Evidence of metabotropic glutamate receptor subtypes found on catfish horizontal and bipolar retinal neurons

We have used electrophysiological, pharmacological and immunological techniques to determine which classes of metabotropic glutamate receptors exist on cone horizontal cells in the catfish retina. Patch-clamp recordings in acutely dissociated cone horizontal cells provide evidence that group I and III metabotropic glutamate receptors exist, and are linked to modulation of a voltage-gated calcium current. Group II metabotropic glutamate receptor agonists did not affect the calcium current. Immunocytochemical techniques were used to study the localization of metabotropic glutamate receptor subtypes found in the catfish retina. Antibodies raised against group I (metabotropic glutamate receptor 1, metabotropic glutamate receptor 5), group II (metabotropic glutamate receptor 2/3) and group III (metabotropic glutamate receptor 6) metabotropic glutamate receptor subtypes were used to label acutely dissociated horizontal, bipolar and Müller cells. Results from immunostaining provide evidence that cone horizontal cells express group I (metabotropic glutamate receptor 1, metabotropic glutamate receptor 5) and group III (metabotropic glutamate receptor 6), but not group II (metabotropic glutamate receptor 2/3) receptor subtypes, consistent with our electrophysiological results. Cone horizontal cells exposed to anti-metabotropic glutamate receptor 1, 5 or 6 antibodies all demonstrated diffuse overall staining, with patches of dark immunostaining found on both dendritic processes and cell somata. In catfish bipolar cells, all four of the anti-metabotropic glutamate receptor antibodies stained the processes and cell bodies of bipolar cells homogeneously. There was no evidence for a group of bipolar cells that did not stain with the anti-metabotropic glutamate receptor antibodies, although the densest immunostaining occurred when bipolar cells were incubated with the anti-metabotropic glutamate receptor 6 antibody. Müller cells did not show immunostaining against any anti-metabotropic glutamate receptor antibody. Our non-immune controls confirmed that immunostaining was specific for the antigen, and immunoblots were performed to demonstrate the specificity of the antibodies in catfish retina.

These results support the hypothesis that group I and III metabotropic glutamate receptor subtypes are found on catfish horizontal cells, and group I, II and III metabotropic glutamate receptor subtypes are expressed on catfish bipolar cells. The metabotropic glutamate receptors on catfish cone horizontal cells act to modulate the voltage-gated sustained calcium current found on these cells.     

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.     

Synaptic pathways that shape the excitatory drive in an OFF retinal ganglion cell

Different types of retinal ganglion cells represent distinct spatiotemporal filters that respond selectively to specific features in the visual input. Much about the circuitry and synaptic mechanisms that underlie such specificity remains to be determined. This study examines how N-methyl-d-aspartate (NMDA) receptor signaling combines with other excitatory and inhibitory mechanisms to shape the output of small-field OFF brisk-sustained ganglion cells (OFF-BSGCs) in the rabbit retina. We used voltage clamp to separately resolve NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and inhibitory inputs elicited by stimulation of the receptive field center. Three converging circuits were identified. First is a direct glutamatergic input, arising from OFF cone bipolar cells (CBCs), which is mediated by synaptic NMDA and AMPA receptors. The NMDA input was saturated at 10% contrast, whereas the AMPA input increased monotonically up to 60% contrast. We propose that NMDA inputs selectively enhance sensitivity to low contrasts. The OFF bipolar cells, mediating this direct excitatory input, express dendritic kainate (KA) receptors, which are resistant to the nonselective AMPA/KA receptor antagonist, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt (NBQX), but are suppressed by a GluK1- and GluK3-selective antagonist, (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxy-thiophene-3-yl-methyl)-5-methylpyrimidine-2,4-dione (UBP-310). The second circuit entails glycinergic crossover inhibition, arising from ON-CBCs and mediated by AII amacrine cells, which modulate glutamate release from the OFF-CBC terminals. The third circuit also comprises glycinergic crossover inhibition, which is driven by the ON pathway; however, this inhibition impinges directly on the OFF-BSGCs and is mediated by an unknown glycinergic amacrine cell that expresses AMPA but not KA receptors.     

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.     

