Biomedical subjects

D V Pow

Publications and source records attributed to D V Pow.

At least 19 recordsLinked to original sources

PubMed2005

Expression of pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R) in the ewe hypothalamus: distribution and colocalization with tyrosine hydroxylase-immunoreactive neurones.

We have examined the distribution of the pituitary adenylate cyclase activating polypeptide type I receptor (PAC1R) in the ewe hypothalamus by reverse transcription-polymerase chain reaction, in situ hybridization and immunohistochemistry. PAC1R mRNA was highly expressed in the mediobasal hypothalamus of the ewe, particularly in the arcuate nucleus and ventromedial hypothalamus, compared to other hypothalamic regions. Similar results were obtained from immunohistochemistry using a specific PAC1R antibody. Intense immunolabelling was observed in the arcuate nucleus, external zone of the median eminence and ventromedial hypothalamus. Only relatively weak immunolabelling was observed in other hypothalamic regions, including the paraventricular nucleus and supraoptic nucleus. In the ewe, PACAP acts via the arcuate nucleus to suppress prolactin secretion. Therefore we examined whether PAC1R was present on the tuberoinfundibular dopamine (TIDA) neurones in this nucleus. Dual immunofluorescence labelling for PAC1R and tyrosine hydroxylase revealed that 21.2 +/- 1.7% of dopaminergic neurones in the arcuate nucleus (A12 cell group) also stained for PAC1R. By contrast, other hypothalamic dopaminergic cell groups (A11, A13, A14 and A15) exhibited little (< 3%) or no colocalization. Overall, our results indicate that, in the ewe hypothalamus, PAC1R is most concentrated in the arcuate nucleus, where it is localized on a substantial proportion of dopaminergic neurones. These observations, together with previous in vivo studies, suggest that PACAP could act directly on TIDA neurones via PAC1R to increase dopamine release and consequently inhibit prolactin secretion in the sheep.

Alternative Splicing↗

PubMed2001

Projections from the substantia nigra pars reticulata to the motor thalamus of the rat: single axon reconstructions and immunohistochemical study.

This is a study in the rat of the distribution of specific neurotransmitters in neurones projecting from the substantia nigra reticulata (SNR) to the ventrolateral (VL) and ventromedial (VM) thalamic nuclei. Individual axons projecting from the SNR to these thalamic nuclei have also been reconstructed following small injection of the anterograde tracer dextran biotin into the the SNR. Analysis of reconstructions revealed two populations of SNR neurones projecting onto the VL and VM thalamic nuclei. One group projects directly onto the VM and VL, and the other projects to the VM/VL and to the parafascicular nucleus. In another set of experiments Fluoro-Gold was injected into the VL/VM to label SNR projection neurones retrogradely, and immunohistochemistry was performed to determine the distribution of choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), gamma-aminobutyric acid (GABA), and glutamate in Fluoro-Gold-labelled SNR projection neurones. Most SNR-VL/VM thalamic projection neurones were immunoreactive to acetylcholine or glutamate, whereas only 25% of the projection neurones were found to be immunoreactive to GABA.

Animals↗

PubMed2001

Visualising the activity of the cystine-glutamate antiporter in glial cells using antibodies to aminoadipic acid, a selectively transported substrate.

The cystine-glutamate antiporter is a transport system that facilitates the uptake of cystine, concomitant with the release of glutamate. The cystine accumulated by this transporter is generally considered for use in the formation of the cysteine-containing antioxidant glutathione, which is abundant in many glial cells. This study used the simple strategy of generating an antibody to aminoadipic acid, a selective substrate for the cystine-glutamate antiporter. Stereospecific accumulation of aminoadipic acid into specific cell types in rat brain slice preparations was detected immunocytochemically. Strong accumulation was detected in astroglial cells in all brain regions studied including those in white matter tracts. Strong accumulation into radial glial cells, including the retinal Müller cells and the Bergmann glial cells was also observed. Glial accumulation was observed not only in cells within the blood brain barrier, but also outside such; anterior pituitary folliculostellate cell and intermediate lobe pituitary glial cells exhibited strong accumulation of aminoadipic acid. Interestingly, some glial cells such as the posterior pituitary glial cells (pituicytes) exhibited very little if any accumulation of aminoadipic acid. Within the brain labelling was not uniform. Particularly strong labelling was noted in some regions, such as the glial cells surrounding the CA1 pyramidal cells. By contrast, neurons never exhibited uptake of aminoadipic acid. Because cystine uptake is associated with glutamate release, it is suggested that this antiporter might contribute to release of glutamate from glial cells under some pathophysiological conditions.

