New anatomical insights into the inputs and outputs from hypothalamic magnocellular neurons.
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Afferent Pathways↗
Biomedical subjects
D V Pow
Publications and source records attributed to D V Pow.
Immunocytochemical evidence for somatic repair of the mutant AVP allele in heterozygous Brattleboro rats.
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Alleles↗
Extremely high titre polyclonal antisera against small neurotransmitter molecules: rapid production, characterisation and use in light- and electron-microscopic immunocytochemistry.
We have produced polyclonal antibodies against the small amino acid neurotransmitters, GABA, glutamate, glycine and taurine, with a simple new technique using antigens co-adsorbed with an adjuvant peptide to gold particles, which causes rapid and massive immune responses in all animals that we have studied. These antibodies are all of extremely high titre; they are typically used in immunocytochemistry at dilutions from 1 in 250,000 to 1 in 1,000,000 which represents an increase in titre of at least two orders of magnitude compared to standard antibody production techniques. Such very high dilutions result in minimal background labeling and a high signal-to-noise ratio when applied to sections of aldehyde-fixed, epoxy resin-embedded tissues at both light- and electron-microscopic levels. Each antibody displays minimal cross-reactivity with other neurotransmitter molecules. We suggest that our technique may be broadly applicable for raising antibodies against a wide variety of antigens of interest to neuroscientists, particularly those that normally elicit weak immune responses. The technique may also assist in clonal expansion prior to generation of monoclonal antibodies and may be viable, with modifications, for use in human immunisations.
Adjuvants, Immunologic↗
Immunocytochemistry of amino-acids in the rodent pituitary using extremely specific, very high titre antisera.
The compartmental interrelationships of the metabolically related amino-acids glutamate, GABA and glutamine and the metabolically unrelated amino-acids taurine and glycine in the rodent pituitary, were investigated by light microscopic immunocytochemistry using highly specific antisera. Glutamate-like immunoreactivity was abundant in astrocytes in the posterior pituitary. Glutamine immunoreactivity was present only at low levels in the posterior pituitary, but was abundant in astrocytes within the intermediate lobe. Other glia-like cells in the anterior pituitary were also glutamine-immunoreactive. GABA immunoreactivity was abundant in the intermediate lobe but absent from anterior and posterior lobes. The GABA immunoreactivity mainly took the form of small punctata, the majority of which were in intimate apposition to the glutamine-immunoreactive glia. Strong taurine immunoreactivity was present in astrocytes in the posterior pituitary but only weak labelling was present in intermediate and anterior lobes of the pituitary. Specific glycine immunoreactivity was not detected in the pituitary. These results suggest that glutamate-immunoreactive astrocytes in the posterior pituitary, unlike glia in loci such as the retina, do not convert much, if any, of their glutamate content into glutamine (or if they do, it is rapidly further metabolized to another compound), whereas those astrocytes in the intermediate lobe do contain glutamine. The spatial association of GABAergic fibres with glutamine-positive astrocytes raises the possibility that astrocytes in the intermediate lobe receive a GABAergic innervation. Glutamate, glutamine and taurine (or their metabolites) may have roles as neuroactive substances regulating pituitary secretion.
Amino Acids↗
Tunicamycin, puromycin and brefeldin A influence the subcellular distribution of neuropeptides in hypothalamic magnocellular neurones of rat.
Magnocellular neurones in the supraoptic nuclei of normal Long Evans and homozygous Brattleboro rats were examined electron-microscopically after intracisternal injections of tunicamycin, puromycin, or brefeldin A. Moderate (50 micrograms) or high (200 micrograms) doses of tunicamycin caused the formation of electron-dense filamentous accretions in the endoplasmic reticulum (ER) cisterns of vasopressin neurones, but only the high dose of tunicamycin also caused accretions to form in the ER of some oxytocin neurones. Immunogold labelling of ultrathin sections from tunicamycin-treated rats revealed that, in about 5% of vasopressin neurones, the accretions could be immunogold-labelled for vasopressin and its associated neurophysin. However, in the majority of vasopressin neurones, the sections required trypsinisation before immunolabelling of the accretions could be detected. Small accretions in the ER of oxytocin neurones did not label for oxytocin or its neurophysin without prior trypsinisation, whereas larger accretions in other oxytocin cells could be labelled without prior trypsin treatment. Administration of puromycin resulted in the formation of small ER accretions in both vasopressin and oxytocin neurones. These accretions were immunolabelled with antisera, respectively, to vasopressin and oxytocin, but neurophysin-immunoreactivity was in most cases absent and was not revealed by treatment with trypsin, suggesting that neurophysin-immunoreactive epitopes were absent from truncated peptides forming the accretions. Brefeldin A caused dilatation of ER cisterns and disruption of the Golgi apparatus in both oxytocin and vasopressin neurones, but did not cause accretions to form in the ER.
