Salamander Retina Research Articles

Suppressive Actions of Betaxolol on Ionic Currents in Retinal Ganglion Cells May Explain its Neuroprotective Effects

Betaxolol, a β1-selective adrenoceptor antagonist, is widely used in the treatment of glaucoma. In addition to its ocular hypotensive effects, betaxolol has been suggested to act as a retinal neuroprotective agent (27). To investigate possible mechanisms underlying the neuroprotective effects, we tested the actions of betaxolol on ion channels and calcium signaling in isolated retinal ganglion cells. Betaxolol (50μM) reduced by about 20% the high-voltage-activated (HVA) Ca channel currents in ganglion cells isolated from tiger salamander retina. In contrast, the β1-adrenoceptor antagonists propranolol (10μM) and timolol (50μM) had no inhibitory actions on HVA Ca channel currents. The L -type Ca channel antagonist, nisoldipine, blocked the HVA Ca channel current partially and the remaining current was not inhibited by betaxolol. Outward current was inhibited in the presence of betaxolol. Both iberiotoxin (IBTX; 10n M), a selective inhibitor of large-conductance Ca-activated K channels, and Cd2+(100μM), which suppresses Ca-activated K channels subsequent to its block of Ca channels, reduced outward current and the remaining current was not blocked significantly with betaxolol. In the presence of betaxolol, Na channel currents were reduced by about 20%, as were currents evoked by glutamate (10m M) and GABA (1m M). Current clamp recordings from isolated ganglion cells showed that betaxolol had several effects on excitability: spike height decreased, repetitive spike activity was suppressed, spike width increased and hyperpolarization following spikes was reduced. Calcium imaging in isolated rat retinal ganglion cells revealed that betaxolol inhibited glutamate-induced increases in [Ca2+]i. These results suggest that betaxolol has a diversity of suppressive actions on ganglion cell ion channels and that, as a consequence, one of the net actions of the drug is to reduce Ca2+influx. The subsequent reduction in [Ca2+]imay contribute to the apparent neuroprotective actions of betaxolol in promoting ganglion cell survival following ischemic insult, as may occur in glaucoma and retinal disease.

Mechanisms of Glutamate Metabolic Signaling in Retinal Glial (Müller) Cells

Retinal Müller (glial) cells metabolize glucose to lactate, which is preferentially taken up by photoreceptor neurons as fuel for their oxidative metabolism. We explored whether lactate supply to neurons is a glial function controlled by neuronal signals. For this, we used subcellular fluorescence imaging and either amperometric or optical biosensors to monitor metabolic responses simultaneously from mitochondrial and cytosolic compartments of individual Müller cells from salamander retina. Our results demonstrate that lactate production and release is controlled by the combined action of glutamate and NH(4)(+), both at micromolar concentrations. Transport of glutamate by a high-affinity carrier can produce in Müller cells a rapid rise of glutamate concentration. In our isolated Müller cells, glutamine synthetase (GS) converted transported glutamate to glutamine that was released. This reaction, predominant when enough NH(4)(+) is available, was limited at micromolar concentrations of NH(4)(+), and more glutamate entered then as substrate into the mitochondrial tricarboxylic acid cycle (TCA). Increased production of glutamine by GS leads to increased utilization of ATP, some of which is generated glycolytically. Methionine sulfoximine, a specific inhibitor of GS, suppressed the stimulatory effect of glutamate and NH(4)(+) on glycolysis and induced massive entry of glutamate into the TCA cycle. The rate of glutamine production also determined the amount of pyruvate transaminated by glutamate to alanine. Lactate, alanine, and glutamine can be taken up and metabolized by photoreceptor neurons. We conclude that a major function of Müller glial cells is to nourish retinal neurons and to metabolize the neurotoxic ammonia and glutamate.

