Coupling from AII amacrine cells to ON cone bipolar cells is bidirectional.

The AII amacrine cell is a critical interneuron in the rod pathway of the mammalian retina. Rod signals pass into cone pathways by means of gap junctions between AII amacrine cells and ON cone bipolar cells. Filling AII amacrine cells with Neurobiotin produces labeling of cone bipolar cells by means of these gap junctions. However, tracer injections into bipolar cells do not produce labeling of the AII network (Vaney [1997] Invest Ophthalmol Vis Sci. 38:267-273), which suggests that the AII/bipolar gap junctions allow the passage of tracer in only one direction. This mechanism stands in contrast to physiological results, which indicate that light adapted signals can pass from ON cone bipolar cells into the AII network (Xin and Bloomfield [1999] Vis Neurosci. 16:653-665). Here, we report that a variety of ON and OFF bipolar cells are sometimes anomalously coupled to the A-type horizontal cell network. These relatively rare examples do not result from dye injection errors, but seem to represent minor developmental errors. However, this provides a method to obtain Neurobiotin-filled cone bipolar cells without the necessity of impaling them with a microelectrode. Under these conditions, Neurobiotin spreads from ON cone bipolar cells into neighboring AII amacrine cells. The dye-coupled AII amacrine cells, positively identified by double labeling with an antibody against calretinin, were centered around anomalously coupled ON bipolar cells. These results indicate that AII/bipolar cell gap junctions allow tracer coupling in both directions, consistent with previous physiological results. The previous failure to detect the passage of neuronal tracer from injected bipolar cells to AII amacrine cells may reflect electrode damage or perhaps the asymmetrical voltage sensitivity of a heterotypic gap junction.

Rod pathways in the mammalian retina use connexin 36.

Many neurons in the mammalian retina are coupled by means of gap junctions. Here, we show that, in rabbit retina, an antibody to connexin 36 heavily labels processes of AII amacrine cells, a critical interneuron in the rod pathway. Image analysis indicates that Cx36 is primarily located at dendritic crossings between overlapping AII amacrine cells. This finding suggests that Cx36 participates in homotypic gap junctions between pairs of AII amacrine cells. Cx36 was also found at AII/cone bipolar contacts, previously shown to be gap junction sites. This finding suggests that Cx36 participates at gap junctions that may be heterotypic. These results place an identified neuronal connexin in the context of a well-defined retinal circuit. The absence of Cx36 in many other neurons known to be coupled suggests the presence of additional unidentified connexins in mammalian neurons. Conversely, Cx36 labeling in other regions of the retina is not associated with AII amacrine cells, indicating some other cell types use Cx36.

A series of biotinylated tracers distinguishes three types of gap junction in retina.

Gap junctions serve many important roles in various tissues, but their abundance and diversity in neurons is only beginning to be understood. The tracer Neurobiotin has revealed many different networks interconnected by gap junctions in retina. We compared the relative permeabilities of five different retinal gap junctions by measuring their permeabilities to a series of structurally related tracers. When large tracers were injected, the staining of coupled cells fell off more rapidly in some networks than others relative to Neurobiotin controls. Three distinctly different permeability profiles were found, suggesting that multiple neuronal connexin types were present. The most permeant to large molecules were gap junctions from A-type horizontal cells. The permeability of gap junctions of two types of amacrine cell were not distinguishable from those from B-type horizontal cells. The lowest permeability was found for gap junctions between cone bipolar cells and the AII amacrine cells to which they are coupled. Because only a single neural connexin type has been identified in retina, our results suggest more types remain to be found. To determine whether the unitary permeability of channels is altered by channel modulators, we reduced permeability with octanol and a cAMP analog. Although net permeability was substantially diminished, the proportion by which it declined was constant across tracer size. This suggests that these agents act only to close channels rather than alter individual channel permeabilities. This tracer series can therefore be used to contrast permeability properties of gap junctions in intact circuits, even at the level of individual channels.

