Protein kinase C gamma associates directly with the GluR4 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit. Effect on receptor phosphorylation.
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Ionotropic glutamate receptors mediate the majority of excitatory synaptic transmission in the brain and are thought to be involved in learning and memory formation. The activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors can be regulated by direct phosphorylation of their subunits, which affects the electrophysiological properties of the receptor, and the receptor association with numerous proteins that modulate membrane traffic and synaptic targeting of the receptor. In the present study we investigated the association of protein kinase C (PKC) gamma isoform with the GluR4 AMPA receptor subunit. PKC gamma was co-immunoprecipitated with GluR4 AMPA receptor subunit in rat cerebellum and in cultured chick retina cell extracts, and immunocytochemistry experiments showed co-localization of GluR4 and PKC gamma in cultured chick retinal neurons. Pull-down assays showed that native PKC gamma binds the GluR4 C-terminal membrane-proximal region, and recombinant PKC gamma was retained by GST-GluR4 C-terminal fusion protein, suggesting that the kinase binds directly to GluR4. Furthermore, GST-GluR4 C-terminal protein was phosphorylated on GluR4 Ser-482 by bound kinases, retained by the fusion protein, including PKC gamma. The GluR4 C-terminal segment that interacts with PKC gamma, which lacks the PKC phosphorylation sites, inhibited histone H1 phosphorylation by PKC, to the same extent as the PKC pseudosubstrate peptide 19-31, indicating that PKC gamma bound to GluR4 preferentially phosphorylates GluR4 to the detriment of other substrates. Additionally, PKC gamma expression in GluR4 transfected human embryonic kidney 293T cells increased the amount of plasma membrane-associated GluR4. Our results suggest that PKC gamma binds directly to GluR4, thereby modulating the function of GluR4-containing AMPA receptors. (+info)
Starburst cells nondirectionally facilitate the responses of direction-selective retinal ganglion cells.
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The mechanism of direction selectivity in retinal ganglion cells remains controversial. An important issue is how the starburst amacrine cells, which are known to provide a major synaptic input to the direction-selective ganglion cells, participate in the directional discrimination. Here, we present evidence that the cholinergic outputs of the starburst cells affect the responses of the ganglion cells symmetrically; they provide a feedforward excitation that facilitates the response of the ganglion cells to movement in both the preferred and null directions. This seems to place a constraint on models of the directional discrimination in which the starburst cells participate, namely, that their cholinergic synapses be nondirectional in their effects on the ganglion cells. (+info)
Electrical synapses mediate signal transmission in the rod pathway of the mammalian retina.
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In the retina, AII (rod) amacrine cells are essential for integrating rod signals into the cone pathway. In addition to being interconnected via homologous gap junctions, these cells make extensive heterologous gap junctions with ON-cone bipolar cells (BCs). These gap junctions are the pathway for transfer of rod signals to the ON-system. To investigate the functional properties of these gap junctions, we performed simultaneous whole-cell recordings from pairs of AII amacrine cells and ON-cone bipolar cells in the in vitro slice preparation of the rat retina. We demonstrate strong electrical coupling with symmetrical junction conductance (approximately 1.2 nS) and very low steady-state voltage sensitivity. However, signal transmission is more effective in the direction from AII amacrine cells to ON-cone bipolar cells than in the other direction. This functional rectification can be explained by a corresponding difference in membrane input resistance between the two cell types. Signal transmission has low-pass filter characteristics with increasing attenuation and phase shift for increasing stimulus frequency. Action potentials in AII amacrine cells evoke distinct electrical postsynaptic potentials in ON-cone bipolar cells. Strong and temporally precise synchronization of subthreshold membrane potential fluctuations are commonly observed. (+info)
Confocal analysis of reciprocal feedback at rod bipolar terminals in the rabbit retina.
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Amacrine cells in the mammalian retina are famously diverse in shape and function. Here, we show that two wide-field GABA amacrine cells, S1 and S2, have stereotyped synaptic contacts with the appropriate morphology and distribution to perform specific functions. S1 and S2 both supply negative feedback to rod bipolar terminals and thus provide a substrate for lateral inhibition in the rod pathway. Synapses are specialized structures, and the presynaptic compartment is normally characterized by a swelling or varicosity. Each S1 amacrine cell has approximately 280 varicosities, whereas an S2 cell has even more, approximately 500 per cell. Confocal analysis shows that essentially all varicosities aggregate around rod bipolar terminals where they are apposed by postsynaptic GABA receptors. Each rod bipolar terminal is contacted by varicosities from approximately 25 different S1 and 50 different S2 amacrine cells. In fact, rod bipolar cells are the only synaptic target for S1 and S2 amacrine cells: all of the output from these two wide-field GABA amacrine cells goes to rod bipolar terminals. It has long been a puzzle why two amacrine cells, apparently with the same connections, are required. However, an analysis of the distribution of varicosities suggests that S1 and S2 amacrine cells provide different signals. S2 amacrine cells dominate within 200 mu from a rod bipolar terminal and can provide an inhibitory input with spatial characteristics that match the size of the surround signal recorded from AII amacrine cells in the rod pathway. In contrast, the larger, better-coupled S1 amacrine cells may provide a more distant network signal. (+info)
Cholecystokinin-like immunoreactive amacrine cells in the rat retina.
