Two-Photon Excitation of Fluorescent Voltage-Sensitive Dyes: Monitoring Membrane Potential in the Infrared.

Functional imaging microscopy based on voltage-sensitive dyes (VSDs) has proven effective for revealing spatio-temporal patterns of activity in vivo and in vitro. Microscopy based on two-photon excitation of fluorescent VSDs offers the possibility of recording sub-millisecond membrane potential changes on micron length scales in cells that lie upwards of one millimeter below the brain's surface. Here we describe progress in monitoring membrane voltage using two-photon excitation (TPE) of VSD fluorescence, and detail an application of this emerging technology in which action potentials were recorded in single trials from individual mammalian nerve terminals in situ. Prospects for, and limitations of this method are reviewed.

[Morpho-functional characteristics of conducting airways and the respiratory portion of rat lung under the conditions of intratracheal infection from the positions of the neuroendocrine regulation of reparative histogeneses].

With the use of light and electron microscopy and immunohistochemistry, the morpho-functional changes in the lungs, the hypothalamus and the neurohypophysis were studied in 45 outbred albino male rats 1, 3, 7 and 14 days after the intratracheal infusion of Staphylococcus aureus strains either possessing anti-lactoferrin activity (ALfA(+)) or lacking it (ALfA(-)). After the infusion of ALfA(+) bacteria, the bronchial wall and the respiratory portion of the lungs demonstrated the destructive changes of tissues, sclerosis phenomena, disturbances of regeneration processes (polypoid outgrowth, metaplasia), while in the neurohypophysis a delay in the release of neurosecretion into the blood from the terminals of nonapeptidergic neurosecretory cells took place. These phenomena were not observed after the infection with ALfA(-)bacteria. The results obtained indicate the disturbances of the structural-functional homeostasis of pulmonary tissues associated with bacterial ALfa, taking place together with the limitations of the hypothalamic neurosecretion.

Differential modulation of N-type calcium channels by micro-opioid receptors in oxytocinergic versus vasopressinergic neurohypophysial terminals.

Opioids modulate the electrical activity of magnocellular neurons (MCN) and inhibit neuropeptide release at their terminals in the neurohypophysis. We have previously shown that micro-opioid receptor (MOR) activation induces a stronger inhibition of oxytocin (OT) than vasopressin (AVP) release from isolated MCN terminals. This higher sensitivity of OT release is due, at least in part, to the selective targeting of R-type calcium channels. We now describe the underlying basis for AVP's weaker inhibition by MOR activation and provide a more complete explanation of the complicated effects on neuropeptide release. We found that N-type calcium channels in AVP terminals are differentially modulated by MOR; enhanced at lower concentrations but increasingly inhibited at higher concentrations of agonists. On the other hand, N-type calcium channels in OT terminals were always inhibited. The response pattern in co-labeled terminals was analogous to that observed in AVP-containing terminals. Changes in intracellular calcium concentration and neuropeptide release corroborated these results as they showed a similar pattern of enhancement and inhibition in AVP terminals contrasting with solely inhibitory responses in OT terminals to MOR agonists. We established that fast translocation of Ca(2+) channels to the plasma membrane was not mediating current increments and thus, changes in channel kinetic properties are most likely involved. Finally, we reveal a distinct Ca-channel beta-subunit expression between each type of nerve endings that could explain some of the differences in responses to MOR activation. These results help advance our understanding of the complex modulatory mechanisms utilized by MORs in regulating presynaptic neuropeptide release.

Voltage-dependent kappa-opioid modulation of action potential waveform-elicited calcium currents in neurohypophysial terminals.

Release of neurotransmitter is activated by the influx of calcium. Inhibition of Ca(2+) channels results in less calcium influx into the terminals and presumably a reduction in transmitter release. In the neurohypophysis (NH), Ca(2+) channel kinetics, and the associated Ca(2+) influx, is primarily controlled by membrane voltage and can be modulated, in a voltage-dependent manner, by G-protein subunits interacting with voltage-gated calcium channels (VGCCs). In this series of experiments we test whether the kappa- and micro-opioid inhibition of Ca(2+) currents in NH terminals is voltage-dependent. Voltage-dependent relief of G-protein inhibition of VGCC can be achieved with either a depolarizing square pre-pulse or by action potential waveforms. Both protocols were tested in the presence and absence of opioid agonists targeting the kappa- and micro-receptors in neurohypophysial terminals. The kappa-opioid VGCC inhibition is relieved by such pre-pulses, suggesting that this receptor is involved in a voltage-dependent membrane delimited pathway. In contrast, micro-opioid inhibition of VGCC is not relieved by such pre-pulses, indicating a voltage-independent diffusible second-messenger signaling pathway. Furthermore, relief of kappa-opioid inhibition during a physiologic action potential (AP) burst stimulation indicates the possibility of activity-dependent modulation in vivo. Differences in the facilitation of Ca(2+) channels due to specific G-protein modulation during a burst of APs may contribute to the fine-tuning of Ca(2+)-dependent neuropeptide release in other CNS terminals, as well.

