RESUMO
Transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of a cell-specific promoter have been used with great success to identify and label specific cell types of the retina. We studied the expression of EGFP in the retina of mice making use of four transgenic mouse lines. Expression of EGFP driven by the calretinin promoter was found in amacrine, displaced amacrine and ganglion cells. Comparison of the EGFP expression and calretinin immunolabeling showed that many but not all cells appear to be double labeled. Expression of EGFP under the control of the choline acetyltransferase promoter was found in amacrine cells; however, the cells did not correspond to the well known cholinergic (starburst) cells of the mouse retina. The expression of EGFP under the control of the parvalbumin promoter was restricted to amacrine cells of the inner nuclear layer and to cells of the ganglion cell layer (displaced amacrine cells and ganglion cells). Most of the cells were also immunoreactive for parvalbumin, however, differences in labeling intensity were observed. The expression of EGFP driven by the promoter for the 5-HT3 A receptor (5-HTR3A) was restricted to type 5 bipolar cells. In contrast, immunostaining for 5-HTR3A was found in synaptic hot spots in sublamina 1 of the inner plexiform layer and was not related to type 5 bipolar cells. The results show that these transgenic mice are very useful for future electrophysiological studies of specific types of amacrine and bipolar cells that express EGFP and thus permit directed microelectrode targeting under microscopic control.
Assuntos
Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Neurônios Retinianos/metabolismo , Acetilcolina/metabolismo , Células Amácrinas/metabolismo , Animais , Calbindina 2 , Colina O-Acetiltransferase/genética , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Parvalbuminas/genética , Regiões Promotoras Genéticas , Receptores 5-HT3 de Serotonina/genética , Células Bipolares da Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Proteína G de Ligação ao Cálcio S100/genética , Especificidade da EspécieRESUMO
Amacrine cells are known to express strychnine-sensitive glycine receptors (GlyRs), however, it is not known which of the four GlyRalpha subunits (alpha1-4) are expressed in this diverse group of cells. Herein, we studied the presence of glycine activated currents and spontaneous inhibitory postsynaptic currents (sIPSCs) of amacrine cells in the mouse retina. By recording glycinergic currents in retinal slices of wildtype mice and of mice deficient in GlyRalpha subunits (Glra1spd-ot, Glra2-/-, Glra3-/-), we could classify AII and narrow-field amacrine cells (NF, Types 5, 6, 7) on the basis of their alpha-subunit composition. Glycinergic sIPSCs of AII cells displayed medium fast kinetics (mean decay time constant tau=11+/-2 ms), which were completely absent in the Glra3-/- mouse, indicating that synaptic GlyRs of AII cells mainly contain the alpha3 subunit. Glycinergic sIPSCs of NF cells had slow kinetics (tau=27+/-6.8 ms) that were significantly prolonged in Glra2-/- mice (tau=69+/-16 ms). These data show that morphologically distinct amacrine cells express different sets of GlyRs.
Assuntos
Células Amácrinas/fisiologia , Glicina/fisiologia , Receptores de Glicina/deficiência , Retina/citologia , Células Amácrinas/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Interações Medicamentosas , Estimulação Elétrica/métodos , Antagonistas GABAérgicos/farmacologia , Glicina/farmacologia , Glicinérgicos/farmacologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Potenciais Pós-Sinápticos Inibidores/efeitos da radiação , Modelos Lineares , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Potenciais da Membrana/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Patch-Clamp/métodos , Estricnina/farmacologiaRESUMO