Dihydropyridine-sensitive calcium currents in bipolar cells of salamander retina are inhibited by reductions in extracellular chloride

Dihydropyridine-sensitive calcium currents ( I Ca) in photoreceptors are unusual in that they can be inhibited by reductions in extracellular chloride. The present study examined whether I Ca in retinal bipolar cells, which as in photoreceptors mediates sustained neurotransmission, is also inhibited by reductions in chloride. Nystatin-perforated patch, whole cell recordings were obtained from bipolar cells in a retinal slice preparation of larval tiger salamander. In the presence of Ba 2+, voltage steps above −40 mV evoked sustained inward currents, which were enhanced by the dihydropyridine, (−)BayK8644, and inhibited by nisoldipine. Similar to photoreceptors, replacing Cl − with gluconate or CH 3SO 4 inhibited bipolar cell I Ca and produced a negative shift in the current/voltage relationship. Thus, sensitivity to Cl − may be a more general property of L-type Ca 2+ channel subtypes that mediate sustained neurotransmission.

Somatostatin Modulates Voltage-Gated K+and Ca2+Currents in Rod and Cone Photoreceptors of the Salamander Retina

We investigated the cellular localization in the salamander retina of one of the somatostatin [or somatotropin release-inhibiting factor (SRIF)] receptors, sst(2A), and studied the modulatory action of SRIF on voltage-gated K(+) and Ca(2+) currents in rod and cone photoreceptors. SRIF immunostaining was observed in widely spaced amacrine cells, whose perikarya are at the border of the inner nuclear layer and inner plexiform layer. sst(2A) immunostaining was seen in the inner segments and terminals of rod and cone photoreceptors. Additional sst(2A) immunoreactivity was expressed by presumed bipolar and amacrine cells. SRIF, at concentrations of 100-500 nM, enhanced a delayed outwardly rectifying K(+) current (I(K)) in both rod and cone photoreceptors. SRIF action was blocked in cells pretreated with pertussis toxin (PTX) and was substantially reduced by intracellular GDP(beta)S. Voltage-gated L-type Ca(2+) currents in rods and cones were differently modulated by SRIF. SRIF reduced Ca(2+) current in rods by 33% but increased it in cones by 40%, on average. Both effects were mediated via G-protein activation and blocked by PTX. Ca(2+)-imaging experiments supported these results by showing that 500 nM SRIF reduced a K(+)-induced increase in intracellular Ca(2+) in rod photoreceptor terminals but increased it in those of cones. Our results suggest that SRIF may play a role in the regulation of glutamate transmitter release from photoreceptors via modulation of voltage-gated K(+) and Ca(2+) currents.

GABACReceptors Control Adaptive Changes in a Glycinergic Inhibitory Pathway in Salamander Retina

We studied the role of GABA in adaptive changes in a lateral inhibitory system in the tiger salamander retina. In dark-adapted retinal slice preparations picrotoxin caused a slow enhancement of glycine-mediated IPSCs in ganglion cells. The enhancement of glycinergic IPSCs developed slowly over the course of 5-20 min, even though picrotoxin blocked both GABA(A) and GABA(C) receptors within a few seconds. The slow enhancement of glycinergic IPSCs by picrotoxin was much weaker in light-adapted preparations. The slow enhancement of glycinergic inhibitory inputs was not produced by bicuculline, indicating that it involved GABA(C) receptors. The responses of ganglion cells to direct application of glycine were not enhanced by picrotoxin, indicating that the enhancement was not caused by an action on glycine receptors. In dark-adapted eyecup preparations picrotoxin caused a slow enhancement of glycinergic IPSPs and transient lateral inhibition produced by a rotating windmill pattern, similar to the effect of light adaptation. The results suggest that the glycinergic inhibitory inputs are modulated by an unknown substance whose synthesis and/or release is inhibited in dark-adapted retinas by GABA acting at GABA(C) receptors.

GABA(A) receptor binding and localization in the tiger salamander retina.

