Proceedings of the National Academy of Sciences of the United States of America, Dec 10, 1996
Expression of G protein-regulated phospholipase C (PLC) 4 in the retina, lateral geniculate nucl... more Expression of G protein-regulated phospholipase C (PLC) 4 in the retina, lateral geniculate nucleus, and superior colliculus implies that PLC 4 may play a role in the mammalian visual process. A mouse line that lacks PLC 4 was generated and the physiological significance of PLC 4 in murine visual function was investigated. Behavioral tests using a shuttle box demonstrated that the mice lacking PLC 4 were impaired in their visual processing abilities, whereas they showed no deficit in their auditory abilities. In addition, the PLC 4-null mice showed 4-fold reduction in the maximal amplitude of the rod a-and b-wave components of their electroretinograms relative to their littermate controls. However, recording from single rod photoreceptors did not reveal any significant differences between the PLC 4-null and wild-type littermates, nor were there any apparent differences in retinas examined with light microscopy. While the behavioral and electroretinographic results indicate that PLC 4 plays a significant role in mammalian visual signal processing, isolated rod recording shows little or no apparent deficit, suggesting that the effect of PLC 4 deficiency on the rod signaling pathway occurs at some stage after the initial phototransduction cascade and may require cell-cell interactions between rods and other retinal cells.
We have investigated the morphology of the giant interneurons (GIs) and the main sensory projecti... more We have investigated the morphology of the giant interneurons (GIs) and the main sensory projections to these interneurons in the American cockroach. These neurons are thought to mediate the animal's escape behavior. We describe here the dendritic branching pattern of each of the 14 GIs (7 bilateral pairs) in the terminal ganglion, the pattern of projection of the cercal sensory nerve, and the overlap of the cercal projections with the dendrites of the GIs. Visualization of the GIs and cercal nerve projection was accomplished by single cell injection and axonal backfilling with cobalt. Comparisons of the same identified GI in different animals show the position of the soma and the locations and orientations of the major processes are characteristic for each GI. The axons of the cercal nerve project to a well-defined ipsilateral region of the terminal ganglion. After entering the terminal ganglion, the cercal afferents split into lateral and medial tracts. The projections of the lateral cercal tract overlap extensively with the dendritic fields of the GIs. In contrast, the medial tract does not overlap the dendritic fields of the GIs in the posterior portion of the ganglion and shows only a small degree of overlap in the anterior portion. Correlations between physiological properties of the GIs and cercal afferents are discussed in relation to our anatomical findings.
Heterotrimeric G-proteins, comprising G␣ and G␥ subunits, couple metabotropic receptors to vario... more Heterotrimeric G-proteins, comprising G␣ and G␥ subunits, couple metabotropic receptors to various downstream effectors and contribute to assembling and trafficking receptor-based signaling complexes. A G-protein  subunit, G 3 , plays a critical role in several physiological processes, as a polymorphism in its gene is associated with a risk factor for several disorders. Retinal ON bipolar cells express G 3 , and they provide an excellent system to study its role. In the ON bipolar cells, mGluR6 inverts the photoreceptor's signal via a cascade in which glutamate released from photoreceptors closes the TRPM1 channel. This cascade is essential for vision since deficiencies in its proteins lead to complete congenital stationary night blindness. Here we report that G 3 participates in the G-protein heterotrimer that couples mGluR6 to TRPM1. G 3 deletion in mouse greatly reduces the light response under both scotopic and photopic conditions, but it does not eliminate it. In addition, G 3 deletion causes mislocalization and downregulation of most cascade elements and modulators. Furthermore, G 3 may play a role in synaptic maintenance since in its absence, the number of invaginating rod bipolar dendrites is greatly reduced, a deficit that was not observed at 3 weeks, the end of the developmental period.