Amacrine cells are a heterogeneous class of interneurons that modulate the transfer of the light signals through the retina. In addition to ionotropic glutamate receptors, amacrine cells express two types of inhibitory receptors, GABA(A) receptors (GABA(A)Rs) and glycine receptors (GlyRs). To characterize the functional contribution of these different receptors, spontaneous postsynaptic currents (sPSCs) were recorded with the whole cell configuration of the patch-clamp technique in acutely isolated slices of the adult mouse retina. All amacrine cells investigated (n = 47) showed spontaneous synaptic activity. In six amacrine cells, spontaneous excitatory postsynaptic currents could be identified by their sensitivity to kynurenic acid. They were characterized by small amplitudes [mean: -13.7 +/- 1.5 (SE) pA] and rapid decay kinetics (mean tau: 1.35 +/- 0.16 ms). In contrast, the reversal potential of sPSCs characterized by slow decay kinetics (amplitude-weighted time constant, tau(w), >4 ms) was dependent on the intracellular Cl(-) concentration (n = 7), indicating that they were spontaneous inhibitory postsynaptic currents (sIPSCs). In 14 of 34 amacrine cells sIPSCs were blocked by bicuculline (10 microM), indicating that they were mediated by GABA(A)Rs. Only four amacrine cells showed glycinergic sIPSCs that were inhibited by strychnine (1 microM). In one amacrine cell, sIPSCs mediated by GABA(A)Rs and GlyRs were found simultaneously. GABAergic sIPSCs could be subdivided into one group best fit by a monoexponential decay function and another biexponentially decaying group. The mean amplitude of GABAergic sIPSCs (-42.1 +/- 5.8 pA) was not significantly different from that of glycinergic sIPSCs (-28.0 +/- 8.5 pA). However, GlyRs (mean T10/90: 2.4 +/- 0.08 ms) activated significantly slower than GABA(A)Rs (mean T10/90: 1.2 +/- 0.03 ms). In addition, the decay kinetics of monoexponentially decaying GABA(A)Rs (mean tau(w): 20.3 +/- 0.50), biexponentially decaying GABA(A)Rs (mean tau(w): 30.7 +/- 0.95), and GlyRs (mean tau(w) = 25.3 +/- 1.94) were significantly different. These differences in the activation and decay kinetics of sIPSCs indicate that amacrine cells of the mouse retina express at least three types of functionally different inhibitory receptors: GlyRs and possibly two subtypes of GABA(A)Rs.
Assuntos
Células Amácrinas/fisiologia , Sinapses/fisiologia , Animais , Bicuculina/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Antagonistas GABAérgicos/farmacologia , Glicina/fisiologia , Glicinérgicos/farmacologia , Técnicas In Vitro , Camundongos , Inibição Neural/fisiologia , Técnicas de Patch-Clamp , Estricnina/farmacologia , Ácido gama-Aminobutírico/fisiologiaRESUMO
Rod bipolar (RB) cells of the mammalian retina release glutamate in a graded, light-dependent fashion from 20 to 40 ribbon synapses (dyads). At the dyads, two classes of amacrine cells, the AI and AII cells, are the postsynaptic partners. We examined the glutamate receptors (GluRs) that are expressed by AI and AII cells using immunocytochemistry with specific antibodies against GluR subunits. Sections of macaque monkey and rabbit retina were examined by confocal microscopy. AII amacrine cells were selectively labeled for calretinin, and AI cells in rabbits were labeled for 5-HT uptake. Thus, double- and triple-labeling for these markers and GluR subunits was possible. Electron microscopy using postembedding immunocytochemistry and double-labeling was applied to show the synaptic expression of GluRs. We also studied the synaptic localization of the two postsynaptic density proteins PSD-95 and glutamate receptor-interacting protein (GRIP). We found that AII amacrine cells express the AMPA receptor subunits GluR2/3 and GluR4 at the RB cell dyads, and they are clustered together with PSD-95. In contrast, AI amacrine cells express the delta1/2 subunits that appear to be associated with kainate receptor subunits and to be clustered together with GRIP. The RB cell dyad is therefore a synapse that initiates two functionally and molecularly distinct pathways: a "through conducting" pathway based on AMPA receptors and a modulatory pathway mediated by a combination of delta1/2 subunits and kainate receptors.