2-Aminoadipic Acid↗

PubMed2001

Glyt-1 expression in cultured human Müller cells and intact retinae.

In this study, we demonstrate that Müller cells cultured from human retinas are capable of strongly expressing the glycine transporter Glyt-1 as assessed by immunocytochemistry. By contrast, intact normal and pathological human retinas exhibit Glyt-1 immunoreactivity only in neurons. These data suggest that Glyt-1 expression in cultured Müller cells is an epiphenomenon associated with culturing in vitro, rather than a normal physiological or even pathophysiological phenomenon in vivo.

Aged↗

PubMed2001

Localization of neurotransmitters and calcium binding proteins to neurons of salamander and mudpuppy retinas.

We wished to identify the different types of retinal neurons on the basis of their content of neuroactive substances in both larval tiger salamander and mudpuppy retinas, favored species for electrophysiological investigation. Sections and wholemounts of retinas were labeled by immunocytochemical methods to demonstrate three calcium binding protein species and the common neurotransmitters, glycine, GABA and acetylcholine. Double immunostained sections and single labeled wholemount retinas were examined by confocal microscopy. Immunostaining patterns appeared to be the same in salamander and mudpuppy. Double and single cones, horizontal cells, some amacrine cells and ganglion cells were strongly calbindin-immunoreactive (IR). Calbindin-IR horizontal cells colocalized GABA. Many bipolar cells, horizontal cells, some amacrine cells and ganglion cells were strongly calretinin-IR. One type of horizontal cell and an infrequently occurring amacrine cell were parvalbumin-IR. Acetylcholine as visualized by ChAT-immunoreactivity was seen in a mirror-symmetric pair of amacrine cells that colocalized GABA and glycine. Glycine and GABA colocalized with calretinin, calbindin and occasionally with parvalbumin in amacrine cells.

Acetylcholine↗

PubMed2001

Amino acids and their transporters in the retina.

This review provides an overview of the distributions, properties and roles of amino acid transport systems in normal and pathological retinal tissues and discusses the roles of specific identified transporters in the mammalian retina. The retina is used in this context as a vehicle for describing neuronal and glial properties, which are in some, but not all cases comparable to those found elsewhere an the brain. Where significant departures are noted, these are discussed in the context of functional specialisations of the retina and its relationship to adjacent supporting tissues such as the retinal pigment epithelium. Specific examples are given where immunocytochemical labelling for amino acid transporters may yield inaccurate results, possibly because of activity-dependent conformation changes of epitopes in these proteins which render the epitopes more or less accessible to antibodies.

Amino Acid Transport Systems↗

PubMed2001

Differential perturbation of neuronal and glial glutamate transport systems in retinal ischaemia.