Dye coupling in horizontal cells of developing rabbit retina.

In the mature rabbit retina, two classes of horizontal cells, A type and B type, provide lateral inhibition in the outer plexiform layer (OPL) and spatially modify the activation of bipolar cells by photoreceptors. Gap junctions connecting homologous horizontal cells determine the extent to which this inhibitory activity spreads laterally across the OPL. Little is currently known about the expression of gap junctions in horizontal cells during postnatal development or how cell-cell coupling might contribute to subsequent maturational events. We have examined the morphological attributes and coupling properties of developing A and B type horizontal cells in neonatal rabbit retina using intracellular injections of Lucifer Yellow and Neurobiotin. Prelabeling with DAPI permitted the targeting of horizontal cell bodies for intracellular injection in perfused preparations of isolated retina. A and B type horizontal cells were identifiable at birth although their dendritic field sizes had not reached adult proportions and their synaptic contacts in the OPL were minimal. Both cell types exhibited homologous dye coupling at birth. Similar to that seen in the adult, no heterologous coupling was observed, and homologous coupling among A type cells was stronger than that observed among B type cells. The spread of tracer compounds through gap junctions of morphologically immature horizontal cells suggests that ions and other small, bioactive compounds may likewise spread through coupled, horizontal networks to coordinate the subsequent maturational of emerging outer plexiform layer pathways.

Gap junctions between AII amacrine cells and calbindin-positive bipolar cells in the rabbit retina.

Electrical synapses or gap junctions occur between many retinal neurons. However, in most cases, the gap junctions have not been visualized directly. Instead, their presence has been inferred from tracer spread throughout the network of cells. Thus, tracer coupling is taken as a marker for the presence of gap junctions between coupled cells. AII amacrine cells are critical interneurons in the rod pathway of the mammalian retina. Rod bipolar cell output passes to AII amacrine cells, which in turn make conventional synapses with OFF cone bipolar cells and gap junctions with ON cone bipolar cells. Injections of biotinylated tracers into AII amacrine cells reveals coupling between the AII amacrine cell network and heterologous coupling with a variety of ON cone bipolar cells, including the calbindin-positive cone bipolar cell. To directly visualize gap junctions in this network, we prepared material for electron microscopy that was double labeled with antibodies to calretinin and calbindin to label AII amacrine cells and calbindin-positive cone bipolar cells, respectively. AII amacrine cells were postsynaptic to large vesicle-laden rod bipolar terminals, as previously reported. Gap junctions were identified between AII amacrine cells and calbindin-positive cone bipolar cell terminals identified by the presence of immunostaining and ribbon synapses. This represents direct confirmation of gap junctions between two different yet positively identified cells, which are tracer coupled, and provides additional evidence that tracer coupling with Neurobiotin indicates the presence of gap junctions. These results also definitively establish the presence of gap junctions between AII amacrine cells and calbindin bipolar cells which can therefore carry rod signals to the ON alpha ganglion cell.

Antibody to calretinin stains AII amacrine cells in the rabbit retina: double-label and confocal analyses.

The AII or rod amacrine cell is a critical interneuron in the rod pathway of mammalian retinae. In this report, it is shown that commercially available antibodies to the calcium binding protein calretinin may be used to label the population of AII amacrine cells selectively. Calretinin-positive amacrine cells had the morphological attributes of AII amacrine cells. Double-labeling procedures showed that calretinin-positive somata were surrounded by dopaminergic varicosities and that calretinin-positive dendrites enclosed rod bipolar terminals, both as previously described for AII amacrine cells. By analyzing the surrounding kernel for each labeled pixel in the rod bipolar image, it is shown here that AII processes are adjacent to rod bipolar terminals at a level that far exceeds the random overlap present in images in which one label was rotated out of phase. Such a spatial relationship is indicative of synaptic connections, as well described for rod bipolar input to AII amacrine cells. AII amacrine cells also were double-labeled for calretinin and parvalbumin; however, a scattergram analysis of red versus green intensity showed that the parvalbumin antibody stained additional unidentified amacrine cells. In conclusion, at the appropriate dilution, calretinin antibodies are a useful marker for AII amacrine cells in the rabbit retina.