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the retina and also appears to act as a trophic factor regulating photoreceptor development and regeneration. Although the tiger salamander is a major model system for the study of retinal circuitry and regeneration, our understanding of GABA receptors in this species is almost exclusively based on the results of physiological studies. Therefore, we have examined the pharmacological binding properties of GABA(A) receptors and their anatomical localization in the tiger salamander retina. Radioligand-binding studies showed that specific 3H-GABA binding to GABA(A) receptors was dominated by a single high-affinity binding site (Kd = 15.6+/-6.9 nM). Specific binding of 3H-GABA was almost completely eliminated by muscimol (Ki = 105+/-62 nM) and bicuculline (Ki = 14.3+/-2.2 microM); however, SR-95531 only displaced about 40% of specific 3H-GABA binding (Ki = 35.0+/-3.8 nM). These data indicate that there are at least two subtypes of GABA(A) receptors present in the salamander retina that can be distinguished by their antagonist binding properties: one sensitive to both bicuculline and SR-95531, and one sensitive to bicuculline but insensitive to SR-95531. Because localization of GABA receptors in the salamander retina by immunocytochemistry is problematic, GABA(A) receptors were localized by fluorescent ligand binding combined with immunocytochemical labeling for cell specific markers. Binding of fluorescently labeled muscimol to GABA(A) receptors was present in both plexiform layers and on photoreceptor cell bodies. GABA(A) receptors in the outer plexiform layer were localized to both photoreceptor terminals and horizontal cell processes.

Amacrine cells of the anuran retina: morphology, chemical neuroanatomy, and physiology.

Amacrine cells are third-order retinal interneurons, projecting their processes into the inner plexiform layer. Historically, they were not considered as neurons first. By the middle of the 20th century, their neuronal nature was confirmed, and their enormous diversity established. Amacrine cells have been most successfully subdivided into morphological categories based on two parameters: diameter of the dendritic field and ramification pattern in the inner plexiform layer. Works combining anatomy, physiology, and neurochemistry are scarce and in the case of the anuran retina, the situation is even worse. Correlation between morphology, neurochemistry, and physiology is little studied. Here we try to build up a database and pinpoint some of the missing data. Obtaining those could help to better understand retinal function. Sporadic attempts did not make it possible to develop a comprehensive catalog of morphologically distinct amacrine cell types in the anuran retina. The number of morphologically identified amacrine cells currently stands at 16. The list of neurochemically identified distinct cell types can be given as follows: five types GABA-containing cell types with secondary markers and at least one without; two glycinergic cell types and one interplexiform cell where glycine colocalizes with somatostatin; one dopaminergic amacrine cell and also a variant of this with interplexiform morphology; two types of serotoninergic cells; three NADPHdiaphorase-positive cells, one substance P-positive cell type without identified second marker; one CCK-positive cell type without identified second marker and the calbindin positive cells (at least one but potentially more types). This adds up to 19 cell types, out of which two are interplexiform in character. This is more than that could be identified by purely morphological means. Out of Cajal's original 13 amacrine cell types described in the frog retina, 5 parallel unequivocally with neurons defined by neurochemistry. Three others have one close match each, but their exact identity is uncertain. The remaining amacrine cells have more than one potential matches. At the same time, on one hand the amacrine cell named two-layered by Cajal so far has no match among the neurochemically identified amacrine cells. On the other hand, the interplexiform subtype of the dopaminergic cell, the somatostatin-containing glycinergic interplexiform cell, the starburst cell, and the bistratified neuropeptide Y-immunoreactive cell have no match among Cajal's cells. All in all, the number of known amacrine and interplexiform cells now stands at at least 21 in the anuran retina. Physiological characterization of amacrine cells shows that their general features seem to be rather similar to those described in tiger salamander retina. In Xenopus retina, morphologically and physiologically identified amacrine cells responded to light stimulation most frequently with ON-OFF characteristics. Immunhistochemical identification of the recorded and dye injected cells showed that amacrine cells of the "same physiological type" might have different morphology. In other words, amacrine cells with different morphology can respond similarly to illumination. Even so, small differences between almost identical responses may reflect that the cell they stem from indeed belongs to different cell types.

Cannabinoid CB1 receptors and ligands in vertebrate retina: localization and function of an endogenous signaling system.

