Membrane retrieval by vacuoles after exocytosis in the neural lobe of Brattleboro rats

The possible role of microvesicles and vacuoles in the recapture of membrane after pituitary hormone release by exocytosis has been studied in homozygous Brattleboro rats. These mutant animals are unable to synthesize vasopressin and exhibit a steady state hypersecretion of oxytocin from the neural lobe as a result of the osmotic imbalance caused by their diabetes insipidus. This can be converted to a second steady state which approximates to the rate of secretion found in normal Long Evans rats by the administration of exogenous vasopressin daily for 30 days. In the Brattleboro rat, presumptive oxytocinergic nerve endings contain typical 160–170 nm diameter neurosecretory granules; other magnocellular nerve endings contain a population of smaller ( ~ 100 nm diameter) dense-cored granules.The number of dense-cored granules was reduced in both types of nerve ending in the hypersecreting Brattleboro rats, but increased as a result of vasopressin treatment to levels which, for the classical neurosecretory granules, approximated that found in Long Evans rats. The microvesicle population of the nerve endings was essentially similar in quantitative terms in all the three groups (i.e. hypersecreting Brattleboro rats; vasopressin-treated Brattleboro rats and Long Evans controls). The number of vacuoles, on the other hand, was increased in nerve endings in the hypersecreting animals but reduced to levels found in Long Evans rats in the Brattleboro animals treated with vasopressin. Furthermore, the size of the vacuoles was comparable to the size of the dense-cored granules contained in the nerve endings. These changes in the vacuole population are exactly those that would be predicted for an organelle responsible for recapture of the granule membrane.We threfore conclude that membrane retrieval after exocytosis of neurosecretory granules in the neural lobe is achieved by vacuoles and that these organelles probably retrieve the membrane of the granule intact.     

Neurosecretory granule release and endocytosis during prolonged stimulation of the rat neurohypophysis in vitro

The nature of the readily releasable pool of neurohypophysial hormone and the recapture of membrane which occurs after hormone release have been investigated using a radioimmunoassay and stereological analysis of electron micrographs. Prolonged stimulation of the rat neurohypophysis in vitro with high potassium ion concentration (high-K⁺) gives rise to hormone release which declines with time. A second increase in hormone release is observed when veratridine (an agent which increases intracellular Ca²⁺ concentration independent of K⁺-produced depolarization) is added to the high-K⁺ containing medium at the time when the decline in hormone release has occurred. There is a depletion of neurosecretory granules from the nerve endings associated with both phases of hormone release and the time course of granule release and hormone release are similar. There is no quantitative change in the microvesicle population either during the high-K⁺-or the veratridine-stimulated release. There is, however, a large increase in the volumetric density occupied by endocytotic vacuoles associated with both phases of release, and the time course of the appearance of vacuoles closely parallels that of the decrease in the granules.These findings indicate that the hormone in both the ‘readily releasable’ pool mobilized by high-K⁺ and that in the pool released by veratridine is contained within granules and that therefore the decline in hormone release during prolonged stimulation is unlikely to be due to the exhaustion of a readily releasable pool of granules defined by their position within the nerve endings. They also indicate that, in the neurohypophysis, vacuoles are the major route for membrane retrieval after hormone release and that microvesicles are unlikely to arise from the division of vacuoles.     

Endocytosis in glial cells (pituicytes) of the rat neurohypophysis demonstrated by incorporation of horseradish peroxidase.

The uptake and intracellular disposition of an extracellular marker, horseradish peroxidase, was traced at various time intervals in glial cells of the neurohypophysis, the pituicytes. Glands from normal rats and rats in which neurohypophysial secretion was activated by various stimuli were studied.Following an intravenous injection of the enzyme, reaction product rapidly appeared in the pituicyte cytoplasm, within numerous membrane-bound vacuoles of various size and morphology. The vacuoles presumably arose from large bristle coated invaginations of the plasmalemma that were marked by the tracer as soon as 5 min after injection. By 24 h, the reaction product was sequestered mainly in lysosomal multivesicular bodies and dense bodies.The observations suggest that endocytosis normally occurs in pituicytes. Furthermore, this endocytotic activity appears to be related to neurohypophysial secretion: morphometric analysis indicated that at each time period studied, the relative volume occupied by the peroxidase-labelled structures in cells from stimulated preparations was significantly greater than their corresponding controls. The possible role of uptake of extracellular fluid and accompanying plasma membrane that result from endocytosis is discussed.     

