Search PubMed⌕ Search

Biomedical subjects

D V Pow

Publications and source records attributed to D V Pow.

At least 37 records · Page 2Linked to original sources
PubMed1998

Neurotransmitter coupling through gap junctions in the retina.

Although all bipolar cells in the retina probably use the excitatory transmitter glutamate, approximately half of the cone bipolar cells also contain elevated levels of the inhibitory transmitter glycine. Some types of cone bipolar cells make heterologous gap junctions with rod amacrine cells, which contain elevated levels of glycine, leading to the hypothesis that the bipolar cells obtain their glycine from amacrine cells. Experimental support for this hypothesis is now provided by three independent lines of evidence. First, the glycine transporter GLYT1 is expressed by the glycine-containing amacrine cells but not by the glycine-containing bipolar cells, suggesting that only the amacrine cells are functionally glycinergic. Second, the gap-junction blocker carbenoxolone greatly reduces exogenous 3H-glycine accumulation into the bipolar cells but not the amacrine cells. Moreover, when the endogenous glycine stores in both cell classes are depleted by incubating the retina with a glycine-uptake inhibitor, carbenoxolone blocks the subsequent glycine replenishment of the bipolar cells but not the amacrine cells. Third, intracellular injection of rod amacrine cells with the gap-junction permeant tracer Neurobiotin secondarily labels a heterogenous population of cone bipolar cells, all of which show glycine immunoreactivity. Taken together, these findings indicate that the elevated glycine in cone bipolar cells is not derived by high-affinity uptake or de novo synthesis but is obtained by neurotransmitter coupling through gap junctions with glycinergic amacrine cells. Thus transmitter content may be an unreliable indicator of transmitter function for neurons that make heterologous gap junctions.

Amino Acid Transport Systems, Neutral↗
PubMed1998

Glycinergic amacrine cells of the rat retina.

Physiological studies of neurons of the inner retina, e.g., of amacrine cells, are now possible in a mammalian retinal slice preparation. The present anatomical study characterizes glycinergic amacrine cells of the rat retina and thus lays the ground for such future physiological and pharmacological experiments. Rat retinae were immunolabeled with antibodies against glycine and the glycine transporter-1 (GLYT-1), respectively. Glycine immunoreactivity was found in approximately 50% of the amacrine and 25% of the bipolar cells. GLYT-1 immunoreactivity was restricted to glycinergic amacrine cells. They were morphologically characterized by the intracellular injection of Lucifer Yellow followed by GLYT-1 immunolabeling. Eight different types of glycinergic amacrine cells could be distinguished. They were all small-field amacrine cells with bushy dendritic trees terminating at different levels within the inner plexiform layer. The well-known AII amacrine cell was encountered most frequently. From our measurements of the dendritic field sizes and the density of glycinergic cells, we estimate that there are enough glycinergic amacrine cells available to make sure that all eight types and possibly more tile the retina regularly with their dendritic fields.

Amino Acid Transport Systems, Neutral↗
PubMed1998

Functions of the perikaryon and dendrites in magnocellular vasopressin-secreting neurons: new insights from ultrastructural studies.

Magnocellular hypothalamic neurosecretory neurons secreting vasopressin or oxytocin provide a robust model system for the investigation and understanding of many aspects of peptidergic neuronal function. Many of their functions and the cellular organelles involved are well understood. However, recent ultrastructural studies have thrown new light on various aspects of magnocellular neurosecretory function which have not previously received much attention. This review concerns two of these: the effects of mutations in the vasopressin gene on the handling of the translated peptide by the rough endoplasmic reticulum; and the role of the magnocellular dendrites in the production, secretion and localisation of peptides. Investigation of the synthesis of proteins derived from vasopressin genes which have undergone various mutations has at the moment provided more answers than questions: Why do some abnormal products accumulate as masses of peptide in the rough endoplasmic reticulum while others do not? Why do accumulations in humans appear to be damaging to the neurons while those in the rat do not? Investigations of the role of dendrites in the production and release of peptides show that the dendrites have all the machinery needed for protein translation and appear to synthesize locally proteins required for dendritic function. Of particular interest is the possibility that various transmitter receptor proteins could be synthesized in the dendrites close to the synapses in which they become localized. Precisely how such membrane proteins are inserted into the synaptic complex is, however, unclear, because the most part of the dendrites lack any form of the Golgi packaging organelle that can be recognised as such either by immunocytochemistry or electron microscopy. Better established is the ability of magnocellular dendrites to secrete either vasopressin or oxytocin in response to a variety of stimuli including sex steroids. This local release of peptide into the magnocellular nuclei has important but as yet incompletely defined effects on the functioning of the neurons.