Tissue plasminogen activator and plasminogen are critical for osmotic homeostasis by regulating vasopressin secretion.

Systemic osmotic homeostasis is regulated mainly by neuroendocrine system of arginine-vasopressin (AVP) in mammalians. In the present study, we demonstrated that the immunoreactivity of tissue plasminogen activator (tPA) was observed specifically at neurosecretory granules of AVP-positive magnocellular terminals and that of plasminogen was seen at astrocytes in the neurohypophysis (NH). Both tPA and plasminogen knockout (KO) mice revealed higher plasma osmolarity upon water deprivation, a chronic osmotic stimulation, as compared with their wild-type (WT) animals, indicating abnormal osmotic control in these KO mice. tPA KO mice but not plasminogen ones revealed lower ability in secreting AVP into the blood circulation upon an acute osmotic stimulation. Both tPA and plasminogen KO animals showed lower ability in secreting AVP into the blood circulation upon a chronic osmotic stimulation. The recombinant tPA was able to promote the release of AVP from isolated NH. Chronic osmotic stimulation decreased the laminin expression level of neurohypophysial microvessel in WT mice but not in plasminogen KO ones. We suggest that AVP secretion is critically regulated by tPA-dependent facilitation of AVP release from terminals and plasminogen-dependent increase of AVP permeability across microvessels possibly via laminin degradation.

A freeze-fracture study of membrane events during neurohypophysial secretion.

Freeze-fracture was used to study the membrane events taking place during neurosecretory granule discharge (exocytosis) and subsequent membrane internalization (endocytosis) in axons of neurohypophyses from control and water-deprived rats. En face views of the cytoplasmic leaflet (P face) of the split axolemma reveal circular depressions that represent the secretory granule membranes fused with the plasma membrane during exocytosis. These depressions often contain granule core material in the process of extrusion into the extracellular space. The membrane surrounding some of the exocytotic openings shows a decreased number of intramembrane particles (mean diameter, 8 nm) which are elsewhere more numerous and evenly distrubuted on the fracture face. Endocytotic sites appear as smaller plasma membrane invaginations, with associated intramembrane particles. Moreover, such invaginations often contain large particles (mean diameter, 12 nm) that appear as clusters on en face views of the membrane leaflet. Quantitative analysis indicates that the number of exocytotic images increases significantly in glands from water-deprived rats. Concomitantly, the number of endocytotic figures per unit area of membrane is raised as is the number of clusters of large particles. The observations demonstrate that, in the neurohypophysis, it is possible to distinguish exocytosis morphologically from endocytosis and that the two events can be assessed quantitatively.

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.

Dynorphin-A-(1-8) is contained within vasopressin neurosecretory vesicles in rat pituitary.

Dynorphin-A-(1-8), an opioid peptide widely distributed in the rat central nervous system, is present in vasopressin-containing neurosecretory cells terminating in the neural lobe of the pituitary. Electron microscopic immunocytochemistry reveals that dynorphin-A-(1-8) is contained within the same neurosecretory vesicles as vasopressin and vasopressin-associated neurophysin in the neural lobe of the rat. The results indicate that dynorphin may be released in the pituitary concomitantly with vasopressin during the antidiuretic response.

Possible role during exocytosis of a Ca(2+)-activated channel in neurohypophysial granules.

Ion channels from bovine neurohypophysial granules were incorporated into artificial lipid bilayers. The larger amplitude channel is permeable to cations and exhibits multiple conductances. The channel opens only in the presence of free Ca2+, but is inhibited by relatively high Ca2+ concentrations. Release of vasopressin from permeabilized neurohypophysial terminals also shows a similar biphasic dependence on Ca2+. Release is selectively inhibited by low concentrations of the long-chain alcohol octanol, but not by high concentrations of ethanol, as is the neurosecretory granule Ca(2+)-activated cation channel. Furthermore, Ca(2+)-evoked release and channel activity are both inhibited by the long-chain tetraethylammonium analogs decamethonium and decyl-triethyl ammonium bromide. The close correlation between channel and release properties lead us to conclude that the Ca(2+)-activated channel is involved in peptide secretion.

