Fine structure of the median eminence and the pars nervosa of the bullfrog,Rana catesbeiana

Summary The hypothalamic neurosecretory system of the bullfrog, Rana catesbeiana, was studied with light- and electron microscopy. The median eminence is roughly divided into two portions. The upper portion mostly consists of ependymal cells, glial cells and preoptico-hypophysial nerve tract, whereas in the lower portion, neurosecretory axons, glial cells, processes of glial and ependymal cells, and fine blood vessels of the hypothalamic portal vein are located. A part of the neurosecretory axons of the preoptico-hypophysial tract proceeds to the lower portion of the median eminence. These axons are arranged perpendicularly to the capillaries of the hypothalamic portal vein. The glial cells are densely located in the area of the median eminence where neurosecretory material is abundant. The neurosecretory material in the neurosecretory cells, their axons, the median eminence and the pars nervosa of the bullfrog shows a positive reaction to PAS treatment.The neurohemal area of the median eminence is occupied by many neurosecretory and non-neurosecretory axons, containing neurosecretory granules and/or synaptic vesicles. The axonal portions with the synaptic vesicles which are considered to be the nerve endings abut on the capillaries of the portal system. The size of synaptic vesicles in the axon terminals containing few neurosecretory granules is larger than those in the endings with many neurosecretory granules. Infrequently glial and ependymal processes are interposed between the nerve endings and the capillary wall.In the hilar region of the infundibulum, synapses are frequently observed between the thin fibers with or without neurosecretory granules and dendrites of non-neurosecretory neurons. The probable functions of these synapses are briefly discussed on the basis of our findings. Both in the hilar region of the infundibulum and in the pars nervosa, electron-dense neurosecretory granules of two different sizes were observed. The median eminence contains only one type of granules.The fine structure of the pars nervosa shows similar structures to those of the median eminence. Both in the median eminence and the pars nervosa, the fenestrated endothelium of the capillaries was frequently observed. The thick perivascular connective tissue space containing fibroblasts and collagen fibrils was observed both in the median eminence and the pars nervosa. Vesicles in the cytoplasm of the endothelial cells which appear to take a part in the transendothelial transport were observed.This investigation was supported in part by United States Public Health Service Research Grant, No. A-3678, to Hideshi Kobayashi from the National Institute of Arthritis and Metabolic Diseases and partly by a grant for Fundamental Scientific Research from the Ministry of Education of Japan. The authors wish to express their thanks to Prof. K. Takewaki for his kind encouragement.     

Synaptic and Synaptoid Vesicles Constitute a Single Category of Inclusions. New Evidence From ZIO Impregnation

Parallel observations on central synaptic and neurohaemal terminals of the same types of neurosecretory fibres in the polychaete annelid Nereis diversicolor reveal that their respective populations of inclusions exhibit identical, highly distinctive patterns of affinity for the zinc iodide-osmium tetroxide (ZIO) reagent. The method highlights the duality of possible secretory inclusions in nerve terminals. Many typical synaptic/synaptoid vesicles have ZIO-positive contents, but intermingle with unreactive vesicles. Both positively and negatively reacting vesicles contribute to the unusual dense clusters associated with sites of release of neurochemical mediators, characteristic of polychaete nervous systems. Fewer dense-cored synaptic/synaptoid vesicles have reactive cores. The larger ‘storage granules’ typically have unreactive contents, but dense deposits form within a small minority. A possible cytophysical, in contradistinction from a cytochemical, basis of affinity for ZIO is discussed. The results further support the postulated fundamental identity of synaptic and synaptoid vesicles.     

The zinc iodide-osmium (ZIO) reaction in the central nervous system: localization and relations to functional activity.

