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Recent studies have localized the glutamatergic cell marker type-2 vesicular glutamate transporter (VGLUT2) to distinct peptidergic neurosecretory systems that regulate hypophysial functions in rats. The present studies were aimed to map the neuronal sources of VGLUT2 in the median eminence and the posterior pituitary, the main terminal fields of hypothalamic neurosecretory neurons. Neurons innervating these regions were identified by the uptake of the retrograde tract-tracer Fluoro-Gold (FG) from the systemic circulation, whereas glutamatergic perikarya of the hypothalamus were visualized via the radioisotopic in situ hybridization detection of VGLUT2 mRNA. The results of dual-labeling studies established that the majority of neurons accumulating FG and also expressing VGLUT2 mRNA were located within the paraventricular, periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area. In contrast, only few FG-accumulating cells exhibited VGLUT2 mRNA signal in the arcuate nucleus. Dual-label immunofluorescent studies of the median eminence and posterior pituitary to determine the subcellular location of VGLUT2, revealed the association of VGLUT2 immunoreactivity with SV2 protein, a marker for small clear vesicles in neurosecretory endings. Electron microscopic studies using pre-embedding colloidal gold labeling confirmed the localization of VGLUT2 in small clear synaptic vesicles. These data suggest that neurosecretory neurons located mainly within the paraventricular, anterior periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area secrete glutamate into the fenestrated vessels of the median eminence and posterior pituitary. The functional aspects of the putative neuropeptide/glutamate co-release from neuroendocrine terminals remain to be elucidated.

A single base deletion (delta G) in the vasopressin gene is the cause of diabetes insipidus in the homozygous Brattleboro rat (di/di). The resulting frameshift leads to the expression of an aberrant vasopressin precursor which is unable to enter the secretory pathway, thereby preventing vasopressin biosynthesis. In a small number of solitary magnocellular hypothalamic neurons within the supraoptic and paraventricular nuclei, the reading frame is restored by a dinucleotide (delta GA) frameshift mutation, at two separate GAGAG motifs downstream of the original G-deletion. This results in two + 1 di-vasopressin precursors that are still partially mutated within the neurophysin region. The present study provides immunocytochemical evidence which demonstrates that, within magnocellular solitary neurons of the supraoptic and paraventricular nuclei of the di/di rat, the + 1 di-vasopressin precursors can enter the secretory pathway followed by their enzymatic processing into vasopressin during axonal transport to the neural lobe. However, the cellular characteristics of biosynthesis are different from those of wild-type rats. Immunoelectron microscopical localization of vasopressin gene products in the neural lobe of did/di rats revealed their presence in neurosecretory granules, the diameter of which is intermediate (116 nm) between those of the neurosecretory granules in the di/di (80-100 nm) and wild-type (160 nm) rats.

In the present experiments, we examined the effect of ovariectomy on the increases in litter weight and structural plasticity of MNCs in the supraoptic nucleus (SON) during lactation. Female rats were ovariectomized 2 days after parturition, and the increases in litter weight were measured as the index of milk let-down from dams during lactation. The lactation period was elongated up to 6 weeks by providing new litter to obtain more apparent effects of the ovariectomy. There was no significant difference in the increases in litter weight between non-operated and ovariectomized females. After lactation for 6 weeks, the ultrastructures such as juxtaposition (surface membrane apposition) and multiple synapses (terminals contacting with two or more postsynaptic elements) of MNCs in the SON in nonoperated and ovariectomized females were examined to compare with those of virgins. The percentage of juxtaposition and the number of multiple synapses significantly increased in nonoperated lactating females as compared with those of virgins. Ovariectomized rats showed similar structural changes to those of nonoperated females during lactation. Therefore, we conclude that ultrastructural plasticity of MNCs in the SON is maintained even in the absence of an ovary, and direct or indirect actions of suckling stimulation may be important in maintaining the plasticity during lactation.