Dark-adapted response threshold of OFF ganglion cells is not set by OFF bipolar cells in the mouse retina

The nervous system frequently integrates parallel streams of information to encode a broad range of stimulus strengths. In mammalian retina it is generally believed that signals generated by rod and cone photoreceptors converge onto cone bipolar cells prior to reaching the retinal output, the ganglion cells. Near absolute visual threshold a specialized mammalian retinal circuit, the rod bipolar pathway, pools signals from many rods and converges on depolarizing (AII) amacrine cells. However, whether subsequent signal flow to OFF ganglion cells requires OFF cone bipolar cells near visual threshold remains unclear. Glycinergic synapses between AII amacrine cells and OFF cone bipolar cells are believed to relay subsequently rod-driven signals to OFF ganglion cells. However, AII amacrine cells also make glycinergic synapses directly with OFF ganglion cells. To determine the route for signal flow near visual threshold, we measured the effect of the glycine receptor antagonist strychnine on response threshold in fully dark-adapted retinal cells. As shown previously, we found that response threshold for OFF ganglion cells was elevated by strychnine. Surprisingly, strychnine did not elevate response threshold in any subclass of OFF cone bipolar cell. Instead, in every OFF cone bipolar subclass strychnine suppressed tonic glycinergic inhibition without altering response threshold. Consistent with this lack of influence of strychnine, we found that the dominant input to OFF cone bipolar cells in darkness was excitatory and the response threshold of the excitatory input varied by subclass. Thus, in the dark-adapted mouse retina, the high absolute sensitivity of OFF ganglion cells cannot be explained by signal transmission through OFF cone bipolar cells.     

Expression and localization of ionotropic glutamate receptor subunits in the goldfish retina--an in situ hybridization and immunocytochemical study

The expression and distribution of AMPA, kainate and NMDA glutamate receptor subunits was studied in the goldfish retina. For the immunocytochemical localization of the AMPA receptor antisera against GluR2, GluR2/3 and GluR4 were used, and for in situ hybridization rat specific probes for GluR1 and GluR2 and goldfish specific probes for GluR3 and GluR4 were used. The localization of the low affinity kainate receptor and NMDA receptor was studied using antisera against GluR5-7 and NR1. All AMPA receptor subtypes were demonstrated to be present in the goldfish retina both by immunocytochemistry and in situ hybridization. In situ hybridization revealed expression of all AMPA receptors subunit at the inner border of the INL. Only GluR3 was also strongly expressed in the outer border of the INL. Some of the ganglion cells displayed a strong signal for GluR1, GluR3 and GluR4. GluR1-immunoreactivity was present in subsets of bipolar, amacrine, and ganglion cells. GluR2 and GluR2/3-immunoreactivity was mainly localized in the outer plexiform layer. GluR2 and GluR2/3-immunoreactivity are associated with the photoreceptor synaptic terminals. GluR4-immunoreactivity is present on Müller cells in the inner retina and on dendrites of bipolar cells in the OPL, whereas GluR5-7-immunoreactivity was prominently present on horizontal cell axon terminals. Finally, NR1-immunoreactivity was confined to amacrine cells, the inner plexiform layer and ganglion cells. This study shows that there is a strong heterogeneity of glutamate receptor subunit expression in the various layers of the retina. Of the AMPA receptor subunits GluR3 seems to be expressed the most widely in all layers with strong glutamatergic synaptic interactions whereas all the other subunits seem to have a more restricted expressed pattern.     

Synaptic inhibition by glycine acting at a metabotropic receptor in tiger salamander retina

Glycine is the lone fast neurotransmitter for which a metabotropic pathway has not been identified. In retina, we found a strychnine-insensitive glycine response in bipolar and ganglion cells. This glycine response reduced high voltage-activated calcium current. It was G-protein mediated and protein kinase A dependent. The EC₅₀ of the metabotropic glycine response is 3 μ m , an order of magnitude lower than the ionotropic glycine receptor in the same retina. The bipolar cell glutamatergic input to ganglion cells was suppressed by metabotropic glycine action. The synaptic output of about two-thirds of bipolar cells and calcium current in two-thirds of ganglion cells are sensitive to the action of glycine at metabotropic receptors, suggesting this signal regulates specific synaptic pathways in proximal retina. This study resolves the curious absence of a metabotropic glycine pathway in the nervous system and reveals that the major fast inhibitory neurotransmitters, GABA and glycine, both activate G-protein-coupled pathways as well.     

GABA_A和GABA_C受体各亚单位基因在鲫鱼视网膜内的分布──原位杂交组织化学法研究

应用原位杂交组织化学技术，利用同位素［￣（３５）Ｓ］－ｄＡＴＰ标记的寡核苷酸探针，在鲫鱼视网膜观察了含ＧＡＢＡＡ受体α１、α３、α４、α６，β１－３，γ１－２及ＧＡＢＡＣ受体ρ１亚单位ｍＲＮＡ的神经元分布。在外核层，所有测试的亚单位均无表达；而在内核层和神经节细胞层，除α４和γ２亚单位外，均有不同程度的表达。在不同区域标记神经元的数量和标记强度各不相同，α１亚单位广泛分布在内核层的远端、中部及神经节细胞层，呈强阳性；α３亚单位相对稀少，主要分布在内核层近端和神经节细胞层，呈中等阳性；α４和α６亚单位几乎无阳性表达，仅α６亚单位在神经节细胞层呈弱阳性。β１和β２亚单位在内核层及神经节细胞层呈中等阳性；β３亚单位主要分布在内核层，在神经节细胞层标记细胞较少，呈弱阳性。γ１亚单位分布在整个内核层，在神经节细胞层有零星阳性表达。ＧＡＢＡＣ受体主要分布在内核层，ρ１亚单位主要分布在内核层的远端及中间部分，呈强阳性，而在神经节细胞层表达相对较弱。这种独特的表达型式与其功能密切相关。     