The fine structural characteristics of ZIO reaction was studied in the cerebral and cerebellar cortex and olfactory bulb of the rat and in synaptosomes prepared from rat cerebellar cortex. It was concluded that: 1. Organelles of different nerve cell types exhibit different ZIO reactions provided that the impregnation was carried out under standardized conditions. 2. 6...10 times more synaptic vesicles were stained by ZIO in the inhibitory terminals than in the excitatory ones. 3. ZIO positivity was found in all types of synaptosomes prepared from cerebral cortex. Following electrical or chemical (KCl) depolarization there was a decrease in the number of ZIO positive synaptic vesicles, which decrease was directly proportional to the parameters of stimulation. 4. By x-ray microanalysis Os, Zn and Ca were consistently detected in the ZIO precipitates. Iodine, however, could not always be found. After stimulation the presence of Ca was observed even in those synaptosomes in which the ZIO reaction product was absent. 5. On the basis of the staining characteristics the reaction, under standard conditions, can reflect certain functional states of the nerve terminals.     

Ultrastructure of the hypothalamic neurosecretory system of the dog

Summary The hypothalamic neurosecretory system of normal dogs was studied by light and electron microscopy after perfusion-fixation. In the supraoptic nucleus most neurons are loaded with elementary neurosecretory granules having a content of low electron density. Neurons with less neurosecretory material and signs of enhanced synthetic activity, as recognized by the changes in the endoplasmic reticulum, were also observed.The vesiculated neurons ofJewell were studied under the electron microscope and various stages of development were described. It was postulated that they originate by a localized process of cytoplasmic cytolysis which ends in the formation of a large aqueous intracellular cavity limited by a plasma membrane. The possible significance of these vesiculated neurones is discussed. Some few myelinated neurosecretory axons are found in the supraoptic nucleus.The neurons of the paraventricular nucleus are smaller and contain less neurosecretory material. This is abundant and very pale in the axons. The median eminence consists of an inner zone, mainly occupied by the neurosecretory axons of the hypothalamic-neurohypophysial tracts, and an outer zone in which some neurosecretory axons end on the capillary of the portal system. This outer zone contains numerous axons with the axoplasm rich in neurofilaments and some containing granulated and non-granulated synaptic vesicles. Some neurons with granulated vesicles were observed in this region. The adrenergic nature of these neurons and axons is postulated.The infundibular process of the neurohypophysis shows small axons with discrete amounts of elementary granules and vesicles of synaptic type at the endings. Some enlarged axons having, in addition, large polymorphic bodies are observed and related to the Herring bodies.The size and morphology of the granules are analyzed along the entire hypothalamic-neurohypophysial system. The changes in diameter and electron density are related to the maturation of the granules and the possible significance of such evolution.Supported by grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas and by the Air Force Office of Scientific Research (AF-AFOSR 963-66).     

Ultrastructure of the peptidergic and monoaminergic neurons in the hypothalamic neurosecretory system of Anuran Batracians

Summary In the toad Bufo arenarum Hensel the following regions of the hypothalamic — neurohypophyseal system were studied under the electronmicroscope: preoptic and paraventricular nuclei, median eminence and infundibular process of the neurohypophysis.Neuronal perikarya of the preoptic nucleus are loaded with typical neurosecretory granules of peptidergic nature having a mean diameter of 1660 Å. While most neurons of the winter toad are in a storage stage a few show signs of a more active synthetic activity. A distinctive feature of preoptic neurons is the presence of large lipid droplets. The paraventricular nucleus contains small neurons containing granulated vesicles with a mean diameter of 800-1000 Å. In the region extending between these two nuclei and the median eminence axons containing either neurosecretory elementary granules or granulated vesicles are observed.The inner zone of the median eminence is occupied by axons of the preoptic neurohypophyseal tract; two types of axons, according to the size and density of the neurosecretory granules, may be recognized. The outer zone of the median eminence contains mainly axons and nerve terminals containing granulated vesicles of probable monoaminergic nature and only a few with granules of peptidergic type.The neurohypophysis contains two kinds of axons: one with more dense granules of 1800 Å and the other with granules of lesser electron density and 2100 Å. At the ending proper small clear vesicles of synaptic type are found.A progressive increase in volume of the peptidergic granules along the axon is demonstrated. This is of the order of 218% from the preoptic perikarya down to the infundibular process. The physiological significance of the two neurosecretory systems — i.e. the monoaminergic and the peptidergic — and the probable nature of the two types of peptidergic axons is discussed.Supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas and by the Air Force Office of Scientific Research (AF-AFOSR 963-67).The authors want to express their gratitude to Mrs. Defilippi-Novoa and Mr. Alberto Sáenz for their skillful assistance.     

Microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of presynaptic nerve terminals

Nerve endings of the posterior pituitary are densely populated by dense- core neurosecretory granules which are the storage sites for peptide neurohormones. In addition, they contain numerous clear microvesicles which are the same size as small synaptic vesicles of typical presynaptic nerve terminals. Several of the major proteins of small synaptic vesicles of presynaptic nerve terminals are present at high concentration in the posterior pituitary. We have now investigated the subcellular localization of such proteins. By immunogold electron microscopy carried out on bovine neurohypophysis we have found that three of these proteins, synapsin I, Protein III, and synaptophysin (protein p38) were concentrated on microvesicles but were not detectable in the membranes of neurosecretory granules. In addition, we have studied the distribution of the same proteins and of the synaptic vesicle protein p65 in subcellular fractions of bovine posterior pituitaries obtained by sucrose density centrifugation. We have found that the intrinsic membrane proteins synaptophysin and p65 had an identical distribution and were restricted to low density fractions of the gradient which contained numerous clear microvesicles with a size range the same as that of small synaptic vesicles. The peripheral membrane proteins synapsin I and Protein III exhibited a broader distribution extending into the denser part of the gradient. However, the amount of these proteins clearly declined in the fractions preceding the peak of neurosecretory granules. Our results suggest that microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of all other nerve terminals and argue against the hypothesis that such vesicles represent an endocytic byproduct of exocytosis of neurosecretory granules.     

Fine structure of islet-cell innervation in the pancreas of normal and alloxan-treated rats

Summary The innervation of the islets of Langerhans of normal albino rats and of albino rats treated with several daily doses of 125 mg/kg of alloxan was studied by electron microscopy. In the normal rat, nerve endings containing either agranular vesicles (200–400 Å) alone or in combination with large granular vesicles (500–800 Å) were found on both alpha and beta cells. Infrequently a third type of nerve ending containing small granular synaptic vesicles could be observed. Bundles of unmyelinated axons were also seen, as were typical autonomic ganglion cells. Similar normal neural elements were noted in rats treated with alloxan. However, islets of alloxan-treated animals also possess large elliptical profiles which appear to be dystrophic nerve terminals. These structures most frequently contact degranulated beta cells. Islets of Langerhans fixed with zinc iodide-osmium (ZIO) reported to specifically impregnate synaptic vesicles were also studied. Synaptic vesicles of normal axons and nerve endings as well as of the dystrophic structures were filled with ZIO reactive material. These studies suggest that alloxan may induce autonomic nerve ending changes in the rat endocrine pancreas. This may result from neuronal hyperactivity in an attempt to secrete insulin from the post-alloxan insulin-depleted beta cell.     

Ultrastructure of the neurosecretory cells of the anterior commissural nucleus of the hypothalamus in rats

The ultrastructure of neurosecretory cells of the anterior commissural nucleus of rat hypothalamus is similar to that of the supraoptic nucleus and of the "magnocellular" part of the paraventricular nucleus. The only difference is a less expressed granular endoplasmatic reticulum and a smaller diameter of elementary neurosecretory granules (80-150 nm in diameter). Such elementary granules are characteristic of neurosecretory terminals located in the external zone of the median eminence. It is suggested that neurosecretory cells of the anterior commissural nucleus project to this neurohemal region.     