Animals↗
PubMed1998

Transport is the primary determinant of glycine content in retinal neurons.

This study demonstrates that in mammalian and nonmammalian species it is possible to deplete selectively and reversibly retinal glycinergic neurons of their content of glycine by exposure to sarcosine, a competitive inhibitor of glycine transporter 1 (glyt-1). This observation was used as a tool to test the hypothesis that uptake of glycine rather than de novo synthesis is the main determinant of glycine content in retinal neurons. Isolated retinae were depleted of immunocytochemically detectable pools of glycine. Thereafter retinae were exposed either to physiological medium containing glycine or to medium lacking glycine but containing precursors for the synthesis of glycine. Retinae exposed to glycine-containing medium rapidly recovered their content of glycine, whereas retinae exposed to medium lacking glycine but containing serine, a substrate for synthesis of glycine, showed only a slow recovery of immunoreactivity for glycine in a few amacrine cells. These data indicate that uptake of glycine is the primary determinant of glycine content in most retinal glycinergic neurons. The origins of the extracellular pools of glycine remain to be identified; however, it is suggested that such glycine may be derived from the vitreous humor and that in turn this glycine may be derived from the peripheral circulation.

Animals↗
PubMed1997

Tryptophan is present in glial cells and photoreceptors in the chicken retina.

Tryptophan is a large neutral amino acid which is utilized in the biosynthesis of neuroactive substances such as serotonin and melatonin. However, it has been unclear where pools of tryptophan might be localized. Using a specific antiserum against tryptophan, we demonstrate that in the chicken retina tryptophan is present in radial glial cells and photoreceptors, but not in other neuronal elements. These data suggest that serotonergic neurones are probably dependent upon the transfer of tryptophan from the glial cells in order to manufacture serotonin and other tryptophan derivatives in the brain. If glia do supply tryptophan to neurones then this process will have significant practical implications for our basic understanding of and pharmacological manipulation of serotonergic systems.

Animals↗
PubMed1997

Immunocytochemical analysis of the transport of arginine analogues into nitrergic neurons and other cells in the retina and pituitary.

Nitric oxide is formed by the action of nitric oxide synthase upon l-arginine. The efficacy of some exogenously applied arginine analogues in inhibiting nitric oxide synthase and thus nitrergic transmission indicates that neurons producing nitric oxide may possess an arginine transport system. To investigate whether arginine analogues are preferentially transported into nitric oxide-utilising cells or into cells making other neurochemicals, we have raised highly specific antisera against a number of arginine analogues including NG-methyl arginine, D-arginine, NGnitro-L-arginine, NG-nitro-L-arginine methyl ester and canavanine. Retinae were incubated in physiological media containing these analogues and rats were given intraperitoneal injections of the analogues to study the pituitary. Immunocytochemistry and NADPH-diaphorase histochemistry revealed that many of these analogues could be transported preferentially, but not exclusively, into nitric oxide-generating cells. However, some nitric oxide-producing cells apparently lacked the ability to take up some arginine analogues. We conclude that nitric oxide-generating cells in the retina and pituitary possess one or more arginine transporters. Other subsets of neurons that use GABA or glutamate as a neurotransmitter may also accumulate arginine analogues, possibly as a substrate for formation of these neurochemicals.

Animals↗
PubMed1996

GABA transamination regulates neuronal glutamate content in the retina.