CB1, a cannabinoid receptor enriched in neuronal tissue, was found in high concentration in retinas of rhesus monkey, mouse, rat, chick, goldfish, and tiger salamander by using a subtype-specific polyclonal antibody. Immunolabeling was detected in the two synaptic layers of the retina, the inner and outer plexiform layers, of all six species examined. In the outer plexiform layer, CB1 was located in and/or on cone pedicles and rod spherules. Labeling was detected in some amacrine cells of all species and in the ganglion cells and ganglion cell axons of all species except fish. In addition, sparse labeling was found in the inner and/or outer segments of the photoreceptors of monkey, mouse, rat, and chick. Using GC/MS to detect possible endogenous cannabinoids, we found 3 nmol of 2-arachidonylglycerol per g of tissue, but no anandamide was detectable. Cannabinoid receptor agonists induced a dramatic reduction in the amplitude of voltage-gated L-type calcium channel currents in identified retinal bipolar cells. The presence and distribution of the CB1 receptor, the large amounts of 2-arachidonylglycerol found, and the effects of cannabinoids on calcium channel activity in bipolar cells suggest a substantive role for an endogenous cannabinoid signaling system in retinal physiology, and perhaps vision in general.

Modulation of horizontal cell function by GABA(A) and GABA(C) receptors in dark- and light-adapted tiger salamander retina.

The physiological function of GABA transporters and GABA receptors in retinal horizontal cells (HCs) under dark-and light-adapted conditions were studied by whole-cell voltage clamp and intracellular recording techniques in retinal slices and whole-mounted isolated retinas of the larval tiger salamander. Puff application of GABA in picrotoxin elicited a NO-711 (a potent GABA transporter blocker)-sensitive inward current that did not exhibit a reversal potential in the physiological range, consistent with the idea that these HCs contain electrogenic GABA transporters. Application of GABA in NO-711 elicited a chloride current in HCs; about half of the current was suppressed by bicuculline or I4AA (a GABA(C) receptor antagonist), and the remaining half was suppressed by bicuculline + I4AA or picrotoxin. In whole-mount retinas, NO-711, bicuculline, I4AA, or picrotoxin hyperpolarized the HCs and enhanced the light responses under dark-adapted conditions, and blocked the time-dependent recovery of HC membrane potential and light responses during background illumination. Based on the parallel conductance model, GABA released in darkness mediates a chloride conductance about three times greater than the leak conductance or the glutamate-gated cation conductance. About half of this chloride conductance is mediated by GABA(A) receptors, and the other half is mediated by GABA(C) receptors. These results suggest that GABA released from HCs through the NO-711-sensitive GABA transporters activates GABA(A) and GABA(C) receptors, resulting in chloride conductance increase which leads to a HC depolarization and reduction of the light response. Additionally, GABA transporters also mediate GABA release in background light that is responsible for the recovery of HC membrane potential and light responses.

Anatomical and electrophysiological evidence for GABAergic bipolar cells in tiger salamander retina

Our previous work showed that about 12% of bipolar cells in salamander retina synthesize and take up γ-aminobutyric acid (GABA), are GABA transporter (GAT)-immunoreactive, and respond with a GAT current to extracellularly applied GABA, suggesting that these bipolar cells use GABA, in addition to glutamate, as a neurotransmitter. Further support for this idea was obtained in this study by use of immunogold electron microscopy and whole-cell patch clamp electrophysiology. Ultrastructural analysis showed that amacrine cell and ganglion cell processes were postsynaptic to GABA-immunoreactive synapses made by bipolar cell axon terminals. Whole-cell recordings were obtained from amacrine and ganglion cells in response to activation of bipolar cells by puffing KCl at their dendrites in the outer plexiform layer. Inhibitory postsynaptic currents were observed in several third order neurons, even after blocking the excitatory postsynaptic responses, generated in the inner plexiform layer, with a combined application of NMDA and non-NMDA receptor antagonists, AP-5 and CNQX. These ultrastructural and electrophysiological data support our previous neurochemical results, and suggest that the retinal through-information pathway in salamander includes both inhibitory GABAergic as well as excitatory glutamatergic synaptic mechanisms.