Retinitis pigmentosa (RP) is a group of inherited photoreceptor degeneration diseases that causes... more Retinitis pigmentosa (RP) is a group of inherited photoreceptor degeneration diseases that causes blindness without effective treatment. The pathogenesis of retinal degeneration involves mainly oxidative stress and inflammatory responses. Zeaxanthin dipalmitate (ZD), a wolfberry‐derived carotenoid, has anti‐inflammatory and anti‐oxidative stress effects. Here we investigated whether these properties of ZD can delay the retinal degeneration in rd10 mice, a model of RP, and explored its underlying mechanism. One shot of ZD or control vehicle was intravitreally injected into rd10 mice on postnatal day 16 (P16). Retinal function and structure of rd10 mice were assessed at P25, when rods degenerate substantially, using a visual behavior test, multi‐electrode‐array recordings and immunostaining. Retinal pathogenic gene expression and regulation of signaling pathways by ZD were explored using transcriptome sequencing and western blotting. Our results showed that ZD treatment improved the v...
Calcium enters the outer segment of a vertebrate photoreceptor through a cGMP-gated channel and i... more Calcium enters the outer segment of a vertebrate photoreceptor through a cGMP-gated channel and is extruded via a Na/Ca, K exchanger. We have identified another element in mammalian cones that might help to control cytoplasmic calcium. Reverse transcription-PCR performed on isolated photoreceptors identified mRNA for the SII Ϫ splice variant of the type I receptor for inositol 1,4,5-triphosphate (IP 3), and Western blots showed that the protein also is expressed in outer segments. Immunocytochemistry showed type I IP 3 receptor to be abundant in red-sensitive and green-sensitive cones of the trichromatic monkey retina, but it was negative or weakly expressed in blue-sensitive cones and rods. Similarly, the green-sensitive cones expressed the receptor in dichromatic retina (cat, rabbit, and rat), but the blue-sensitive cones did not. Immunostain was localized to disk and plasma membranes on the cytoplasmic face. To restore sensitivity after a light flash, cytoplasmic cGMP must rise to its basal level, and this requires cytoplasmic calcium to fall. Cessation of calcium release via the IP 3 receptor might accelerate this fall and thus explain why the cone recovers much faster than the rod. Furthermore, because its own activity of the IP 3 receptor depends partly on cytoplasmic calcium, the receptor might control the set point of cytoplasmic calcium and thus affect cone sensitivity.
Investigative Ophthalmology & Visual Science, Mar 10, 2014
PURPOSE. L-type voltage gated calcium channels in retina localize primarily at the presynaptic ac... more PURPOSE. L-type voltage gated calcium channels in retina localize primarily at the presynaptic active zones of photoreceptors and bipolar cells where they modulate glutamate release. However, the pore forming subunit Cacna1s of certain L-type channels is also expressed postsynaptically at the tips of ON bipolar cell dendrites where it colocalizes with mGluR6, but has an unknown function. At these dendritic tips, the components of the mGluR6 signaling cascade cluster together in a macromolecular complex, and each one's localization often depends on that of the others. Thus, we explored if Cacna1s is part of the mGluR6 complex. METHODS. We determined Cacna1s expression by PCR using an ON bipolar library, by Western blotting, and by standard immunohistochemistry. RESULTS. The PCR amplification confirmed expression of the transcript in ON bipolar cells, and Western blotting showed the expected bands. Immunostaining for Cacna1s was stronger in the dendritic tips of rod bipolar cells than in those of ON cone bipolar cells. This staining severely decreased in mice missing various mGluR6 cascade elements (Grm6 À/À , Gnao1 À/À , Gnb3 À/À , Gng13 À/À , and Trpm1 À/À). During development, the ratio of the number of Cacna1s puncta to the number of presynaptic ribbons followed a sigmoidal pattern, rising rapidly from P13 to P17. The mGluR6 expression preceded that of Cacna1s and RGS11. CONCLUSIONS. Our results show that the localization and stability of Cacna1s depend on the expression of mGluR6 and its cascade components, and they suggest that Cacna1s is part of the mGluR6 complex. We hypothesize that Cacna1s contributes to light adaptation by permeating calcium.
