Neurokinin 1 receptor expression and substance P physiological actions are developmentally regulated in the rabbit retina

We investigated the expression of the substance P (SP) receptor (the neurokinin 1 receptor, NK1 receptor) and SP functional effects in developing rabbit retinas. NK1 receptors in adult retinas were in a population of cone bipolar cells and in dopaminergic amacrine cells, as previously described. In contrast, at birth and at postnatal day (PND) 6, NK1 receptors were exclusively expressed by cholinergic amacrine and displaced amacrine cells. NK1 receptor expression in cholinergic cells was still observed at PND10 (eye opening), while at PND21 it was confined to cholinergic cells of the inner nuclear layer. Starting at PND10, NK1 receptors were also in bipolar cells and in dopaminergic amacrine cells. A fully mature NK1 receptor expression pattern was observed at PND35. Dopamine release was assessed in isolated retinas in the presence of SP, the NK1 receptor agonist GR73632 or the NK1 receptor antagonist GR82334. At PND35, extracellular dopamine was significantly increased by 10 microM SP or 0.01-100 microM GR73632, and it was decreased by 0.01-10 microM GR82334. No effects were detected in developing retinas up to PND21. Ca2+ imaging experiments were performed in single cholinergic cells identified by their "starburst" morphology in perinatal retinas. Intracellular Ca2+ levels were significantly increased by 1 microM SP or GR73632. This effect was reversibly inhibited by 1 microM GR82334. These data demonstrate that both NK1 receptor expression and SP physiological actions are developmentally regulated in the retina. SP neurotransmission in the immature retina may subserve developmental events, and SP is likely to represent an important developmental factor for the maturation of retinal neurons and circuitries.     

Expression of the neurokinin 1 receptor in the rabbit retina

Substance P is the preferred ligand for the neurokinin 1 (NK1) receptor. In vertebrate retinas, substance P is expressed by amacrine, interplexiform and ganglion cells. Substance P influences the activity of amacrine and ganglion cells and it is reported to evoke dopamine release. We investigated NK1 receptor expression in the rabbit retina using affinity-purified NK1 receptor antibodies. NK1 receptors were expressed by two distinct populations of retinal neurons. One is a population of ON-type bipolar cells characterized by axonal arborizations that ramified in the inner plexiform layer near the ganglion cell layer. Double-label studies showed that NK1 receptor-expressing bipolar cells were distinct from rod bipolar cells and from other immunocytochemically identified types of cone bipolar cells. Their density was about 2250 cells/mm2 in the visual streak and 1115 cells/mm2 in ventral mid-periphery. They were distributed in a non-random pattern. In the outer plexiform layer, the dendrites of these bipolar cells converged into heavily immunostained clusters having a punctate appearance. The density of these clusters in mid-peripheral ventral regions (about 13000 clusters/mm2) was similar to the reported cone density [Famiglietti and Sharpe (1995) Vis. Neurosci. 12, 1151-1175], suggesting these dendrites contact all cone photoreceptors. The second NK1 receptor expressing cell population corresponds to the tyrosine hydroxylase-containing amacrine cell population. NK1 receptor immunostaining was localized to the cell body and processes, but not to the processes that form the 'rings' that are known to encircle somata of AII amacrine cells. These findings show that NK1 receptor immunoreactivity is localized to a population of ON-type cone bipolar cells and to dopaminergic amacrine cells, suggesting that substance P acting on NK1 receptors influences multiple retinal circuits in the rabbit retina.     

The neurons of the ground squirrel retina as revealed by immunostains for calcium binding proteins and neurotransmitters