Endocytic vacuoles formed following a short pulse of K+ -stimulation contain a plethora of presynaptic membrane proteins

It is now well established that the membrane of synaptic vesicles is recycled following exocytosis. However, little is known concerning the identity of the primary or secondary endocytic structures and their molecular composition. Using cultured rat cerebellar granule cells we combined uptake of horseradish peroxidase as a fluid phase marker and immunogold labeling for a variety of presynaptic proteins to assess the molecular identity of the stimulation-induced endocytic compartments. Short periods (5 or 30 s) of stimulation with 50 mM KCl were followed by periods of recovery for up to 30 min. Stimulation resulted in the formation of horseradish-peroxidase-filled vacuoles in the axonal varicosities as the apparent primary endocytic compartment. Horseradish peroxidase-filled synaptic vesicles were formed when stimulated cells were allowed to recover in horseradish peroxidase-free culture medium. Horseradish peroxidase-filled vacuoles as wells as vesicles contained the synaptic vesicle membrane proteins VAMP II, synaptotagmin, SV2, and synaptophysin, the vesicle-associated proteins rab 3A and synapsin I, and in addition SNAP-25. No incorporation of vesicle proteins into the plasma membrane was observed. Horseradish peroxidase-filled vesicles and vacuoles generated on incubation of unstimulated granule cells with horseradish peroxidase for prolonged periods of time were equally immunolabeled. Renewed stimulation of prestimulated granule cells with either 100 mM KCl or 30 microM Ca2+ ionophore A23187 resulted in a reduction of horseradish peroxidase-filled vacuoles suggesting that the vacuolar membrane compartment was exocytosis-competent. Our results suggest that varicosities of cultured cerebellar granule cells possess a fast stimulation-induced pathway for recycling the entire synaptic vesicle membrane compartment. The primary endocytic compartment represents not a synaptic vesicle but a somewhat larger vesicle protein-containing vacuolar entity from which smaller vesicles of identical protein composition may be regenerated. Endocytic vacuoles and synaptic vesicles share membrane and membrane-associated proteins and presumably also major functional properties.     

Membrane recapture after hormone release from nerve endings in the neural lobe of the rat pituitary gland

The magnocellular neurosecretory system of mammalia, which produces oxytocin and vasopressin and releases these hormones at a neurohaemal contact area in the neural lobe of the pituitary gland, provides a useful model for the study of release of granule-packaged material from both neurones and endocrine cells. Isolated neural lobes from rats were stimulated to release hormone by incubation for 15min in a depolarizing sodium-free medium containing 56 mM potassium ions. Controls were incubated in a sodium-free medium containing 5.6 mM potassium ions. The amounts of vasopressin and oxytocin released by depolarization together comprised ~83 mU. At the end of the stimulation the glands were fixed for electron microscopy and stereological analysis. Loss of neurosecretory granules from stimulated glands was restricted to the nerve endings. From knowledge of the amount of hormone stored in a single neurosecretory granule, the number of granules lost from the gland could be calculated to contain approximately the amount of hormone released. After stimulation, the nerve endings were unaltered in size or membrane area. There was also no change in the total microvesicle population of the endings, though the microvesicles were redistributed towards the basement membrane contact zone. The vacuole population of the endings was, however, increased three-fold after stimulation.We conclude that, after acute stimulation of hormone release from the neural lobe, neurosecretory granules are lost specifically from the nerve endings and that the excess membrane that results from their exocytosis is to be found primarily in vacuoles.     

A comparison of synaptic protein localization in hippocampal mossy fiber terminals and neurosecretory endings of the neurohypophysis using the cryo-immunogold technique

In central synapses synaptic vesicle docking and exocytosis occurs at morphologically specialized sites (active zones) and requires the interaction of specific proteins in the formation of a SNARE complex. In contrast, neurosecretory terminals lack active zones. Using the cryo-immunogold technique we analyzed the localization of synaptic vesicle proteins and of proteins of the docking complex at active zones. This was compared to the localization of the identical proteins in neurosecretory terminals. In addition we compared the vesicular and granular localization of the proteins investigated. Synaptic vesicles in rat hippocampal mossy fiber synapses and microvesicles in the neurosecretory terminals of the neurohypophysis contained in common the proteins VAMP II (a v-SNARE), SV2, rab3A, and N-type Ca²⁺ channels. Only minor immunolabeling for these proteins was observed at neurosecretory granules. These results support the notion of a close functional identity of microvesicles from neurosecretory endings of the neurohypophysis and of synaptic vesicles. The vesicular pool of N-type Ca²⁺ channels may serve their stimulation-induced translocation into the plasma membrane. We find increased labeling for VAMP II, SNAP-25, N-type Ca²⁺ channels and of rab3A at the active zones of mossy fiber synapses. Labeling at release sites is by far highest for Bassoon, a high molecular weight protein of the active zone. The labeling pattern implies an association of Bassoon with presynaptic dense projections. Bassoon is absent from neurosecretory terminals and VAMP II, SNAP-25, rab3A, and N-type Ca²⁺ channels reveal a scattered distribution over the plasma membrane. The competence of the presynaptic active zone for selective vesicle docking may not primarily result from its contents in SNARE proteins but rather from the preformation of presynaptic dense projections as structural guides for vesicle exocytosis.     