AII amacrine cells limit scotopic acuity in central macaque retina: A confocal analysis of calretinin labeling.

We have used calretinin antibodies to label selectively the mosaic of AII amacrine cells in the macaque retina. Confocal analysis of double-labeled material indicated that AII dendrites spiral down around descending rod bipolar axons before enveloping the synaptic terminals. Processes from a previously observed dopaminergic plexus in the inner nuclear layer were observed to contact the somata of calretinin-positive AII somata. Intracellular neurobiotin injection revealed that AII amacrine cells are tracer coupled to other AII amacrine cells and to some unidentified cone bipolar cells. An analysis of the retinal distribution of macaque AII amacrine cells, including an area in and around the fovea, showed a peak density of approximately 5,000 cells/mm(2) at an eccentricity of 1.5 mm. Staining of AII amacrine cells in central retina with antibodies to calretinin was confirmed by confocal microscopy. These results indicate that calretinin antibodies can be used to label the AII amacrine cell population selectively and that primate AII amacrine cells share many of the features of previously described mammalian AII amacrine cells. The peak AII cell density closely matched the peak sampling rate of scotopic visual acuity. Calculations suggest that, in central macaque retina, where midget ganglion cells are more numerous, AII amacrine cells form the limit of scotopic visual acuity (Wässle et al. [1995] J. Comp. Neurol. 361:537-551). As the ganglion cell density falls rapidly away from the fovea, there is a cross-over point at around 15 degrees eccentricity that matches the inflection point in a psychophysically derived plot of scotopic visual acuity versus eccentricity (Lennie and Fairchild [1994] Vision Res. 34:477-482). The correspondence between the anatomic and psychophysical data supports our interpretation that the anatomic sampling rate of AII amacrine cells limits central scotopic acuity.

Immunocytochemical localization of excitatory and inhibitory neurotransmitters in the zebrafish retina.

The patterns of glutamate, gamma-aminobutyric acid (GABA), and glycine distribution in the zebrafish retina were determined using immunocytochemical localization of antisera at the light-microscope level. The observed GABA immunoreactivity (GABA-IR) patterns were further characterized using antibodies to both isoforms of glutamic acid decarboxylase (GAD65 and GAD67), the synthetic enzyme for GABA. Glutamate-IR was observed in all retinal layers with photoreceptors, bipolar cells, and ganglion cells prominently labeled. Bipolar cells displayed the most intense glutamate-IR and bipolar cell axon terminals were clearly identified as puncta arranged in layers throughout the inner plexiform layer (IPL). These findings suggest the presence of multiple subtypes of presumed OFF- and ON-bipolar cells, including some ON-bipolar cells characterized by a single, large (9 microm X 6 microm) axon terminal. GABA-, GAD-, and glycine-IR were most intense in the inner retina. In general, the observed labeling patterns for GABA, GAD65, and GAD67 were similar. GABA- and GAD-IR were observed in a population of amacrine cells, a few cells in the ganglion cell layer, throughout the IPL, and in horizontal cells. In the IPL, both GABA- and GAD-IR structures were organized into two broad bands. Glycine-IR was observed in amacrine cells, interplexiform cells, and in both plexiform layers. Glycine-positive terminals were identified throughout the IPL, with a prominent band in sublamina 3 corresponding to an immunonegative region observed in sections stained for GAD and GABA. Our results show the distribution of neurons in the zebrafish retina that use glutamate, GABA, or glycine as their neurotransmitter. The observed distribution of neurotransmitters in the inner retina is consistent with previous studies of other vertebrates and suggests that the advantages of zebrafish for developmental studies may be exploited for retinal studies.

Localization of nitric oxide synthase, NADPH diaphorase and soluble guanylyl cyclase in adult rabbit retina.