Ground squirrel retinas were immunostained with antibodies against calcium binding proteins (CBPs) and classical neurotransmitters in order to describe neuronal phenotypes in a diurnal mammalian retina and to then compare these neurons with those of more commonly studied nocturnal retinas like cats' and rabbits'. Double immunostained tissue was examined by confocal microscopy using antibodies against the following: rhodopsin and the CBPs, calbindin, calretinin, parvalbumin, calmodulin and recoverin (CB, CR, PV, CM, RV), glycine, GABA, choline acetyltransferase (CHAT) and tyrosine hydroxylase (TOH).In ground squirrel retina, the traditional cholinergic mirror symmetric amacrine cells colocalize CHAT with PV and GABA and faintly with glycine. A second cholinergic amacrine cell type colocalizes glycine alone. CR is found in at least 3 different amacrine cell types. The CR-immunoreactive (IR) cell population is a mixture of glycinergic and GABAergic types. The dopamine cell type IR to tyrosine hydroxylase has the typical morphology of a wide field cell with dendrites in S1 but the rings seen in cat or rabbit retina are not as numerous. TOH-IR amacrine cells send large club-shaped processes to the outer plexiform layer. CB and CR are in bipolar cells, A- and B-type horizontal cells and several amacrine cell types. Anti-rhodopsin labels the low density rod photoreceptor population in this species. Anti-recoverin labels cones and some bipolar cells while PKC is found in several different bipolar cell types. One ganglion cell with dendritic branching in S3 is strongly CR-IR.We find no evidence for an AII amacrine cell in the ground squirrel, with either anti-CR or anti-PV. An amacrine cell with similarity to the DAP1-3 cell of rabbit is CR-IR and glycine-IR. We discuss this labeling pattern in relationship to other mammalian species. The differences in staining patterns and phenotypes revealed suggest a functional diversity in the populations of amacrine cells according to whether the retinas are rod or cone dominated.     

Characterization of receptors for glutamate and GABA in retinal neurons

Glutamate and gamma-aminobutyric acid (GABA) are major excitatory and inhibitory neurotransmitters in the vertebrate retina, "a genuine neural center" (Ramón y Cajal, 1964, Recollections of My Life, C.E. Horne (Translater) MIT Press, Cambridge, MA). Photoreceptors, generating visual signals, and bipolar cells, mediating signal transfer from photoreceptors to ganglion cells, both release glutamate, which induces and/or changes the activity of the post-synaptic neurons (horizontal and bipolar cells for photoreceptors; amacrine and ganglion cells for bipolar cells). Horizontal and amacrine cells, which mediate lateral interaction in the outer and inner retina respectively, use GABA as a principal neurotransmitter. In recent years, glutamate receptors and GABA receptors in the retina have been extensively studied, using multi-disciplinary approaches. In this article some important advances in this field are reviewed, with special reference to retinal information processing. Photoreceptors possess metabotropic glutamate receptors and several subtypes of GABA receptors. Most horizontal cells express AMPA receptors, which may be predominantly assembled from flop slice variants. In addition, these cells also express GABAA and GABAC receptors. Signal transfer from photoreceptors to bipolar cells is rather complicated. Whereas AMPA/KA receptors mediate transmission for OFF type bipolar cells, several subtypes of glutamate receptors, both ionotropic and metabotropic, are involved in the generation of light responses of ON type bipolar cells. GABAA and GABAC receptors with distinct kinetics are differentially expressed on dendrites and axon terminals of both ON and OFF bipolar cells, mediating inhibition from horizontal cells and amacrine cells. Amacrine cells possess ionotropic glutamate receptors, whereas ganglion cells express both ionotropic and metabotropic glutamate receptors. GABAA receptors exist in amacrine and ganglion cells. Physiological data further suggest that GABAC receptors may be involved in the activity of these neurons. Moreover, responses of these retinal third order neurons are modulated by GABAB receptors, and in ganglion cells there exist several subtypes of GABAB receptors. A variety of glutamate receptor and GABA receptor subtypes found in the retina perform distinct functions, thus providing a wide range of neural integration and versatility of synaptic transmission. Perspectives in this research field are presented.     

