Biomedical subjects

R G Pourcho

Publications and source records attributed to R G Pourcho.

At least 19 recordsLinked to original sources

PubMed2001

Cellular and subcellular distribution of NMDA receptor subunit NR2B in the retina.

Immunocytochemical studies showed the presence of staining for the N-methyl-D-aspartate (NMDA)-R2B glutamate receptor subunit at multiple sites in the cat retina. Reaction product in photoreceptor cells was localized at the inner/outer segment junction and in the axon terminals. Staining within the inner retina was limited to ganglion cells and their dendrites ramifying throughout the inner plexiform layer. These cells were seen to receive synaptic input from cone bipolar cells in both sublaminae. As with other glutamate receptor subunits, this immunoreactivity was typically confined to a single postsynaptic element at a cone bipolar dyad complex. Immunocytochemical localization of the NMDA-R1 subunit, considered to be an essential component of functional receptors, showed a widespread distribution across the retina including all the sites where NMDA-R2B staining was seen. Immunoprecipitation and Western blot analysis were used to confirm the presence of the NR2B receptor protein and its association with the NR1 subunit in both proximal and distal retinal layers. The findings suggest that NMDA-R2B subunits are positioned for multiple functions within the retina.

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PubMed2001

Immunocytochemical localization of kainate-selective glutamate receptor subunits GluR5, GluR6, and GluR7 in the cat retina.

Localizations of the kainate-selective glutamate receptor subunits GluR5, 6, and 7 were studied in the cat retina by light and electron microscopic immunocytochemistry. GluR5 immunoreactivity was observed in the cell bodies and dendrites of numerous cone bipolar cells and ganglion cells. The labeled cone bipolar cells make basal or flat contacts with cone pedicles in the outer plexiform layer, leading to their identification as OFF-center bipolar cells. Reaction product within the inner plexiform layer was observed in processes of ganglion cells at their sites of input from cone bipolar cells. Staining for GluR6 was localized to A- and B-type horizontal cells, numerous amacrine cells, and an occasional cone bipolar cell. The larger ganglion cells were also immunoreactive. As with other GluR molecules, labeling was usually confined to one of the two postsynaptic elements at a cone bipolar dyad contact. Immunoreactivity for GluR7 was very limited and was seen only in a few amacrine and displaced amacrine cells. Findings of this study are consistent with a major role for kainate receptors in mediating OFF pathways in the outer retina with participation in both OFF and ON pathways in the inner retina.

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PubMed1999

Transmitter-specific input to OFF-alpha ganglion cells in the cat retina.

The synaptic input to OFF-center alpha ganglion cells in the cat retina was analyzed by electron microscopic reconstruction to quantify the bipolar and amacrine cell input and to determine the neurotransmitter content of the presynaptic cells. Cone bipolar cells were found to comprise 11% of the total input with their dyad synapses distributed across the dendritic tree. The remaining contacts were conventional synapses indicative of amacrine cells; postembedding immunogold labeling was used to characterize these cells as either GABA- or glycine-immunoreactive. Results showed the amacrine input to be equally divided between GABA and glycinergic contacts at each order of dendritic branching of the alpha cells. Among the GABA-positive neurons were A19 amacrine cells, the processes of which are characterized by a dense array of neurotubules. A major source of glycinergic input was from lobular appendages of AII amacrine cells with lesser contributions from other glycine-positive amacrine cells. The physiological role(s) of these amino acids must be interpreted in view of the multiple subpopulations of amacrine cells, which provide input to OFF-alpha cells, and the diversity in receptors at their synapses.

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PubMed1999

Localization of metabotropic glutamate receptors mGluR1alpha and mGluR2/3 in the cat retina.