Nitric oxide (NO) acts as a neuronal messenger which activates soluble guanylyl cyclase (SGC) in neighboring cells and produces a wide range of physiological effects in the central nervous system (CNS). Using immunocytochemical and histochemical stains, we have characterized the NO/SGC system in the rabbit retina and to a lesser extent, in monkey retina. Based on staining patterns observed with an antibody to nitric oxide synthase (NOS) type I and a histochemical marker for NADPH diaphorase, a metabolic intermediate required for NOS activity, three major classes of neurons appear to generate NO in the rabbit retina. These include two subclasses of sparsely distributed wide field amacrine cells, rod and cone photoreceptors, and a subpopulation of ganglion cells. Equivalent cell populations were labeled in monkey retina. An antibody to SGC (tested only in rabbit retina), labeled large arrays of cone photoreceptors in the outer nuclear layer, both amacrine and bipolar cells in the inner nuclear layer (INL), as well as populations of neurons in the ganglion cell layer. These data suggest that the ability to generate NO is restricted to relatively few neurons in the inner retina and to photoreceptor cells in the outer retina; while presumptive target cells, containing pools of SGC, are widespread and form contiguous fields across the inner and outer nuclear layers (ONL) as well as the ganglion cell layer.

The kinetics of tracer movement through homologous gap junctions in the rabbit retina.

Observation of the spread of biotinylated or fluorescent tracers following injection into a single cell has become one of the most common methods of demonstrating the presence of gap junctions. Nevertheless, many of the fundamental features of tracer movement through gap junctions are still poorly understood. These include the relative roles of diffusion and iontophoretic current, and under what conditions the size of the stained mosaic will increase, asymptote, or decline. Additionally, the effect of variations in amount of tracer introduced, as produced by variation in electrode resistance following cell penetration, is not obvious. To examine these questions, Neurobiotin was microinjected into the two types of horizontal cell of the rabbit retina and visualized with streptavidin-Cy3. Images were digitally captured using a confocal microscope. The spatial distribution of Neurobiotin across the patches of coupled cells was measured. Adequate fits to the data were obtained by fitting to a model with terms for diffusion and amount of tracer injected. Results indicated that passive diffusion is the major source of tracer movement through gap junctions, whereas iontophoretic current played no role over the range tested. Fluorescent visualization, although slightly less sensitive than peroxidase reactions, produced staining intensities with a more useful dynamic range. The rate constants for movement of Neurobiotin between A-type horizontal cells was about ten times greater than that for B-type horizontal cells. Although direct extrapolation to ion conductances cannot be assumed, tracer movement can be used to give an estimate of relative coupling rates across cell types, retinal location, or modulation conditions in intact tissue.

Contributions of GABAA receptors and GABAC receptors to acetylcholine release and directional selectivity in the rabbit retina.

GABA is a major inhibitory neurotransmitter in the mammalian retina and it acts at many different sites via a variety of postsynaptic receptors. These include GABAA receptors and bicuculline-resistant GABAC receptors. The release of acetylcholine (ACh) is inhibited by GABA and strongly potentiated by GABA antagonists. In addition, GABA appears to mediate the null inhibition which is responsible for the mechanism of directional selectivity in certain ganglion cells. We have used these two well-known examples of GABA inhibition to compare three GABA antagonists and assess the contributions of GABAA and GABAC receptors. All three GABA antagonists stimulated ACh release by as much as ten-fold. By this measure, the ED50s for SR-95531, bicuculline, and picrotoxin were 0.8, 7.0, and 14 microM, respectively. Muscimol, a potent GABAA agonist, blocked the effects of SR-95531 and bicuculline, but not picrotoxin. This indicates that SR-95531 and bicuculline are competitive antagonists at the GABAA receptor, while picrotoxin blocks GABAA responses by acting at a different, nonreceptor site such as the chloride channel. In the presence of a saturating dose of SR-95531 to completely block GABAA receptors, picrotoxin caused a further increase in the release of ACh. This indicates that picrotoxin potentiates ACh release by a mechanism in addition to the block of GABAA responses, possibly by also blocking GABAC receptors, which have been associated with bipolar cells. All three GABA antagonists abolished directionally selective responses from ON/OFF directional-selective (DS) ganglion cells. In this system, the ED50S for SR-95531, bicuculline, and picrotoxin were 0.7 microM, 8 microM, and 94.6 microM, respectively. The results with SR-95531 and bicuculline indicate that GABAA receptors mediate the inhibition responsible for directional selectivity. The addition of picrotoxin to a high dose of SR-95531 caused no further increase in firing rate. The comparatively high dose required for picrotoxin also suggests that GABAC receptors do not contribute to directional selectivity. This in turn suggests that feedforward GABAA inhibition, as opposed to feedback at bipolar terminals, is responsible for the null inhibition underlying directional selectivity.