Retinal Ganglion Cell Dysfunction Induced by Hypoxia and Glutamate: Potential Neuroprotective Effects of β-Blockers

The objective of this study was to examine the effects of hypoxia, glutamate, and beta-blockers on the electrical activities of retinal ganglion cells. Single-unit extracellular and whole-cell voltage clamp recording techniques were used to record electrical activities from ganglion cells in the tiger salamander retina. This was performed under physiologic conditions, hypoxia, or elevated exogenous or endogenous glutamate levels. Light-evoked spike activities, glutamate-induced currents, and voltage-gated sodium and calcium currents were measured in the presence of the beta-1 selective antagonist betaxolol or the nonselective antagonist timolol. Hypoxia resulted in suppressing or blocking the OFF responses in the majority of ON-OFF ganglion cells tested, whereas the ON responses were only slightly affected. The presence of increased glutamate had similar findings and demonstrated an increase in the spontaneous firing rate of retinal ganglion cells. Betaxolol (2–50 μM) reduced the rate of spontaneous firing of retinal ganglion cells induced by glutamate. At 2 to 50 μM, betaxolol reversibly reduced the voltage-gated sodium currents and calcium currents in retinal ganglion cells. Timolol (up to 100 μM) did not demonstrate any detectable action on these currents. The physiologic responses of retinal ganglion cells to hypoxia or elevated glutamate levels in this animal model appear to be very similar. Although short-term exposure to hypoxia and glutamate used in this study exerts reversible actions on ganglion cells and does not induce permanent cell damage, such initial physiologic actions are likely to be precursors of permanent cell damage. Thus, hypoxia and elevated glutamate levels in the retina may represent a final pathway in diseases affecting retinal ganglion cells, such as glaucoma. Similar damage could result from different factors, such as decreased perfusion-induced ischemia or anomalous neuronal processing of glutamate. Betaxolol exerts its primary neuronal actions on retinal ganglion cells. It reversibly blocked voltage-gated calcium current and reduced the spontaneous firing rate by suppressing glutamate-gated currents and sodium currents in ganglion cells. These actions may protect ganglion cells from damage caused by ischemia or elevated glutamate levels.

Spatial heterogeneity and function of voltage- and ligand-gated ion channels in retinal amacrine neurons.

The spatial distribution of ion channels within amacrine cells of the tiger salamander retina was studied using patch recording in the retinal slice preparation. By focally puffing kainate, GABA and glycine at amacrine cell processes in the inner plexiform layer, it was determined that the cell's glutamate receptors were located in a confined region of the processes near the soma, while glycine and GABA receptors were located throughout the processes. Likewise, similar techniques in conjunction with voltage steps demonstrated that voltage-gated sodium channels were located throughout the cell and were shown to generate sodium-dependent spikes, while only the processes contained voltage-gated calcium channels. These results suggest that this form of transient amacrine cell collects its excitatory synaptic inputs in a region confined to a central annular region near the soma, that the signal is actively propagated throughout its processes by voltage-gated sodium channels and that calcium-dependent neurotransmitter release of glycine from this neuron can occur throughout its processes. Thus, excitatory signals are collected in the processes near the soma, inhibitory signals throughout the processes and excitation is probably propagated throughout the processes of the amacrine cell.

Metabotropic GABA receptors facilitate L-type and inhibit N-type calcium channels in single salamander retinal neurons.