Animals↗
Immuno-electron microscopic evidence for two different types of partial somatic repair of the mutant Brattleboro vasopressin gene.
In homozygous Brattleboro rats a frame-shift mutation in the vasopressin gene prevents secretion of vasopressin by magnocellular neurosecretory neurons and thus causes diabetes insipidus. Whereas most "vasopressin" neurons in Brattleboro homozygotes apparently lack vasopressin and its associated neurophysin and glycopeptide, some isolated cells overcome the mutation and "revert" to producing readily detectable amounts of vasopressin. We describe here two morphologically and immunocytochemically distinct subsets of such "revertant" cells. One subset contain, in their rough endoplasmic reticulum cisterns, electron-dense aggregates immunoreactive for vasopressin, for parts of oxytocin-neurophysin, and for CP14 (a peptide with a sequence deduced from the mutated precursor), but not for vasopressin-associated glycopeptide ("glycopeptide") or vasopressin-neurophysin. In Brattleboro heterozygotes, which have one mutant and one normal copy of the vasopressin gene, morphologically similar revertant cells exist; the aggregates in the rough endoplasmic reticulum of these cells do not immuno-label for CP14, but the cells do produce 160-nm neurosecretory granules immunoreactive for vasopressin, vasopressin-neurophysin and glycopeptide. In Brattleboro homozygotes, the second, more abundant subset of neurons which recover vasopressin immunoreactivity also express vasopressin-associated glycopeptide and CP14 but not oxytocin-neurophysin; both glycopeptide and CP14 are restricted to the rough endoplasmic reticulum but do not form aggregates. We conclude that two different somatic repairs of the Brattleboro mutation can occur. We propose that, in aggregate-containing neurons, exons B and C have been exchanged between the vasopressin and oxytocin genes; glycopeptide-immunoreactive neurons have either undergone mismatch repair or exchanged exon B.
Animals↗
Neuropeptide accretions in the endoplasmic reticulum of oxytocinergic neurons in cats, monkeys and rabbits: a widespread phenomenon.
Light microscopic observations using Nomarski optics on the aldehyde-fixed hypothalamus of normal adult cats, monkeys and rabbits revealed the presence of cells in the supraoptic, paraventricular and periventricular nuclei which possessed yellow birefringent inclusions. Immunogold labelling showed that in each species the cells displayed oxytocin-like immunoreactivity, both in electron-dense inclusions within some (but not all) cisterns of rough endoplasmic reticulum and in secretory granules. The cells in cats and rabbits were in all respects indistinguishable from the homologous 'birefringent' cells previously described in rats, but in monkeys, cells frequently contained additional inclusions in cisterns of rough endoplasmic reticulum which did not display oxytocin or vasopressin-like immunoreactivity, even after trypsin, pepsin or chymotrypsin treatment of sections. Observations on cats and rabbits using fluorescence microscopy revealed that the birefringent cells possessed bright autofluorescence which facilitated the identification of more cells than were seen using Nomarski optics alone. Autofluorescence was abolished when sections were mounted in glycerol, or when exposed to light for protracted periods of time. Attempts to label for monoamines in these cells were not successful, suggesting that the fluorescence is not due to aldehyde-induced amine fluorescence. It is not clear why neuropeptides are retained in some rough endoplasmic reticulum cisterns. It is possible that these birefringent cells contain a peptide, or peptides, which are abnormal in some manner, or which may be other members of the oxytocin gene family. Alternatively, the processing of neuropeptides to permit their export to the Golgi apparatus may be deficient. Acetylcholinesterase (AChE) histochemistry revealed that, unlike other oxytocin neurons, cells with intracellular accretions lacked detectable acetyl cholinesterase. As AChE is a known peptidase, it may be involved in regulating peptide export from the rough endoplasmic reticulum.
Acetylcholinesterase↗
Widespread release of peptides in the central nervous system: quantitation of tannic acid-captured exocytoses.