Neurosecretory brain cells from embryonic locusts cultured in serum-free medium failed to show any visible signs of growth. In contrast, the same neurons co-cultured with CNS explants (brain-retrocerebral complexes and thoracic ganglia) show excellent axonal growth: sprouting occurs after one day of co-culture and increases within the first week. These results indicate the production of an active neurite outgrowth stimulating factor by co-cultured CNS explants. The similarity of the stimulating effects by the two explants on neurite outgrowth rule out brain neurohormones as probable candidates for the stimulating factor. In addition, neither insulin nor neuroparsin added to the culture medium to test their trophic effect improves the growth of the cells. Conditioned medium derived from cultures of brain-retrocerebral complexes produced no neurite outgrowth, suggesting that the active factor released in the medium by brain explants does not remain free in solution but binds to the substratum. Finally, neurons co-cultured with CNS explants attached to the bottom of the culture dish develop neurites only when in close proximity to the explants. This observation strongly suggests the binding of an active neurite outgrowth stimulating factor to the substratum in the vicinity of the explants. As a control for CNS explants, the action of non-nervous tissue was tested: a similar, but less extensive neurotrophic effect, was observed with esophagus segments co-cultured with neurosecretory brain cells. These results demonstrate that locust neurosecretory neurons isolated in cell culture require combined explants for elaborating processes and suggest that the neurite promoting effect is mediated by a substrate-associated molecule(s).(ABSTRACT TRUNCATED AT 250 WORDS)

Previous work has demonstrated that intravenous injection of neuronal tracers, e.g. horseradish peroxidase or Fast Blue, can retrogradely label neurons in brain areas that project outside the blood-brain barrier, e.g. magnocellular neuroendocrine neurons of the hypothalamus. Here we have shown that 24 h after intravenous injection of the fluorescent retrograde tracer Evans Blue, the same population of magnocellular neuroendocrine neurons is labeled in the paraventricular, supraoptic and accessory magnocellular nuclei. Parvicellular neuroendocrine cells in the paraventricular nuclei are also labeled. Most Evans Blue-labeled magnocellular neuroendocrine cells in the supraoptic nucleus could be stained immunocytochemically for neurophysins, suggesting that these neurons continue to produce their peptide hormones after taking up the fluorescent dye. Ultrastructural observation of supraoptic cells retrogradely labeled with Evans Blue shows that 95% of the neurons appeared healthy. There was no ultrastructural evidence of degeneration, hyperstimulation, or interruption of the axoplasmic flow. Labeling the neuroendocrine cells with Evans Blue did not alter the size of magnocellular cells, the animal's fluid balance or ingestive behavior. Following enzymatic/mechanical dissociation of the supraoptic nucleus from animals that had been injected with Evans Blue 24 h previously, phase-bright neurons that often contained fluorescent material were observed, thus identifying these neurons as neuroendocrine. Recording from identified neuroendocrine cells showed that these neurons generated spontaneous or current-evoked overshooting action potentials with an afterhyperpolarization and had negative resting membrane potentials. Action potential broadening, a feature of magnocellular neurons, was observed during bursts of action potentials elicited by depolarizing current injection. Taken together, this work would suggest that Evans Blue is non-toxic at the doses used and that it provides a method to identify single neuroendocrine cells in primary cell cultures made from adult hypothalamus for voltage-clamp recordings.

The cerebral neuroendocrine Light Green Cells of the pulmonate snail Lymnaea stagnalis, which control body growth and associated processes, stain positively with an affinity-purified antiserum raised to a large part of the C-chain of pro-molluscan insulin-related peptides. At the ultrastructural level, the rough endoplasmic reticulum is immunonegative, the Golgi apparatus is slightly positive and secretory granules in the process of budding from the Golgi apparatus are strongly positive. These observations indicate that the Light Green Cells synthesize molluscan insulin-related peptides, which are processed before packing by the Golgi apparatus into secretory granules. The two morphologically distinct secretory granule types, i.e. with pale and dark contents, respectively, are equally immunoreactive with antiserum raised to the C-chain of molluscan insulin-related peptides. Secretory granules within lysosomal structures reveal various degrees of immunoreactivity, indicating their graded breakdown. The Light Green Cells release secretory material by the process of exocytosis into the haemolymph from neurohaemal axon terminals located in the periphery of the median lip nerve. The electron-dense (tannic acid method) released contents are clearly immunopositive. The same holds for secretory granule contents released from Light Green Cells axon profiles in the centre of the lip nerve. Some immunoreactivity is also present in the intercellular space between these axon profiles. It is concluded that molluscan insulin-related peptides may act in two ways, namely (1) as neurohormones via the haemolymph at peripheral targets and (2) in a non-synaptic (paracrine) fashion at targets within the central nervous system.