Photoperiodic and osmotic influences on the ultrastructure of the hypothalamic neurosecretory system of the White-crowned sparrow,Zonotrichia leucophrys gambelii

Summary An attempt was made to correlate functional changes in the neurohypophysis of the White-crowned Sparrow, Zonotrichia leucophrys gambelii, with morphologic features on the light- and electron-microscope levels. The aldehyde-fuchsin-staining anterior median eminence possesses essentially the same ultrastructural features as the non-staining posterior median eminence. The axon terminals are characterized by the presence of a large number of small vesicles (approximately 400 Å in diameter) and occasional electron-dense granules. The more-or-less depleted anterior median eminence occasionally evident in the photosensitive bird showing testicular development is indistinguishable ultrastructurally from the more intensely staining median eminence generally characteristic of the photorefractory bird. In the median eminence, stainability and functional state do not seem to be correlated with changes in the type, size or number of vesicles. A slight increase in the number of granules was noted in the photorefractory bird but this was considered insufficient basis to account for differences in stainability.The pars nervosa, on the other hand, responded to osmotic stimuli (saline drinking water) by loss of stainability and decrease in numbers of elementary neurosecretory granules. Small vesicles are also present in the pars nervosa axon terminals, but are intermingled with neurosecretory granules in normal birds. Acute-osmotic birds, however, had axon terminals almost entirely occupied by small vesicles.It is to be emphasized that the pars nervosa and the median eminence are two structurally very different regions of the neurohypophysis. The basis for aldehyde-fuchsin staining in the median eminence appears to differ from that in the pars nervosa. The implications of these findings are considered in regard to hypothalamic control over gonadotropic activity in the White-crowned Sparrow.Dedicated to Professor Dr. W. Bargmann in honor of his 60th birthday.This investigation was supported by grant GB-2484 from the National Science Foundation to Professor Bern, grant GB-2819 from the National Science Foundation to Professor Mewaldt, and grant NB-01353 from the National Institutes of Health to Professor Farner. The authors wish to express their appreciation of the technical assistance of Mrs. Irene Brown, Mr. John Butchart, Sally S. Kibby, Mrs. Carol Nicoll, and Mr. John Striffler. Mrs. Emily Reid kindly prepared the histograms.     

Direct projection from the medial preoptic area to the median eminence of the cat.

The projection from the medial preoptic area to the median eminence of the cat was clarified by electron microscopy. After placing the electrolytic lesion in the preoptic area several kinds of degenerating neuronal processes and terminals were observed in the external layer of the median eminence. The one was dark shrunk terminals containing dense cored vesicles, the other was the dark ones containing myeline figure-like structure. The relationship between catecholamine-containing nerve endings and RH/IH-containing endings in the external layer of the median eminence was discussed.     

Ecologic-histophysiological analysis of the neurohypophysis-metaadenohypophysis complex of the sazan female before and after spawning]

Much neurosecretory material (4.4 +/- 0.1 units) is seen in the neurohypophysis of sazan females before spawning. A1, A2, and B type terminals contain numerous neurosecretory granules, in particular, elementary granules. Synaptic vesicles are not numerous. The amount of neurosecretory material decreases during and shortly after spawning down to 3.6 +/- 0.3 units. Neurosecretory terminals have less elementary granules. The number of granulated, disintegrating, residual granules and synaptic vesicles somewhat increases. It is hypothesized that mainly peptide neurohormones are discharged from A1 and A2 terminals in the common circulation, and that both peptide neurohormones and monoamines reach glandular cells of the metaadenohypophysis in sazan females during and immediately spawning. The role played by neurohormones and monoamines in controlling the function of both the metaadenohypophysis glandular cells and visceral organs involved in the general adaptive reactions of the fish organism is discussed.     

ISOLATION OF NERVE ENDINGS FROM THE POSTERIOR PITUITARY GLAND : Electron Microscopy of Fractions Obtained by Centrifugation

Bovine posterior pituitary glands were homogenized in 10 per cent sucrose and fractionated by differential centrifugation. The following centrifugation procedure resulted in the most satisfactory separation: 1000 g for 15 minutes—nuclei, connective tissue, basement membranes with associated endothelium, giant nerve endings, and whole pituicytes; 4200 g for 15 minutes—free nerve endings, including Herring bodies; 17,000 g for 15 minutes—mitochondria; 68,000 g for 15 minutes—neurosecretory granules. Electron microscopic examination was carried out on whole tissue and on the isolated fractions. Isolated nerve endings were examined also by negative staining techniques. Isolated nerve endings retain an apparently normal complement of mitochondria, neurosecretory granules, and microvesicles ("synaptic" vesicles). The free nerve endings closely resemble those observed in sections of intact posterior pituitary tissue. Free microvesicles were not observed in any of the fractions isolated and apparently sediment at centrifugal forces higher than those employed in this study.     