Tannic acid methods have been applied to capture the exocytosis of peptide-containing granules from peptidergic neurons. The captured exocytoses have been quantitated to assess the proportion and amount of peptide released at different parts of the neuronal membrane. Examination of hypothalamic synaptic boutons shows that only about one-half of the peptidergic vesicles is exocytosed into the synaptic cleft and also that exocytosis also occurs from undilated peptidergic axons. Study of the magnocellular neurosecretory system reveals that all parts of their extensive terminal arborization appear to be equally capable to exocytose peptide. Only about one-half of their peptide is released from their nerve endings, which about the capillaries. The remainder is released much deeper in the lobules of secretory tissue where its principal target(s) could be the pituicytes and/or neurosecretory axons. Dendrites of magnocellular neurons are also capable of releasing peptide by exocytosis and dendrites could release sufficient oxytocin and vasopressin to account for the peptide known to be released into the hypothalamus. We conclude that peptidergic neurons release substantial amounts of peptides from all of their processes and that this must be taken into account when considering what functions those peptides might serve.
Animals↗
Membrane routing during exocytosis and endocytosis in neuroendocrine neurones and endocrine cells: use of colloidal gold particles and immunocytochemical discrimination of membrane compartments.
The hypothesis that the retrieval of membranes of neurohypophysial neurosecretory granules (NSG) and small electron-lucent microvesicles occurs by different routes was tested by incubating neurohypophysial neurosecretosomes with colloidal gold particles of various sizes. Neurosecretosomes derived from normal Long Evans rats and incubated in media of normal ionic composition endocytosed a few small (less than 25 nm) gold particles into 40-50 nm electron-lucent microvesicles. After depolarisation, more small gold particles were found in microvesicles, and small and large (greater than 25 nm) gold particles in vacuoles. Oxytocin-containing neurosecretosomes derived from Brattleboro rats, which contain 160 nm-diameter NSG, endocytosed gold particles in a pattern indistinguishable from that of neurosecretosomes from Long Evans rats. However, neurosecretosomes derived from defective vasopressin neurones of Brattleboro rats, which contain microvesicles, small vacuoles, and a few 100 nm dense-cored vesicles, but no 160 nm NSG, endocytosed only small colloidal gold particles. Early after depolarisation the gold particles were present only in microvesicles, but later some could be found in vacuoles and lysosome-like structures. Immunogold cytochemistry using a polyclonal antiserum raised against microvesicle-rich neurosecretosomes derived from Brattleboro rats labelled microvesicles in the posterior pituitary strongly, NSG weakly, and vacuoles to a variable extent. These data together indicate that, after exocytosis, the membranes of NSG are recaptured as large vacuoles. Microvesicles are exocytosed and endocytosed separately.
Animals↗
'Neurosecretion' by synaptic terminals in the locust corpus cardiacum: is non-synaptic exocytosis part of the regulated or the constitutive pathway?
Nerve fibres form conventional synaptic junctions with gland cells in the corpus cardiacum of the locust, Schistocerca gregaria. They contain synaptic vesicles whose contents are normally electron-lucent, but which react positively to cytochemical tests for amines (eg, incubation in the false transmitter 5-OHDA). Secretory granules are also present in the terminals and such inclusions are known to contain neuropeptides. The granules undergo non-synaptic exocytosis and this process has been visualized by the application of tannic acid. Granule exocytosis gives clear signs of being part of a regulated secretory pathway: it is elevated in vivo by flight--a natural stimulus known to activate the gland (this effect is blocked by prior injection of trehalose); its incidence is closely correlated with a postsynaptic response, suggesting a role for the materials discharged in short-term signalling; and when induced in vitro by high K+, it is Ca(2+)-dependent. However, a low level of exocytosis was encountered under all the conditions employed, suggesting the existence of a constitutive component. It is postulated that the regulated and constitutive patterns of discharge of neuropeptides are related to the roles of these materials as neurotransmitters/modulators and neurotrophic substances, respectively.
Animals↗
Peptide accretions in the endoplasmic reticulum of magnocellular neurosecretory neurons in normal and experimentally manipulated rats.
Light microscopic observations using Nomarski interference contrast optics or darkfield optics on unstained aldehyde-fixed vibratome sections of hypothalami from normal young adult male and female Long Evans rats and from vasopressin-deficient Brattleboro rats, revealed the presence of cells in the supraoptic, paraventricular and periventricular nuclei which possessed yellow birefringent inclusions of globular or filamentous appearance in their somata. These inclusions were morphologically distinct from the large lipid droplets present in vasopressinergic magnocellular neurons of diabetes insipidus mice. Small portions of the vibratome sections containing the birefringent cells were excised and prepared for correlative electron microscopy. This revealed that the birefringent inclusions represented electron-dense material within cisterns of endoplasmic reticulum in magnocellular neurons. Antibodies to oxytocin or oxytocin-associated neurophysin immunolabelled the intracisternal electron-dense material and neurosecretory granules in resin-embedded ultrathin sections. Antibodies to vasopressin or vasopressin-associated neurophysin, and a panel of lectins did not label the intracisternal material. Quantitation revealed a small increase in the numbers of birefringent cells in aged rats and in rats drinking saline for 3 days. Subcutaneous injection of oestradiol benzoate for 7 days prior to fixation caused a large increase. After cessation of oestradiol administration the numbers of birefringent cells decreased; observations on the remaining cells showed that the endoplasmic reticulum cisterns were frequently fused with the plasmalemma, resulting in direct release of neurosecretory material into the extracellular spaces.
