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G R Holstein

Publications and source records attributed to G R Holstein.

At least 19 recordsLinked to original sources
PubMed2005

A role for nitric oxide in sensory-induced neurogenesis in an adult insect brain.

In the adult cricket, neurogenesis occurs in the mushroom bodies, the main integrative structures of the insect brain. Mushroom body neuroblast proliferation is modulated in response to environmental stimuli. However, the mechanisms underlying these effects remain unspecified. In the present study, we demonstrate that electrical stimulation of the antennal nerve mimics the effects of olfactory activation and increases mushroom body neurogenesis. The putative role of nitric oxide (NO) in this activity-regulated neurogenesis was then explored. In vivo and in vitro experiments demonstrate that NO synthase inhibition decreases, and NO donor application stimulates neuroblast proliferation. NADPH-d activity, anti-L-citrulline immunoreactivity, as well as in situ hybridization with a probe specific for Acheta NO synthase were used to localize NO-producing cells. Combining these three approaches we clearly establish that mushroom body interneurons synthesize NO. Furthermore, we demonstrate that experimental interventions known to upregulate neuroblast proliferation modulate NO production: rearing crickets in an enriched sensory environment induces an upregulation of Acheta NO synthase mRNA, and unilateral electrical stimulation of the antennal nerve results in increased L-citrulline immunoreactivity in the corresponding mushroom body. The present study demonstrates that neural activity modulates progenitor cell proliferation and regulates NO production in brain structures where neurogenesis occurs in the adult insect. Our results also demonstrate the stimulatory effect of NO on mushroom body neuroblast proliferation. Altogether, these data strongly suggest a key role for NO in environmentally induced neurogenesis.

Afferent Pathways↗
PubMed2004

Synapsin-like immunoreactivity is present in hair cells and efferent terminals of the toadfish crista ampullaris.

The synapsins are presynaptic membrane-associated proteins involved in neurotransmitter release. They are differentially expressed in tissues and cells of the central and peripheral nervous system. In vestibular end organs of mammals, synapsin I-like immunoreactivity has been reported in efferent and afferent terminals and in afferent nerve calyces surrounding type I hair cells. In addition, synapsin I has recently been described in several non-neural cell lines. The present study was conducted to locate synapsin-like immunoreactivity in the neuronal and non-neuronal cells of the fish crista ampullaris, to examine the possibility that the non-neuronal sensory receptor cells express synapsins in vivo. Synapsin-like immunostaining was visualized by immunofluorescence detection in wholemounts of the toadfish crista ampullaris using multiphoton laser scanning microscopy and by electron microscopic visualization of post-embedding immunogold labeling. The results demonstrate that synapsin-like immunoreactivity is present in vestibular hair cells and efferent boutons of the toadfish crista ampullaris. Afferent endings are not labeled. Staining in hair cells is not associated with the synaptic ribbons, suggesting that there is an additional, non-synaptic role for the synapsins in some non-neuronal cells of vertebrates. Moreover, while the cristae of amniote and anamniote species share many functional attributes, differences in their synaptic vesicle-associated protein profiles appear to reflect their disparate hair cell populations.

Animals↗
PubMed2004

Hair-cell versus afferent adaptation in the semicircular canals.

The time course and extent of adaptation in semicircular canal hair cells was compared to adaptation in primary afferent neurons for physiological stimuli in vivo to study the origins of the neural code transmitted to the brain. The oyster toadfish, Opsanus tau, was used as the experimental model. Afferent firing-rate adaptation followed a double-exponential time course in response to step cupula displacements. The dominant adaptation time constant varied considerably among afferent fibers and spanned six orders of magnitude for the population ( approximately 1 ms to >1,000 s). For sinusoidal stimuli (0.1-20 Hz), the rapidly adapting afferents exhibited a 90 degrees phase lead and frequency-dependent gain, whereas slowly adapting afferents exhibited a flat gain and no phase lead. Hair-cell voltage and current modulations were similar to the slowly adapting afferents and exhibited a relatively flat gain with very little phase lead over the physiological bandwidth and dynamic range tested. Semicircular canal microphonics also showed responses consistent with the slowly adapting subset of afferents and with hair cells. The relatively broad diversity of afferent adaptation time constants and frequency-dependent discharge modulations relative to hair-cell voltage implicate a subsequent site of adaptation that plays a major role in further shaping the temporal characteristics of semicircular canal afferent neural signals.