Assuntos
Receptores de Glutamato/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Calbindina 2 , Proteínas de Transporte/metabolismo , Imuno-Histoquímica , Macaca fascicularis , Microscopia Eletrônica , Proteínas do Tecido Nervoso/metabolismo , Subunidades Proteicas , Coelhos , Receptores de AMPA/metabolismo , Receptores de Ácido Caínico/metabolismo , Retina/citologia , Retina/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Proteína G de Ligação ao Cálcio S100/metabolismo , Serotonina/metabolismo , Serotonina/farmacocinéticaRESUMO
In the macaque monkey retina cone pedicles, the output synapses of cone photoreceptors, contain between 20 and 45 ribbon synapses (triads), which are the release sites for glutamate, the cone transmitter. Several hundred postsynaptic dendrites contact individual cone pedicles, and we studied the glutamate receptors expressed and clustered at these contacts, particularly the kainate receptor subunits GluR5, GluR6/7, and KA2. Pre- and postembedding immunocytochemistry and electron microscopy were used to localize GluR5 and GluR6/7 to specific synaptic contacts at the cone pedicle base. The GluR5 subunit was aggregated at bipolar cell flat contacts. The GluR6/7 subunit was aggregated at bipolar cell flat contacts and at the desmosome-like junctions formed by horizontal cell processes underneath the cone pedicles. KA2 immunoreactivity was observed at the invaginating dendritic tips of ON-cone and rod bipolar cells, which we interpret as a cross-reactivity of the KA2 antiserum with some other, unknown protein of the monkey retina. Kainate receptors are preferentially expressed by OFF-cone bipolar cells and to a lesser extent by horizontal cells. We also performed double-labeling experiments with the ribbon-specific marker bassoon and with antibodies against GluR5 and GluR6/7 in order to define the position of the flat bipolar cell contacts with respect to the triads. There was a tendency of GluR6/7 clusters to represent triad-associated contacts, whereas GluR5 clusters represented non-triad-associated contacts. The GluR5 and GluR6/7 subunits were clustered at different bipolar cell contacts. We studied a possible cone-selective expression of the kainate receptor subunits by double labeling cone pedicles for the S-cone opsin and for the different receptor subunits. We observed a reduced expression of both GluR5 and GluR6/7 at the S-cone pedicles. The reduced expression of GluR6/7 was analyzed in more detail and it appears to be a consequence of a horizontal cell-specific expression: H1 horizontal cells express GluR6/7, whereas H2 horizontal cells, which preferentially innervate S-cones, show no expression of GluR6/7.
Assuntos
Receptores de Ácido Caínico/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Animais , Dendritos/metabolismo , Dendritos/ultraestrutura , Imuno-Histoquímica , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Imunoeletrônica , Retina/ultraestrutura , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Sinapses/metabolismo , Sinapses/ultraestrutura , Receptor de GluK2 CainatoRESUMO
Retinal ganglion cells show direction selectivity in their responses to moving stimuli. The circuitry necessary to generate directional selectivity in these cells has been long debated. Yoshida et al. (2001) use immunotoxin-mediated cell ablation to demonstrate that the starburst amacrine cell is at the core of this computation.
Assuntos
Percepção de Movimento/fisiologia , Células Ganglionares da Retina/fisiologia , Visão Binocular/fisiologia , AnimaisRESUMO
The receptive field (RF) of retinal ganglion cells (RGCs) consists of an excitatory central region, the RF center, and an inhibitory peripheral region, the RF surround. It is still unknown in detail which inhibitory interneurons (horizontal or amacrine cells) and which inhibitory circuits (presynaptic or postsynaptic) generate the RF surround. To study surround inhibition, light-evoked whole-cell currents were recorded from RGCs of the isolated, intact rabbit retina. The RFs were stimulated with light or dark spots of increasing diameters and with annular light stimuli. Direct inhibitory currents could be isolated by voltage clamping ganglion cells close to the Na(+)/K(+) reversal potential. They mostly represent an input from GABAergic amacrine cells that contribute to the inhibitory surround of ganglion cells. This direct inhibitory input and its physiological function were also investigated by recording light-evoked action potentials of RGCs in the current-clamp mode and by changing the intracellular Cl(-) concentration. The excitatory input of the ganglion cells could be isolated by voltage clamping ganglion cells at the Cl(-) reversal potential. Large light spots and annular light stimuli caused a strong attenuation of the excitatory input. Both GABA(A) receptors and GABA(C) receptors contributed to this inhibition, and picrotoxinin was able to completely block it. Together, these results show that the RF surround of retinal ganglion cells is mediated by a combination of direct inhibitory synapses and presynaptic surround inhibition.