Animals↗
'Neurosecretion' by synaptic terminals and glandular discharge in the endocrine pancreas. Application of tannic acid to the teleost Xiphophorus helleri.
Nerve fibers within the large pancreatic islet of Xiphophorus helleri are apparently of a single type and form typical, well-differentiated synapses with the endocrine gland cells. A histochemical test for cholinesterases generates reaction product in association with the outer surfaces of the neuronal membranes. Two categories of presumptive secretory inclusions are present in the fiber terminals: secretory granules usually 80-120 nm in diameter have a wide distribution within the fibers, whereas electron-lucent synaptic vesicles 30-60 nm across often cluster adjacent to membrane thickenings and differentiated clefts. Infusion of tannic acid in high-K+ saline enabled sites of granule exocytosis to be visualized. These presumed sites of neuropeptide discharge were associated with morphologically undifferentiated regions of the terminal membranes. Tannic acid also facilitated the detection of exocytosis, involving granules of endocrine gland cells; discharged secretory material exposed to this agent retains immunoreactivity at a reduced level.
Animals↗
Dendrites of hypothalamic magnocellular neurons release neurohypophysial peptides by exocytosis.
Exocytosis of neurosecretory granules from dendrites of magnocellular neurons can be visualized electron microscopically after incubation of hypothalamic brain slices in media containing 1.2 mM tannic acid, which stabilizes extracellular peptidergic granule cores, and permits their immunocytochemical identification. Morphometric analysis of stimulated slices demonstrates that exocytosis of neurosecretory granules from the dendrites of magnocellular neurons can account for the vasopressin and oxytocin known to be released into the hypothalamus. Exocytosis from cell bodies of magnocellular neurons was not observed in stimulated slices from normal rats but, when granules had been caused to accumulate in the neuronal somata by prior administration of colchicine, exocytosis of granules from the somata was unambiguously identified. These data demonstrate exocytosis from dendrites and cell bodies of a mammalian peptidergic neuron, and show that all parts of their plasmalemma are competent for exocytosis of granules.
Animals↗
Differential distribution of acetylcholinesterase activity among vasopressin- and oxytocin-containing supraoptic magnocellular neurons.
Acetylcholinesterase activity was demonstrated histochemically at light- and electron-microscopic levels, in Vibratome sections of the supraoptic nucleus of fixed hypothalami derived from osmotically stimulated and unstimulated Long Evans rats, from homozygous Brattleboro rats with hypothalamic diabetes insipidus, from lactating rats, from normal adult male house mice (Mus musculus) and from mice with hereditary nephrogenic diabetes insipidus (di/di). Reaction product was located in supraoptic magnocellular neurons; in dorsal and rostral aspects of the supraoptic nuclei lightly stained cells predominate, whereas in ventral and caudal regions densely staining perikarya predominate. Pre- and post-embedding immunocytochemical detection of oxytocin-neurophysin or vasopressin-neurophysin, combined with acetylcholinesterase histochemistry, showed that the lightly staining cells are oxytocinergic, and the densely staining cells vasopressinergic. Osmotic stimulation of the animals, either by substitution of drinking water for 3 days with 2.5% saline or reason of genetic defects which result in diabetes insipidus, enhanced the acetylcholinesterase activity of the vasopressin neurons but had little effect on the weekly acetylcholinesterase-reactive oxytocin cells. Acetylcholinesterase activity was particularly marked in the hypertrophied abnormal magnocellular neurons of homozygous Brattleboro rats which do not release significant amounts of vasopressin. The increased acetylcholinesterase activity in osmotically stimulated animals cannot, therefore, be a function of vasopressin. Acetylcholinesterase activity was also detected in large multipolar neurons lying dorsolateral to the supraoptic nucleus, and in their fine axonal processes which project towards the supraoptic nucleus. A very few synaptic boutons surrounded by acetylcholinesterase reaction product were found in contact with magnocellular neuron basal dendrites. However, much of the punctate acetylcholinesterase reactivity observed at the light microscopic level and previously interpreted as representing the loci of cholinergic synaptic boutons was shown to be intracellular, and probably caused by acetylcholinesterase activity in some large, secondary lysosomes.