1. Whole-cell voltage clamp experiments were performed on isolated spiking retinal neurons from the salamander retina. Calcium channel currents were studied using barium as the charge carrier while potassium and sodium currents were suppressed with TEA and TTX, respectively. 2. Baclofen, a metabotropic GABA receptor agonist, both enhanced and suppressed high-voltage-activated calcium channel current. Baclofen facilitated an L-type channel current, and this effect was not voltage dependent. As reported previously, baclofen inhibited an N-type channel current and this action was voltage dependent. 3. While the suppressive effect was mediated by a fast-acting, direct G-protein action, the facilitatory effect was slower and was blocked by inhibitors of protein kinase C (PKC), either GF-109203x or the PKC (19-36) sequence fragment. 4. The pharmacology of the inhibitory and facilitatory responses differed. Commonly used antagonists of metabotropic GABA receptors, CGP35348 and CGP55845, were more potent antagonists of the inhibitory response. Similarly, a selective agonist at the metabotropic GABA receptor, APMPA, was also more effective in eliciting the inhibitory response. 5. These observations indicate that there may be two baclofen-sensitive metabotropic GABA receptors with opposing effects on calcium channel current. This is the first description of a facilitatory action of GABAB receptors and indicates that GABA may not function exclusively as an inhibitory transmitter.

The Metabotropic Receptor mGluR6 May Signal Through Go, But Not Phosphodiesterase, in Retinal Bipolar Cells

Bipolar cells are retinal interneurons that receive synaptic input from photoreceptors. Glutamate, the photoreceptor transmitter, hyperpolarizes On bipolar cells by closing nonselective cation channels, an effect mediated by the metabotropic receptor mGluR6. Previous studies of mGluR6 transduction have suggested that the receptor couples to a phosphodiesterase (PDE) that preferentially hydrolyzes cGMP, and that cGMP directly gates the nonselective cation channel. This hypothesis was tested by dialyzing On bipolar cells with nonhydrolyzable analogs of cGMP. Whole-cell recordings were obtained from On bipolar cells in slices of larval tiger salamander retina. Surprisingly, On bipolar cells dialyzed with 8-(4-chlorophenylthio)-cyclic GMP (8-pCPT-cGMP), or 8-bromo-cyclic GMP (8-Br-cGMP) responded normally to glutamate or L-2-amino-4-phosphonobutyrate (L-APB). Response amplitudes and kinetics were not significantly altered compared with cells dialyzed with cGMP alone. Comparable results were obtained with the PDE inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) or with 8-pCPT-cGMP and IBMX together, indicating that PDE is not required for mGluR6 signal transduction. Addition of the G-protein subunit G(o)alpha to the pipette solution suppressed the cation current and occluded the glutamate response, whereas dialysis with G(i)alpha or with transducin Gbetagamma had no significant effect on either the cation current or the response. Dialysis of an antibody directed against G(o)alpha also reduced the glutamate response, indicating a functional role for endogenous G(o)alpha. These results indicate that mGluR6 may signal through G(o), rather than a transducin-like G-protein.

Impulse encoding across the dendritic morphologies of retinal ganglion cells.

Nerve impulse entrainment and other excitation and passive phenomena are analyzed for a morphologically diverse and exhaustive data set (n = 57) of realistic (3-dimensional computer traced) soma-dendritic tree structures of ganglion cells in the tiger salamander (Ambystoma tigrinum) retina. The neurons, including axon and an anatomically specialized thin axonal segment that is observed in every ganglion cell, were supplied with five voltage- or ligand-gated ion channels (plus leakage), which were distributed in accordance with those found in a recent study that employed an equivalent dendritic cylinder. A wide variety of impulse-entrainment responses was observed, including regular low-frequency firing, impulse doublets, and more complex patterns involving impulse propagation failures (or aborted spikes) within the encoder region, all of which have been observed experimentally. The impulse-frequency response curves of the cells fell into three groups called FAST, MEDIUM, and SLOW in approximate proportion as seen experimentally. In addition to these, a new group was found among the traced cells that exhibited an impulse-frequency response twice that of the FAST category. The total amount of soma-dendritic surface area exhibited by a given cell is decisive in determining its electrophysiological classification. On the other hand, we found only a weak correlation between the electrophysiological group and the morphological classification of a given cell, which is based on the complexity of dendritic branching and the physical reach or "receptive field" area of the cell. Dendritic morphology determines discharge patterns to dendritic (synaptic) stimulation. Orthodromic impulses can be initiated on the axon hillock, the thin axonal segment, the soma, or even the proximal axon beyond the thin segment, depending on stimulus magnitude, soma-dendritic membrane area, channel distribution, and state within the repetitive impulse cycle. Although a sufficiently high dendritic Na-channel density can lead to dendritic impulse initiation, this does not occur with our "standard" channel densities and is not seen experimentally. Even so, impulses initiated elsewhere do invade all except very thin dendritic processes. Impulse-encoding irregularities increase when channel conductances are reduced in the encoder region, and the F/I properties of the cells are a strong function of the calcium- and Ca-activated K-channel densities. Use of equivalent dendritic cylinders requires more soma-dendritic surface area than real dendritic trees, and the source of the discrepancy is discussed.