Immunocytochemical localization of synaptophysin (protein p38) and synapsin I in nerve terminals of rat neurohypophysis

Synaptophysin and synapsin I, the synaptic vesicle-associated proteins, were demonstrated immunocytochemically in nerve terminals of the neurohypophysis of rats. Although both are associated with microvesicles 50-60 nm in diameter, they are not localized on or around the large neurosecretory granules nor the clear vacuoles, 100-200 nm in diameter. These findings strongly suggest that the microvesicles in the nerve terminal of the neurohypophysis are, for the most part, not the structures involved in the retrieval of the limiting membranes of the released neurosecretory granules, but rather typical synaptic vesicles. The clear vacuoles, which are negative for synaptophysin and synapsin I, are considered to be related to the retrieval of the limiting membranes of the released neurosecretory granules.     

Chloride and magnesium dependence of vasopressin release from rat permeabilized neurohypophysial nerve endings

The role of Cl- and Mg+ ions has been studied on the secretory mechanism leading to the release of vasopressin from digitonin permeabilized nerve endings isolated from the rat neurohypophysis. Secretion was triggered by challenging the permeabilized nerve endings with 1.1 microM free Ca2+. Magnesium enhances secretion and its maximal effect occurred at a concentration of about 2 mM. Further increase of this divalent cation concentration however led to an inhibition of secretion. Chloride ions are necessary for the final steps in exocytosis and this effect of Cl- was inhibited by the chloride channel antagonist N144. It is concluded that in neurosecretory nerve endings magnesium and chloride ions are crucial components for exocytosis to occur.     

Nonclathrin-coated vesicles are involved in endocytosis in kidney collecting duct intercalated cells

Intercalated cells of the kidney collecting duct are able to modify the structure of their apical plasma membrane in response to different physiological conditions. It has been proposed that this process involves the transfer of membrane components (including a proton-pumping ATPase) to and from the apical membrane by a specialized population of tubulovesicles that are found in the apical cytoplasm of these cells. These vesicles have a prominent cytoplasmic coat of regularly arranged dense studs that we have recently shown to be immunocytochemically and morphologically distinct from clathrin. In this study, we have examined the function of these vesicles by using horseradish peroxidase as a tracer of endocytosis at the light and electron microscopic levels. Following the intravenous injection of rats with the tracer, we found a massive labeling of the tubulovesicle compartment of intercalated cells, providing direct evidence that these nonclathrin-coated vesicles are involved in endocytotic events in this cell type. This novel membrane coating material could contain the cytoplasmic domains of molecules transported to and from the plasma membrane by these vesicles (e.g., and H+ ATPase) or it could be a molecule that is involved in vesicle function, by analogy with clathrin.     

Compensatory endocytosis in chromaffin cells

Exocytosis occurs via fusion of secretory granules with the cell membrane, whereupon the granule content is at least partially released and the granule membrane is temporarily added to the plasma membrane. Exocytosis is balanced by compensatory endocytosis to achieve net equilibrium of the cell surface area and to recycle and redistribute components of the exocytosis machinery. The underlying molecular mechanisms remain a matter of debate. In this review, we summarize and discuss recent progress in the understanding of compensatory endocytosis, with the focus on chromaffin cells as a useful model for studying mechanisms of regulated secretion.     

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.     

Ultrastructural demonstration that melanin-concentrating hormone-like and alpha-melanocyte-stimulating hormone-like immunoreactive molecules coexist in the same neurosecretory granules

Using immunocytochemical methods at the electron microscope level, immunoreactivity for both melanin-concentrating hormone (MCH) and alpha-melanocyte-stimulating hormone (alpha-MSH) has been demonstrated in the carp neurohypophysis. A double-labelling technique, using colloidal gold probes of different sizes showed that immunoreactivity to both molecules coexists within the same neurosecretory granules in some neurones, while in other neurones the granules exhibit only MCH-like immunoreactivity. These observations suggest that the two immunoreactivities are attributable to separate molecules; if they are derived from the same precursor molecule, then this must be cleaved differently in the two sets of neurones. The absence of adrenocorticotropic hormone (ACTH)-like immunostaining in any neurosecretory granule might suggest the alpha-MSH-like molecule is not derived from the conventional pro-opiomelanocortin precursor.     