PCP2, a member of the GoLoco domain-containing family, is present exclusively in cerebellar Purki... more PCP2, a member of the GoLoco domain-containing family, is present exclusively in cerebellar Purkinje cells and retinal ON bipolar cells. Its function in these tissues is unknown. Biochemical and expression system studies suggest that PCP2 is a guanine nucleotide dissociation inhibitor, although a guanine nucleotide exchange factor has also been suggested. Here, we studied the function of PCP2 in ON bipolar cells because their light response depends on G␣ o1 , which is known to interact with PCP2. We identified a new splice variant of PCP2 (Ret-PCP2) and localized it to rod bipolar and ON cone bipolar cells. Electroretinogram recordings from PCP2-null mice showed a normal a-wave but a slower falling phase of the b-wave (generated by the activity of ON bipolar cells) relative to the wild type. Whole-cell recordings from rod bipolar cells showed, both under Ames medium and after blocking GABA A/C and glycine receptors, that PCP2-null rod bipolar cells were more depolarized than wild-type cells with greater inward current when clamped to Ϫ60 mV. Also under both conditions, the rise time of the response to intense light was slower by 28% (Ames) and 44% (inhibitory blockers) in the null cells. Under Ames medium, we also observed Ͼ30% longer decay time in the PCP2-null rod bipolar cells. We conclude that PCP2 facilitates cation channels closure in the dark, shortens the rise time of the light response directly, and accelerates the decay time indirectly via the inhibitory network. These data can most easily be explained if PCP2 serves as a guanine nucleotide exchange factor.
Retinal ganglion cells comprise about 10 morphological types that also differ functionally. To de... more Retinal ganglion cells comprise about 10 morphological types that also differ functionally. To determine whether functional differences might arise partially from differences in excitatory input, we quantified the distributions of ribbon contacts to four mammalian ganglion cell types [brisk-transient (BT), brisk-sustained (BS), local edge (LE), directionally selective (DS)], comparing small vs. large and "sluggish" vs. "brisk." Cells in guinea pig retina were filled with fluorescent dye, immunostained for synaptic ribbons, and reconstructed with their ribbon contacts by confocal microscopy. False-positive contacts were corrected by performing the same analysis on processes that lack synapses: glial stalks and rod bipolar axons. All types shared a domed distribution of membrane that was well fit by a Gaussian function (R 2 ϭ 0.96 Ϯ 0.01); they also shared a constant density of contacts on the dendritic membrane, both across each arbor and across cell types (19 Ϯ 1 contacts/100 m 2 membrane). However, the distributions of membrane across the retina differed markedly in width (BT Ͼ DS Ϸ BS Ͼ LE) and peak density (BS Ͼ DS Ͼ LE Ͼ BT). Correspondingly, types differed in peak density of contacts (BS Ͼ DS Ϸ LE Ͼ BT) and total number (BS Ϸ BT Ͼ DS Ͼ LE). These differences between cell types in spatial extent and local concentration of membrane and synapses help to explain certain functional differences.
The distribution of GABAA receptor in the outer plexiform layer of cat retina was studied by immu... more The distribution of GABAA receptor in the outer plexiform layer of cat retina was studied by immunocytochemistry with monoclonal antibodies. Staining was observed at the base of the cone pedicle, extracellularly, in association with the “triad” synaptic complex. Some bipolar dendrites and the basal processes that interconnect the cone pedicles were also stained. Rod spherules and horizontal cells were negative. The findings support the idea that the cone horizontal cells are GABAergic.
Synaptic transmission from photoreceptors to depolarizing bipolar cells is mediated by the APB gl... more Synaptic transmission from photoreceptors to depolarizing bipolar cells is mediated by the APB glutamate receptor. This receptor apparently is coupled to a G-protein which activates cGMP-phosphodiesterase to modulate cGMP levels and thus a cGMP-gated cation channel. We attempted to localize this system immunocytochemically using antibodies to various components of the rod phototransduction cascade, including G, (transducin), phosphodiesterase, the cGMP-gated channel, and arrestin. All of these antibodies reacted strongly with rods, but none reacted with bipolar cells. Antibodies to a different G-protein, G o , reacted strongly with rod bipolar cells of three mammalian species (which are depolarizing and APB-sensitive). Also stained were subpopulations of cone bipolar cells but not the major depolarizing type in cat (b^. G o antibody also stained certain salamander bipolar cells. Thus, across a wide range of species, G o is present in retinal bipolar cells, and at least some of these are depolarizing and APB-sensitive.