Pharmacology of directionally selective ganglion cells in the rabbit retina.

In this report we describe extracellular recordings made from ON and ON-OFF directionally selective (DS) ganglion cells in the rabbit retina during perfusion with agonists and antagonists to acetylcholine (ACh), glutamate, and gamma-aminobutyric acid (GABA). Nicotinic ACh agonists strongly excited DS ganglion cell in a dose-dependent manner. Dose-response curves showed a wide range of potencies, with (+/-)-exo-2-(6-chloro-3pyridinyl)-7-azabicyclo[2.2.1] heptane dihydrochloride (epibatidine) > > > nicotine > 1,1-dimethyl-4-phenylpiperazinium iodide = carbachol. In addition, the mixed cholinergic agonist carbachol produced a small excitation, mediated by muscarinic receptors, that could be blocked by atropine. The specific nicotinic antagonists hexamethonium bromide (100 microM), dihydro-beta-erythroidine (50 microM), mecamylamine (50 microM), and tubocurarine (50 microM) blocked the responses to nicotinic agonists. In addition, nicotinic antagonists reduced the light-driven input to DS ganglion cells by approximately 50%. However, attenuated responses were still DS. We deduce that cholinergic input is not required for directional selectivity. These experiments reveal the importance of bipolar cell input mediated by glutamate. N-methyl-D-aspartic acid (NMDA) excited DS ganglion cells, but NMDA antagonists did not abolish directional selectivity. However, a combined cholinergic and NMDA blockade reduced the responses of DS ganglion cells by > 90%. This indicates that most of the noncholinergic excitatory input appears to be mediated by NMDA receptors, with a small residual made up by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate (KA) receptors. Responses to AMPA and KA were highly variable and often evoked a mixture of excitation and inhibition due to the release of ACh and GABA. Under cholinergic blockade AMPA/KA elicited a strong GABA-mediated inhibition in DS ganglion cells. AMPA/KA antagonists, such as 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo (F)quinoxaline dione and GYKI-53655, promoted null responses and abolished directional selectivity due to the blockade of GABA release. We conclude that GABA release, mediated by non-NMDA glutamate receptors, is an essential part of the mechanism of directional selectivity. The source of the GABA is unknown, but may arise from starburst amacrine cells.

A calbindin-immunoreactive cone bipolar cell type in the rabbit retina.