The distribution of metabotropic glutamate receptors 1alpha (mGluR1alpha) and mGluR2/3 in the cat retina was studied through the use of preembedding immunocytochemistry for light and electron microscopy. Staining for mGluR1alpha in the outer plexiform layer was seen in numerous punctate structures that were identified as rod spherules. Cone pedicles remained unlabeled. A number of amacrine and ganglion cell somata also were stained with processes ramifying throughout the inner plexiform layer. These processes were postsynaptic to cone bipolar cells in both sublaminae, where they comprised one but not both of the postsynaptic elements at dyad contacts. Immunostaining for mGluR2/3 was observed in horizontal cells as well as in numerous amacrine and displaced amacrine cells. Labeled amacrine processes were postsynaptic to cone bipolar cells in both sublaminae but, similar to mGluR1alpha, comprised only one of the postsynaptic elements. Staining for mGluR2/3 also was seen in amacrine processes postsynaptic to rod bipolar terminals; these processes were identified as belonging to type A17 amacrine cells. The distribution patterns indicate that both mGluR1alpha and mGluR2/3 are positioned for postsynaptic function, whereas mGluR1alpha also may contribute to the presynaptic regulation of glutamate release from rod photoreceptors.

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PubMed1996

Distribution of AMPA-selective glutamate receptor subunits in the cat retina.

Immunocytochemical techniques were used to localize AMPA-selective glutamate receptor subunits in the cat retina. The antisera employed recognize GluR1, GluR2/3 or GluR4 subunits. Each antiserum produced a distinctive staining pattern which included horizontal cells, cone bipolar cells, and amacrine and ganglion cells. Some cells such as alpha ganglion cells expressed multiple subunits whereas amacrine cells were typically immunoreactive with only one of the antisera.

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PubMed1996

Neurotransmitters in the retina.

Processing of visual information within the retina depends in large measure upon a complement of chemical neurotransmitters which are released at synaptic contacts between individual neurons. Numerous investigators have participated in the identification of many of these transmitters and their assignment to specific neuronal subpopulations. However, it is now clear that the action of each transmitter depends upon the receptor molecules to which it binds. Multidisciplinary studies are underway to characterize these receptors as well as to investigate transporter molecules which may serve not only to inactivate certain neurotransmitters but may also function in their release.

Acetylcholine↗

PubMed1995

Morphological diversity and glutamate immunoreactivity of retinal terminals in the suprachiasmatic nucleus of the cat.

Although the cat visual system has been the subject of intensive investigation, little attention has been given to the morphological features of ganglion cell projections to the suprachiasmatic nucleus. The present study has utilized anterograde transport of horseradish peroxidase and wheat germ agglutinin-conjugated horseradish peroxidase to label ganglion cell terminals in the cat suprachiasmatic nucleus. Visualization of the reaction product was facilitated through the use of gold-substituted silver intensification. Ganglion cell terminals were found to be morphologically diverse, making both asymmetric and symmetric contacts with postsynaptic processes. Synaptic vesicles were either scattered or densely packed, sometimes forming paracrystalline arrays. In contrast to other retinorecipient areas in which ganglion cell terminals have been characterized by the presence of lightly staining mitochondria, many of the retinal terminals in the suprachiasmatic nucleus were seen to contain darkly stained mitochondria. Postembedding antiglutamate immunocytochemistry was used to evaluate the level of endogenous glutamate in these ganglion cell terminals. Although morphologically diverse, all of the retinal terminals in the suprachiasmatic nucleus were glutamate positive, consistent with the postulated role of glutamate as the neurotransmitter of retinal ganglion cells.

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PubMed1992

Hydrolysis of substance P in the rabbit retina: I. Involvement of acetylcholine and acetylcholinesterase. An in vivo study.