Adaptation, Physiological↗
PubMed2004

Ultrastructural observations of efferent terminals in the crista ampullaris of the toadfish, Opsanus tau.

The present study was conducted to visualize the ultrastructural features of vestibular efferent boutons in the oyster toadfish, Opsanus tau. The crista ampullaris of the horizontal semicircular canal was processed for and examined by routine transmission electron microscopy. The results demonstrate that such boutons vary in size and shape, and contain a heterogeneous population of lucent vesicles with scattered dense core vesicles. Efferent contacts with hair cells are characterized by local vesicle accumulations in the presynaptic terminal and a subsynaptic cistern in the postsynaptic region of the hair cell. Serial efferent to hair cell to afferent synaptic arrangements are common, particularly in the central portion of the crista. However, direct contacts between efferent terminals and afferent neurites were not observed in our specimens. The existence of serial synaptic contacts, often with a row of vesicles in the efferent boutons lining the efferent-afferent membrane apposition, suggests that the efferent influence on the crista may involve both synaptic and nonsynaptic, secretory mechanisms. Further, it is suggested that differences in more subtle aspects of synaptic architecture and/or transmitter and receptor localization and interaction may render the efferent innervation of the peripheral crista less effective in influencing sensory processing.

Animals↗
PubMed2003

Ultrastructural observations of efferent terminals in the crista Ampullaris of the toadfish, opsanus tau.

The present study was conducted to visualize the ultrastructural features of vestibular efferent boutons in the oyster toadfish, Opsanus tau. The crista ampullaris of the horizontal semicircular canal was processed for and examined by routine transmission electron microscopy. The results demonstrate that such boutons vary in size and shape, and contain a heterogeneous population of lucent vesicles with scattered dense core vesicles. Efferent contacts with hair cells are characterized by local vesicle accumulations in the presynaptic terminal and a subsynaptic cistern in the postsynaptic region of the hair cell. Serial efferent to hair cell to afferent synaptic arrangements are common, particularly in the central portion of the crista. However, direct contacts between efferent terminals and afferent neurites were not observed in our specimens. The existence of serial synaptic contacts, often with a row of vesicles in the efferent boutons lining the efferent-afferent membrane apposition, suggests that the efferent influence on the crista may involve both synaptic and nonsynaptic, secretory mechanisms. Further, it is suggested that differences in more subtle aspects of synaptic architecture and/or transmitter and receptor localization and interaction may render the efferent innervation of the peripheral crista less effective in influencing sensory processing.

Animals↗
PubMed2002

L-citrulline immunostaining identifies nitric oxide production sites within neurons.