Activation of mGluR5 modulates GABA(A) receptor function in retinal amacrine cells

Amacrine cells in the vertebrate retina receive glutamatergic input from bipolar cells and make synapses onto bipolar cells, ganglion cells, and other amacrine cells. Recent studies indicate that amacrine cells express metabotropic glutamate receptors (mGluRs) and that signaling within the inner plexiform layer (IPL) of the retina might be modulated by mGluR activity. This study tests the hypothesis that activation of mGluR5 modulates GABA(A) receptor function in retinal amacrine cells. Whole cell voltage-clamp recordings were combined with pharmacology to establish the identity of the ionotropic GABA receptors expressed in primary cultures of chick amacrine cells and to determine how mGluR5 activity affected the behavior of those receptors. Application of GABA (20 microM) produced currents that reversed at -58.2 +/- 0.9 mV, near the predicted Cl(-) reversal potential of -59 mV. The GABA(A) receptor antagonist, bicuculline (50 microM), completely blocked the GABA-gated currents. cis-4-Aminocrotonic acid (CACA; 100 microM), a GABA(C) receptor agonist, produced small currents that were not blocked by the GABA(C) antagonist, (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid (TPMPA; 20 microM), but were completely blocked by bicuculline. These results indicate that cultured amacrine cells express GABA(A) receptors exclusively. Activating mGluR5 with (RS)-2-chloro-5-hydroxyphenylglycine (CHPG; 300 microM) enhanced GABA-gated currents by 10.0 +/- 1.5%. Buffering internal Ca(2+) with BAPTA (10 mM) blocked the CHPG-dependent enhancement. Activation of PKC with the cell-permeable PKC activators (-)-7-octylindolactam V, phorbol 12-myristate 13 acetate (PMA), or 1-oleoyl-2-acetyl-sn-glycerol (OAG) also enhanced GABA-gated currents in a dose-dependent manner. Preactivation of PKC occluded the mGluR5-dependent enhancement, and inhibition of Ca-dependent PKC isotypes with G?6976 (35 nM) suppressed the effects of mGluR5 activation, suggesting that mGluR5 and PKC are part of the same pathway. To determine if mGluR5-dependent enhancement occurred at synaptic GABA(A) receptors, postsynaptic currents were recorded in the presence of CHPG. On average, the mean amplitudes of the quantal events were increased by about 18% when mGluR5 was activated. These results indicate that activation of mGluR5 enhances GABA-gated current in cultured amacrine cells in a manner that is both Ca(2+)- and PKC-dependent. These results support the possibility that glutamate released from bipolar cells can modulate the function of GABAergic amacrine cells and alter signaling in the inner plexiform layer.     

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. Anat Rec 255:363–373, 1999. © 1999 Wiley‐Liss, Inc.     

A molecular phenotype atlas of the zebrafish retina

The rasborine cyprinid Danio rerio (the zebrafish) has become a popular model of retinal function and development. Its value depends, in part, on validation of homologies with retinal cell populations of cyprinine cyprinids. This atlas provides raw and interpreted molecular phenotype data derived from computationally classified sets of small molecule signals from different cell types in the zebrafish retina: L-alanine, L-aspartate, L-glutamine, L-glutamate, glutathione, glycine, taurine and -aminobutyrate. This basis set yields an 8-dimensional signature for every retinal cell and formally establishes molecular signature homologies with retinal neurons, glia, epithelia and endothelia of other cyprinids. Zebrafish photoreceptor classes have been characterized previously: we now show their metabolic profiles to be identical to those of the corresponding photoreceptors in goldfish. The inner nuclear layer is partitioned into precise horizontal, bipolar and amacrine cell layers. The horizontal cell layer contains at least three and perhaps all four known classes of cyprinine horizontal cells. Homologues of cyprinid glutamatergic ON-center and OFF-center mixed rod-cone bipolar cells are present and it appears likely that all five classes are present in zebrafish. The cone bipolar cells defy simple analysis but comprise the largest fraction of bipolar cells, as in all cyprinids. Signature analysis reveals six molecular phenotypes in the bipolar cell cohort: most are superclasses. The amacrine cell layer is composed of 64% GABA+ and 35% glycine+ amacrine cells, with the remainder being sparse dopaminergic interplexiform cells and other rare unidentified neurons. These different amacrine cell types are completely distinct in the dark adapted retina, but light adapted retinas display weak leakage of GABA signals into many glycinergic amacrine cells, suggesting widespread heterocellular coupling. The composition of the zebrafish ganglion cell layer is metabolically indistinguishable from that in other cyprinids, and the signatures of glial and non-neuronal cells display strong homologies with those in mammals. As in most vertebrates, zebrafish Müller cells possess a high glutamine, low glutamate signature and contain the dominant pool of glutathione in the neural retina. The retinal pigmented epithelium shows a general mammalian signature but also has exceptional glutathione content (5–10 mM), perhaps required by the unusually high oxygen tensions of teleost retinas. The optic nerve and the marginal zone of the retina reveal characteristic metabolic specializations. The marginal zone is strongly laminated and its nascent neurons display their characteristic signatures before taking their place in the retina proper.     