Correlated firing in rabbit retinal ganglion cells.

A ganglion cell's receptive field is defined as that region on the retinal surface in which a light stimulus will produce a response. While neighboring ganglion cells may respond to the same stimulus in a region where their receptive fields overlap, it generally has been assumed that each cell makes an independent decision about whether to fire. Recent recordings from cat and salamander retina using multiple electrodes have challenged this view of independent firing by showing that neighboring ganglion cells have an increased tendency to fire together within +/-5 ms. However, there is still uncertainty about which types of ganglion cells fire together, the mechanisms that produce coordinated spikes, and the overall function of coordinated firing. To address these issues, the responses of up to 80 rabbit retinal ganglion cells were recorded simultaneously using a multielectrode array. Of the 11 classes of rabbit ganglion cells previously identified, coordinated firing was observed in five. Plots of the spike train cross-correlation function suggested that coordinated firing occurred through two mechanisms. In the first mechanism, a spike in an interneuron diverged to produce simultaneous spikes in two ganglion cells. This mechanism predominated in four of the five classes including the ON brisk transient cells. In the second mechanism, ganglion cells appeared to activate each other reciprocally. This was the predominant pattern of correlated firing in OFF brisk transient cells. By comparing the receptive field profiles of ON and OFF brisk transient cells, a peripheral extension of the OFF brisk transient cell receptive field was identified that might be produced by lateral spike spread. Thus an individual OFF brisk transient cell can respond both to a light stimulus directed at the center of its receptive field and to stimuli that activate neighboring OFF brisk transient cells through their receptive field centers.

Automatic characterization and classification of ganglion cells from the salamander retina.

The classification of retinal ganglion cells according to their morphological features is addressed by using a comprehensive set of shape measures and several clustering strategies. The morphological features considered include many common measures (such as dendritic radii and the number of dendritic segments) and three new quantifiable measures: 1) the area of influence of the dendritic tree as calculated in an operator-independent manner by using Minkowski sausages; 2) the complexity of tortuousity along each dendritic segment as represented by the 3D bending energy; and 3) the coverage factor as calculated by using the Bouligand-Minkowski fractal dimension, which is more accurate than the commonly used box-counting algorithm. We evaluated four clustering approaches including the k-means and Ward's hierarchical clustering methods. By using these highly quantifiable methods to group the cells into classes, the present work has extended and reassessed the analysis of 68 ganglion cells from the tiger salamander previously classified by Toris et al. ([1995] J. Comp. Neurol. 352:535-559). Though substantiating the number of classes (5) previously proposed by Toris et al., the results obtained here indicate a number of discrepancies among the members of each class, especially regarding the border between two classes, originally called the medium simple and the medium complex cells. Such an effect has motivated the proposal of new names for the medium simple and medium complex classes, now called small highly complex and medium cells, respectively. Also included in the present article are comprehensive statistics of each class, correlations among all the adopted shape measures, and examples of the cells from each class. The resultant classes that emerged were compared using their electrotonic characteristics and physiological profiles.

Rapid desensitization converts prolonged glutamate release into a transient EPSC at ribbon synapses between retinal bipolar and amacrine cells.