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High levels of endogenous cholecystokinin (CCK) are present in the rat retina (Eskay & Beinfeld, 1982), but the cellular localization and physiological actions of CCK in the rat retina are uncertain. The goals of this study were to characterize the cells containing CCK, identify cell types that interact with CCK cells, and investigate the effects of CCK on rod bipolar cells. Rat retinas were labeled with antibody to gastrin-CCK (gCCK) using standard immunofluorescence techniques. Patch-clamp methods were used to record from dissociated rod bipolar cells from rats and mice. Gastrin-CCK immunoreactive (-IR) axons were evenly distributed throughout the retina in stratum 5 of the inner plexiform layer of the rat retina. However, the gCCK-IR somata were only detected in the ganglion cell layer in the peripheral retina. The gCCK-IR cells contained glutamate decarboxylase, and some of them also contained immunoreactive substance P. Labeled axons contacted PKC-IR rod bipolar cells, and recoverin-IR ON-cone bipolar cells. CCK-octapeptide inhibits GABA(C) but not GABA(A) mediated currents in dissociated rod bipolar cells. (+info)
Determination of retinal cell fates is affected in the absence of extraocular striated muscles.
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Neural retinas of genetically modified mouse embryos and fetuses entirely lacking extraocular striated muscles (designated as Myf5-/-:MyoD-/- or amyogenic) are used to study in vivo the role of extraocular muscle (i.e., fetal ocular movements) in the genesis of retinal cell diversity. Although retinal lamination and the total number of cells per retinal layer appeared unaffected in amyogenic fetuses, electron microscopy and histochemistry revealed the absence of cholinergic amacrine cell type. By contrast, the amounts of other amacrine cell subpopulations (calretinin-, tyrosine hydroxylase-, and parvalbumin-expressing) were increased, whereas the amounts of Islet1/2-expressing retinal ganglion cells were decreased. Surprisingly, it was not possible to detect any change in proliferation or cell death. Consistently, the number of progenitors for retinal ganglion cells (nestin-expressing precursors) were increased, whereas the amounts of precursors for amacrine cells (syntaxin- and VC1.1-expressing precursors) were decreased in the mutant retinas. The difference in requirements for extraocular muscle support in regulation of precise ratios of retinal neuronal cell types suggests an essential role of extrinsic cues in the determination of retinal cell fates. Taken together, it appears that patterning mechanisms intrinsic to the neural retina specify the basic organization of retinal spatial organization (e.g., retinal layers and total number of cells). However, extrinsic cues seem to change intrinsic properties (e.g., competence) of retinal progenitor cells and influence the ratios of the differentiated cell types (i.e., cell fate choice) they produce. (+info)
Particular types of amacrine cells of the vertebrate retina show oscillatory membrane potentials (OMPs) in response to light stimulation. Historically it has been thought the oscillations arose as a result of circuit properties. In a previous study we found that in some amacrine cells, the ability to oscillate was an intrinsic property of the cell. Here we characterized the ionic mechanisms responsible for the oscillations in wide-field amacrine cells (WFACs) in an effort to better understand the functional properties of the cell. The OMPs were found to be calcium (Ca2+) dependent; blocking voltage-gated Ca2+ channels eliminated the oscillations, whereas elevating extracellular Ca2+ enhanced them. Strong intracellular Ca2+ buffering (10 mM EGTA or bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid) eliminated any attenuation in the OMPs as well as a Ca2+-dependent inactivation of the voltage-gated Ca2+ channels. Pharmacological and immunohistochemical characterization revealed that WFACs express L- and N-type voltage-sensitive Ca2+ channels. Block of the L-type channels eliminated the OMPs, but omega-conotoxin GVIA did not, suggesting a different function for the N-type channels. The L-type channels in WFACs are functionally coupled to a set of calcium-dependent potassium (K(Ca)) channels to mediate OMPs. The initiation of OMPs depended on penitrem-A-sensitive (BK) K(Ca) channels, whereas their duration is under apamin-sensitive (SK) K(Ca) channel control. The Ca2+ current is essential to evoke the OMPs and triggering the K(Ca) currents, which here act as resonant currents, enhances the resonance as an amplifying current, influences the filtering characteristics of the cell membrane, and attenuates the OMPs via CDI of the L-type Ca2+ channel. (+info)
Differential expression and distribution of Kir5.1 and Kir4.1 inwardly rectifying K+ channels in retina.
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Kir5.1 is an inwardly rectifying K+ channel subunit whose functional role has not been fully elucidated. Expression and distribution of Kir5.1 in retina were examined with a specific polyclonal antibody. Kir5.1 immunoreactivity was detected in glial Muller cells and in some retinal neurons. In the Kir5.1-positive neurons the expression of glutamic acid decarboxylase (GAD65) was detected, suggesting that they may be GABAergic-amacrine cells. In Muller cells, spots of Kir5.1 immunoreactivity distributed diffusely at the cell body and in the distal portions, where Kir4.1 immunoreactivity largely overlapped. In addition, Kir4.1 immunoreactivity without Kir5.1 was strongly concentrated at the endfoot of Muller cells facing the vitreous surface or in the processes surrounding vessels. The immunoprecipitant obtained from retina with anti-Kir4.1 antibody contained Kir5.1. These results suggest that heterotetrameric Kir4.1/Kir5.1 channels may exist in the cell body and distal portion of Muller cells, whereas homomeric Kir4.1 channels are clustered in the endfeet and surrounding vessels. It is possible that homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels play different functional roles in the K+-buffering action of Muller cells. (+info)