The main developmental events in the subfornical organ take place between 17 fetal days (fd) and 5 days post natum (dpn) at which time it possesses most of its mature fine structural characteristics. The surface regional characteristics of ependymal cells differentiate primarily during this time as well, while the ependymal cellular fine structure, shape and relationship with neurons and the vascularity are well established prior to birth. Undifferentiated neurons contain glycogen prior to 19 fd and then differentiate by developing processes and organelles characteristic of neurons. By 5 dpn, the various types of neurons found in the mature subfornical organ are all present, except for giant vacuolated cells. Synapses containing only electron-lucent vesicles are first present at 20 fd, those containing additional electron-dense vesicles at 3 dpn. Microglial cells are first identifiable at 17 fd, and the first protoplasmic astrocytes are recognizable at 21 fd, while fibrous astrocytes are not detectable prior to 7 dpn. By 5 dpn, the cytological elements of the subfornical organ are all in place, and further developmental changes leading to adult fine structural characteristics by 30 dpn are essentially quantitative in nature.

The fine structure of the axons of the cerebral, egg-laying stimulating caudodorsal cells of the snail Lymnaea stagnalis has been studied with various light and electron microscope techniques. Special attention was paid to exocytotic release of secretory material (demonstrated with the tanic acid method) from nonsynaptic release sites in the cerebral commissure. This phenomenon has been compared with neurohaemal release. The commissure consists of two morphological compartments, separated by a sheath of glial cells. The outer compartment is formed by the neurohaemal area of the caudodorsal cells, the inner consists of thousands of, mainly unidentified, axons. Furthermore, ventral caudodorsal cells send axons through the inner compartment. These give rise to collaterals, which divide into smaller collaterals, forming an extensive network ("collateral system") throughout the inner compartment. Eventually, collaterals end blindly within the inner compartment. They contain the same three morphological types of secretory granule as the neurohaemal axon terminals. The collaterals never form synaptic contacts; exocytotic release of the contents of secretory granules takes place at nonsynaptic release sites. These sites occur rather dispersed and do not face one particular type of neighbouring neural element. As in the neurohaemal area, both single and multiple exocytoses occur. Widened intercellular spaces, filled with flocculent, electron-dense material, occur near highly active nonsynaptic release sites. The spaces are often bordered by glial cells and may facilitate diffusion of released secretory material through the inner compartment. Apparently, a ventral caudodorsal cell releases secretory material in two fashions: from neurohaemal axon terminals into the haemolymph, and nonsynaptically, from the collaterals into the intercellular space of the central nervous system. Possible functions of the glial sheath between the neurohaemal area and the inner compartment are proposed. Most likely, the collateral system enables the caudodorsal cells to communicate with targets within the central nervous system in a nonsynaptic fashion. A possible target is the cerebral Ring Neuron, which sends an axon branch through the inner compartment and, as was previously shown neurophysiologically, is controlled by the caudodorsal cells in a nonsynaptic fashion.

Neurosecretory PC12 cells were exposed in a variety of experimental conditions to nanomolar concentrations of alpha-latrotoxin purified from the venom of the black widow spider. When applied in a modified Ringer medium containing millimolar Ca2+ the toxin rapidly elicited a marked stimulation of exocytosis, as indicated by the appearance of typical images of granule-plasmalemma interaction and by the decreased density (number/unit area) of secretion granules in the cytoplasm. Without Ca2+ in the medium this early toxin effect was delayed and evolved less rapidly, but was still clearly appreciable. These morphological results appear in good quantitative agreement with the biochemical data on dopamine release reported in the preceding article. The stimulation of exocytosis was followed after a short delay by a stimulation of endocytosis, as revealed by an increased accumulation of the extracellular tracer, [14C]sucrose, within the toxin-treated cells. At later times after the application of alpha-latrotoxin other effects appeared, but only in the presence of Ca2+: these included changes in cell shape; focal alterations of the mitochondrial matrix (clear discrete areas and dense precipitates) and frank signs of cytotoxicity (rupture of the plasmalemma, clearing of the cytoplasmatic matrix). The toxin-induced cell death was studied quantitatively by using trypan blue exclusion as well as the 51Cr test, and was found to be dependent on alpha-latrotoxin concentration, temperature of incubation and Ca2+ concentration in the medium. Ionic substitutions concerning anions as well as cations other than Ca2+ had minor or no consequences. Thus, the early effect of alpha-latrotoxin in PC12 cells (stimulation of exocytosis, at least partially Ca2+-independent) can be dissociated from the late 'toxic' effect (strictly Ca2+-dependent).