The laminar patterns of acetylcholinesterase (AChE) activity and substance P (SP) immunoreactivity within the inner plexiform layer (IPL) of the rabbit retina show striking similarities. Discrete bands of SP-immunoreactivity were seen at 1-7%, 40-48% and 85-95% depth of IPL. AChE activity was present throughout the entire thickness of the IPL with moderately stained bands in each sublamina (3-24% in sublamina a and 62-89% in sublamina b depth IPL). These bands were bordered on both sides by bands of even greater density (in sublamina a 0-3% and 24-34% and in sublamina b 55-62% and 89-100% depth IPL). Cell processes staining for choline acetyltransferase (ChAT) have previously been shown to ramify at 19-24% and 63-79% depth levels. Thus, SP- and ChAT-immunoreactive bands are located in both sublaminae, positioned within regions of moderate AChE activity and flanked by bands with greater AChE activity. This strong morphological correspondence and reported interactions between acetylcholine (ACh), AChE and SP in vitro provide the basis for the present study to determine whether such interactions can be demonstrated in vivo. Retinas infused with ACh showed a 60% average increase in SP-IR as compared with untreated retinas from the same animals. Treatment with diisopropylfluorophosphate (DFP) also resulted in a 56% increase in SP-IR. The ability of ACh to induce increased levels of SP was not inhibited by CoCl2, atropine or mecamylamine, ruling out the possibilities of polysynaptic transmission or involvement of muscarinic or nicotinic receptors. Infusion of ACh did not increase the levels of preprotachykinin-mRNA indicating that the increase in SP-IR is not due to de novo synthesis but rather to inhibition of the enzyme(s) responsible for SP degradation. Whether AChE functions alone or in concert with other enzymes to hydrolyze SP cannot be determined from these experiments but is addressed in a separate study.

Acetylcholine↗

PubMed1992

Hydrolysis of substance P in the rabbit retina: II. The role of a membrane-associated acetylcholine-sensitive metalloendopeptidase. An in vitro study.

Studies in the rabbit retina have shown that infusion of exogenous acetylcholine (ACh) into the vitreal chamber leads to an increase in the amount of substance P (SP) immunoreactivity (Goebel and Pourcho, submitted). This increase was determined to be independent of new peptide synthesis, suggesting that the elevated level of SP is the result of ACh inhibition of an SP-degrading protease. This phenomenon has now been confirmed in vitro in both tissue slice and retinal homogenate assays. These studies have shown that ACh decreases the rate of SP hydrolysis in a concentration dependent manner. Recovery of SP hydrolytic activity following ACh inhibition was found to be directly proportional to the amount of acetylcholinesterase (AChE) activity in the membrane fraction. Specific protease inhibitors were used to determine the relative contributions of membrane associated retinal enzymes to SP-hydrolysis. In the presence of 1 mM 1,10-phenanthroline or p-chloromercuribenzenesulfonic acid all SP-hydrolytic activity was abolished, indicating that the enzyme(s) responsible for the degradation of the peptide is a metallopeptidase. The ACh sensitive retinal enzyme was found to be concentrated in the membrane fraction where it accounts for approximately 70% of the SP hydrolytic activity. Although the precise identity of this enzyme remains to be determined, the present evidence indicates that it shares many of the characteristics of the enzyme substance P-degrading endopeptidase (Endo et al. 1988, 1989). Enkephalinase activity was also found, contributing to 28% of the hydrolytic activity in the membrane fraction. However, the activity of this enzyme was insensitive to elevated levels of ACh. After initial cleavage of SP by the primary hydrolytic enzymes, further degradation of the fragments appears to be carried out by membrane associated serine protease(s). The activity exhibited by this class of enzymes was inhibited by DFP treatment and was not sensitive to ACh. Although AChE does not make a major contribution to the hydrolysis of SP, it does participate in peptide degradation via its esterase activity which controls the level of ACh, thereby modulating the primary SP-hydrolytic enzyme.

Acetylcholine↗

PubMed1992

Immunocytochemical evidence for the involvement of glycine in sensory centers of the rat brain.

Glycine-like immunoreactivity was localized to a number of sites in the rat brain which are involved in processing sensory information. In the auditory and vestibular systems, glycine immunoreactivity was seen in dorsal and ventral cochlear nuclei, superior olive, trapezoid body, medial and lateral vestibular nuclei, and inferior colliculus. Staining in the visual system was seen in retina, dorsal lateral geniculate nucleus, and superior colliculus. The olfactory system exhibited staining in the olfactory bulb and accessory olfactory formation. Somatosensory centers with glycine immunoreactivity included the dorsal column nuclei, spinal trigeminal nucleus, principal sensory nucleus of V, reticular formation, and periaqueductal gray. Glycine-immunoreactive neurons were also seen in cerebellar cortex, deep cerebellar nuclei, hippocampus, cerebral cortex, and striatum. The distribution of staining indicates that glycine plays a major role in sensory centers with actions at both strychnine-sensitive and strychnine-insensitive receptors.