Assuntos
Inibição Neural/fisiologia , Retina/fisiologia , Células Ganglionares da Retina/metabolismo , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Potenciais de Ação/fisiologia , Animais , Cloretos/metabolismo , Dendritos/classificação , Antagonistas GABAérgicos/farmacologia , Antagonistas de Receptores de GABA-A , Técnicas In Vitro , Luz , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Inibição Neural/efeitos dos fármacos , Técnicas de Patch-Clamp , Estimulação Luminosa/métodos , Picrotoxina/análogos & derivados , Picrotoxina/farmacologia , Potássio/metabolismo , Coelhos , Receptores de GABA/efeitos dos fármacos , Receptores de GABA/metabolismo , Receptores de GABA-A/metabolismo , Receptores de Glicina/antagonistas & inibidores , Receptores de Glicina/metabolismo , Retina/citologia , Retina/efeitos dos fármacos , Células Ganglionares da Retina/classificação , Células Ganglionares da Retina/efeitos dos fármacos , Sesterterpenos , Sódio/metabolismo , Tetrodotoxina/farmacologiaRESUMO
The expression of GABA receptors (GABARs) was studied in frog and turtle retinae. Using immunocytochemical methods, GABA(A)Rs and GABA(C)Rs were preferentially localized to the inner plexiform layer (IPL). Label in the IPL was punctate indicating a synaptic clustering of GABARs. Distinct, but weaker label was also present in the outer plexiform layer. GABA(A)R and GABA(C)R mediated effects were studied by recording electroretinograms (ERGs) and by the application of specific antagonists. Bicuculline, the GABA(A)R antagonist, produced a significant increase of the ERG. Picrotoxin, when co-applied with saturating doses of bicuculline, caused a further increase of the ERG due to blocking of GABA(C)Rs. The putative GABA(C)R antagonist Imidazole-4-acidic acid (I4AA) failed to antagonize GABA(C)R mediated inhibition and, in contrast, appeared rather as an agonist of GABARs.
Assuntos
Rana ridibunda/fisiologia , Receptores de GABA/fisiologia , Retina/fisiologia , Tartarugas/fisiologia , Animais , Bicuculina/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Eletrofisiologia , Eletrorretinografia/efeitos dos fármacos , Agonistas GABAérgicos/farmacologia , Antagonistas GABAérgicos/farmacologia , Imidazóis/farmacologia , Imuno-Histoquímica , Picrotoxina/farmacologiaRESUMO
Cone pedicles, the output synapses of cone photoreceptors, transfer the light signal onto the dendrites of bipolar and horizontal cells. Cone pedicles contain between 20 and 45 ribbon synapses (triads) which are the release sites for glutamate, the cone transmitter. Several hundred postsynaptic dendrites contact individual cone pedicles, and we studied the glutamate receptors expressed and clustered at these contacts, particularly the AMPA receptor subunits. Using immunocytochemistry and confocal imaging we were able to resolve individual triads within the cone pedicles by light microscopy. We studied their differences in L/M- and S-cones, and we counted the number of triads per pedicle across the retina. The presynaptic matrix protein bassoon, the synapse-associated membrane protein P84, and peanut agglutinin were used to specifically label synaptic ribbons, invaginating dendrites of horizontal cells and invaginating dendrites of ON-cone bipolar cells, respectively. Pre- and post-embedding immunocytochemistry and electron microscopy were used to localize the AMPA receptor subunits at the cone pedicle base. They were aggregated at three different postsynaptic sites: at horizontal cell invaginating contacts, at bipolar cell flat contacts, and at desmosome-like junctions underneath the cone pedicles. We also performed double-labeling experiments with the triad-specific markers and the antibodies against the AMPA receptor subunits. AMPA receptors were preferentially expressed by horizontal cells, and to a lesser extent by OFF-cone bipolar cells. We did not observe any cone-selective expression of AMPA receptor subunits postsynaptic to L/M- or S-cones, suggesting AMPA receptors are not the key to understanding trichromatic signaling in the primate retina.
Assuntos
Macaca/metabolismo , Receptores de AMPA/fisiologia , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Sinapses/metabolismo , Animais , Dendritos/metabolismo , Dendritos/fisiologia , Imuno-Histoquímica , Macaca/fisiologia , Microscopia Confocal , Primatas/metabolismo , Primatas/fisiologia , Receptores de AMPA/metabolismo , Retina/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Sinapses/fisiologiaRESUMO
Microtubule-associated proteins such as MAP1B have long been suspected to play an important role in neuronal differentiation, but proof has been lacking. Previous MAP1B gene targeting studies yielded contradictory and inconclusive results and did not reveal MAP1B function. In contrast to two earlier efforts, we now describe generation of a complete MAP1B null allele. Mice heterozygous for this MAP1B deletion were not affected. Homozygous mutants were viable but displayed a striking developmental defect in the brain, the selective absence of the corpus callosum, and the concomitant formation of myelinated fiber bundles consisting of misguided cortical axons. In addition, peripheral nerves of MAP1B-deficient mice had a reduced number of large myelinated axons. The myelin sheaths of the remaining axons were of reduced thickness, resulting in a decrease of nerve conduction velocity in the adult sciatic nerve. On the other hand, the anticipated involvement of MAP1B in retinal development and gamma-aminobutyric acid C receptor clustering was not substantiated. Our results demonstrate an essential role of MAP1B in development and function of the nervous system and resolve a previous controversy over its importance.