The neurotransmitter used by horizontal cells in mammals has not been identified. GABA has been t... more The neurotransmitter used by horizontal cells in mammals has not been identified. GABA has been the leading candidate, but doubt has remained because of failure to clearly demonstrate the GABA synthetic enzyme, glutamic acid decarboxylase (GAD) in these cells. Because GAD was recently shown to exist as two isoforms, 65 kDa and 67 kDa, we considered whether there might be a mismatch between the forms of GAD expressed in horizontal cells and the probes used to detect it. Accordingly, we stained sections of mammalian retina with antibodies specific for each isoform. Cat horizontal cells of both types (A and B) were immunoreactive for GAD 67 but negative for GAD 65 ; monkey horizontal cells of both types (H, and H n) were positive for GAD 65 and negative for GAD 67. The findings reconcile previous, apparently conflicting, observations and strengthen considerably the hypothesis that mammalian horizontal cells are GABAergic.
The subcellular distribution of GABAA receptor in the macaque and human retina was studied by imm... more The subcellular distribution of GABAA receptor in the macaque and human retina was studied by immunocytochemistry with monoclonal antibodies for the • and ~O subunits with a particular focus on bipolar cells. Immunoreactivity to GABAA receptor was present on dendritic tips of all bipolar cells. The stain was strongest on bipolar membranes in apposition to horizontal cell processes. Stain was concentrated on the tips of flat and invaginating cone bipolar cells at the base of the cone pedicle and on the invaginating tips of rod bipolar cells. Stain on the cone pedicle membrane was restricted to sites of apposition to stained bipolar dendrites; pedicle membrane in apposition to horizontal cell processes was unstained. Stain was also present on bipolar axon terminals in both on and off strata of the inner plexiform layer. All bipolar cell somas stained faintly; horizontal and Miiller cell somas were unstained. The ~ and/I subunits distributed similarly in monkey and human retina. Presence of GABA A receptor on the bipolar dendritic tips suggests that horizontal cells directly affect bipolar cells. Thus, GABAA receptor might mediate the receptive field surround of both off and on bipolar cells. Presence of GABAA receptor on bipolar axon terminals suggests that much of the inhibition feeding back from GABAergic amacrine to bipolar cells is GABAA-mediated. GABA Cone pedicle Horizontal cells Interplexiform cell Feedforward inhibition GABA depolarization
Proceedings of the National Academy of Sciences of the United States of America, Dec 10, 1996
Expression of G protein-regulated phospholipase C (PLC) 4 in the retina, lateral geniculate nucl... more Expression of G protein-regulated phospholipase C (PLC) 4 in the retina, lateral geniculate nucleus, and superior colliculus implies that PLC 4 may play a role in the mammalian visual process. A mouse line that lacks PLC 4 was generated and the physiological significance of PLC 4 in murine visual function was investigated. Behavioral tests using a shuttle box demonstrated that the mice lacking PLC 4 were impaired in their visual processing abilities, whereas they showed no deficit in their auditory abilities. In addition, the PLC 4-null mice showed 4-fold reduction in the maximal amplitude of the rod a-and b-wave components of their electroretinograms relative to their littermate controls. However, recording from single rod photoreceptors did not reveal any significant differences between the PLC 4-null and wild-type littermates, nor were there any apparent differences in retinas examined with light microscopy. While the behavioral and electroretinographic results indicate that PLC 4 plays a significant role in mammalian visual signal processing, isolated rod recording shows little or no apparent deficit, suggesting that the effect of PLC 4 deficiency on the rod signaling pathway occurs at some stage after the initial phototransduction cascade and may require cell-cell interactions between rods and other retinal cells.
We have investigated the morphology of the giant interneurons (GIs) and the main sensory projecti... more We have investigated the morphology of the giant interneurons (GIs) and the main sensory projections to these interneurons in the American cockroach. These neurons are thought to mediate the animal's escape behavior. We describe here the dendritic branching pattern of each of the 14 GIs (7 bilateral pairs) in the terminal ganglion, the pattern of projection of the cercal sensory nerve, and the overlap of the cercal projections with the dendrites of the GIs. Visualization of the GIs and cercal nerve projection was accomplished by single cell injection and axonal backfilling with cobalt. Comparisons of the same identified GI in different animals show the position of the soma and the locations and orientations of the major processes are characteristic for each GI. The axons of the cercal nerve project to a well-defined ipsilateral region of the terminal ganglion. After entering the terminal ganglion, the cercal afferents split into lateral and medial tracts. The projections of the lateral cercal tract overlap extensively with the dendritic fields of the GIs. In contrast, the medial tract does not overlap the dendritic fields of the GIs in the posterior portion of the ganglion and shows only a small degree of overlap in the anterior portion. Correlations between physiological properties of the GIs and cercal afferents are discussed in relation to our anatomical findings.