Glutamate is the major excitatory neurotransmitter in the retina and is removed from the extracellular space by an energy-dependent process involving neuronal and glial cell transporters. The radial glial Müller cells express the glutamate transporter, GLAST, and preferentially accumulate glutamate. However, during an ischaemic episode, extracellular glutamate concentrations may rise to excitotoxic levels. Is this catastrophic rise in extracellular glutamate due to a failure of GLAST? Using immunocytochemistry, we monitored the transport of the glutamate transporter substrate, D-aspartate, in the retina under normal and ischaemic conditions. Two models of compromised retinal perfusion were compared: (1) Anaesthetised rats had their carotid arteries occluded for 7 days to produce a chronic reduction in retinal blood flow. Retinal function was assessed by electroretinography. D-aspartate was injected into the eye for 45 min. Following euthanasia, the retina was processed for D-aspartate, GLAST and glutamate immunocytochemistry. Although reduced retinal perfusion suppresses the electroretinogram b-wave, neither retinal histology, GLAST expression, nor the ability of Müller cells to uptake D-aspartate is affected. As this insult does not appear to cause excitotoxic neuronal damage, these data suggest that GLAST function and glutamate clearance are maintained during periods of reduced retinal perfusion. (2) Occlusion of the central retinal artery for 60 min abolishes retinal perfusion, inducing histological damage and electroretinogram suppression. Although GLAST expression appears to be normal, its ability to transport D-aspartate into Müller cells is greatly reduced. Interestingly, D-aspartate is transported into neuronal cells, i.e. photoreceptors, bipolar and ganglion cells. This suggests that while GLAST is vitally important for the clearance of excess extracellular glutamate, its capability to sustain inward transport is particularly susceptible to an acute ischaemic attack. Manipulation of GLAST function could alleviate the degeneration and blindness that result from ischaemic retinal disease.

Amino Acid Transport System X-AG↗

PubMed2001

Selective down-regulation of the astrocyte glutamate transporters GLT-1 and GLAST within the medial thalamus in experimental Wernicke's encephalopathy.

Although earlier studies on thiamine deficiency have reported increases in extracellular glutamate concentration in the thalamus, a vulnerable region of the brain in this disorder, the mechanism by which this occurs has remained unresolved. Treatment with pyrithiamine, a central thiamine antagonist, resulted in a 71 and 55% decrease in protein levels of the astrocyte glutamate transporters GLT-1 and GLAST, respectively, by immunoblotting in the medial thalamus of day 14 symptomatic rats at loss of righting reflexes. These changes occurred prior to the onset of convulsions and pannecrosis. Loss of both GLT-1 and GLAST transporter sites was also confirmed in this region of the thalamus at the symptomatic stage using immunohistochemical methods. In contrast, no change in either transporter protein was detected in the non-vulnerable frontal parietal cortex. These effects are selective; protein levels of the astrocyte GABA transporter GAT-3 were unaffected in the medial thalamus. In addition, astrocyte-specific glial fibrillary acidic protein (GFAP) content was unchanged in this brain region, suggesting that astrocytes are spared in this disorder. Loss of GLT-1 or GLAST protein was not observed on day 12 of treatment, indicating that down-regulation of these transporters occurs within 48 h prior to loss of righting reflexes. Finally, GLT-1 content was positively correlated with levels of the neurofilament protein alpha-internexin, suggesting that early neuronal drop-out may contribute to the down-regulation of this glutamate transporter and subsequent pannecrosis. A selective, focal loss of GLT-1 and GLAST transporter proteins provides a rational explanation for the increase in interstitial glutamate levels, and may play a major role in the selective vulnerability of thalamic structures to thiamine deficiency-induced cell death.

ATP-Binding Cassette Transporters↗

PubMed2000

Study of projections from the entopeduncular nucleus to the thalamus of the rat.

The entopeduncular nucleus (EP) is a major outflow nucleus of the basal ganglia and innervates the lateral habenula, parafascicular, pedunculopontine, ventrolateral (VL), ventromedial (VM), and mediodorsal thalamic nuclei. This study investigated the morphology of single axons of entopeduncular neurons projecting to the motor thalamus by placing small injections of dextran biotin into the EP and reconstructing drawings of single axons from serial sections. There were two populations of entopeduncular-thalamic projection axons: those that projected only to the motor thalamus (VL and VM) and those that projected to both the motor thalamus and other nuclei (e.g., the habenula). The neurochemistry of EP neurons projecting to the thalamus was investigated by injecting the retrograde tracer FluoroGold into the VL and VM thalamic nuclei to retrogradely fill entopeduncular projection neurons. These were subsequently immunohistochemically labeled for choline acetyl transferase, gamma-aminobutyric acid (GABA), and glutamate. Consistent with previous studies, significant proportions of these neurons were GABA immunoreactive. In addition, approximately half of the entopeduncular-thalamic projecting neurons were found to be cholinergic. This excitatory input is most likely derived from axons that branch as they pass through the motor thalamus to the lateral habenula.