We have used quantitative immunocytochemistry to examine the content of GABA and glutamate in rabbit retinae where the enzyme GABA transaminase has been selectively inhibited. Inhibition of GABA breakdown led not only to the expected rise in GABA levels in neurones and glial cells but also to a reduction in neuronal pools of glutamate, particularly in neuronal elements in the inner plexiform layer. We suggest that a significant proportion of the glutamate pool in nerve terminals is derived from GABA via the GABA shunt. This observation is of practical significance since GABA transaminase inhibitors are used in the treatment of epilepsy; accordingly GABA-transaminase inhibitors may modify uncontrolled excitatory episodes in the brain both by raising levels of GABA, and reducing levels of the excitatory transmitter, glutamate.

Animals↗
PubMed1996

Direct immunocytochemical evidence for the transfer of glutamine from glial cells to neurons: use of specific antibodies directed against the d-stereoisomers of glutamate and glutamine.

We have raised antibodies against D-stereoisomers of the amino acids glutamate and glutamine. These stereoisomers are not naturally occurring in mammals but can be taken up into cells by transporters that normally handle the endogenous L-amino acids. Exposure of isolated rabbit retinae to 50 microM D-glutamate resulted in a strong accumulation of D-glutamate, and hence immunoreactivity for D-glutamate in radial glial cells (Müller cells). By contrast the glutamatergic ganglion cells exhibited no immunoreactivity for D-glutamate. D-Glutamate can be converted into D-glutamine by the glial enzyme glutamine synthetase. Immunolabelling for D-glutamine revealed the presence of D-glutamine in somata of subsets of neurons including the glutamatergic ganglion cells. Labelling was also present in the inner plexiform layer, possibly indicating labelling of neuronal processes. These data indicate that after D-glutamate has been taken up into glial cells it is converted into D-glutamine. This D-glutamine is then exported from the glial cells and taken up by a subset of neurons, including the glutamatergic ganglion cells.

Animals↗
PubMed1996

Activity-dependent transport of GABA analogues into specific cell types demonstrated at high resolution using a novel immunocytochemical strategy.

We have raised antisera against the GABA analogues gamma-vinyl GABA, diaminobutyric acid and gabaculine. These analogues are thought to be substrates for high-affinity GABA transporters. Retinae were exposed to micromolar concentrations of these analogues in the presence or absence of uptake inhibitors and then fixed and processed for immunocytochemistry at the light and electron microscopic levels. Immunolabelling for gamma-vinyl GABA revealed specific labelling of GABAergic amacrine cells and displaced amacrine cells in retinae of rabbits, cats, chickens, fish and a monkey. GABA-containing horizontal cells of cat and monkey retinae failed to exhibit labelling for gamma-vinyl GABA, suggesting that they lacked an uptake system for this molecule. In light-adapted fish, gamma-vinyl GABA was readily detected in H1 horizontal cells; similar labelling was also observed in light-adapted chicken retinae. The pattern of labelling in the fish and chicken retinae was modified by dark adaptation, when labelling was greatly reduced in the horizontal cells, indicating the activity dependence of GABA (analogue) transport. Intraperitoneal injection of gamma-vinyl GABA into rats resulted in its transport across the blood-brain barrier and subsequent uptake into populations of GABAergic neurons. The other analogues investigated in this study exhibited different patterns of transport; gabaculine was taken up into glial cells, whilst diaminobutyric acid was taken up into neurons, glial cells and retinal pigment epithelia. Thus, these analogues are probably substrates for different GABA transporters. We conclude that immunocytochemical detection of the high-affinity uptake of gamma-vinyl GABA permits the identification of GABAergic neurons which are actively transporting GABA, and suggest that this novel methodology will be a useful tool in rapidly assessing the recent activity of GABAergic neurons at the cellular level.

Adaptation, Ocular↗
PubMed1995

Immunocytochemical evidence for the presence of high levels of reduced glutathione in radial glial cells and horizontal cells in the rabbit retina.

Reduced glutathione is an antioxidant; it is thought to be essential for normal functioning of the central nervous system. We have examined by means of immunocytochemistry, the distribution of reduced glutathione in the retina of the rabbit. Strong immunoreactivity was present in the radial glial cells (Müller cells) and in the horizontal cells. Other neuronal elements contained only low, or no detectable levels of immunoreactivity for reduced glutathione. The presence of an abundance of reduced glutathione in glial cells suggests that glia play a critical role in regulating the content of potentially damaging oxidative species in the central nervous system.