Glutamatergic innervation of the hypothalamic median eminence and posterior pituitary of the rat.

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.

Ultrastructural study of infectious bronchitis virus infection in infundibulum and magnum of commercial laying hens.

The infundibulum and magnum of the oviduct were examined in hens in full lay which were infected with two Australian strains of infectious bronchitis virus (IBV). The ultramicroscopic changes in the infundibulum and magnum were compared with control hens which had eggs at different positions in the oviduct. The ciliated and granular cells of the surface epithelia and secretory epithelial cells of the tubular glands were the target cells of IBV. No pathological changes were recorded during 2-8 days post-infection (p.i.). Patchy loss of cilia occurred at 10-14 days p.i. Between 16 and 24 days p.i., there was no cilia loss and lymphoid nodules were observed in the muscularis layer of the infundibulum and magnum of some hens from both infected groups. Virus particles were detected mostly in the rough endoplasmic reticulum (RER) and Golgi complex between 10 and 12 days p.i. Cytopathology was noticed in various cell organelles between the 10th and 14th days p.i. There was an increase in RER deposits in infected cells, irrespective of egg position in the oviduct. The magnum was more affected than the infundibulum. Cellular changes were more severe in the infundibulum and magnum of T-infected hens as compared to N1/88-infected hens. Eggs with watery whites which were laid by infected hens could be attributed to cytopathological changes in the granular epithelial cells and tubular gland epithelial cells of the magnum resulting in reduced synthesis of albumen proteins. IBV can cause pathology in parts of the fully functional oviduct which may persist up to the 30th day p.i. However, both the challenge strains of IBV can cause a small number of hens to cease production. Loss of cilia in both the infundibulum and magnum pose a potential threat of secondary bacterial infection and also may affect fertility in breeder hens.

Possible involvement of brain-derived neurotrophic factor (BDNF) in the innervation of dopaminergic neurons from the rat periventricular nucleus to the pars intermedia.

Developing neurons are guided to their appropriate targets by specific guidance substances that have neurotrophic actions. The aim of the present study was to elucidate the mechanism by which hypothalamic neurons reach the pars intermedia (PI) by correlating the development of dopaminergic (DA) neurons arising in the periventricular nucleus (PeV) of fetal rats with the expression of brain-derived neurotrophic factor (BDNF) in the rat pituitary. The differentiation of DA neurons was observed by immunohistochemistry using an antibody against tyrosine hydroxylase (TH), whereas the ontogenesis of BDNF mRNA in the PI was examined by in situ hybridization and RT-PCR. Immunoreactive TH-neurons were first observed in the PeV at embryonic day (E) 16.5, following which time their axons elongated toward the pituitary. TH-positive reactions were observed in the connective tissue between the PI and the pars nervosa at E20.5. Innervation of the PI by TH-positive neurons was determined at postnatal day (P) 1.5; however, BDNF mRNA was first detected in the PI cells at E17.5, with an increase in its expression clearly visible at E21.5 and continuing high expression levels in the PI thereafter. These results suggest that BDNF is a specific guidance cue for DA neurons elongating from the PeV to the PI.

Scanning electron microscopic observations on the third ventricular floor of the rat following cervical sympathectomy.

Various investigators have shown that unilateral ganglionectomy or transection of the internal and external carotid nerves leads to a regenerative response in the ipsilateral superior cervical ganglion and to uninjured mature sympathetic neurons sprouting into bilaterally innervated shared target organs. In this study changes in the supraependymal neuronal network following unilateral and bilateral cervical sympathectomy on the infundibular floor of the third ventricle were studied by scanning electron microscopy in comparison with normal and sham-operated control animals. After unilateral cervical sympathectomy there was a great increase in the number of varicose nerve fibres on the infundibular floor as compared to the normal and sham-operated control animals. Not only was there an increase in the number of nerve fibres, but also their varicosities were substantially larger than those normally present on the ependymal surface. This study indicates the possible sympathetic projections from the superior cervical ganglia to the ependymal surface of the third cerebral ventricle.

Structural Reconstruction of the Perivascular Space in the Adult Mouse Neurohypophysis During an Osmotic Stimulation.

