin and NSM were both present in the hypothalamo-supraoptico-neurohypophysial system—the pathway taken by oxytocin, vasopressin and neurophysin from the hypothalamus to the posterior pituitary lobe.Whereas the tuberoinfundibular tract of the median eminence was almost devoid of NSM, the presence of neurophysin-like material was clearly evident using immunoperoxidase histochemistry. The significance of a protein in the external median eminence possessing determinants cross-reactive against anti-neurophysin serum is discussed.This work was financed by a grant from the Medical Research Council of New Zealand.     

Electron microscopic histochemistry of cholinesterase in the posterior,intermediate, and anterior lobes of the rat pituitary

Summary The three lobes of the pituitary gland of the rat were examined histochemically for specific (AChE) and non-specific (BuChE) cholinesterase at the light and electron microscopic levels. Acetylthiocholine was utilized in conjunction with ethopropazine to demonstrate AChE, and butyrylthiocholine with BW284C51 to demonstrate BuChE. Using the histochemical method of Lewis and Shute, only BuChE was detected in the posterior pituitary by both light and electron microscopy; the enzyme was localized to certain pituicytes, including the endoplasmic and nuclear membranes of these cells and the pituicyte-neurosecretory neuron junctions. Endothelial cells of the posterior pituitary were also BuChE-positive. In the intermediate lobe, AChE was localized to the polygonal glandular cells, whereas BuChE was localized to cells of the interlobular septa and to elongated, densely staining cells which penetrate the lobules. In the anterior lobe, cells were positive for AChE, whereas follicular cells were positive for BuChE.Supported by the Medical Research Council of Canada.Medical Research Associate of the MRC of Canada.The authors wish to acknowledge the technical assistance of Mrs. Maria Prasher.     

Vacant postsynaptic densities on supraoptic dendrites of adult rats diminish in number with chronic stimuli

Summary In earlier ultrastructural studies of the supraoptic nucleus in adult rats we noted free and incompletely covered postsynaptic densities (collectively referred to here as vacant postsynaptic densities) on dendritic shafts. Free postsynaptic densities have been reported in other parts of the central nervous system of normal rodents. We investigated the possibility that physiological activation of the supraoptic cells, which produces changes in many aspects of their morphology, would alter the incidence of the free or incompletely covered postsynaptic densities on dendrites in the supraoptic basal dendritic zone. The cells of the supraoptic nucleus are activated to increase cell firing and secretion of oxytocin and/or vasopressin in response to dehydration, gestation, and lactation. We have examined: (i) untreated virgin females; (ii) untreated males; (iii) 24 h water-deprived males; (iv) prepartum (21st day of gestation) females; (v) postpartum females (on the day of parturition); (vi) lactating females (14 days of suckling); (vii) mothers 10 days after weaning their pups; (viii) females given 2% saline to drink (dehydrated) for 10 days; and females or males given 2% saline to drink for 10 days, then given tap water for (ix) 2 or (x) 5 weeks to allow rehydration. Only long-term activation of the supraoptic nucleus by lactation or by drinking saline for 10 days brought about significant decreases in the percentage of dendrites with vacant postsynaptic densities. These densities did not reappear in saline treated rats which had been rehydrated for 2 weeks, but did return in both the 5-week rehydration and the 10-day postweaning groups. Short-term activation of the supraoptic nucleus, such as occurs at parturition or in acute dehydration, did not affect the vacant postsynaptic densities. Analysis of semiserial thin sections indicated that presynaptic elements facing the incompletely covered postsynaptic densities contain predominantly clear round vesicles and also that apparently free postsynaptic densities were usually at least partially contacted by a presynaptic ending in adjacent sections.     

Release of a neurosecretory protein from the corpora cardiaca of Locusta migratoria induced by high potassium saline and compound action potentials

Electrical stimulation of nervus corpus cardiacum I (NCCI) resulted in the propagation of a compound action potential into the storage and glandular lobes of the corpus cardiacum (CC) of Locusta migratoria. The compound action potential was abolished in the presence of both sodium-free saline and tetrodotoxin (TTX). Calcium-free saline had variable effects.The release of a neurosecretory protein (estimated following precipitation with an antiserum directed against neurosectetory protein) was examined after treatment with high potassium saline and electrical stimulation of NCC I. Release was induced by elevated potassium salines and by the propagation of a compound action potential along NCC I. The release was calcium-dependent. TTX and sodium-free saline abolished the electrically-induced release of protein. Concomitant with the release of protein was the release of a factor with diuretic activity, illustrating that hormones are also being released along with the neurosecretory protein. The release of this protein was dependent upon the frequency of electrical stimulation (up to approx. 5 Hz) and the patterning of electrical stimulation. This neurosecretory protein which has previously been shown to be very similar in both size and amino acid composition to the pituitary neurophysins, now also shares the characteristic of being released along with hormone.     

Isolation and characterization of neurosecretory granules of the rat posterior pituitary gland

Summary Neurosecretory granules (NSG) of rat posterior pituitary glands were prepared by differential centrifugation techniques mainly according to the procedure as described by Barer, Heller and Lederis (1963). As revealed by electron microscopy, the recovery of neurophysin and the contents of enzymes, purified NSG were obtained in a pellet at 30 000 g/60 min (0.44 M sucrose). Eighteen h after injection of (³⁵S) cysteine into the supraoptic nucleus 60% of the recovered radioactivity in the neural lobe was found in the NSG, whereas 20% was found in the final supernatant (100 000 g/120 min). Sixteen days after injection the NSG and the final supernatant fraction contained fairly equal amount of (³⁵S) cysteine (approximately 40%). It is suggested that after a period of intragranular maturation neurophysin is extruded into an extragranular pool of neurosecretory material.With the use of conventional polyacrylamide-gel electrophoresis it was shown that the predominating proportion of radioactivity in the NSG after a hypothalamic injection of (³⁵S) cysteine was located within the neurophysin fraction A and in fraction B. Fraction B is suggested to be partly bound to the NSG membranes. When the NSG soluble and NSG insoluble proteins, obtained after lysis of NSG, were separated on polyacrylamide gels in the presence of sodium dodecylsulphate, the highly radioactive soluble protein was shown to consist of two components with average molecular weights of 12 300 and 14 600. Most of the proteins in the lysate were found in the NSG membranes, though less radioactive. A component with a mol.wt. of 37 000 was enriched in the membrane fraction. At longer times after isotope injection the high mol.wt. proteins, particularly those of the NSG membranes, contained increased amounts of radioactivity.Abbreviations NSG neurosecretory granule - NSM neurosecretory material - SON supraoptic nucleus The present investigation was supported by grants from Svenska Livförsäkringsbolags nämnd för medicinsk forskning from Svenska Sällskapet för Medicinsk forskning, from the Medical Faculty, University of Göteborg, and from the Swedish Medical Research Council (B 72-12X-2543-04A).We are indebted to Mrs. Marie-Louise Eskilsson, Mrs. Wally Holmberg and Mrs. Ulla Svedin for technical assistance, and to Miss Gull Grönstedt for careful secreterial work.     

Neurophysin and the neurosecretory fibres of the sheep infundibulum

Summary A cross-species reactive antiserum was used to study the cellular localization of neurophysin in the sheep hypothalamus and neurohypophysis. The immunofluorescent technique was used to follow the neurosecretory fibres emanating from the magnocellular nuclei to the lower infundibular stem. It was confirmed that neurophysin was present in both the internal and external zone of the infundibulum.This work was supported by a research grant from the New Zealand Medical Research Council.