The Role of Cholesterol in the Exo- and Endocytosis of Synaptic Vesicles in Frog Motor Nerve Endings

Experiments on frog neuromuscular preparations using electrophysiological (two-electrode voltage clamping) and optical (with the fluorescent endocytic stain FM1-43) methods were performed to study the importance of membrane cholesterol in the exo- and endocytic cycle of synaptic vesicles (SV) in motor nerve endings in conditions of prolonged rhythmic stimulation of the motor nerve (20 impulses/sec, 3 min). Extraction of cholesterol from the superficial plasma membranes using methyl-β-cyclodextrin (1 mM) led to marked changes in SV recycling. There was weakening of SV exocytosis and suppression of processes leading to the recovery of SV populations with rapid readiness to release neurotransmitter. When cholesterol was leached from the outer membranes and the membranes of SV undergoing recycling, these effects were supplemented by impairments to SV endocytosis and recycling. Thus, plasma membrane cholesterol plays a key role in the processes of exocytosis, while the efficiency of endocytosis depends on cholesterol in SV membranes.     

Synaptic vesicle endocytosis at a CNS nerve terminal: faster kinetics at physiological temperatures and increased endocytotic capacity during maturation

Synaptic vesicle membrane must be quickly retrieved and recycled after copious exocytosis to limit the depletion of vesicle pools. The rate of endocytosis at the calyx of Held nerve terminal has been measured directly using membrane capacitance measurements from immature postnatal day P7-P10 rat pups at room temperature (RT: 23-24 degrees C). This rate has an average time constant of tens of seconds and becomes slower when the amount of exocytosis (measured as capacitance jump) increases. Such slow rates seem paradoxical for a synapse that can operate continuously at high-input frequencies. Here we perform time-resolved membrane capacitance measurements from the mouse calyx of Held in brain stem slices at physiological temperature (PT: 35-37 degrees C), and also from more mature calyces after the onset of hearing (P14-P18). Our results show that the rate of endocytosis is strongly temperature dependent, whereas the endocytotic capacity of a nerve terminal is dependent on developmental stage. At PT we find that endocytosis accelerates due to the addition of a kinetically fast component (time constant: tau = 1-2 s) immediately after exocytosis. Surprisingly, we find that at RT the rate of endocytosis triggered by short (1- to 5-ms) or long (> or =10-ms) depolarizing pulses in P14-P18 mice are similar (tau approximately 15 s). Furthermore, this rate is greatly accelerated at PT (tau approximately 2 s). Thus endocytosis becomes faster and less saturable during synaptic maturation, making the calyceal terminal more capable of sustaining prolonged high-frequency transmitter release.     

Selective uptake and anterograde transport of horseradish peroxidase by hippocampal granule cells

Introduction of horseradish peroxidase into the ventriculocisternal system results in selective labeling of the granule cells of the dentate gyrus and their axons, the mossy fibers. This labeling pattern is not seen after direct injections of horseradish peroxidase into the dorsal hippocampus. The density of the granule cell labeling appears to be related to their proximity to the site of highest horseradish peroxidase concentration. The combined distribution of horseradish peroxidase in the granule cells and mossy fibers strongly suggests that the latter element is labeled as the result of anterograde transport of horseradish peroxidase taken up by the granule cell perikarya or dendrites. This labeling was found in the absence of injury to the hippocampus, suggesting that neuronal damage is not necessary for anterograde transport horseradish peroxidase to occur.     

Kiss-and-Run Quantal Secretion in Frog Nerve-Muscle Synapse

Electrophysiological and optical methods were used to study exo- and endocytosis of synaptic vesicles underlying secretion of the neurotransmitter from motor nerve terminals in frog sternocutaneous muscle. Increase in extracellular concentration of K+ or sucrose produced similar increase in the frequency of miniature endplate currents recorded by extracellular microelectrode. Fluorescent microscopy revealed bright spots in nerve terminal during stimulation of secretion with high-potassium solutions in the presence of endocytosis marker FM1-43. These spots corresponded to clusters of synaptic vesicles that passed through the cycles of exo- and endocytosis. Subsequent high-potassium stimulation of exocytosis in normal Ringer solution led to disappearance of marker spots, while in hyperosmotic saline the spots were preserved. No spots were seen after stimulation of neurotransmitter secretion with sucrose in the presence of FM1-43. It is concluded that quantal secretion of the neurotransmitter in frog motor nerve endings can be realized via both complete exocytosis of synaptic vesicles with subsequent endocytosis and kiss-and-run mechanism with the formation of a temporary pore.     