The cellular and subcellular localization of L-citrulline was analyzed in the adult rat brain and compared with that of traditional markers for the presence of nitric oxide synthase. Light, transmission electron, and confocal laser scanning microscopy were used to study tissue sections processed for immunocytochemistry employing a monoclonal antibody against L-citrulline or polyclonal anti-neuronal nitric oxide synthase sera, and double immunofluorescence to detect neuronal nitric oxide synthase and L-citrulline co-localization. The results demonstrate that the same CNS regions and cell types are labeled by neuronal nitric oxide synthase polyclonal antisera and L-citrulline monoclonal antibodies, using both immunocytochemistry and immunofluorescence. Short-term pretreatment with a nitric oxide synthase inhibitor reduces L-citrulline immunostaining, but does not affect neuronal nitric oxide synthase immunoreactivity. In the vestibular brainstem, double immunofluorescence studies show that many, but not all, neuronal nitric oxide synthase-positive cells co-express L-citrulline, and that local intracellular patches of intense L-citrulline accumulation are present in some neurons. Conversely, all L-citrulline-labeled neurons co-express neuronal nitric oxide synthase. Cells expressing neuronal nitric oxide synthase alone are interpreted as neurons with the potential to produce nitric oxide under other stimulus conditions, and the subcellular foci of enhanced L-citrulline staining are viewed as intracellular sites of nitric oxide production. This interpretation is supported by ultrastructural observations of subcellular foci with enhanced L-citrulline and/or neuronal nitric oxide synthase staining that are located primarily at postsynaptic densities and portions of the endoplasmic reticulum. We conclude that nitric oxide is produced and released at focal sites within neurons that are identifiable using L-citrulline as a marker.

Animals↗
PubMed2001

Monoclonal L-citrulline immunostaining reveals nitric oxide-producing vestibular neurons.

Nitric oxide is an unstable free radical that serves as a novel messenger molecule in the central nervous system (CNS). In order to understand the interplay between classic and novel chemical communication systems in vestibular pathways, the staining obtained using a monoclonal antibody directed against L-citrulline was compared with the labeling observed using more traditional markers for the presence of nitric oxide. Brainstem tissue from adult rats was processed for immunocytochemistry employing a monoclonal antibody directed against L-citrulline, a polyclonal antiserum against neuronal nitric oxide synthase, and/or NADPH-diaphorase histochemistry. Our findings demonstrate that L-citrulline can be fixed in situ by vascular perfusion, and can be visualized in fixed CNS tissue sections by immunocytochemistry. Further, the same vestibular regions and cell types are labeled by NADPH-diaphorase histochemistry, by the neuronal nitric oxide synthase antiserum, and by our anti-L-citrulline antibody. Clusters of L-citrulline-immunoreactive neurons are present in subregions of the vestibular nuclei, including the caudal portion of the inferior vestibular nucleus, the magnocellular portion of the medial vestibular nucleus, and the large cells in the ventral tier of the lateral vestibular nucleus. NADPH-diaphorase histochemical staining of these neurons clearly demonstrated their multipolar, fusiform and globular somata and long varicose dendritic processes. These results provide support for the suggestion that nitric oxide serves key roles in both vestibulo-autonomic and vestibulo-spinal pathways.

Animals↗
PubMed1999

Paradoxical locomotor behavior of dopamine D1 receptor transgenic mice.

The behavioral effects of augmenting dopamine D1 receptor expression in the brain were investigated in mice incorporating additional copies of the mouse D1 receptor gene. Two transgenic lines showed increases in brain D1 receptor binding sites, which were greatest in extrastriatal regions. The full D1 agonist SKF 81297, when administered systemically to control animals, stimulated a dose-dependent increase in locomotor activity. In contrast, in D1 receptor overexpressing transgenic mice, this drug caused a marked suppression of locomotion due to a decrease in the frequency of movement initiation. Amphetamine and cocaine induced comparable locomotor activation in both transgenic animals and their control littermates. In the transgenic animals, D1 agonist-induced rearing and climbing behaviors were suppressed. However, on rotarod testing, the agonist-treated transgenic and control mice performed comparably, indicating that sensorimotor coordination was unaffected. These studies demonstrate that altering the levels of D1 receptor expression reverses the effects of D1 agonism on locomotor initiation and rearing.

Animals↗
PubMed1999

Ultrastructural features of non-commissural GABAergic neurons in the medial vestibular nucleus of the monkey.