Assuntos
Encéfalo/patologia , Comunicação Celular/fisiologia , Corpo Caloso/patologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Sistema Nervoso Periférico/patologia , Animais , Axônios/fisiologia , Encéfalo/embriologia , Corpo Caloso/embriologia , Genes Letais , Heterozigoto , Homozigoto , Camundongos , Camundongos Mutantes , Bainha de Mielina/patologia , Sistema Nervoso Periférico/embriologia , Receptores de GABA/isolamento & purificação , Retina/embriologia , Deleção de SequênciaRESUMO
Clustering of neurotransmitter receptors in postsynaptic densities involves proteins that aggregate the receptors and link them to the cytoskeleton. In the case of glycine and GABA(A) receptors, gephyrin has been shown to serve this function. However, it is unknown whether gephyrin is involved in the clustering of all glycine and GABA(A) receptors or whether it interacts only with specific isoforms. This was studied in the retinae of mice, whose gephyrin gene was disrupted, with immunocytochemistry and antibodies that recognize specific subunits of glycine and GABA(A) receptors. Because homozygous (geph -/-) mutants die around birth, an organotypic culture system of the mouse retina was established to study the clustering of gephyrin and the receptors in vitro. We found that all gephyrin and all glycine receptor clusters (hot spots) were abolished in the geph (-/-) mouse retina. In the case of GABA(A) receptors, there was a significant reduction of clusters incorporating the gamma2, alpha2, and alpha3 subunits; however, a substantial number of hot spots was still present in geph (-/-) mutant retinae. This shows that gephyrin interacts with all glycine receptor isoforms but with only certain forms of GABA(A) receptors. In heterozygous geph (+/-) mutants, no reduction of hot spots was observed in the retina in vivo, but a significant reduction was found in the organotypic cultures. This suggests that mechanisms may exist in vivo that allow for the compensation of a partial gephyrin deficit.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Receptores de GABA/metabolismo , Receptores de Glicina/metabolismo , Retina/metabolismo , Sinapses/metabolismo , Animais , Proteínas de Transporte/genética , Feminino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Técnicas de Cultura de Órgãos , Gravidez , Receptores de GABA/genética , Receptores de Glicina/genética , Sinapses/genéticaRESUMO
To further characterize the H1 and H2 horizontal cell populations in macaque monkey retinae, cells were injected with the tracer Neurobiotin following intracellular recordings. Tracer coupling between cells of the same type revealed all H1 or H2 cells in small patches around the injected cell. The mosaics of their cell bodies and the tiling of the retina with their dendrites were analyzed. Morphological differences between the H1 and H2 cells observable in Neurobiotin-labeled patches made it possible to recognize H1 and H2 cells in retinae immunolabeled for the calcium-binding proteins parvalbumin and calbindin, and thus to study their relative spatial densities across the retina. These data, together with the intracellularly stained patches, show that H1 cells outnumber H2 cells at all eccentricities. There is, however, a change in the relative proportions of H1 and H2 cells with eccentricity: close to the fovea the ratio of H1 to H2 cells is approximately 4 to 1, in midperipheral retina approximately 3 to 1, and in peripheral retina approximately 2 to 1. In both the Neurobiotin-stained and the immunostained retinae, about 3-5% of the H2 cells were obviously misplaced into the ganglion cell layer. Several features of the morphology of the misplaced H2 cells suggest that they represent the so-called "biplexiform ganglion cells" previously described in Golgi studies of primate retina.