The mechanisms underlying the conversion of prolonged glutamate release from ribbon synapses in bipolar cells to sustained and transient excitatory postsynaptic responses in identified retinal amacrine cells were studied in tiger salamander (Ambystoma tigrinum) retina. A retina slice preparation with whole cell patch recording techniques under voltage- and current-clamp conditions was used to assay the electrical properties of bipolar and amacrine cells. Amacrine cells were categorized into two basic forms: (i) transient amacrine cells that respond to a step of light with a burst of spikes only at the transitions of the step; and (ii) sustained amacrine cells that respond with continuous spiking during the entire light step. The two cell types each had a characteristic morphology: transient amacrine cells possessed wide dendritic fields (chi = 375 microns), while sustained cells had much more narrowly confined dendritic fields (chi = 85 microns). Whole cell voltage-gated currents of the transient and sustained cell types were not significantly different. Both cell types had spikes that were sensitive to tetradotoxin (TTX, 0.3 microM) with voltage deflections of up to 100 mV. Light-evoked excitatory synaptic currents relaxed rapidly in transient neurons (tau 1/2 = 100 ms) and more slowly in sustained neurons (tau 1/2 = 1.2 s). EPSCs in both cells reversed near 0 mV. Rapid application of glutamate or kainate elicited rapidly desensitizing ionic currents (tau 1/2 = 85 ms) followed by a slowly desensitizing component. Cyclothiazide, a drug that eliminates rapid desensitization, lengthened the time course of the glutamate gated current from tau 1/2 = 85 ms to about 3 s, and the relaxation kinetics of the glutamatergic EPSC from tau 1/2 = 85 ms to about 1.0 s. These data suggest that a key determinant in forming transient versus sustained responses in amacrine cells of vertebrate retina is the differences in their excitatory, glutamatergic synaptic inputs, and that rapid desensitization of glutamate receptors plays a role in converting the presynaptic signal associated with sustained glutamate release into a postsynaptic, transient signal at the ribbon synapse.

Calcium-independent release of neurotransmitter in the retina: a “Copernican” viewpoint change

The release of synaptic transmitter in chemical synapses is brought about by Ca2+ influx through voltage-dependent Ca2+ channels opened by depolarisation of presynaptic terminals. However, in some preparations transmitter release persists or increases in low-Ca2+ media, and it has therefore been proposed that transmitter release could also occur through a Ca2+-independent, carrier mediated process. In particular it has been suggested that this may be the case for synaptic transmission between photoreceptors and second order neurones of the vertebrate retina. From our recent experiments on synaptic transmission from photoreceptors to horizontal cells of turtle and salamander retinas, it appears that lowering extracellular Ca2+ can actually promote Ca2+ influx through voltage-activated Ca2+ channels via a modification of surface potential of plasma membranes. On the basis of this apparently paradoxical effect of low Ca2+ media, it is possible to reaccommodate the so-called Ca2+-independent release within the framework of Ca2+-dependent synaptic transmission without invoking unconventional mechanisms.

Cloning and localization of RPE65 mRNA in salamander cone photoreceptor cells

RPE65 is a potential retinoid-processing protein expressed in the retinal pigment epithelium. Mutations in the RPE65 gene have been shown to cause certain inherited retinal dystrophies. Previous studies have shown that salamander cone photoreceptor cells have a unique retinoid processing mechanism which is distinct from that of rods. To determine whether RPE65 is expressed in photoreceptors, the RPE65 cDNA was cloned from a salamander retinal cDNA library. The deduced protein consists of 533 amino acids and is 85% identical to human and bovine RPE65. The RPE65 mRNA was detected in all of the single cone cells isolated from the salamander retina, as well as in the retinal pigment epithelium by RT-PCR, but not in the isolated rods. The RT-PCR products have been confirmed to be RPE65 by DNA sequencing. The results indicate that this potential retinoid processing protein is expressed in the cone photoreceptor cells but not in rods. Therefore, this protein may contribute to the unique retinoid processing capabilities in salamander cones.