This review of the subfornical organ, with special emphasis on the rat, summarizes the fine structural characteristics of the capillaries, the access route for blood-borne substances, the ependyma through which cerebrospinal fluid-borne substances penetrate the organ, neuronal perikarya, and types of synapses and axons, together with a brief discussion of the principal as yet unresolved problems.

The caudal neurosecretory system of Poecilia sphenops (molly) is an isolated population of neurosecretory cells located in the caudal most aspect of the teleost spinal cord. The structure of this neuroendocrine system is favorable for studies on the synaptic control of neurosecretory mechanisms. Little is known about the detailed synaptology of the system. Morphological and electrophysiological reports have shown that the caudal neurosecretory system is linked to higher brain centers by descending spinal projections. To examine the synaptology of the descending synaptic input, surgical deafferentation was performed by microsuction removal of a segment of spinal cord rostral to the caudal system. The degeneration of axon terminals was studied at various times following deafferentation and compared to control synaptology. Based on vesicle content and morphology, three axon terminal types were found in the caudal neurosecretory system. These terminals formed axosomatic, axodendritic, and axoaxonic synaptic contacts. Following deafferentation, axon terminals with dense-cored vesicles and boutons with round clear vesicles degenerated as evidenced by the electron dense dark reaction and the electron lucent reaction respectively. This suggested that at least two different types of axon terminals arise from the descending projection to the caudal neurosecretory system and that two different neurotransmitters may be influencing the neurosecretory activity of these cells.

A gradation of small rostral to larger caudal neurosecretory cells is found in the preoptic area of fish. Regional variations in ultrastructural features were assessed in this region of fish ranging widely in size (1.7-283.1 g), some of which had received intraperitoneal injections of horseradish peroxidase in order to label neurons projecting beyond the blood-brain barrier (i.e. neurosecretory cells). In small (less than or equal to 6.6 g) fish, neurosecretory cells of the caudal preoptic area were 10-15 microns in diameter and contained dense core vesicles. More rostral smaller cells had few or no dense core vesicles, but were labeled following intraperitoneal horseradish peroxidase injections. Cells in both areas were progressively larger, with larger nuclei and more granular reticulum in larger fish. In large fish, neurons that contained dense core vesicles ranged from 10 microns in diameter rostrally to 70 microns in diameter caudally. Some intermediate and large cells were extensively vacuolated and contained extremely convoluted and possibly multiple nuclei. Degenerating material was seen in apparently normal large fish. A few neurosecretory cells were extensively surrounded by perineuronal electron-dense glial cells. Most neurosecretory cells were involved in extensive soma-somatic apposition, which was especially pronounced in fish which were removed from cold water in late fall. Gap junctions were also clearly present on the somata of preoptic neurosecretory cells of these fall fish. These data imply that increased capacity for hormone secretion in large fish may be accomplished in part by neuronal hypertrophy. Maturational and seasonal variation in structure suggest that physiological characteristics are also variable in this nucleus.

The rat neurohypophysis contains a population of microglial cells, the majority of which occupy a pericapillary position in the resting gland. The microglia are immunocytochemically identifiable by the presence of macrophage-associated antigens and resemble microglia of the CNS. Morphometry at light and electron microscopic levels reveals that such cells constitute approximately 19% of the intrinsic cell population, excluding the endothelial cells. Two other populations of neurohypophysial glial cells, parenchymatous pituicytes and fibrous pituicytes, do not express macrophage-associated antigens. The microglia have long processes which surround and, in some cases, engulf apparently viable portions of the magnocellular neurosecretory nerve terminals. A sequence of stages of selective endocytosis and degradation of the engulfed nerve terminals can be visualized within pericapillary microglia. Some phagosomes and secondary lysosomes contain morphologically intact neurosecretory granules; others contain partially destroyed neurosecretory granules or amorphous material all of which are identifiable as originating from the magnocellular neurosecretory terminals by their immunoreactivity for oxytocin- or vasopressin-neurophysin. This finding indicates a novel role for the microglial cells in remodelling terminal aborizations of neurosecretory neurons and in processing or degrading hormones and peptides they contain. Because of their close and selective associations with other cellular elements of the neurohypophysis, any substances produced by microglia also have the potential to influence hormone secretion, pituicyte proliferation and neurohypophysial vasculature.