Assuntos
Biotina/análogos & derivados , Interneurônios/citologia , Retina/citologia , Células Ganglionares da Retina/citologia , Animais , Biotina/metabolismo , Calbindinas , Contagem de Células , Dendritos/metabolismo , Proteínas do Olho/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Técnicas Imunoenzimáticas , Interneurônios/metabolismo , Macaca fascicularis , Macaca mulatta , Macaca nemestrina , Parvalbuminas/metabolismo , Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismoRESUMO
Cone pedicles, the synaptic terminals of cone photoreceptors, are connected in the macaque monkey retina to several hundred postsynaptic dendrites. Using light and electron microscopy, we found underneath each cone pedicle a laminated distribution of dendritic processes of bipolar and horizontal cells. Superimposed were three strata of glutamate receptor (GluR) aggregates, including a novel layer of glutamate receptors clustered at desmosome-like junctions. They are, most likely, postsynaptic densities on horizontal cell dendrites. GABA(A) and GABA(C) receptors are aggregated on bipolar cell dendrites in a narrow band underneath the cone pedicle. Glutamate released from cone pedicles and GABA released from horizontal cell dendrites act not only through direct synaptic contacts but also (more so) through diffusion to the appropriate receptors.
Assuntos
Receptores de Glutamato/fisiologia , Retina/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Sinapses/fisiologia , Animais , Dendritos/fisiologia , Dendritos/ultraestrutura , Desmossomos/fisiologia , Eletrofisiologia , Imuno-Histoquímica , Técnicas In Vitro , Junções Intercelulares/fisiologia , Macaca fascicularis , Microscopia Confocal , Microscopia Eletrônica , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Receptores de GABA/metabolismo , Retina/ultraestrutura , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Sinapses/ultraestrutura , Ácido gama-Aminobutírico/metabolismoRESUMO
gamma-Aminobutyric acid type A receptors (GABA(A)Rs) are ligand-gated chloride channels that exist in numerous distinct subunit combinations. At postsynaptic membrane specializations, different GABA(A)R isoforms colocalize with the tubulin-binding protein gephyrin. However, direct interactions of GABA(A)R subunits with gephyrin have not been reported. Recently, the GABA(A)R-associated protein GABARAP was found to bind to the gamma2 subunit of GABA(A)Rs. Here we show that GABARAP interacts with gephyrin in both biochemical assays and transfected cells. Confocal analysis of neurons derived from wild-type and gephyrin-knockout mice revealed that GABARAP is highly enriched in intracellular compartments, but not at gephyrin-positive postsynaptic membrane specializations. Our data indicate that GABARAP-gephyrin interactions are not important for postsynaptic GABA(A)R anchoring but may be implicated in receptor sorting and/or targeting mechanisms. Consistent with this idea, a close homolog of GABARAP, p16, has been found to function as a late-acting intra-Golgi transport factor.
Assuntos
Proteínas de Transporte/metabolismo , Canais de Cloreto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Receptores de GABA-A/metabolismo , Sinapses/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Transporte Biológico , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Humanos , Invertebrados/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Neurônios/metabolismo , Células PC12 , Ratos , Retina/metabolismo , Homologia de Sequência de Aminoácidos , Frações SubcelularesRESUMO
Transgenic mice provide a new approach for studying the structure and function of the mammalian retina. In the past, the cellular organization of the mammalian retina was investigated preferentially in primates, cats, and rats but rarely in mice. In the current study, the authors applied 42 different immunocytochemical markers to sections of the mouse retina and studied their cellular and synaptic localization by using confocal microscopy. The markers applied were from three major groups: 1) antibodies against calcium-binding proteins, such as calbindin, parvalbumin, recoverin, or caldendrin; 2) antibodies that recognize specific transmitter systems, such as glycine, gamma-aminobutyric acid, or acetylcholine; and 3) antibodies that recognize transmitter receptors and show their aggregation at specific synapses. Only a few markers labeled only one cell type: Most antibodies recognized specific groups of neurons. These were analyzed in more detail in double-labeling experiments with different combinations of the antibodies. In light of their results, the authors offer a list of immunocytochemical markers that can be used to detect possible changes in the retinal organization of mutant mice.