We have studied the distribution of the calcium-binding protein calbindin in the adult rabbit retina by using a commercially available antibody and immunocytochemical methods. The most heavily labeled cells are A-type horizontal cells, but B-type horizontal cells are also lightly labeled by this antibody. Among the horizontal cells, there is a mosaic of small, well-labeled somata, which we have identified as a subset of ON cone bipolar cells. In addition, some wide-field amacrine cells and a few large ganglion cells are also labeled for calbindin. The calbindin bipolar cells form a regular mosaic with a peak density of approximately 1,700 cells/mm2, falling to 550 cells/mm2 in the periphery. They account for about one-twelfth of cone bipolar cells, and they are narrowly stratified deep in sublamina 4 of the inner plexiform layer immediately above the rod bipolar terminals. Double-label experiments using an antibody to protein kinase C (PKC) indicate that the calbindin bipolar cells are completely distinct from the population of rod bipolar cells. Rod bipolar cells outnumber the calbindin cone bipolar cells by a factor of four to five. Further double-label experiments show that the calbindin bipolar cells are also labeled for recoverin. The calbindin bipolar cells are well coupled to AII amacrine cells, and they account for roughly 23% of the AII coupled bipolar cells. This suggests that there are three to four additional ON cone bipolar cell types that are coupled to AII amacrine cells. The calbindin cone bipolar cell described in this paper shares many characteristics with a reconstructed cone bipolar cell that forms the most gap junctions with AII amacrine cells (Strettoi et al. [1994] J. Comp. Neurol. 347:139-149). We conclude that these different methodologies provide complementary descriptions of the same cone bipolar cell type. The calbindin antibody defines a subset of cone bipolar cells in the rabbit retina. The cells in this subset are almost certainly the deepest of the cone bipolar cells. The tight stratification of the calbindin cone bipolar cell suggests that the inner plexiform layer is stratified according to depth, with narrow functional divisions within the broad partition of sublamina b, where ON signals are processed. The strength of coupling between the calbindin cone bipolar cells and AII amacrine cells suggests this pathway plays a major role under scotopic conditions.

Homocysteate-evoked release of acetylcholine from the rabbit retina.

The cholinergic amacrine cells of the rabbit retina can be labeled with [3H]choline and the activity of the cholinergic population monitored by following the release of [3H]acetylcholine. It has been proposed that L-homocysteate may be the main endogenous transmitter released onto cholinergic amacrine cells by bipolar cells. Therefore, we have examined the effects of the isomers of homocysteate on the release of [3H]acetylcholine. In magnesium-free medium, D-homocysteate was slightly more potent than the L-isomer. The addition of magnesium, which blocks responses mediated by NMDA receptors, preferentially reduced but did not eliminate, the response to L-homocysteate. 2-Amino-7-phosphonoheptanoate, a potent NMDA antagonist, preferentially blocked L-homocysteate evoked responses. 6,7-Dinitroquinoxaline-2,3-dione, a potent kainate antagonist, preferentially blocked D-homocysteate-evoked responses. Therefore, in the rabbit retina, L-homocysteate is an NMDA-preferring agonist, whereas D-homocysteate is a kainate-preferring agonist. In addition, we found that L-homocysteate can activate the physiologically activated kainate receptor but only when used in millimolar concentrations and under conditions that minimize NMDA-receptor activation. However, the low potency of L-homocysteate combined with low affinity for the glutamate transporter, lack of immunocytochemical localization in bipolar cells, and low retinal content place serious limitations on the role of L-homocysteate at the bipolar-to-cholinergic amacrine cell synapse.

Differential properties of two gap junctional pathways made by AII amacrine cells.

The retina is sensitive to light stimuli varying over more than 12 log units in intensity. It accomplishes this, in part, by switching between rod-dominated circuits designed for maximum utilization of scarce photons and cone circuits designed for greater acuity. Rod signals are integrated into the cone pathways through AII amacrine cells, which are connected by gap junctions both to other AII amacrine cells and to cone bipolar cells. To determine the relative permeabilities of the two junctional pathways, we have measured the distribution of biotinylated tracers across this heterologous cell assembly after injecting a single AII amacrine cell. We found that neurobiotin (relative molecular mass, 286) passed easily through both types of gap junctions, but that biotin-X cadaverine (relative molecular mass, 442) passed through AII/bipolar cell gap junctions poorly compared to AII/AII gap junctions. Thus, the AII/bipolar cell channel has a lower permeability to large molecules than does the AII/AII amacrine cell channel. The two pathways are also regulated differently. Dopamine and cyclic AMP agonists, known to diminish AII-AII coupling, did not change the relative labelling intensity of AII to bipolar cells. However, nitric oxide and cGMP agonists selectively reduced labelling in bipolar cells relative to AII amacrine cells, perhaps by acting at the bipolar side of this gap junction. This suggests that increased cGMP controls the network switching between rod and cone pathways associated with light adaptation.