Animals↗

PubMed2000

The dendritic architecture of the cholinergic plexus in the rabbit retina: selective labeling by glycine accumulation in the presence of sarcosine.

The cholinergic amacrine cells in the rabbit retina slowly accumulate glycine to very high levels when the tissue is incubated with excess sarcosine (methylglycine), even though these cells do not normally contain elevated levels of glycine and do not express high-affinity glycine transporters. Because the sarcosine also depletes the endogenous glycine in the glycine-containing amacrine cells and bipolar cells, the cholinergic amacrine cells can be selectively labeled by glycine immunocytochemistry under these conditions. Incubation experiments indicated that the effect of sarcosine on the cholinergic amacrine cells is indirect: sarcosine raises the extracellular concentration of glycine by blocking its re-uptake by the glycinergic amacrine cells, and the excess glycine is probably taken-up by an unidentified low-affinity transporter on the cholinergic amacrine cells. Neurobiotin injection of the On-Off direction-selective (DS) ganglion cells in sarcosine-incubated rabbit retina was combined with glycine immunocytochemistry to examine the dendritic relationships between the DS ganglion cells and the cholinergic amacrine cells. These double-labeled preparations showed that the dendrites of the DS ganglion cells closely follow the fasciculated dendrites of the cholinergic amacrine cells. Each ganglion cell dendrite located within the cholinergic strata is associated with a cholinergic fascicle and, conversely, there are few cholinergic fascicles that do not contain at least one dendrite from an On-Off DS cell. It is not known how the dendritic co-fasciculation develops, but the cholinergic dendritic plexus may provide the initial scaffold, because the dendrites of the On-Off DS cells commonly run along the outside of the cholinergic fascicles.

Animals↗

PubMed2000

Developmental expression of excitatory amino acid transporter 5: a photoreceptor and bipolar cell glutamate transporter in rat retina.

Excitatory amino acid transporter 5 (EAAT5) is a retina-specific glutamate transporter which has an associated chloride conductance. Thus it is comparable in its functional properties to the glutamate transport systems previously described in photoreceptors and some bipolar cells. We have raised antibodies to the carboxyl- and amino-terminal regions of EAAT5. Labeling for both of these antisera was developmentally regulated: weak labeling appeared in photoreceptors around P7; by P10 strong labeling was present in photoreceptors and by P21 a population of bipolar elements were also weakly labeled. In adult retinae both antisera heavily immunolabeled all photoreceptors as well as a heterogeneous population of bipolar cell somata and their proximal axonal processes: synaptic terminals of these cells were also labeled after partial proteolytic digestion of the tissues. The positions and morphology of these terminals suggests that they are the terminals of both rod and cone rod bipolar cells. We conclude that in rat retina, EAAT5 is a photoreceptor and bipolar cell glutamate transporter.

Amino Acid Sequence↗

PubMed2000

Inhibition of Müller cell glutamine synthetase rapidly impairs the retinal response to light.

It is widely assumed that neurones have sufficient metabolic reserves to allow them to function independently of glial cells for extended periods. The present study investigates the length of time taken before retinal neurones no longer respond normally to light after the inhibition of glial enzymes that are involved in the synthesis of precursors of neuronal glutamate. The glutamine synthetase inhibitor methionine sulfoximine, when injected intraocularly in Wistar rats, caused a time- and dose-dependent suppression of the scotopic electroretinogram b-wave. At the highest dosage (40 mM) the b-wave was significantly reduced within 2 min of injection. Because the b-wave is an indicator of neurotransmission in the retina, it is deduced that inhibition of glutamine synthetase rapidly blocks glutamatergic neurotransmission. Immunohistochemistry revealed a depletion of neuronal glutamate and an accumulation of glutamate in Müller glial cells, in a time course that matched the b-wave suppression. The b-wave was quickly restored by injection of glutamine (4 mM). The rapid reduction of glutamatergic transmission after methionine sulfoximine administration challenges the view that neurones have sufficient reserves to allow them to function independently for extended periods; instead, it indicates that glia are essential for the moment-to-moment sustenance of neuronal function.