The ultrastructural characteristics of non-degenerating GABAergic neurons in rostrolateral medial vestibular nucleus were identified in monkeys following midline transection of vestibular commissural fibers. In the previous papers, we reported that most degenerated cells and terminals in this tissue were located in rostrolateral medial vestibular nucleus, and that many of these neurons were GABA-immunoreactive. In the present study, we examined the ultrastructural features of the remaining neuronal elements in this medial vestibular nucleus region, in order to identify and characterize the GABAergic cells that are not directly involved in the vestibular commissural pathway related to the velocity storage mechanism. Such cells are primarily small, with centrally-placed nuclei. Axosomatic synapses are concentrated on polar regions of the somata. The proximal dendrites of GABAergic cells are surrounded by boutons, although distal dendrites receive only occasional synaptic contacts. Two types of non-degenerated GABAergic boutons are distinguished. Type A terminals are large, with very densely-packed spherical synaptic vesicles and clusters of large, irregularly-shaped mitochondria with wide matrix spaces. Such boutons form symmetric synapses, primarily with small GABAergic and non-GABAergic dendrites. Type B terminals are smaller and contain a moderate density of round/pleomorphic vesicles, numerous small round or tubular mitochondria, cisterns and vacuoles. These boutons serve both pre- and postsynaptic roles in symmetric contacts with non-GABAergic axon terminals. On the basis of ultrastructural observations of immunostained tissue, we conclude that at least two types of GABAergic neurons are present in the rostrolateral portion of the monkey medial vestibular nucleus: neurons related to the velocity storage pathway, and a class of vestibular interneurons. A multiplicity of GABAergic bouton types are also observed, and categorized on the basis of subcellular morphology. We hypothesize that "Type A" boutons correspond to Purkinje cell afferents in rostrolateral medial vestibular nucleus, "Type B" terminals represent the axons of GABAergic medial vestibular nucleus interneurons, and "Type C" boutons take origin from vestibular commissural neurons of the velocity storage pathway.

Animals↗
PubMed1999

The ultrastructure of GABA-immunoreactive vestibular commissural neurons related to velocity storage in the monkey.

The purpose of the present study was to visualize the synaptic interactions of GABAergic neurons involved in the mediation of velocity storage. In the previous report, ultrastructural studies of degenerating neurons were conducted following midline section of rostral medullary commissural fibers with subsequent behavioral testing. The midline lesion caused functionally discrete damage to the velocity storage component, but not to the direct pathway, of the angular vestibulo-ocular reflex, and the degenerating neurons were interpreted as potential participants in the velocity storage network. We concluded that at least some of the commissural axons mediating velocity storage originate from clusters of neurons in the lateral crescents of the rostral medial vestibular nucleus. In the present report, immunocytochemical evidence is presented that many vestibular commissural neurons, putatively involved in mediating velocity storage, are GABAergic. These cells have large nuclei, small round or narrow tubular mitochondria, occasional cisterns and vacuoles, but few other organelles. Their axons are thinly-myelinated, and terminate in boutons containing mitochondria of similar ultrastructural appearance and a moderate density of round/pleomorphic synaptic vesicles. Such terminals often form axoaxonic synapses, and less frequently axodendritic contacts, with non-GABAergic elements. On the basis of the present results, we conclude that a portion of the commissural neurons of the velocity storage pathway is GABAergic. The observation of GABAergic axoaxonic synapses in this pathway is interpreted as a structural basis for presynaptic inhibition of medial vestibular nucleus circuits by velocity storage-related commissural neurons. Conversely, substantial ultrastructural evidence for postsynaptic inhibition of non-GABAergic commissural cells argues for a dual role for GABAergic terminals mediating velocity storage: presynaptic inhibition of non-GABAergic vestibular cells by GABAergic velocity storage commissural axons, and postsynaptic inhibition of non-GABAergic velocity storage cells by GABAergic axons. Both pre- and postsynaptic inhibitory arrangements could provide the morphologic basis for disinhibitory activation of the velocity storage network within local neuronal circuits.

Animals↗
PubMed1999

Ultrastructure of vestibular commissural neurons related to velocity storage in the monkey.