Animals↗
PubMed1995

The immunocytochemical detection of amino-acid neurotransmitters in paraformaldehyde-fixed tissues.

In this study, we show that specific antibodies can be raised against paraformaldehyde conjugates of amino acids, including the neurotransmitters glycine, gamma-amino-butyric acid and glutamate, and a non-neuroactive amino acid, glutamine. These antibodies against paraformaldehyde conjugates specifically detect the above amino acids in paraformaldehyde-fixed tissues. The penetration of antibodies into paraformaldehyde-fixed tissues is much superior to the penetration of antibodies into glutaraldehyde-fixed tissues; hence good labeling can be observed through the depth of the tissues. Unlike glutaraldehyde, fixation with paraformaldehyde does not give rise to high levels of tissue autofluorescence and, thus, these antibodies are very effective for immunofluorescence studies. Furthermore we suggest that the ability of these antibodies to detect amino acids in paraformaldehyde-fixed tissues will permit their use in situations where it is necessary to detect other other fixation-sensitive antigens, such as neurotransmitter receptors and transporters.

Amino Acids↗
PubMed1995

Production of hybrid oxytocin/vasopressin precursors and accumulation of oxytocin precursors in the rough endoplasmic reticulum of rat magnocellular neurons.

Most magnocellular hypothalamic neurons synthesize the precursor for either vasopressin (AVP) or oxytocin (OT). The AVP precursor is cleaved to give AVP, AVP-associated neurophysin (AVP-NP) and a glycopeptide (GP), whereas the OT precursor gives OT and OT-NP. In Brattleboro rats a frame-shift mutation in the AVP-NP-encoding region of the gene prevents the secretion of AVP by the cells and, in most AVP neurons, AVP itself is virtually undetectable. A small number of magnocellular neurons in homozygous Brattleboro rats contain very large accumulations of peptide in distended saccules of rough endoplasmic reticulum (RER), and this peptide is immunoreactive for AVP and C-terminal OT-NP, but not for OT, AVP-NP or GP (Pow et al., 1992). We have now shown that this results from somatic non-homologous crossing over of the AVP and OT genes, resulting in the production of hybrid mRNA molecules with the 5'end of the AVP sequence and the 3' end of the OT sequence (AVP/OT transcripts). In most cases, the crossing over occurs within the highly homologous B exons (Mohr et al., 1994). In addition to the production of AVP/OT hybrid transcripts, polymerase chain reaction (PCR) amplification of mRNA from the hypothalami of homozygous rats also reveals OT/AVP hybrid transcripts, with 5' OT sequences and 3' AVP sequences. Furthermore, both types of hybrid transcript are not restricted to homozygous Brattleboro rats but can also be found in normal Long Evans animals. To date, we have not been able to locate cells in which the OT/AVP hybrids are produced; all the magnocellular neurons with hybrid peptide accumulations in the RER so far studied have been shown by immunocytochemistry to be of the AVP/OT type. In both normal and homozygous Brattleboro rats large accumulations of peptide do occur in the RER of OT-producing neurons but the peptide is immunoreactive for OT and OT-NP but not for AVP, AVP-NP or GP. Such cells increase in number 10-fold after injection of 20 micrograms estradiol daily for 7 days (Pow et al., 1991). Why this apparently normal gene product accumulates within the RER remains to be determined.

Aging↗
PubMed1994

Immunocytochemical evidence for a glial localisation of arginine, and a neuronal localisation of citrulline in the rat neurohypophysis: implications for nitrergic transmission.

Nitric oxide (NO) is used as a neurochemical mediator in the rodent hypothalamo-neurohypophysial system. Nitric oxide synthase uses arginine to form both NO and citrulline. In this study immunocytochemistry was used to determine the distributions of arginine and citrulline in the neurohypophysis. Arginine was localised within glia whilst citrulline was present in the nitrergic neurones. Aspartate, an amino acid involved in the recycling of citrulline back to arginine, was localised only in the glia. These findings suggest that nitrergic transmission may be dependent on a cyclical process (analogous to the glutamate-glutamine cycle) based on the transfer of arginine from glia to neurones and the subsequent return of citrulline from nerve terminals to glia for aspartate-dependent conversion back into arginine.