Heterotrimeric G-proteins, comprising G␣ and G␥ subunits, couple metabotropic receptors to vario... more Heterotrimeric G-proteins, comprising G␣ and G␥ subunits, couple metabotropic receptors to various downstream effectors and contribute to assembling and trafficking receptor-based signaling complexes. A G-protein  subunit, G 3 , plays a critical role in several physiological processes, as a polymorphism in its gene is associated with a risk factor for several disorders. Retinal ON bipolar cells express G 3 , and they provide an excellent system to study its role. In the ON bipolar cells, mGluR6 inverts the photoreceptor's signal via a cascade in which glutamate released from photoreceptors closes the TRPM1 channel. This cascade is essential for vision since deficiencies in its proteins lead to complete congenital stationary night blindness. Here we report that G 3 participates in the G-protein heterotrimer that couples mGluR6 to TRPM1. G 3 deletion in mouse greatly reduces the light response under both scotopic and photopic conditions, but it does not eliminate it. In addition, G 3 deletion causes mislocalization and downregulation of most cascade elements and modulators. Furthermore, G 3 may play a role in synaptic maintenance since in its absence, the number of invaginating rod bipolar dendrites is greatly reduced, a deficit that was not observed at 3 weeks, the end of the developmental period.
Retinitis pigmentosa (RP) is a group of inherited photoreceptor degeneration diseases that causes... more Retinitis pigmentosa (RP) is a group of inherited photoreceptor degeneration diseases that causes blindness without effective treatment. The pathogenesis of retinal degeneration involves mainly oxidative stress and inflammatory responses. Zeaxanthin dipalmitate (ZD), a wolfberry‐derived carotenoid, has anti‐inflammatory and anti‐oxidative stress effects. Here we investigated whether these properties of ZD can delay the retinal degeneration in rd10 mice, a model of RP, and explored its underlying mechanism. One shot of ZD or control vehicle was intravitreally injected into rd10 mice on postnatal day 16 (P16). Retinal function and structure of rd10 mice were assessed at P25, when rods degenerate substantially, using a visual behavior test, multi‐electrode‐array recordings and immunostaining. Retinal pathogenic gene expression and regulation of signaling pathways by ZD were explored using transcriptome sequencing and western blotting. Our results showed that ZD treatment improved the v...
Calcium enters the outer segment of a vertebrate photoreceptor through a cGMP-gated channel and i... more Calcium enters the outer segment of a vertebrate photoreceptor through a cGMP-gated channel and is extruded via a Na/Ca, K exchanger. We have identified another element in mammalian cones that might help to control cytoplasmic calcium. Reverse transcription-PCR performed on isolated photoreceptors identified mRNA for the SII Ϫ splice variant of the type I receptor for inositol 1,4,5-triphosphate (IP 3), and Western blots showed that the protein also is expressed in outer segments. Immunocytochemistry showed type I IP 3 receptor to be abundant in red-sensitive and green-sensitive cones of the trichromatic monkey retina, but it was negative or weakly expressed in blue-sensitive cones and rods. Similarly, the green-sensitive cones expressed the receptor in dichromatic retina (cat, rabbit, and rat), but the blue-sensitive cones did not. Immunostain was localized to disk and plasma membranes on the cytoplasmic face. To restore sensitivity after a light flash, cytoplasmic cGMP must rise to its basal level, and this requires cytoplasmic calcium to fall. Cessation of calcium release via the IP 3 receptor might accelerate this fall and thus explain why the cone recovers much faster than the rod. Furthermore, because its own activity of the IP 3 receptor depends partly on cytoplasmic calcium, the receptor might control the set point of cytoplasmic calcium and thus affect cone sensitivity.