Effect of ON pathway blockade on directional selectivity in the rabbit retina.

1. In this report we describe extracellular recordings made from directionally selective (DS) ganglion cells in the rabbit retina during perfusion with 2-amino-4-phosphonobutyric acid (APB) to block ON channels through the retina. 2. Application of 100 microM APB selectively and reversibly abolished the responses of ON ganglion cells in the rabbit retina. In addition, 100 microM APB completely and reversibly blocked ON component responses of ON-OFF DS ganglion cells to both stationary and moving stimuli. These results are consistent with the idea that APB blocks ON pathways through the retina. 3. Under ON pathway blockade with APB, OFF component responses of ON-OFF DS ganglion cells remained DS. DS OFF responses retained the same preferred direction as the pre-APB ON-OFF responses and could be driven using either normal or reversed contrast stimuli. 4. Extracellular responses of ON DS ganglion cells were completely blocked by APB. Under APB, these cells showed no response to stationary or moving stimuli. 5. Application of the gamma-aminobutyric acid-A (GABAA) antagonist 2-(3-Carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide (SR95531) reversibly abolished directional selectivity of ON DS and ON-OFF DS ganglion cells in the rabbit retina. This finding is consistent with previous data for picrotoxin. 6. Application of SR95531 during ON channel blockade by APB caused OFF component responses of ON-OFF DS ganglion cells to lose their directional selectivity. Under these conditions, OFF responses to movement in the preferred and null directions became virtually identical. 7. These results indicate that simultaneous ON and OFF layer input is not required to generate directional responses in ON-OFF DS ganglion cells. In addition, it appears that a GABAA-dependent mechanism for directional selectivity may operate independently in the two separate dendritic layers of the ON-OFF DS ganglion cell.

Distribution and coverage of A- and B-type horizontal cells stained with Neurobiotin in the rabbit retina.

Both A- and B-type horizontal cells in the rabbit retina were labeled by brief in vitro incubations of the isolated retina in the blue fluorescent dye 4,6-diamino-2-phenylindole. Intracellular injection of Lucifer Yellow into the somata revealed the morphology of the individual cells. Dye-coupling with Lucifer Yellow was seen only between A-type horizontal cells. By contrast, injection of the tracer Neurobiotin showed dye-coupling between both A- and B-type horizontal cells. There also appeared to be coupling between the axon terminals of B-type horizontal cells. The extensive dye-coupling seen following injection of Neurobiotin into a single horizontal cell soma can be used to obtain population counts of each cell type. Staining of large numbers of each cell type across the retina showed that each type increased in number and declined in dendritic diameter as the visual streak was approached, such that relatively constant coverage across the retina was maintained. In the visual streak, A-type horizontal cells numbered 555 cells/mm2 and averaged 120 microns in diameter, compared to 1375 cells/mm2 and 100 microns for B-type horizontal cells. In the periphery, the A- and B-types numbered 250 cells/mm2 and 400 cells/mm2, respectively. The average diameters of the dendritic trees at these locations were 225 microns for the A-type and 175 microns for the B-type. Coverage across the retina averaged almost six for A-type horizontal cells and 8-10 for the B-type. A-type horizontal cells in the visual streak whose elliptical dendritic fields were shown by Bloomfield (1992) to correlate physiologically with orientation bias were shown to be dye-coupled to cells with symmetrical dendritic fields.

All indoleamine-accumulating cells in the rabbit retina contain GABA.