The angular vestibulo-ocular reflex maintains gaze during head movements. It is thought to be mediated by two components: direct and velocity storage pathways. The direct angular vestibulo-ocular reflex is conveyed by a three neuron chain from the labyrinth to the ocular motoneurons. The indirect pathway involves a more complex neural network that utilizes a portion of the vestibular commissure. The purpose of the present study was to identify the ultrastructural characteristics of commissural neurons in the medial vestibular nucleus that are related to the velocity storage component of the angular vestibulo-ocular reflex. Ultrastructural studies of degenerating medial vestibular nucleus neurons were conducted in monkeys following midline section of rostral medullary commissural fibers with subsequent behavioral testing. After this lesion, oculomotor and vestibular functions attributable to velocity storage were abolished, whereas the direct angular vestibulo-ocular reflex pathway remained intact. Since this damage was functionally discrete, degenerating neurons were interpreted as potential participants in the velocity storage network. Ultrastructural observations indicate that commissural neurons related to velocity storage are small and medium sized cells having large nuclei with deep indentations and relatively little cytoplasm, which are located in the lateral crescents of rostral medial vestibular nucleus. The morphology of degenerating dendritic profiles varied. Some contained numerous round or tubular mitochondria in a pale cytoplasmic matrix with few other organelles, while others had few mitochondria but many cisterns and vacuoles in dense granular cytoplasm. The commissural nature of these cells was further suggested by the presence of two different types of degenerating axon terminals in the rostral medial vestibular nucleus: those with a moderate density of large spherical synaptic vesicles, and those with pleomorphic, primarily ellipsoid synaptic vesicles. The recognition of two types of degenerating terminals further supports our interpretation that at least two morphological types of commissural neurons participate in the velocity storage network. The degenerating boutons formed contacts with a variety of postsynaptic partners. In particular, synapses were observed between degenerating boutons and non-degenerating dendrites, and between intact terminals and degenerating dendrites. However, degenerating pre- and postsynaptic elements were rarely observed in direct contact, suggesting that additional neurons are interposed in the indirect pathway commissural system. On the basis of these ultrastructural observations, it is concluded that vestibular commissural neurons involved in the mediation of velocity storage have distinguishing ultrastructural features and synaptology, that are different from those of direct pathway neurons.

Animals↗
PubMed1999

Presenilin-1 forms complexes with the cadherin/catenin cell-cell adhesion system and is recruited to intercellular and synaptic contacts.

In MDCK cells, presenilin-1 (PS1) accumulates at intercellular contacts where it colocalizes with components of the cadherin-based adherens junctions. PS1 fragments form complexes with E-cadherin, beta-catenin, and alpha-catenin, all components of adherens junctions. In confluent MDCK cells, PS1 forms complexes with cell surface E-cadherin; disruption of Ca(2+)-dependent cell-cell contacts reduces surface PS1 and the levels of PS1-E-cadherin complexes. PS1 overexpression in human kidney cells enhances cell-cell adhesion. Together, these data show that PS1 incorporates into the cadherin/catenin adhesion system and regulates cell-cell adhesion. PS1 concentrates at intercellular contacts in epithelial tissue; in brain, it forms complexes with both E- and N-cadherin and concentrates at synaptic adhesions. That PS1 is a constituent of the cadherin/catenin complex makes that complex a potential target for PS1 FAD mutations.

Animals↗
PubMed1999

Anatomical observations of the rat cerebellar nodulus after 24 hr of spaceflight.