Investigative Ophthalmology & Visual Science, Mar 10, 2014
PURPOSE. L-type voltage gated calcium channels in retina localize primarily at the presynaptic ac... more PURPOSE. L-type voltage gated calcium channels in retina localize primarily at the presynaptic active zones of photoreceptors and bipolar cells where they modulate glutamate release. However, the pore forming subunit Cacna1s of certain L-type channels is also expressed postsynaptically at the tips of ON bipolar cell dendrites where it colocalizes with mGluR6, but has an unknown function. At these dendritic tips, the components of the mGluR6 signaling cascade cluster together in a macromolecular complex, and each one's localization often depends on that of the others. Thus, we explored if Cacna1s is part of the mGluR6 complex. METHODS. We determined Cacna1s expression by PCR using an ON bipolar library, by Western blotting, and by standard immunohistochemistry. RESULTS. The PCR amplification confirmed expression of the transcript in ON bipolar cells, and Western blotting showed the expected bands. Immunostaining for Cacna1s was stronger in the dendritic tips of rod bipolar cells than in those of ON cone bipolar cells. This staining severely decreased in mice missing various mGluR6 cascade elements (Grm6 À/À , Gnao1 À/À , Gnb3 À/À , Gng13 À/À , and Trpm1 À/À). During development, the ratio of the number of Cacna1s puncta to the number of presynaptic ribbons followed a sigmoidal pattern, rising rapidly from P13 to P17. The mGluR6 expression preceded that of Cacna1s and RGS11. CONCLUSIONS. Our results show that the localization and stability of Cacna1s depend on the expression of mGluR6 and its cascade components, and they suggest that Cacna1s is part of the mGluR6 complex. We hypothesize that Cacna1s contributes to light adaptation by permeating calcium.
PCP2, a member of the GoLoco domain-containing family, is present exclusively in cerebellar Purki... more PCP2, a member of the GoLoco domain-containing family, is present exclusively in cerebellar Purkinje cells and retinal ON bipolar cells. Its function in these tissues is unknown. Biochemical and expression system studies suggest that PCP2 is a guanine nucleotide dissociation inhibitor, although a guanine nucleotide exchange factor has also been suggested. Here, we studied the function of PCP2 in ON bipolar cells because their light response depends on G␣ o1 , which is known to interact with PCP2. We identified a new splice variant of PCP2 (Ret-PCP2) and localized it to rod bipolar and ON cone bipolar cells. Electroretinogram recordings from PCP2-null mice showed a normal a-wave but a slower falling phase of the b-wave (generated by the activity of ON bipolar cells) relative to the wild type. Whole-cell recordings from rod bipolar cells showed, both under Ames medium and after blocking GABA A/C and glycine receptors, that PCP2-null rod bipolar cells were more depolarized than wild-type cells with greater inward current when clamped to Ϫ60 mV. Also under both conditions, the rise time of the response to intense light was slower by 28% (Ames) and 44% (inhibitory blockers) in the null cells. Under Ames medium, we also observed Ͼ30% longer decay time in the PCP2-null rod bipolar cells. We conclude that PCP2 facilitates cation channels closure in the dark, shortens the rise time of the light response directly, and accelerates the decay time indirectly via the inhibitory network. These data can most easily be explained if PCP2 serves as a guanine nucleotide exchange factor.
Retinal ganglion cells comprise about 10 morphological types that also differ functionally. To de... more Retinal ganglion cells comprise about 10 morphological types that also differ functionally. To determine whether functional differences might arise partially from differences in excitatory input, we quantified the distributions of ribbon contacts to four mammalian ganglion cell types [brisk-transient (BT), brisk-sustained (BS), local edge (LE), directionally selective (DS)], comparing small vs. large and "sluggish" vs. "brisk." Cells in guinea pig retina were filled with fluorescent dye, immunostained for synaptic ribbons, and reconstructed with their ribbon contacts by confocal microscopy. False-positive contacts were corrected by performing the same analysis on processes that lack synapses: glial stalks and rod bipolar axons. All types shared a domed distribution of membrane that was well fit by a Gaussian function (R 2 ϭ 0.96 Ϯ 0.01); they also shared a constant density of contacts on the dendritic membrane, both across each arbor and across cell types (19 Ϯ 1 contacts/100 m 2 membrane). However, the distributions of membrane across the retina differed markedly in width (BT Ͼ DS Ϸ BS Ͼ LE) and peak density (BS Ͼ DS Ͼ LE Ͼ BT). Correspondingly, types differed in peak density of contacts (BS Ͼ DS Ϸ LE Ͼ BT) and total number (BS Ϸ BT Ͼ DS Ͼ LE). These differences between cell types in spatial extent and local concentration of membrane and synapses help to explain certain functional differences.