Mature eosinophils stimulated to develop in human-cord blood mononuclear cell cultures supplemented with recombinant human interleukin-5. II. Vesicular transport of specific granule matrix peroxidase, a mechanism for effecting piecemeal degranulation.

The mechanism of piecemeal degranulation by human eosinophils was investigated. Mature eosinophils that developed in rhIL-5-containing conditioned media from cultured human cord blood mononuclear cells were prepared for ultrastructural studies using a combined technique to image eosinophil peroxidase by cytochemistry in the same sections on which postembedding immunogold was used to demonstrate Charcot-Leyden crystal protein. Vesicular transport of eosinophil peroxidase from the specific granule matrix compartment to the cell surface was associated with piecemeal degranulation. This process involved budding of eosinophil peroxidase-loaded vesicles and tubules from specific granules. Some eosinophil peroxidase that was released from eosinophils remained bound to the cell surface; some was free among the cultured cells. Macrophages and basophils bound the released eosinophil peroxidase to their plasma membranes, internalized it in endocytotic vesicles, and stored it in their respective phagolysosomes and secretory granules. Charcot-Leyden crystal protein was diffusely present in the nucleus and cytoplasm of IL-5-stimulated mature eosinophils. Extensive amounts were generally present in granule-poor and subplasma membrane areas of the cytoplasm in contrast to eosinophil peroxidase, which was secreted and bound to the external surface of eosinophil plasma membranes. These studies establish vesicular transport as a mechanism for emptying the specific eosinophil granule matrix compartment during IL-5-associated piecemeal degranulation.     

Immunoreactivity to P2X(6) receptors in the rat hypothalamo-neurohypophysial system: an ultrastructural study with extravidin and colloidal gold-silver labelling

The distribution of the purine receptor P2X(6) subtype was studied in the rat hypothalamo-neurohypophysial system at the electron microscope level. Receptors were visualised with ExtrAvidin peroxidase conjugate and immunogold-silver pre-embedding immunocytochemistry using a polyclonal antibody against an intracellular domain of the receptor. Application of ExtrAvidin labelling revealed P2X(6) receptors in subpopulations of: (i) neurosecretory cell bodies, neurosecretory and non-neurosecretory axons and dendrites of neurones in the paraventricular and supraoptic nuclei; and (ii) pituicytes and neurosecretory axons of the neurohypophysis. Some of the neurosecretory granules observed in the supraoptic and paraventricular nuclei neurone cell bodies, dendrites and axons as well as those in neurohypophysial axons were also positive for the P2X(6) receptors. In the paraventricular nucleus, some axons and dendrites of non-neurosecretory neurones positive for P2X(6) receptors formed synapses between themselves. Using the immunogold-silver method, the electron-dense particles labelling P2X(6) receptors were found in neurosecretory cell bodies of the supraoptic and paraventricular nuclei, in relation to the cytoplasm, endoplasmic reticulum, Golgi complex and neurosecretory granules. The particles indicative of P2X(6) receptors were also located in neurosecretory and non-neurosecretory axons including axonal buttons making synapses with P2X(6)-negative dendrites. In the neurohypophysis, the electron-dense particles were localised in a subpopulation of pituicytes and neurosecretory axons. In neurohypophysial axons, particles were at times seen over the membrane of some neurosecretory granules (immunogold label) or microvesicles (immunoperoxidase label).We speculate that the P2X(6) receptors at the neurohypophysial level may be implicated not only in hormone release from the axon terminals, but also in membrane recycling of the granular vesicles and microvesicles.     

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.     

Differential modulation of short-term synaptic dynamics by long-term potentiation at mouse hippocampal mossy fibre synapses

After exocytosis, synaptic vesicle components are selectively retrieved by clathrin-mediated endocytosis and then re-used in future rounds of transmitter release. Under some conditions, synaptic terminals in addition perform bulk endocytosis of large membranous sacs. Bulk endocytosis is less selective than clathrin-mediated endocytosis and probably internalizes components normally targeted to the plasma membrane. Nonetheless, this process plays a major role in some tonic ribbon-type synapses, which release neurotransmitter for prolonged periods of time. We show here, that large endosomes formed after strong and prolonged stimulation undergo stimulated exocytosis in retinal bipolar neurons. The result suggests how cells might return erroneously internalized components to the plasma membrane, and also demonstrates that synaptic vesicles are not the only neuronal organelle that stains with styryl dyes and undergoes stimulated exocytosis.