Oxytocin (OXT) and arginine vasopressin (AVP) neuropeptides in the neurohypophysis (NH) control lactation and body fluid homeostasis, respectively. Hypothalamic neurosecretory neurones project their axons from the supraoptic and paraventricular nuclei to the NH to make contact with the vascular surface and release OXT and AVP. The neurohypophysial vascular structure is unique because it has a wide perivascular space between the inner and outer basement membranes. However, the significance of this unique vascular structure remains unclear; therefore, we aimed to determine the functional significance of the perivascular space and its activity-dependent changes during salt loading in adult mice. The results obtained revealed that pericytes were the main resident cells and defined the profile of the perivascular space. Moreover, pericytes sometimes extended their cellular processes or 'perivascular protrusions' into neurohypophysial parenchyma between axonal terminals. The vascular permeability of low-molecular-weight (LMW) molecules was higher at perivascular protrusions than at the smooth vascular surface. Axonal terminals containing OXT and AVP were more likely to localise at perivascular protrusions than at the smooth vascular surface. Chronic salt loading with 2% NaCl significantly induced prominent changes in the shape of pericytes and also increased the number of perivascular protrusions and the surface area of the perivascular space together with elevations in the vascular permeability of LMW molecules. Collectively, these results indicate that the perivascular space of the NH acts as the main diffusion route for OXT and AVP and, in addition, changes in the shape of pericytes and perivascular reconstruction occur in response to an increased demand for neuropeptide release.

Tomosyn Negatively Regulates Arginine Vasopressin Secretion in Embryonic Stem Cell-Derived Neurons.

Arginine vasopressin (AVP) is secreted via exocytosis; however, the precise molecular mechanism underlying the exocytosis of AVP remains to be elucidated. To better understand the mechanisms of AVP secretion, in our study we have identified proteins that bind with a 25 kDa synaptosomal-associated protein (SNAP25). SNAP25 plays a crucial role in exocytosis, in the posterior pituitary. Embryonic stem (ES) cell-derived AVP neurons were established to investigate the functions of the identified proteins. Using glutathione S-transferase (GST)-pulldown assays and proteomic analyses, we identified tomosyn-1 (syntaxin-binding protein 5) as a SNAP25-binding protein in the posterior pituitary. Coimmunoprecipitation assays indicated that tomosyn formed N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes with SNAP25 and syntaxin1. Immunohistochemistry showed that tomosyn localized to the posterior pituitary. Mouse ES cells self-differentiated into AVP neurons (mES-AVP) that expressed tomosyn and two transmembrane SNARE proteins, including SNAP25 and syntaxin1. KCl increased AVP secretion in mES-AVP, and overexpression of tomosyn-1 reduced KCl-stimulated AVP secretion. Downregulation of tomosyn-1 with siRNA increased KCl-stimulated AVP secretion. These results suggested that tomosyn-1 negatively regulated AVP secretion in mES-AVP and further suggest the possibility of using mES-AVP culture systems to evaluate the role of synaptic proteins from AVP neurons.

Pulsed laser imaging of Ca(2+) influx in a neuroendocrine terminal.

The surge of Ca(2+) that triggers vesicle fusion is shaped by the distribution of Ca(2+) channels and the physical relationship between those channels and the exocytotic apparatus. Although channels and the release apparatus are thought to be tightly associated at fast synapses, the arrangement at neuroendocrine cells is less clear. The distribution of Ca(2+) influx near release sites is difficult to determine because of spatial and temporal limitations on Ca(2+) imaging techniques. We now present spatially resolved images of Ca(2+) influx into rat neuroendocrine terminals on a millisecond time scale. Images of voltage-dependent Ca(2+) influx into neurohypophysial terminals were captured after excitation of Ca(2+)-sensitive dyes with pulses of laser light lasting a fraction of a microsecond. Submembranous Ca(2+) increases were detected during the first millisecond of an evoked Ca(2+) tail current. Steep gradients of Ca(2+) were evident, with concentrations near the membrane reaching above 1 microM during a 30 msec depolarization. Ca(2+) influx appeared evenly distributed, even when diffusion was restricted with an exogenous Ca(2+) chelator. During longer depolarizations, mean and peak Ca(2+) concentrations reached an asymptote in parallel, suggesting that Ca(2+) binding proteins near the membrane rapidly buffer Ca(2+) and do not become saturated during prolonged influx. These data support the hypothesis that exocytosis is activated in these terminals by the summation of influx through multiple, randomly spaced Ca(2+) channels.