Neurochemical differentiation of horizontal and amacrine cells during transformation of the sea lamprey retina

The sea lamprey is a modern representative of the earliest vertebrates (the agnathans) in which development of the eye and retina shows unique patterns. In larval stages the retina is poorly developed, and although a small central region has developed glutamatergic vertical pathways, there is no evidence of chemical differentiation of amacrine and horizontal cells in the central or lateral larval retina [Villar-Cervi?o, V., Abalo, X.M., Villar-Cheda, B., Meléndez-Ferro, M., Pérez-Costas, E., Holstein, G.R., Martinelli, G.P., Rodicio, M.C., Anadón, R., 2006. Presence of glutamate, glycine, and gamma-aminobutyric acid in the retina of the larval sea lamprey: comparative immunohistochemical study of classical neurotransmitters in larval and postmetamorphic retinas. J. Comp. Neurol. 499, 810-827.]. However, in adults all the retina was differentiated and both amacrine and horizontal cells are well developed. Present immunocytochemical results show that the horizontal and amacrine cells of the retina begin their neurochemical differentiation during metamorphosis, when they start to express GABA, glycine, serotonin and dopamine; this occurs several years after the onset of development. Immunoreactivity for GABA, glycine and serotonin was found at early metamorphic stages, while expression of the markers of catecholaminergic amacrine cells, dopamine and tyrosine hydroxylase, was found to be delayed until intermediate metamorphic stages. GABA, which is found in some amacrine and horizontal cells of adults, was first observed in amacrine cells during early stages of transformation and then in horizontal cells during middle stages. All cells immunoreactive to serotonin or tyrosine hydroxylase/dopamine were amacrine cells. Interestingly, all these markers began expression before the appearance of opsin-immunoreactive photoreceptors in the lateral retina. The pattern of chemical differentiation of amacrine and horizontal cells was compared with that of other vertebrates and their significance was discussed.     

Modulation of excitatory synaptic transmission by GABA(C) receptor-mediated feedback in the mouse inner retina.

In many vertebrate CNS synapses, the neurotransmitter glutamate activates postsynaptic non-N-methyl-D-aspartate (NMDA) and NMDA receptors. Since their biophysical properties are quite different, the time course of excitatory postsynaptic currents (EPSCs) depends largely on the relative contribution of their activation. To investigate whether the activation of the two receptor subtypes is affected by the synaptic interaction in the inner plexiform layer (IPL) of the mouse retina, we analyzed the properties of the light-evoked responses of ON-cone bipolar cells and ON-transient amacrine cells in a retinal slice preparation. ON-transient amacrine cells were whole cell voltage-clamped, and the glutamatergic synaptic input from bipolar cells was isolated by a cocktail of pharmacological agents (bicuculline, strychnine, curare, and atropine). Direct puff application of NMDA revealed the presence of functional NMDA receptors. However, the light-evoked EPSC was not significantly affected by D(-)-2-amino-5-phosphonopentanoic acid (D-AP5), but suppressed by 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) or 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466). These results indicate that the light-evoked EPSC is mediated mainly by AMPA receptors under this condition. Since bipolar cells have GABA(C) receptors at their terminals, it has been suggested that bipolar cells receive feedback inhibition from amacrine cells. Application of (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), a specific blocker of GABA(C) receptors, suppressed both the GABA-induced current and the light-evoked feedback inhibition observed in ON-cone bipolar cells and enhanced the light-evoked EPSC of ON-transient amacrine cells. In the presence of TPMPA, the light-evoked EPSC of amacrine cells was composed of AMPA and NMDA receptor-mediated components. Our results suggest that photoresponses of ON-transient amacrine cells in the mouse retina are modified by the activation of presynaptic GABA(C) receptors, which may control the extent of glutamate spillover.     