视觉信息在视网膜中传递和调控

视觉信息在视网膜中传递和调控杨雄里（中国科学院，上海生理研究所，上海生命科学联合开放实验室，上海２００３１）光感受器（视杆和视锥）信号在视网膜中是通过各种信息通道（ｃｈａｎｎｅｌ）进行传递的。这些通道包括：视杆一视锥通道，颜色信号通道，给光一撤光（Ｏ...     

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.     

牛蛙视网膜内γ-氨基丁酸免疫阳性反应的分布

用免疫组织化学ABC法．研究了GABA免疫阳性反应在牛蛙视网膜的分布。证明光感受器内段（主要是视锥细胞）呈棕褐色的GABA反应;在外核层未见GABA标记的胞体,但在靠近外网层处偶见GABA标记的终末;在内核层,大量无长突细胞呈GABA反应阳性,并可鉴别出胞体染色较深和淡的两个亚群,一些双极细胞和个别水平细胞的胞体及它们的突起呈较弱的GABA反应阳性,偶见双极细胞轴突终末以膨体紧密贴附在GABA标记的无长突细胞上。在节细胞层,一些神经节细胞和散在的移位无长突细胞呈GABA反应阳性。此外．外网层和内网层均呈GABA反应阳性。上述结果表明,GABA广泛分布于牛蛙视网膜的各层,提示它在视觉信号的传递过程中发挥着重要作用。     

Intracellular calcium is regulated by different pathways in horizontal cells of the mouse retina

Horizontal cells modulate the output of the photoreceptor to bipolar cell synapse, thereby providing the first level of lateral information processing in the vertebrate retina. Because horizontal cells do not generate sodium-based action potentials, calcium is likely to play an important role for graded potential changes as well as for intracellular events involved in the modulatory role of horizontal cells within the retinal network. Therefore we wanted to determine how the activation of glutamate receptors, voltage-gated calcium channels, and release of calcium from internal stores shape the calcium signal in horizontal cells. All horizontal cells responded to depolarizing voltage steps with sustained inward currents, which activated at around -20 mV, reached a peak amplitude of -79.1 pA at 5 mV, and reversed sign at around 66 mV. The current was insensitive to tetrodotoxin, and it was partially blocked by the L-type channel antagonists verapamil and nifedipine. The N-type channel blocker omega-conotoxin GVIA induced an additional reduction of current amplitudes. Calcium influx through ionotropic glutamate receptors was mediated by both AMPA and kainate but not by N-methyl-D-aspartate receptors. Two agonists at group I metabotropic glutamate receptor, trans-1-amino-1,3-cyclopentanedicarboxylic acid and quisqualate, had no effect. However, intracellular calcium was increased by caffeine, indicating release of calcium from internal stores via ryanodine receptors. These data show that intracellular calcium in horizontal cells is regulated by voltage-dependent L- and N-type calcium channels, ionotropic AMPA and kainate receptors, and release of calcium from internal stores after activation of ryanodine receptors.     

Glutamate modulation of GABA transport in retinal horizontal cells of the skate

Transport of the amino acid GABA into neurons and glia plays a key role in regulating the effects of GABA in the vertebrate retina. We have examined the modulation of GABA-elicited transport currents of retinal horizontal cells by glutamate, the likely neurotransmitter of vertebrate photoreceptors. Enzymatically isolated external horizontal cells of skate were examined using whole-cell voltage-clamp techniques. GABA (1 m m ) elicited an inward current that was completely suppressed by the GABA transport inhibitors tiagabine (10 μ m ) and SKF89976-A (100 μ m ), but was unaffected by 100 μ m picrotoxin. Prior application of 100 μ m glutamate significantly reduced the GABA-elicited current. Glutamate depressed the GABA dose-response curve without shifting the curve laterally or altering the voltage dependence of the current. The ionotropic glutamate receptor agonists kainate and AMPA also reduced the GABA-elicited current, and the effects of glutamate and kainate were abolished by the ionotropic glutamate receptor antagonist 6-cyano-7-nitroquinoxaline. NMDA neither elicited a current nor modified the GABA-induced current, and metabotropic glutamate analogues were also without effect. Inhibition of the GABA-elicited current by glutamate and kainate was reduced when extracellular calcium was removed and when recording pipettes contained high concentrations of the calcium chelator BAPTA. Caffeine (5 m m ) and thapsigargin (2 n m ), agents known to alter intracellular calcium levels, also reduced the GABA-elicited current, but increases in calcium induced by depolarization alone did not. Our data suggest that glutamate regulates GABA transport in retinal horizontal cells through a calcium-dependent process, and imply a close physical relationship between calcium-permeable glutamate receptors and GABA transporters in these cells.     