The indoleamine-accumulating amacrine cells of the rabbit retina are wide-field and numerous. They form a dense plexus in sublamina 5 of the inner plexiform layer where they make reciprocal synapses with rod bipolar cells. To provide a quantitative test for the colocalization of serotonin (5-HT) and gamma-aminobutyric acid (GABA) in the rabbit retina, we designed two parallel double-label experiments. In the first series, the indoleamine-accumulating cells were labeled with 5,7-dihydroxytryptamine (5,7-DHT), which was subsequently visualized by photooxidation in the presence of diaminobenzidine. This was combined with autoradiography for 3H-muscimol. In the second and complementary series, 3H-5-HT uptake was combined with postembedding GABA immunocytochemistry. These two experiments provided essentially identical results: over 98% of the indoleamine-accumulating amacrine cells were double-labeled. This means that, within the limit of experimental error, all the indoleamine-accumulating amacrine cells are GABAergic. The indoleamine-accumulating amacrine cells account for 15-20% of a large diverse group of GABA amacrine cells. In addition, the rare type 3 indoleamine-accumulating cells and fine processes running in the optic fiber layer were double-labeled. If there is insufficient 5-HT to support a transmitter role in the rabbit retina, our results suggest that the indoleamine-accumulating cells may use GABA as a neurotransmitter. Thus, rod bipolar cells, in common with other bipolar cell types, receive extensive negative feedback at GABA-mediated reciprocal synapses.

Morphology of bipolar cells labeled by DAPI in the rabbit retina.

The morphology, distribution, and coverage of certain cone bipolar cell types were investigated in rabbit retina. Brief in vitro incubation of isolated rabbit retina in the fluorescent dye 4,6-diamino-2-phenylindole labeled only a few cell types in the inner nuclear layer. Intracellular injection of Lucifer Yellow into these types showed them to be horizontal cells and cone bipolar cells. All stained bipolar cells ramified in sublamina a of the inner plexiform layer (IPL) and formed three classes. Two types ranged from 20 to 60 microns in diameter in both plexiform layers; the other large bipolar cell was 40-70 microns in diameter in the outer plexiform layer (OPL) and up to 150 microns in diameter in the IPL. The brightest type was narrowly stratified in the outer portion of sublamina a. Its density increased from about 500 cells/mm2 in the periphery to about 2,500 cells/mm2 in the visual streak. Staining of neighboring cells of this type showed that processes in the IPL rarely crossed, but often converged at a common site so as to impart a "honeycomb" appearance to a single sublayer of retina. The other small bipolar cell was similar in density and coverage, but stratified diffusely throughout sublamina a. The large bipolar cell stratified narrowly in the distal portion of sublamina a and was more sparsely distributed. Whether determined by staining adjacent cells or by density vs. area calculations, coverage in the OPL approached 1 for each type, as did coverage in the IPL for the two types with narrow fields.

GABA inhibits ACh release from the rabbit retina: a direct effect or feedback to bipolar cells?

The cholinergic amacrine cells of the rabbit retina may be labeled with [3H]-Ch and the activity of the cholinergic population monitored by following the release of [3H]-ACh. We have tested the effect of muscimol, a potent GABAA agonist, on (1) the light-evoked release of ACh, presumably mediated via bipolar cells, which are known to have a direct input to the cholinergic amacrine cells and (2) ACh release produced by exogenous glutamate analogs that probably have a direct effect on cholinergic amacrine cells. Muscimol blocked the light-evoked release of ACh with an IC50 of 1.0 microM. In contrast, ACh release produced by nonsaturating doses of kainate or NMDA was not reduced even by 100 microM muscimol. Thus, we have been unable to demonstrate a direct effect of GABA on the cholinergic amacrine cells. GABA antagonists, such as picrotoxin, caused a large increase in the base release and potentiated the light-evoked release of ACh. Both these effects were abolished by DNQX, a kainate antagonist that blocks the input to cholinergic amacine cells from bipolar cells. DNQX blocked the effects of picrotoxin even when controls showed that the mechanism of ACh release was still functional. Together, these results imply that the dominant site for the GABA-mediated inhibition of ACh release is on the bipolar cell input to the cholinergic amacrine cells. This is consistent with previous anatomical and physiological evidence that bipolar cells receive negative feedback from GABA amacrine cells.