Assuntos
Camundongos/metabolismo , Retina/metabolismo , Animais , Dendritos/ultraestrutura , Glicina/metabolismo , Imuno-Histoquímica , Camundongos Endogâmicos C57BL , Microscopia Confocal , Receptores de GABA/metabolismo , Receptores de Glutamato/metabolismo , Receptores de Glicina/metabolismo , Receptores de Neurotransmissores/metabolismo , Retina/citologia , Retina/ultraestrutura , Distribuição Tecidual , Ácido gama-Aminobutírico/metabolismoRESUMO
Cyclic nucleotide-gated channels (CNGC) are ligand-gated ion channels that open and close in response to changes in the intracellular concentration of the second messengers, 3;,5;-cyclic adenosine monophosphate and 3;,5;-cyclic guanosine monophosphate. Most notably, they transduce the chemical signal produced by the absorption of light in photoreceptors into a membrane potential change, which is then transmitted to the ascending visual pathway. CNGCs have also been implicated in the signal transduction of other neurons downstream of the photoreceptors, in particular the ON-bipolar cells, as well as in other areas of the central nervous system. We therefore undertook a search for additional cyclic nucleotide-gated channels expressed in the retina. Following a degenerate reverse transcription polymerase chain reaction approach to amplify low-copy number messages, a cDNA encoding a new splice variant of CNGC alpha-subunit was isolated from mouse retina and classified as mCNG3. An antiserum raised against the carboxy-terminal sequence identified the retinal cell type expressing mCNG3 as cone photoreceptors. Preembedding immunoelectron microscopy demonstrated its membrane localization in the outer segments, consistent with its role in phototransduction. Double-labeling experiments with cone-specific markers indicated that all cone photoreceptors in the murid retina use the same or a highly conserved cyclic nucleotide-gated channel. Therefore, defects in this channel would be predicted to severely impair photopic vision.
Assuntos
Canais Iônicos/fisiologia , Retina/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Sequência de Aminoácidos , Animais , Western Blotting , Clonagem Molecular , Canais de Cátion Regulados por Nucleotídeos Cíclicos , DNA Complementar/genética , DNA Complementar/fisiologia , Imuno-Histoquímica , Canais Iônicos/biossíntese , Canais Iônicos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Dados de Sequência Molecular , Retina/citologia , Retina/ultraestrutura , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Segmento Externo da Célula Bastonete/metabolismo , Segmento Externo da Célula Bastonete/ultraestrutura , Sistemas do Segundo Mensageiro/fisiologia , Especificidade da EspécieRESUMO
The distribution and synaptic clustering of N-methyl-D-aspartate (NMDA) receptors were studied in the rat retina by using subunit specific antisera. A punctate immunofluorescence was observed in the inner plexiform layer (IPL) for all subunits tested, and electron microscopy confirmed that the immunoreactive puncta represent labeling of receptors clustered at postsynaptic sites. Double labeling of sections revealed that NMDA receptor clusters within the IPL are composed of different subunit combinations: NR1/NR2A, NR1/NR2B, and in a small number of synapses NR1/NR2A/NR2B. The majority of NMDA receptor clusters were colocalized with the postsynaptic density proteins PSD-95, PSD-93, and SAP 102. Double labeling of the NMDA receptor subunit specific antisera with protein kinase C (PKC), a marker of rod bipolar cells, revealed very little colocalization at the rod bipolar cell axon terminal. This suggests that NMDA receptors are important in mediating neurotransmission within the cone bipolar cell pathways of the IPL. The postsynaptic neurons are a subset of amacrine cells and most ganglion cells. Usually only one of the two postsynaptic processes at the bipolar cell ribbon synapses expressed NMDA receptors. In the outer plexiform layer (OPL), punctate immunofluoresence was observed for the NR1C2; subunit, which was shown by electron microscopy to be localized presynaptically within both rod and cone photoreceptor terminals.
Assuntos
Receptores de N-Metil-D-Aspartato/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Sinapses/metabolismo , Animais , Proteína 4 Homóloga a Disks-Large , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Proteínas do Tecido Nervoso/metabolismo , Ratos , Retina/ultraestrutura , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Sinapses/ultraestruturaRESUMO
In addition to the well-studied AII amacrine cell, there is another amacrine cell type participating in the rod pathway of the mammalian retina. In cat, this cell is called the A17 amacrine cell, and in rabbits, it is called the indoleamine-accumulating amacrine cell (S1 and S2); however, the presence of the corresponding cell type has not yet been described in detail for the rat retina. To this end, we injected amacrine cells with Neurobiotin in vertical retinal slices. After histological processing, we were able to reconstruct the morphology of a wide-field amacrine cell which showed characteristics of A17 and S1/S2 amacrine cells. The rat wide-field amacrine cells exhibited the same stratification pattern, their dendrites bore varicosities and ramified in sublamina 5 of the inner plexiform layer (IPL), and they were dye-coupled to other amacrine cells. To determine whether those amacrine cells shared electrophysiological characteristics as well, we performed whole-cell patch-clamp recordings and examined their voltage-activated currents and neurotransmitter-induced currents. We never observed voltage-gated Na+ currents and spike-like potentials upon depolarization by current injection in these cells. We identified GABA- and glycine-sensitive Cl- currents that could be blocked by bicuculline and strychnine, respectively. We also observed kainate- and AMPA-activated currents, which could be inhibited by the application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Finally, a 400-ms full-field light stimulus was used to characterize the light responses of A17 amacrine cells. The light ON-induced inward current could be suppressed by the application of 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (NBQX), while the majority of the light OFF-induced current was inhibited by bicuculline and reduced to a smaller extent by NBQX. CPP, an NMDA blocker, had no effect on the light response of rat A17 amacrine cells.