Exposure to microgravity causes alterations in postural, locomotor and oculomotor functions. The vestibular abnormalities experienced by astronauts entail immediate reflex motor responses, including postural illusions, sensations of rotation, nystagmus, dizziness and vertigo, as well as space motion sickness. Adaptation to the microgravity environment usually occurs within one week, and a subsequent re-adaptation period of several months is often required upon return to Earth. Some astronauts experience recurrences of dizziness, nausea, and vomiting, as well as marked disturbances in postural equilibrium in the absence of vision during this readaptation period. The mechanisms underlying such adaptation processes remain unclear, although current evidence favors some type of sensory conflict. The purpose of the present study was to explore the structural basis for the reorganization in the central vestibular system that underlies the process of adaptation to altered gravitational environments. Hindbrain tissue was obtained from rats flown on the Neurolab shuttle mission (STS-90) that launched on April 17, 1998. Tissue for the present report was obtained from four adult Fisher 344 rats sacrificed on orbit during flight day 2 (FD2), 24 hr after launch. Equal numbers of vivarium control animals and cage-controls were sacrificed 48 and 96 hr, respectively, after the flight dissections. Following decapitation, each hindbrain was immersion-fixed for 45 min in 4% paraformaldehyde/0.1% glutaraldehyde in 0.1M phosphate buffer pH 7.3, and then transferred to a 4% paraformaldehyde solution in 0.1M phosphate buffer for 18 days at 4 degrees C. After this fixation, the cerebellum was dissected away from the ventral portion of the brainstem by severing the cerebellar peduncles. The entire cerebellum of each rat was cut by Vibratome into 100 micrometers thick sections in the parasagittal plane. These sections were collected serially and processed for electron microscopy by osmication, dehydration in a graded series of methanol solutions, infiltration with resin, and embedment in Epon-Araldite resin between plastic coverslips.

Adaptation, Physiological↗
PubMed1993

Intracellular distribution of transgenic bacterial beta-galactosidase in central nervous system neurons and neuroglia.

Bacterial beta-galactosidase is widely used as a marker for gene expression and in cell tracing experiments. In a survey of three transgenic mouse lines expressing beta-galactosidase in the central nervous system (CNS) under the control of different promoters, we find substantial variation in the intracellular distribution of the lacZ protein. In line M beta P5, transgene beta-galactosidase expression is driven by a promoter/enhancer fragment from the oligodendrocyte-specific myelin basic protein gene; however, electron microscopy of histochemically stained preparations reveals transgene expression not only in oligodendrocytes but also in some neurons. Immunofluorescence and immunoperoxidase staining show the beta-galactosidase protein distributed throughout the perikaryal cytoplasm of oligodendrocytes and in processes reaching to myelin sheaths. By contrast, immunoreactive protein appears restricted in neurons to one or a few small perikaryal immunoreactive granules. The granules are visible in the electron microscope as amorphous inclusion bodies of moderate electron density and lack a limiting membrane. Histochemical staining patterns with X-gal and Bluo-gal echoed the protein distribution: diffuse distribution of enzyme protein yielded cells filled with substrate, while punctate enzyme distribution yielded restricted or punctate histochemical staining. Examination of two other lines using different promoter/enhancers to drive expression in the CNS showed both diffuse and punctate beta-galactosidase immunolocalization and histochemical staining. The amount of protein synthesized or other properties, yet unidentified, intrinsic to the target cells may determine the intracellular distribution of beta-galactosidase. In retroviral marking studies, clone members have been identified as those cells filled with X-gal reaction product. This approach may underestimate both clone size and the minimum number of divisions separating the members of each clone.

Animals↗
PubMed1992

L-baclofen-sensitive GABAB binding sites in the medial vestibular nucleus localized by immunocytochemistry.

L-Baclofen-sensitive GABAB binding sites in the medial vestibular nucleus (MVN) were identified immunocytochemically and visualized ultrastructurally in L-baclofen-preinjected rats and monkeys, using a mouse monoclonal antibody with specificity for the p-chlorophenyl moiety of baclofen. Saline-preinjected animals showed no immunostain. In drug-injected animals, there was evidence for both pre- and postsynaptic GABAergic inhibition in MVN mediated by GABAB receptors. These neural elements could be utilized in control of velocity storage in the vestibulo-ocular reflex.

Animals↗