Analysis of Variance↗

PubMed2000

Cooperative changes in GABA, glutamate and activity levels: the missing link in cortical plasticity.

Different intracortical mechanisms have been reported to contribute to the substantial topographic reorganization of the mammalian primary visual cortex in response to matching lesions in the two retinas: an immediate expansion of receptive fields followed by a gradual shift of excitability into the deprived area and finally axonal sprouting of laterally projecting neurons months after the lesion. To gain insight into the molecular mechanisms of this adult plasticity, we used immunocytochemical and bioanalytical methods to measure the glutamate and GABA neurotransmitter levels in the visual cortex of adult cats with binocular central retinal lesions. Two to four weeks after the lesions, glutamate immunoreactivity was decreased in sensory-deprived cortex as confirmed by HPLC analysis of the glutamate concentration. Within three months normal glutamate immunoreactivity was restored. In addition, the edge of the unresponsive cortex was characterized by markedly increased glutamate immunoreactivity 2-12 weeks postlesion. This glutamate immunoreactivity peak moved into the deprived area over time. These glutamate changes corresponded to decreased spontaneous and visually driven activity in unresponsive cortex and to strikingly increased neuronal activity at the border of this cortical zone. Furthermore, the previously reported decrease in glutamic acid decarboxylase immunoreactivity was found to reflect decreased GABA levels in sensory-deprived cortex. Increased glutamate concentrations and neuronal activity, and decreased GABA concentrations, may be related to changes in synaptic efficiency and could represent a mechanism underlying the retinotopic reorganization that occurs well after the immediate receptive field expansion but long before the late axonal sprouting.

Animals↗

PubMed2000

Taurine blocks spontaneous cone contraction but not horizontal cell dark suppression in isolated goldfish retina.

The objective of this study was to investigate the effects of taurine on cone retinomotor movements and the responses of cone-driven horizontal cells in dark-adapted teleost retina. In isolated goldfish retina preparations maintained in the dark, cones spontaneously contracted, and the responses of horizontal cells were suppressed. Addition of 5 mM taurine to the physiological solution blocked the spontaneous contraction of cones in the dark but did not block the dark-suppression of horizontal cell responses. These results indicate that the mechanism that leads to horizontal cell dark suppression is not sensitive to taurine. Although both cone retinomotor position and horizontal cell responsiveness are known to be modulated by dopamine, the present results do not support the hypothesis that taurine inhibits dopamine release in the dark because only spontaneous cone contraction was affected by taurine. These results also indicate that spontaneous cone contraction in the dark is not the cause of horizontal cell dark suppression because, in the presence of taurine, cones were elongated yet horizontal cell responses were still suppressed. Consequently, these results make it clear that horizontal cell dark suppression is not an artifact produced by incubating isolated teleost retina preparations in taurine-free physiological solution.

Adaptation, Ocular↗

PubMed2000

Antisense knockdown of GLAST, a glial glutamate transporter, compromises retinal function.

PURPOSE: To elucidate the role of the glial glutamate transporter GLAST, in the regulation of retinal function. METHODS: Antisense oligonucleotides to GLAST were injected intravitreally into the left eye of Wistar rats. Sense oligonucleotides (control) were injected into the right eye over a period of 3 days. Scotopic flash electroretinograms were recorded over a 20-day period. To assay whether the antisense oligonucleotides caused a reduction in the expression or the activity of GLAST, retinas were exposed to D-aspartate, a nonendogenous substrate of glutamate transporters. The retinas were immunolabeled with specific antibodies for D-aspartate. Retinal GLAST and glutamate distributions also were determined immunocytochemically. RESULTS: Antisense oligonucleotides markedly suppressed the electroretinogram b-wave, whereas sense oligonucleotides had no significant effect. Significant changes in the electroretinogram were apparent 5 days after injection of antisense oligonucleotide and were sustained for at least 20 days. A marked reduction of D-aspartate uptake into Muller cells of retinas that had been exposed to the antisense oligonucleotides 5 days previously suggests a reduction of GLAST activity. The retinas, however, displayed no evidence of excitotoxic neuronal degeneration, and the distribution of glutamate was unaffected by antisense treatment. CONCLUSIONS: The observed lack of neuronal degeneration suggests that reduced glutamate uptake into Muller cells does not cause excitotoxic tissue damage. A direct perturbation of glutamatergic signaling is more likely, because the rapid clearance of glutamate is necessary for light elicited signaling between photoreceptors and bipolar cells. This suggests that GLAST is essential for the maintenance of normal retinal transmission.