Animals↗
PubMed1994

Rapid postmortem changes in the cellular localisation of amino acid transmitters in the retina as assessed by immunocytochemistry.

We have assessed by means of immunocytochemistry, the cellular distributions of the amino acid transmitters GABA, glycine and glutamate, and the free-radical scavenger taurine, in the retinae of adult rabbits at various times after death. Within 10 min of death, horizontal cells began to display immunoreactivity for GABA, whilst displaced amacrine cells began to display immunoreactivity for glycine. By 40 min postmortem, GABA was present in glial cells. Glutamate, which is not normally detectable in retinal glia, was detected in such glia by 20 min postmortem. By contrast immunocytochemically detectable glycine did not accumulate in glia. There was a gradual diminution of immunoreactivity for taurine in glial cells and photoreceptors. By 2 h postmortem, most immunoreactivity had disappeared from the retina. We conclude that amino acid transmitters show rapid changes in their distributions immediately after death, which may be related to changes in the patterns of transmitter release and uptake, and changes in degradation mechanisms. The rapid changes in cellular localisation of amino acid immunoreactivity illustrated in this study, indicate that the fixation of nervous tissues must be performed rapidly. Moreover, the massive loss of immunoreactivity by 2 h postmortem suggests that any assays for content of these transmitters at this, and subsequent time-points, will bear little resemblance to the values obtained at the time of death.

Amino Acids↗
PubMed1994

Taurine, amino acid transmitters, and related molecules in the retina of the Australian lungfish Neoceratodus forsteri: a light-microscopic immunocytochemical and electron-microscopic study.

The morphology of the retina of the Australian lungfish Neoceratodus forsteri was investigated by means of light- and electron microscopy, whilst immunocytochemical studies were performed to determine the cellular distributions of the major amino acid neurotransmitters and other amino acids. The distributions of glycine and GABA were similar to those previously described for teleost, amphibian and mammalian retinae. Labelling was abundant in amacrine cells, whilst GABA was also present in one layer of horizontal cells and some bipolar cells. Taurine was present in both rods and cones, but, unlike the mammalian or avian retina, was absent from other cellular structures, including glial elements. Unexpectedly, the photoreceptor terminals lacked an apparent content of the excitatory amino acid transmitter glutamate. The glutamate that was present in the rods and cones occupied a crescentic arc corresponding to the location of glycogen-rich paraboloids. Asparagine was also present in rods, albeit in the modified mitochondria that formed the elipsoids of the rod inner segments. Arginine, the precursor for formation of nitric oxide, was present in glial cells, and in the paraboloids of both rods and cones.

Animals↗
PubMed1994

Glutamate in some retinal neurons is derived solely from glia.

Glutamate is the most abundant excitatory neurotransmitter in the vertebrate central nervous system. It is widely assumed that neurons using this transmitter derive it from several sources: (i) synthesizing it themselves from alpha-ketoglutarate or aspartate, (ii) synthesize it from glial-derived glutamine, or (iii) take up glutamate from the extracellular space. By use of immunocytochemistry we show that glutamate is abundant in the retinal ganglion and bipolar cells of the rabbit, but that immunoreactivity for glutamate in these neurons is reduced below immunocytochemical detection limits after the specific inhibition of glutamine synthesis in glial cells by D,L-methionine D,L-sulphoximine. GABA immunoreactivity in retinal amacrine cells was also reduced after inhibition of glutamine synthetase but the patterns and densities of immunoreactivity for taurine and glycine were unaffected. Therefore, this experimental paradigm does not induce generalized metabolic changes in neurons or glia. This study demonstrates that some glutamatergic neurons are dependent on the synthetic processes in glia for their neurotransmitter content.

Animals↗
PubMed1993

Ultrastructural demonstration of exocytosis of neurosecretory granules in the neurohypophysis of the frog Rana temporalia.

The release of neurosecretory granules by exocytosis was ultrastructurally observed in the neurohypophysis pars nervosa (posterior lobe) of the frog Rana temporalia with the tannic acid-Ringer fixation method. Electron microscopic analysis with the immunogold method revealed that many of putative neurosecretory granules displayed oxytocin-like immunoreactivity.

Animals↗