Expression of aquaporin-9 immunoreactivity by catecholaminergic amacrine cells in the rat retina

Aquaporins are involved in the maintenance of ionic and osmotic balance in the central nervous system and in the eye. Whereas the expression of aquaporin-9 immunoreactivity in the brain has been described for catecholaminergic neurons and glial cells, the expression of aquaporin-9 in the neural retina is unclear. We examined the distribution of aquaporin-1 and -9 immunoreactivities in retinas of the rat. Aquaporin-9 immunoreactivity was expressed exclusively by tyrosine hydroxylase (TH) positive amacrine cells, while aquaporin-1 immunoreactivity was expressed by photoreceptor cells and by TH negative amacrine cells. The staining pattern of aquaporin-9 did not change up to 4 weeks after pressure-induced transient retinal ischemia. It is concluded that catecholaminergic, putatively dopaminergic, amacrine cells of the retina express aquaporin-9.     

Localisation of the GABA(C) receptors at the axon terminal of the rod bipolar cells of the mouse retina.

In the vertebrate retina, the rod bipolar cells make reciprocal synapses with amacrine cells at the axon terminal. Amacrine cells may perform a fine control of the transmitter release from rod bipolar cells by means of GABAergic synapses acting on different types of GABA receptors. To clarify this possibility GABA-induced currents were recorded by the patch-clamp whole cell method in rod bipolar cells enzymatically dissociated from the mouse retina. All cells tested showed a desensitising chloride-sensitive GABA-induced current. When GABA 30 microM was applied in presence of 100 microM biccuculine, a blocker of the GABA(A) receptors, a slow-desensitising component of the current still remains. This current was blocked when GABA 30 microM was applied in presence of 100 microM 3-aminopropylphosphonic acid, an antagonist of the GABA(C) receptors. The current mediated by GABA(C) receptors showed an EC50 of less that 5 microM; the ionic current through the GABA(A) receptor showed an EC50 of ca. 30 microM. Two pieces of evidence demonstrated that the GABA(C)-mediated current was localised at the axon terminal of rod bipolar cells: (1) cells lacking the axon terminal only showed the biccuculine-sensitive GABA-induced current; and (2) after mechanical section of the axon terminal, bipolar cells lost the slow-desensitising component of the GABA-induced current. We conclude that the rod bipolar cells express two types of ionotropic GABA receptors, and that the high sensitive GABA(C) receptors are mainly localised at the level of the axon terminal and therefore may contribute to the modulation of the transmitter release from the rod bipolar cell.     

Neuronal expression of P2X3 purinoceptors in the rat retina

P2X3 purinoceptors are involved in fast, excitatory neurotransmission in the nervous system, and are expressed predominantly within sensory neurons. In this study, we examined the cellular and synaptic localization of the P2X3 receptor subunit in the retina of the rat using immunofluorescence immunohistochemistry and pre-embedding immunoelectron microscopy. In addition, we investigated the activity of ecto-ATPases in the inner retina using an enzyme cytochemical method. The P2X3 receptor subunit was expressed in the soma of a subset of GABA immunoreactive amacrine cells, some of which also expressed protein kinase C-alpha. In addition, punctate immunoreactivity was observed within both the inner and outer plexiform layers of the retina. Double labeling studies showed that P2X3 receptor puncta were associated with both rod and cone bipolar cell axon terminals in the inner plexiform layer. Ultrastructural studies indicated that P2X3 receptor subunits were expressed on putative A17 amacrine cells at sites of reciprocal synaptic input to the rod bipolar cell axon terminal. Moreover, we observed P2X3 immunolabeling on amacrine cell processes that were associated with cone bipolar cell axon terminals and other conventional synapses. In the outer retina, P2X3 immunoreactivity was observed on specialized junctions made by putative interplexiform cells. Ecto-ATPase activity was localized to the inner plexiform layer on the extracellular side of all plasma membranes, but was not apparent in the ganglion cell layer or the inner nuclear layer, suggesting that ATP dephosphorylation occurs exclusively in synaptic regions of the inner retina. These data provide further evidence that purines participate in retinal transmission, particularly within the rod pathway.     