The distribution of GABAA receptor in the outer plexiform layer of cat retina was studied by immu... more The distribution of GABAA receptor in the outer plexiform layer of cat retina was studied by immunocytochemistry with monoclonal antibodies. Staining was observed at the base of the cone pedicle, extracellularly, in association with the “triad” synaptic complex. Some bipolar dendrites and the basal processes that interconnect the cone pedicles were also stained. Rod spherules and horizontal cells were negative. The findings support the idea that the cone horizontal cells are GABAergic.
Synaptic transmission from photoreceptors to depolarizing bipolar cells is mediated by the APB gl... more Synaptic transmission from photoreceptors to depolarizing bipolar cells is mediated by the APB glutamate receptor. This receptor apparently is coupled to a G-protein which activates cGMP-phosphodiesterase to modulate cGMP levels and thus a cGMP-gated cation channel. We attempted to localize this system immunocytochemically using antibodies to various components of the rod phototransduction cascade, including G, (transducin), phosphodiesterase, the cGMP-gated channel, and arrestin. All of these antibodies reacted strongly with rods, but none reacted with bipolar cells. Antibodies to a different G-protein, G o , reacted strongly with rod bipolar cells of three mammalian species (which are depolarizing and APB-sensitive). Also stained were subpopulations of cone bipolar cells but not the major depolarizing type in cat (b^. G o antibody also stained certain salamander bipolar cells. Thus, across a wide range of species, G o is present in retinal bipolar cells, and at least some of these are depolarizing and APB-sensitive.
The neurotransmitter used by horizontal cells in mammals has not been identified. GABA has been t... more The neurotransmitter used by horizontal cells in mammals has not been identified. GABA has been the leading candidate, but doubt has remained because of failure to clearly demonstrate the GABA synthetic enzyme, glutamic acid decarboxylase (GAD) in these cells. Because GAD was recently shown to exist as two isoforms, 65 kDa and 67 kDa, we considered whether there might be a mismatch between the forms of GAD expressed in horizontal cells and the probes used to detect it. Accordingly, we stained sections of mammalian retina with antibodies specific for each isoform. Cat horizontal cells of both types (A and B) were immunoreactive for GAD 67 but negative for GAD 65 ; monkey horizontal cells of both types (H, and H n) were positive for GAD 65 and negative for GAD 67. The findings reconcile previous, apparently conflicting, observations and strengthen considerably the hypothesis that mammalian horizontal cells are GABAergic.
The subcellular distribution of GABAA receptor in the macaque and human retina was studied by imm... more The subcellular distribution of GABAA receptor in the macaque and human retina was studied by immunocytochemistry with monoclonal antibodies for the • and ~O subunits with a particular focus on bipolar cells. Immunoreactivity to GABAA receptor was present on dendritic tips of all bipolar cells. The stain was strongest on bipolar membranes in apposition to horizontal cell processes. Stain was concentrated on the tips of flat and invaginating cone bipolar cells at the base of the cone pedicle and on the invaginating tips of rod bipolar cells. Stain on the cone pedicle membrane was restricted to sites of apposition to stained bipolar dendrites; pedicle membrane in apposition to horizontal cell processes was unstained. Stain was also present on bipolar axon terminals in both on and off strata of the inner plexiform layer. All bipolar cell somas stained faintly; horizontal and Miiller cell somas were unstained. The ~ and/I subunits distributed similarly in monkey and human retina. Presence of GABA A receptor on the bipolar dendritic tips suggests that horizontal cells directly affect bipolar cells. Thus, GABAA receptor might mediate the receptive field surround of both off and on bipolar cells. Presence of GABAA receptor on bipolar axon terminals suggests that much of the inhibition feeding back from GABAergic amacrine to bipolar cells is GABAA-mediated. GABA Cone pedicle Horizontal cells Interplexiform cell Feedforward inhibition GABA depolarization
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