Zinc-iodide-osmium procedures as markers of subcellular structures. I. Standardization of staining of transmitter containing vesicles

In the present investigation certain stain properties of the zinc iodide-osmium tetroxide mixture were investigated. It was observed that the type of reaction of certain cell structures with a ZIO mixture largely depended on several factors, namely, the pH of the mixture, aldehyde prefixation and type (s) of buffer (s) used. The standardization of these parameters led to the development of four procedures, each one of them with distinct stain properties. A nomenclature to designate these methods is proposed. The following procedures were applied to material processed for electron microscopy: 1. C.4.4-ZIO-4 degree -18 h: the ZIO mixture was prepared in citric acid-disodium phosphate buffer pH 4.4 and the tissue was incubated at 4 degree C during 18 H; 2. K-P.7.4-C.4.4-ZIO-4 degree -18 h: the tissue was prefixed in Karnovsky fixative prepared in phosphate buffer pH 7.4 and then incubated in C.4.4-ZIO at 4 degree C during 18 h; 3. V.7.4-ZIO-4 degree -18 H: the ZIO was prepared in veronal buffer pH 7.4 and incubation of the tissue was at 4 degree during 18 H; 4.K-P.7.4-V.7.4-ZIO-4 degree -18 h: the tissue was prefixed in Karnovsky fixative prepared in phosphate buffer pH 7.4 and then incubated in V.7.4-ZIO at 4 degree C during 18 h. The chromaffin cells and the cholinergic endings of the rat adrenal medulla and the vas deferens nerves were studied. C.4.4-ZIO-4 degree -18 h: This procedure stained adrenaline and noradrenaline storing granules. Synaptic vesicles at cholinergic endings were not stained. K-P.7.4.4-ZIO-4 degree -18 h: One type of chromaffin granule (probably storing noradrenaline) and both, the small and the granulated synaptic vesicles of cholinergic endings were deeply stained with this method. The aminergic fibres of the vas deferens reacted synaptic vesicles at cholinergic endings were not stained. K-P.7.4.4-ZIO-4 degree -18 h: One type of chromaffin granule (probably storing noradrenaline) and both, the small and the granulated synaptic vesicles of cholinergic endings were deeply stained with this method. The aminergic fibres of the vas deferens reacted synaptic vesicles at cholinergic endings were not stained. K-P.7.4.4-ZIO-4 degree -18 h: One type of chromaffin granule (probably storing noradrenaline) and both, the small and the granulated synaptic vesicles of cholinergic endings were deeply stained with this method. The aminergic fibres of the vas deferens reacted negatively. V.7.4-ZIO-4 degree -18 H: Both types of chromaffin granules and only the small synaptic vesicles of cholinergic endings were revealed with this procedure. In addition, some compartments of the Golgi complex were also stained. K-P.7.4-V.7.4-ZIO-4 degree -18 h: This method did not stain adrenaline and noradrenaline storing granules. Cholinergic synaptic vesicles appeared stained. However, the most striking stain property of this procedure was the staining of many cell organelles. The probable mechanisms by which different factors affect the ZIO reaction are discussed.     

Electronmicroscopic immunocytochemical study on the vasopressin-containing neurons of the thirsting rat