ATP-Binding Cassette Transporters↗

PubMed1999

Immunocytochemical demonstration of reduced glutathione in neurons of rat forebrain.

Histochemical studies show reduced glutathione (GSH) in neuroglia, whereas immunocytochemistry of glutaraldehyde-fixed tissue reveals GSH also in neurons. Using an antibody suitable for formaldehyde-fixed tissue, we find GSH staining in the cytoplasm of neurons throughout the brain. Staining was prominent in large pyramidal neurons of cerebral cortex, in basal ganglia, and in reticular and ventrobasal thalamic nuclei.

Animals↗

PubMed1999

Changing patterns of spatial buffering of glutamate in developing rat retinae are mediated by the Müller cell glutamate transporter GLAST.

The patterns of expression of the glutamate transporter GLAST were compared with the patterns of uptake of exogenous D-aspartate, which is a substrate for all glutamate transporters. At postnatal day 0, fine radial processes and end feet of presumptive Müller cells were weakly immunoreactive for GLAST. At postnatal day 3, intense labelling was associated with astrocytes enveloping newly formed blood vessels on the vitread surface of the retina. Between postnatal days 7 and 10, there was a rapid increase in the intensity of labelling in the Müller cells but clear stratification of GLAST-immunoreactive processes in the inner plexiform layer was not observed until postnatal day 14. By comparison, D-aspartate uptake was initially associated with a wide variety of cellular elements including most neuroblasts, presumptive Müller cells, and astrocytes associated with blood vessels but was absent from the somata of many neurons in the ganglion cell layer and amacrine cell layer. There was a gradual contraction in the numbers of cells that were able to take up D-aspartate, such that, by adulthood, uptake was restricted mainly to Müller cells and astrocytes. We conclude that, during early retinal development, the low levels of GLAST expression by Müller cells permit D-aspartate, and by inference, glutamate, to permeate the retina freely, thus allowing uptake by other glutamate transporters on other cell types. As the retina matures, increased expression of GLAST by Müller cells restricts the access of D-aspartate to other cellular compartments in the retina. This changing pattern of spatial buffering of glutamate by GLAST probably has significant implications regarding our understanding of the role of glutamate during processes such as retinal synaptogenesis.

ATP-Binding Cassette Transporters↗

PubMed1999

Origins of the glycinergic inputs to the rat locus coeruleus and dorsal raphe nuclei: a study combining retrograde tracing with glycine immunohistochemistry.

The amino acid glycine is a major inhibitory neurotransmitter in the brainstem and is likely involved in the tonic inhibition of the monoaminergic neurons during all sleep-waking stages. In order to determine the neurons at the origin of the glycinergic innervation of the two principal monoaminergic nuclei, the locus coeruleus and the dorsal raphe of the rat, we applied a double-labelling technique, combining retrograde transport of cholera-toxin B subunit with glycine immunohistochemistry. Using this technique, we found that the locus coeruleus and dorsal raphe nuclei receive a common glycinergic innervation from the ventral and ventrolateral periaqueductal grey, including the adjacent deep mesencephalic reticular nucleus. Small additional glycinergic inputs to these nuclei originated from the lateral paragigantocellular nucleus and the rostral ventromedial medullary reticular formation. The potential role of these glycinergic inputs in the control of the excitability of the monoaminergic neurons of the locus coeruleus and dorsal raphe nuclei is discussed.

Animals↗