Membrane retrieval in the guinea pig neurohypophysis: biochemical characterization of a retrieval structure.

[3H]Choline and [35S]methionine injected into the guinea pig hypothalamus in vivo were incorporated into the lipids and proteins, respectively, of secretory vesicles transported to the neural lobe. Prolonged in vivo stimulation of hormone secretion by dehydration decreased the [3H]choline content of secretory vesicles, with a concomitant increase in the [3H]choline content of a membrane fraction isolated on sucrose gradients. After stimulation of neural lobes in vitro in the presence of horseradish peroxidase, this extracellular fluid marker was found in the same membrane fraction. SDS electrophoresis of membrane proteins radiolabelled by [35S]methionine in vivo demonstrated that this fraction contained at least one major protein also present in the secretory vesicle membrane. These results suggest that we have isolated a membrane fraction containing the structure(s) involve in membrane retrieval in the neurohypophysis.

Fusion of neurohypophyseal membranes in vitro.

Freeze cleaving electron microscopy has shown that fusion of isolated secretory vesicles from bovine neurohypophyses was induced by Ca2+ in micromolar concentrations. Mg2+ and Sr2+ were ineffective. Mg2+ inhibited Ca2+-induced fusion. In suspensions containing secretory vesicles as well as sheets of cell membrane, release of vasopressin parallel to intervesicular fusion and fusion of secretory vesicles with sheets of cell membrane was observed after exposure to Ca2+. Mg2+ and Sr2+ were ineffective in replacing Ca2+ as trigger for fusion or vasopressin release. Intervesicular fusion and exocytotic profiles were observed when isolated neurohypophyses or neurosecretosomes were exposed to cold.

The destination of the aged, nonreleasable neurohypophyseal peptides stored in the neural lobe is associated to the remodeling of the neurosecretory axon.

The present investigation was designed to investigate the fate of the large pool of neurohypophyseal hormones that is never released into the blood. Normal Sprague-Dawley and taiep mutant rats were investigated under normal water balance, after dehydration and after dehydration-rehydration. Lectin histochemistry and light- and electron-microscopic immunocytochemistry using antibodies against vasopressin, oxytocin, and neurophysins used at low (1:1,000) and high (1:15,000) dilutions allowed to distinguish (1) recently packed immature granules, as those located in the perikaryon; (2) mature; and (3) aged granules. The distribution of these granules within the different domains of the neurosecretory axons located in the neural lobe, namely, undilated segments, swellings, terminals, and Herring bodies, and the response of these compartments to dehydration and dehydration-rehydration allowed to roughly follow the routing of the granules through such axonal domains. It is suggested that granules may move backward and forward between the terminals and the swellings. At variance, aged granules located in Herring body are retained in this compartment and would finally become degraded. Herring bodies displayed distinct lectin binding and immunocytochemical properties, allowing to distinguish them from axonal swellings. After a dehydration-rehydration cycle, immunocytochemistry and electron microscopy revealed that Herring bodies were no longer present in the neural lobe and that several terminals had degenerated. It is concluded that (1) the neurophysin axons may undergo remodeling under appropriate stimuli and (2) Herring bodies are a specialized and plastic domain of the magnocellular neurosecretory neuron involved in the disposal of aged neurosecretory granules. No differences were detected at the neural lobe level between normal and mutant rats subjected to the same experimental conditions.

Colocalization of immunoreactive endothelin-1 and neurohypophysial hormones in the axons of the neural lobe of the rat pituitary.

The intracellular localization of immunoreactive endothelin (ET)-1, and its colocalization with vasopressin (VP) and oxytocin (OT) in the rat neural lobe were investigated by immunogold techniques using specific antisera raised against ET-1, VP, and OT. There were two types of axons: the first contained VP-immunolabeled neurosecretory granules, and the second contained OT-immunolabeled neurosecretory granules. A considerable number of the neurosecretory granules in both types of axon were immunolabeled with antibodies against ET-1, although the VP-immunolabeled granules were more heavily labeled with anti-ET-1 antiserum than OT-immunolabeled ones. Double immunogold labeling clearly demonstrated the intragranular colocalization of immunoreactions for ET-1 and VP and that of immunoreactions for ET-1 and OT. These results suggest that ET in the neural lobe may be released concomitantly with neurohypophysial hormones. Its biological significance remains to be elucidated.