Acetylcholinesterase↗
Microglia in the neurohypophysis associate with and endocytose terminal portions of neurosecretory neurons.
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.
Animals↗
Capturing and quantifying the exocytotic event.
Although exocytosis is now known to be the universal method by which proteins are released from eukaryotic cells, we know surprisingly little of the mechanism by which exocytosis occurs. One reason for this is that it has proved difficult to capture sufficient of these evanescent events to permit their study. The difficulty with which exocytoses can be visualized with standard preparative techniques varies among tissues, but the problem is particularly apparent in the mammalian nervous system. Tannic acid has recently been introduced as an agent by which exocytosed granule cores can be captured and visualized electron-microscopically. Application of tannic acid to the magnocellular neurosecretory system reveals exocytoses from all parts of their terminal arborization within the neural lobe, and also from their dendrites within the hypothalamus. Quantification of the exocytoses in unstimulated tissue and in tissue stimulated by a variety of exogenous and endogenous mechanisms indicates: (a) that exocytosis occurs equally from each unit of membrane of the perivascular nerve endings, and of the axonal swellings that were previously thought to be sites of granule storage, rather than release; (b) that, in the nerve endings, a greater proportion of the stored granules are exocytosed, and thus the endings are specialized for release not by any particular property of their membrane, but by a high surface membrane:volume ratio. Together, the data cast doubt on the hypothesis that exocytosis occurs only at some functionally specialized sites at certain loci in the membrane. Rather, the data favour the hypothesis that magnocellular granules can fuse with any part of the membrane, depending on constraints imposed by the cytoskeleton, and a local increase in cytosolic free calcium level. When applied to hypothalamic central nervous tissue, tannic acid reveals that exocytosis of dense-cored synaptic vesicles occurs preferentially, but not exclusively, at the membrane apposed to the postsynaptic element. However, about half of all exocytoses from synaptic boutons occur at bouton membrane unrelated to the synaptic cleft. In all tissues studied, tannic acid reveals a heterogeneity among secretory cells in the extent of exocytosis that occurs in response to stimulation, and permits an analysis of the degree to which secretion is polarized in any one direction. These results question long-held assumptions concerning the site at which neurones release transmitters and modulators. Tannic acid seems likely to prove a potent tool in the investigation of both the mechanism of exocytosis and the ways in which different types of cells adapt the process to perform their physiol
Animals↗
'Neurosecretion' by a classic cholinergic innervation apparatus. A comparative study of adrenal chromaffin glands in four vertebrate species (teleosts, anurans, mammals).
Nerve terminals forming typical synapses with adrenal chromaffin tissues have been examined in the goldfish, frog (Rana pipiens), hamster and rat. Presumptive secretory inclusions present in the terminals are of two distinct types. Electron-lucent synaptic vesicles 30-50 nm in diameter are densely clustered adjacent to membrane thickenings and presumably discharge their contents into the synaptic clefts. Secretory granules (i.e. large dense-cored vesicles) 60-100 nm in diameter are more abundant in other parts of the terminals. Sites of granule exocytosis have been observed in each of the animals investigated. They are usually encountered within apparently undifferentiated areas of plasmalemma and only rarely occur within synaptic thickenings. Granule exocytosis from within synaptic terminals and chromaffin gland cells is most readily observed in specimens exposed, prior to fixation, to saline solutions containing both tannic acid, and 4-aminopyridine and/or elevated levels of K+. These findings show that the pattern of secretory discharge, involving both synaptic and non-synaptic release, which is widespread in invertebrate central nervous system, is also characteristic of vertebrate, peripheral cholinergic terminals.
Adrenal Medulla↗
'Neurosecretion' by aminergic synaptic terminals in vivo--a study of secretory granule exocytosis in the corpus cardiacum of the flying locust.
Most nerve terminals forming typical synaptic junctions contain both synaptic vesicles and larger 'secretory granules' with electron-dense contents. Visualization of granule exocytosis from within terminals in the corpus cardiacum is facilitated by injection of tannic acid which immobilizes granule cores as they are discharged. The process of discharge is stimulated by flight-induced activation of the neurones and there is a correlated response by the innervated cells. In contrast to synapses with their vesicle clusters, granule discharge is not targeted upon the postsynaptic cells. These findings have general implications for mechanisms of discharge of neuropeptides and other transmitters from synaptic terminals.
Animals↗