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PubMed1991

Connectivity of glycine immunoreactive amacrine cells in the cat retina.

The synaptic relationships of glycine immunoreactive amacrine cells in the cat retina were studied through the use of postembedding immunogold techniques. Glycine immunoreactive amacrine cells were found to synapse extensively with other amacrines and ganglion cells, particularly in strata 1-3 of the inner plexiform layer. This contrasts with GABA immunoreactive amacrine cells which provide major input to bipolar cells in strata 3-5. Glycine containing amacrine terminals exhibited diversity with respect to the morphology of their synaptic vesicles. The three types of terminals which could be distinguished were characterized by small pleomorphic (32-35 nm), medium-sized flattened (38-45 nm), or larger rounded (48-55 nm) vesicles. Comparison of retinal sections processed for glycine immunoreactivity with adjacent sections stained for GABA reactivity revealed a colocalization of glycine and GABA in 3% of the cells in the amacrine layer and approximately 40% of the cells in the ganglion cell layer. The amacrine terminals in which glycine and GABA were colocalized typically contained the small pleomorphic type of vesicles.

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PubMed1991

Glycine receptor immunoreactivity is localized at amacrine synapses in cat retina.

Immunocytochemical techniques were used to localize strychnine-sensitive glycine receptors in cat retina. Light microscopy showed staining in processes ramifying throughout the inner plexiform layer and in cell bodies of both amacrine and ganglion cells. At the electron-microscopic level, receptor immunoreactivity was seen to be clustered at sites postsynaptic to amacrine cells. In contrast, bipolar cells were neither presynaptic nor postsynaptic elements at sites of glycine receptor staining. Double-label studies verified the presence of glycine immunoreactivity in amacrine terminals presynaptic to glycine receptors. These findings support a role for glycine as an inhibitory neurotransmitter in amacrine cells.

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PubMed1990

Colocalization of substance P and GABA in retinal ganglion cells: a computer-assisted visualization.

Ganglion cells in the albino rat retina were retrogradely labeled with the fluorescent dye, diamindino-yellow, from the superior colliculus. Preembedding and postembedding immunocytochemical techniques were employed in conjunction with computer-assisted image processing to visualize SP- and GABA-immunoreactivity. Examination of flatmount and sectioned retinas revealed that approximately 3% of the ganglion cells projecting to the contralateral superior colliculus exhibit SP-immunoreactivity. Moreover, these cells were found to comprise a subpopulation of the GABA-immunoreactive cells projecting to the rat tectum.

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PubMed1989

GABA-immunoreactivity in ganglion cells of the rat retina.

Ganglion cells in the rat retina were labeled with the fluorescent dye, Diamidino-yellow, by retrograde transport from the superior colliculus and subsequently reacted for GABA-like immunoreactivity with a rhodamine-conjugated antiserum. Examination of sectioned retinas by fluorescence microscopy showed double labeling in approximately 6% of the ganglion cells. The presence of GABA in these neurons suggests that they may be involved in providing direct inhibitory input to the rat tectum.

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PubMed1989

Distribution of GABA immunoreactivity in the cat retina: a light- and electron-microscopic study.

The distribution of GABA-like immunoreactivity in the cat retina was studied through the use of preembedding immunocytochemistry for light microscopy and by postembedding immunogold techniques for electron microscopy. Staining was observed in both inner and outer plexiform layers. Approximately 30% of the somata in the amacrine portion of the inner nuclear layer were immunoreactive and included amacrine and interplexiform cells. Horizontal cells and a subpopulation of cone bipolar cells were also stained. In the ganglion cell layer, staining was observed in both small- and medium-sized neurons. GABA-labeled amacrine cells were presynaptic to somata of amacrine cells and to dendrites of amacrine, bipolar, and ganglion cells. Bipolar cells were a major target, receiving more than 60% of all labeled synapses in the inner plexiform layer. Many of these contacts were reciprocal synapses. These findings support a major role for GABA-labeled amacrines in providing feedback inhibition to bipolar cells in the inner retina.