Characterization of histamine projections and their potential cellular targets in the mouse retina

The vertebrate retina receives histaminergic input from the brain via retinopetal axons that originate from perikarya in the posterior hypothalamus. In the nervous system, histamine acts on three G-protein-coupled receptors, histamine receptor (HR) 1, HR2 and HR3. In order to look for potential cellular targets of histamine in the mouse retina, we have examined the retina for the expression of histamine and the presence of these three receptors. Consistent with studies of retina from other vertebrates, histamine was only found in retinopetal axons, which coursed extensively through the ganglion cell and inner plexiform layers. mRNA for all three receptors was expressed in the mouse retina, and immunohistochemical studies further localized HR1 and HR2. HR1 immunoreactivity was observed on dopaminergic amacrine cells, calretinin-positive ganglion cells and axon bundles in the ganglion cell layer. Furthermore, a distinct group of processes in the inner plexiform layer was labeled, which most likely represents the processes of cholinergic amacrine cells. HR2 immunoreactivity was observed on the processes and cell bodies of the primary glial cells of the mammalian retina, the Müller cells. This distribution of histamine and its receptors is consistent with a brain-derived source of histamine acting on diverse populations of cells in the retina, including both neurons and glia.     

GABAA, GABAC and glycine receptor-mediated inhibition differentially affects light-evoked signalling from mouse retinal rod bipolar cells

Rod bipolar cells relay visual signals evoked by dim illumination from the outer to the inner retina. GABAergic and glycinergic amacrine cells contact rod bipolar cell terminals, where they modulate transmitter release and contribute to the receptive field properties of third order neurones. However, it is not known how these distinct inhibitory inputs affect rod bipolar cell output and subsequent retinal processing. To determine whether GABA_A, GABA_C and glycine receptors made different contributions to light-evoked inhibition, we recorded light-evoked inhibitory postsynaptic currents (L-IPSCs) from rod bipolar cells mediated by each pharmacologically isolated receptor. All three receptors contributed to L-IPSCs, but their relative roles differed; GABA_C receptors transferred significantly more charge than GABA_A and glycine receptors. We determined how these distinct inhibitory inputs affected rod bipolar cell output by recording light-evoked excitatory postsynaptic currents (L-EPSCs) from postsynaptic AII and A17 amacrine cells. Consistent with their relative contributions to L-IPSCs, GABA_C receptor activation most effectively reduced the L-EPSCs, while glycine and GABA_A receptor activation reduced the L-EPSCs to a lesser extent. We also found that GABAergic L-IPSCs in rod bipolar cells were limited by GABA_A receptor-mediated inhibition between amacrine cells. We show that GABA_A, GABA_C and glycine receptors mediate functionally distinct inhibition to rod bipolar cells, which differentially modulated light-evoked rod bipolar cell output. Our findings suggest that modulating the relative proportions of these inhibitory inputs could change the characteristics of rod bipolar cell output.     

Morphological and electrophysiological evidence for an ionotropic GABA receptor of novel pharmacology.

Evidence from toxicological studies suggested that an ionotropic GABA receptor of novel pharmacology (picrotoxin-insensitive, bicuculline-sensitive) exists in the chick embryo retina. In this report, we provide direct morphological and electrophysiological evidence for the existence of such an iGABA receptor. Chick embryo retinas (14-16 days old) incubated in the presence of kainic acid showed pronounced histopathology in all retinal layers. Maximal protection from this toxicity required a combination of bicuculline and picrotoxin. Individual application of the antagonists indicated that a picrotoxin-insensitive, bicuculline-sensitive GABA receptor is likely to be present on ganglion and amacrine, but not bipolar, cells. GABA currents in embryonic and mature chicken retinal neurons were measured by whole cell patch clamp. GABA was puffed at the dendritic processes in the IPL. Picrotoxin (500 microM, in the bath) eliminated all (>95%) the GABA current in the majority of ganglion and amacrine cells tested, but many cells possessed a substantial picrotoxin-insensitive component. This current was eliminated by bicuculline (200 microM). This current was not a transporter-associated current, since it was not altered by GABA transport blockers or sodium removal. The current-voltage relation was linear and reversed near E(Cl), as expected for a ligand-gated chloride current. Both pentobarbital and lorazepam enhanced the picrotoxin-insensitive current. We conclude that chicken retinal ganglion and amacrine cells express a GABA receptor that is GABA-A-like, in that it can be blocked by bicuculline, and positively modulated by barbiturates and benzodiazepines, but is insensitive to the noncompetitive blocker picrotoxin. Understanding the molecular properties of this receptor will be important for understanding both physiological GABA neurotransmission and the pathology of GABA receptor overactivation.     