Summary Whereas in thirsting animals the perikarya of the nucleus supraopticus are nearly empty of neurosecretory granules as evidenced by electron microscopic observation, the perikarya are heavily stained by light microscopic immunohistochemical staining. In an attempt to discover the substrate responsible for the positive immunohistochemical staining in thirsting rats, the neurons of the supraoptic nucleus of normal and long-term thirsting animals were compared by electron microscopic immunocytochemistry (indirect PAP-method). In controls all parts of the vasopressin-synthesizing neuron are filled with elementary granules which render a positive and uniform reaction after immunostaining with the indirect PAP-method. The positively reacting fibers in the external zone of the median eminence contain smaller granules than those of the tractus supraoptico-hypophyseus. Within the nucleus suprachiasmaticus, no positive reaction after immunostaining was found. In long-term thirsting animals PAP-complexes as markers of vasopressin are located over the ergastoplasm and over the few small elementary granules. The processes within the nucleus supraopticus and the ballooned axons in the internal zone of the median eminence exhibit free, i.e. non granule-bound, PAP-complexes. Findings in the nucleus suprachiasmaticus and the median eminence of thirsting animals correspond to those in controls. The neurohypophysis is almost completely devoid of PAP-labeled elementary granules.From these results it can be concluded that during thirst vasopressin synthesis is increased in the ergastoplasm and that the hormone is transported partly in a non granule-bound form. Direct contacts between neurosecretory cells and the basal lamina are found more often in thirst-stressed animals and are typical of neurohemal regions. It is discussed whether these neurohemal regions may develop transitionally under stress.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/1) and Stiftung Volkswagenwerk. This work was presented in part at the 72nd meeting of the Anatomische Gesellschaft, Aachen 1977     

Ultrastructural changes of the neurohypophysis of the rat after castration

Summary The infundibular processes of the neurohypophysis of male and female rats were studied after different periods of castration. After seven days an increase in neurosecretory granules was observed. Two types of neurosecretory nerve endings were identified: dark ones, with dense neurosecretory elementary granules of 1600 A, and clear ones, with lighter neurosecretory granules of 1800 A. Protoplasmatic pituicytes showed a large increase in lipid granules together with a general hypertrophy. After one week of castration but with hormonal therapy the protoplasmatic pituicytes appeared normal or even showed less lipid granules than in the controls.With one month of castration the changes already mentioned in the nerve endings and pituicytes were more pronounced and after six months even more accentuated. Two types of neurosecretory nerve endings were clearly identified and the protoplasmatic pituicytes were loaded with lipid granules.The probable significance of the two different neurosecretory axons was discussed in relation to recent studies on the isolation of neurosecretory terminals from the neurohypophysis. The changes in the protoplasmatic pituicytes were considered in relation to the possible significance of the lipid granules.Supported by grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas and by the Air Force Office of Scientific Research (AF-AFOSR 963-67).We are deeply indebted to Mrs. Defilippi-Novoa and Mr. Alberto Saenz for their skillful assistence.Associated Investigator, Consejo Nacional de Investigaliones Científicas y Técnicas, Argentina.     

Electron microscopic and pharmacological studies on the rat median eminence

Summary The rat median eminence contains at least three kinds of granules or vesicles: 1. large electron-dense granules (perhaps carriers of neurohypophysial hormones), 2. small electron-dense granules with or without haloes (perhaps carriers of catecholamines) and 3. synaptic vesicle-like structures (perhaps carriers of acetylcholine). The former two electrondense granules exist in separate axons but they coexist with the latter vesicles in the same axons.The pars nervosa shows basically a similar structure to the median eminence. However, the axons containing the small electron-dense granules are very few. In the pars tuberalis, there are at least two types of cells: the cells of one type contain much cytoplasm with large round nuclei and those of the other type contain a small amount of cytoplasm with polymorphic nuclei. The cells of the former include multivesicular bodies and secretory granules, but those of the latter do not. Some of capillaries of the primary plexus are surrounded by the cells of the pars tuberalis on one side and by neurosecretory axon endings on the other side.The median eminence contains high concentration of acetylcholine or an acetylcholine-like substance and shows neurohypophysial hormone activity.Aided by Grant A-3678 from the United States National Institute of Arthritis and Metabolic Diseases. The authors are indebted to Dr. Welsh, Harvard University, for the kind gift of Mytolon.     