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.     

Presynaptic effects of group III metabotropic glutamate receptors on excitatory synaptic transmission in the retina

Metabotropic glutamate receptors (mGluRs) are located in both plexiform layers in the retina and may modulate transmission between photoreceptors and bipolar cells and between bipolar and ganglion cells. We investigated whether mGluR activation modulates excitatory synaptic input to bipolar cells and ganglion cells in the salamander retinal slice preparation. The group III mGluR agonist L-2-amino-4-phosphonobutyric acid (AP4) inhibited monosynaptic excitatory postsynaptic currents (EPSCs) in ganglion cells evoked by electrical stimuli, whereas group I and group II agonists had no significant effect. AP4 reduced the frequency but not the amplitude of ganglion cell miniature EPSCs, suggesting a presynaptic action at bipolar cell terminals. AP4 also reduced ganglion cell EPSCs evoked by the offset of a light stimulus, suggesting that group III mGluRs modulate release from OFF bipolar cells. Comparison of light-evoked EPSCs in OFF bipolar cells and ganglion cells indicated that AP4 reduced ganglion cell EPSCs by acting primarily at bipolar cell terminals, and to a lesser extent at photoreceptor terminals. The group II/III mGluR antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) blocked the effect of AP4 at bipolar cell terminals, consistent with localization of group III mGluRs at these sites. However, CPPG did not increase EPSCs at light offset, indicating that activation of group III mGluRs by synaptic glutamate does not play a large role in modulating transmission from bipolar cells to ganglion cells.     

Characterization of the spontaneous synaptic activity of amacrine cells in the mouse retina

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.     

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.     

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.     

Spontaneous oscillatory activity of starburst amacrine cells in the mouse retina

Using patch-clamp techniques, we investigated the characteristics of the spontaneous oscillatory activity displayed by starburst amacrine cells in the mouse retina. At a holding potential of -70 mV, oscillations appeared as spontaneous, rhythmic inward currents with a frequency of approximately 3.5 Hz and an average maximal amplitude of approximately 120 pA. Application of TEA, a potassium channel blocker, increased the amplitude of oscillatory currents by >70% but reduced their frequency by approximately 17%. The TEA effects did not appear to result from direct actions on starburst cells, but rather a modulation of their synaptic inputs. Oscillatory currents were inhibited by 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX), an antagonist of AMPA/kainate receptors, indicating that they were dependent on a periodic glutamatergic input likely from presynaptic bipolar cells. The oscillations were also inhibited by the calcium channel blockers cadmium and nifedipine, suggesting that the glutamate release was calcium dependent. Application of AP4, an agonist of mGluR6 receptors on on-center bipolar cells, blocked the oscillatory currents in starburst cells. However, application of TEA overcame the AP4 blockade, suggesting that the periodic glutamate release from bipolar cells is intrinsic to the inner plexiform layer in that, under experimental conditions, it can occur independent of photoreceptor input. The GABA receptor antagonists picrotoxin and bicuculline enhanced the amplitude of oscillations in starburst cells prestimulated with TEA. Our results suggest that this enhancement was due to a reduction of a GABAergic feedback inhibition from amacrine cells to bipolar cells and the resultant increased glutamate release. Finally, we found that some ganglion cells and other types of amacrine cell also displayed rhythmic activity, suggesting that oscillatory behavior is expressed by a number of inner retinal neurons.     

Current views on molecular structure of benzodiazepine receptors]

It is generally accepted that gamma-aminobutyric acid (GABA) is the most important inhibitory transmitter. It interacts with specific receptors. In the central nervous system of the vertebrates there are three types of GABA receptors: GABAA, GABAB, and GABAC receptors. Channel gating at GABAA receptors allosterically is modulated by a wide of neuroactive compounds, including benzodiazepine ligands. The structural and functional bases for the heterogeneity of benzodiazepine receptors are discussed. Investigations of the functional properties of GABAA and benzodiazepine receptors may contribute to progress in neurobiology, neuropathology, neurochemistry, toxicology, and pharmacology.