Localization of glycine uptake and receptors in the cat retina

Immunocytochemistry using a monoclonal antibody against glycine receptors revealed that these receptors in cat retina are confined to the inner plexiform layer (IPL). The outer half of that layer showed strong patchy labelling with some indication of two bands corresponding to the on- and off-sublamina. High-affinity uptake of [3H]glycine followed by autoradiography labelled amacrine cells, bipolar cells and the outer portion of the IPL. Silver grains in the IPL were patchily distributed. These results indicate in the cat retina a close match between presynaptic glycinergic elements labelled by high-affinity uptake and the postsynaptic receptor sites for glycine revealed by immunocytochemistry.     

Effects of GABA receptor antagonists on retinal glycine receptors and on homomeric glycine receptor alpha subunits

Glycinergic and GABAergic inhibition are juxtaposed at one retinal synaptic layer yet likely perform different functions. These functions have usually been evaluated using receptor antagonists. In examining retinal glycine receptors, we were surprised to find that commonly used concentrations of GABA antagonists blocked significant fractions of the glycine current. In retinal amacrine and ganglion cells, the competitive GABAA receptor antagonists (bicuculline and SR95531) were also competitive GlyR antagonists. Picrotoxinin produced a noncompetitive inhibition of retinal GlyRs. [1,2,5,6-tetrahydropyridine-4-yl] methylphosphinic acid, the GABACR antagonist, did not inhibit glycine receptors. All three GABAA receptor antagonists were competitive inhibitors of homomeric alpha1 or alpha2 GlyRs expressed in human embryonic kidney cells (HEK293) cells. Interestingly, bicuculline was much more effective at alpha2 GlyRs and might be used to separate glycine receptor subtypes. Thus commonly used concentrations of GABA antagonists do not unambiguously differentiate GABA and glycine pathways. Picrotoxinin inhibition of GABAC receptors requires two amino acids in the second transmembrane region (TM2): 2' serine and 6' threonine. Although TM2 regions in GABA and glycine receptors are highly homologous, neither 2' serine nor 6' threonine is essential for picrotoxinin sensitivity in glycine receptors.     

GABAergic circuitry in the opossum retina: a GABA release induced by l-aspartate

Glutamate and γ-amino butyric acid (GABA) are the major excitatory and inhibitory neurotransmitters, respectively, in the central nervous system (CNS), including the retina. Although in a number of studies the retinal source of GABA was identified, in several species, as horizontal, amacrine cells and cells in the ganglion cell layer, nothing was described for the opossum retina. Thus, the first goal of this study was to determine the pattern of GABAergic cell expression in the South America opossum retina by using an immunohistochemical approach for GABA and for its synthetic enzyme, glutamic acid decarboxylase (GAD). GABA and GAD immunoreactivity showed a similar cellular pattern by appearing in a few faint horizontal cells, topic and displaced amacrine cells. In an effort to extend the knowledge of the opossum retinal circuitry, the possible influence of glutamatergic inputs in GABAergic cells was also studied. Retinas were stimulated with different glutamatergic agonists and aspartate (Asp), and the GABA remaining in the tissue was detected by immunohistochemical procedures. The exposure of retinas to NMDA and kainate resulted the reduction of the number of GABA immunoreactive topic and displaced amacrine cells. The Asp treatment also resulted in reduction of the number of GABA immunoreactive amacrine cells but, in contrast, the displaced amacrine cells were not affected. Finally, the Asp effect was totally blocked by MK-801. This result suggests that Asp could be indeed a putative neurotransmitter in this non-placental animal by acting on an amacrine cell sub-population of GABA-positive NMDA-sensitive cells.