Assuntos
Biotina/análogos & derivados , Dendritos/fisiologia , Dendritos/ultraestrutura , Retina/citologia , Retina/fisiologia , Visão Ocular/fisiologia , Animais , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Dendritos/efeitos dos fármacos , Corantes Fluorescentes , Isoquinolinas , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp/estatística & dados numéricos , Estimulação Luminosa , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Ratos , Ratos Endogâmicos , Receptores de GABA/efeitos dos fármacos , Receptores de GABA/metabolismo , Receptores de Glutamato/efeitos dos fármacos , Receptores de Glutamato/metabolismo , Receptores de Glicina/efeitos dos fármacos , Receptores de Glicina/metabolismo , Retina/efeitos dos fármacos , Visão Ocular/efeitos dos fármacosRESUMO
Bassoon is a 420-kDa presynaptic protein which is highly concentrated at the active zones of nerve terminals of conventional synapses, both excitatory glutamatergic and inhibitory GABAergic, in rat brain. It is thought to be involved in the organization of the cytomatrix at the site of neurotransmitter release. In the retina, there are two structurally and functionally distinct types of synapses: ribbon and conventional synapses. Antibodies against bassoon were applied to sections of rat and rabbit retina. Strong punctate immunofluorescence was found in the outer and inner plexiform layers. Using pre- and post-embedding immunostaining and electron microscopy, bassoon was localized in the outer plexiform layer at ribbon synapses formed by rods and cones but was absent from basal synaptic contacts formed by cones. In the inner plexiform layer a different picture emerged. As in the brain, bassoon was found at conventional inhibitory GABAergic synapses, made by amacrine cells, but it was absent from the bipolar cell ribbon synapses. These data demonstrate differences in the molecular composition of the presynaptic apparatuses of outer and inner plexiform layer ribbon synapses. Thus, differential equipment with cytomatrix proteins may account for the functional differences observed between the two types of ribbon synapses in the retina.
Assuntos
Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/biossíntese , Células Fotorreceptoras de Vertebrados/química , Terminações Pré-Sinápticas/química , Terminações Pré-Sinápticas/metabolismo , Animais , Biomarcadores , Exocitose/fisiologia , Imunofluorescência , Mamíferos , Microscopia Imunoeletrônica , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Coelhos , Ratos , Ratos Endogâmicos , Receptores de GABA/análise , Dedos de Zinco/fisiologiaRESUMO
gamma-Aminobutyric acid (GABA), is a main source of inhibitory modulation of the rod pathway in the mammalian retina. The authors previously showed that rod bipolar cells express at least three types of ionotropic GABA receptors. Here, the authors sought to determine which neurons are the presynaptic partners at these synapses in the rabbit retina. Indoleamine-accumulating amacrine cells (IACs) were immunolabeled with an antiserum against serotonin (5HT) in vertical sections and wholemounts of rabbit retinae that had been preloaded with 5HT. The tissue was double labeled for the rho subunits of the GABA(C) receptor or the alpha3 subunit of the GABA(A) receptor. Punctate immunofluorescence was observed for both receptor subunits and was found to coincide with the dendrites and varicosities of IACs. The localization of rho subunits was examined at the ultrastructural level by using postembedding techniques on slam-frozen, cryosubstituted tissue. Double labeling at the electron microscopic level revealed that 5HT-immunoreactive processes were presynaptic to rod bipolar cells through GABA(C) receptors. Intracellular injection of the two morphologic subclasses of IAC amacrine cells, S1 and S2, with Lucifer yellow followed by immunolabeling for the alpha3 or rho subunits revealed that varicosities on the dendrites of both cell types were in register with alpha3- and rho-immunoreactive puncta. Taken together, these results suggest that IACs are presynaptic to rod bipolar cells through GABA(C) receptors and possibly through GABA(A) receptors.