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PubMed1988

Substance P-like immunoreactive amacrine cells in the cat retina.

Substance P-like immunoreactivity was localized by immunocytochemical techniques to two subpopulations of amacrine cells in the cat retina. One cell was a unistratified amacrine with processes ramifying within stratum 4 of the inner plexiform layer. The other cell type was a bistratified cell with processes in both stratum 1 (s1) and stratum 4 (s4). Both cell types were seen with their somas displaced to the ganglion cell layer as well as in the conventional amacrine location in the inner nuclear layer. Substance P cells were present in the greatest density within the area centralis and decreased in number toward the periphery. The ratio of amacrine to displaced amacrine cells also decreased peripherally. However, the coverage by immunoreactive fibers in s4 remained three times that seen in s1. Computer-assisted analysis confirmed the location of substance P-containing processes at 5-15% (s1) and 50-70% (s4) depth levels in the inner plexiform layer. A comparison of substance P-like immunoreactivity in light- and dark-adapted cat retinas showed no apparent differences in the distribution of immunoreactivity due to lighting conditions.

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PubMed1988

Localization of glycine-containing neurons in the Macaca monkey retina.

Autoradiography following 3H-glycine (Gly) uptake and immunocytochemistry with a Gly-specific antiserum were used to identify neurons in Macaca monkey retina that contain a high level of this neurotransmitter. High-affinity uptake of Gly was shown to be sodium dependent whereas release of both endogenous and accumulated Gly was calcium dependent. Neurons labeling for Gly included 40-46% of the amacrine cells and nearly 40% of the bipolars. Synaptic labeling was seen throughout the inner plexiform layer (IPL) but with a preferential distribution in the inner half. Bands of labeled puncta occurred in S2, S4, and S5. Both light and postembedding electron microscopic (EM) immunocytochemistry identified different types of amacrine and bipolar cell bodies and their synaptic terminals. The most heavily labeled Gly+ cell bodies typically were amacrine cells having a single, thick, basal dendrite extending deep into the IPL and, at the EM level, electron-dense cytoplasm and prominent nuclear infoldings. This cell type may be homologous with the Gly2 cell in human retina (Marc and Liu: J. Comp. Neurol. 232:241-260, '85) and the AII/Gly2 of cat retina (Famiglietti and Kolb: Brain Res. 84:293-300, '75; Pourcho and Goebel: J. Comp. Neurol. 233:473-480, '85a). Gly+ amacrines synapse most frequently onto Gly- amacrines and both Gly- and Gly+ bipolars. Gly+ bipolar cells appeared to be cone bipolars because their labeled dendrites could be traced only to cone pedicles. The pattern of these labeled dendritic trees indicated that both diffuse and midget types of biopolars were Gly+. The EM distribution of labeled synapses showed Gly+ amacrine synapses throughout the IPL, but these composed only 11-23% of the amacrine population. Most of the Gly+ bipolar terminals were in the inner IPL, where 70% of all bipolar terminals were labeled. These findings are consistent with previous data from cats and humans and suggest that both amacrine and bipolar cells contribute to glycine-mediated neurotransmission in the monkey retina.

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PubMed1988

Colocalization of substance P and gamma-aminobutyric acid in amacrine cells of the cat retina.

Substance P and gamma-aminobutyric acid (GABA) were colocalized by immunocytochemistry in two subpopulations of amacrine cells in the cat retina. All of the cells which stained for substance P also showed GABA reactivity. However, there were many GABA-immunoreactive cells which did not stain for substance P. The presence of neuropeptides provides a basis for additional neurochemical characterization of the multiple populations of GABA immunoreactive cells.

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