Axon terminals with unusual vesicles in the brain of the polychaete,Ophryotrocha puerilis

Summary In the brain of Ophryotrocha puerilis swollen nerve endings filled with electron-lucent vesicles and aggregates of vesicles were observed. The vesicles do not resemble elementary neurosecretory granules. Tests for biogenic amines were negative; no dense-core vesicles were found. The vesicle type described here cannot be related to any of the types thus far found in nerve cells.Supported by Deutsche Forschungsgemeinschaft Pf116/3     

Studies on the transport of secretory granules in the magnocellular hypothalamic neurons of the rat

Intrathecal administration of 20 mug of vincristine sulphate in the rat induced in vivo the formation of paracrystalline inclusions mainly in axonal processes. This is associated with an impairment in the migration of neurosecretory granules as shown by their accumulation in the perikarya of the magnocellular neurons. The granules are intermixed with numerous dense bodies of various shape, sometimes with a fibrillar content, and probably of lysosomal origin. In addition to the impairment of the flow of neurosecretory granules, there is also a striking accumulation of mitochondria and synaptic vesicles, and an apparent proliferation of the smooth endoplasmic reticulum. In the posterior lobe, the axonal endings contain a large number of neurosecretory granules, intermingled with bodies of varying shapes and electron density. Occasionally, a dense membrane surrounding a group of elementary granules is observed, reacting positively for acid phosphatase. This suggests an attempted crinophagia.     

Excitatory Transmission in Insect Neuromuscular Systems

Many, but not all, visceral muscles in insects are innervatedby neurosecretory axons. The neurosecretory junctions with theheart muscle of the American cockroach, Periplaneta americana,show ultrastructural and electrophysiological evidence of chemicallytransmitting synapses, and cytochemical evidence for the presenceof monoamines. Electron microscopy of nerve terminals showsthat synaptic vesicles may be formed directly from electron-dense"neurosecretory" granules Neurotomy of motor axons to skeletal muscles in insects leadsto aggregation and clumping of synaptic vesicles after 48 hours.Treatment of in vitro nerve-muscle preparations with variousrespiratory poisons caused aggregation similar to that developedin neurotomized animals. This suggested that vesicle aggregationin both cases may have resulted from a decrease in availableadenosine triphosphate in the nerve terminal with subsequentalteration in the normal charge density which supports a repulsiveforce between the vesicles.     

ISOLATED NERVE ENDINGS (NEUROSECRETOSOMES) FROM THE POSTERIOR PITUITARY : Partial Separation of Vasopressin and Oxytocin and the Isolation of Microvesicles

Subcellular fractions of the bovine posterior pituitary, including one composed almost exclusively of pinched-off nerve endings (neurosecretosomes), were characterized electron microscopically, hormonally, and enzymically. 15% of the nerve terminals in the gland were isolated as neurosecretosomes, as estimated from determinations of lactic dehydrogenase, a soluble, cytoplasmic enzyme. Neurosecretosomes were subdivided into three fractions by density-gradient centrifugation. The three subfractions, each shown to be nearly homogeneous populations of neurosecretosomes by means of electron microscopic and enzymic criteria, differed from each other in their vasopressin/oxytocin (VP/OT) ratios. The VP/OT ratio increased from the lightest to the densest fraction, indicating that VP is localized to denser and OT to lighter neurosecretosomes; similar results have been obtained previously for subfractions of neurosecretory granules (NSG). No morphological differences were apparent in neurosecretosomes among the three subfractions. Although complete separation of VP and OT was not achieved, the findings suggest that VP and OT are each stored in a different species of nerve ending and support the hypothesis that a given neurosecretory cell synthesizes, stores, and secretes only one of the peptide hormones. Microvesicles, 40–80 mµ diameter and contained in typical neurosecretory cell terminals, are believed to be degradation products of membrane ghosts of depleted NSG; electron micrographs indicative of this transformation are presented. A fraction rich in microvesicles, but containing some NSG membranes, was prepared by density-gradient centrifugation of an osmolysate of neurosecretosomes. Smaller, apparently nonneurosecretory nerve endings, lacking NSG but filled with small vesicles, are occasionally seen in sections from whole gland. The vesicles in these atypical posterior pituitary nerve endings may be true neurohumor-containing, "synaptic" vesicles.