A method for generating precise temporal patterns of retinal spiking using prosthetic stimulation.

The goal of retinal prosthetic devices is to generate meaningful visual information in patients that have lost outer retinal function. To accomplish this, these devices should generate patterns of ganglion cell activity that closely resemble the spatial and temporal components of those patterns that are normally elicited by light. Here, we developed a stimulus paradigm that generates precise temporal patterns of activity in retinal ganglion cells, including those patterns normally generated by light. Electrical stimulus pulses (> or =1-ms duration) elicited activity in neurons distal to the ganglion cells; this resulted in ganglion cell spiking that could last as long as 100 ms. However, short pulses, <0.15 ms, elicited only a single spike within 0.7 ms of the leading edge of the pulse. Trains of these short pulses elicited one spike per pulse at frequencies < or =250 Hz. Patterns of short electrical pulses (derived from normal light elicited spike patterns) were delivered to ganglion cells and generated spike patterns that replicated the normal light patterns. Finally, we found that one spike per pulse was elicited over almost a 2.5:1 range of stimulus amplitudes. Thus a common stimulus amplitude could accommodate a 2.5:1 range of activation thresholds, e.g., caused by differences arising from cell biophysical properties or from variations in electrode-to-cell distance arising when a multielectrode array is placed on the retina. This stimulus paradigm can generate the temporal resolution required for a prosthetic device.

Three levels of lateral inhibition: A space-time study of the retina of the tiger salamander.

The space-time patterns of activity generated across arrays of retinal neurons can provide a sensitive measurement of the effects of neural interactions underlying retinal activity. We measured the excitatory and inhibitory components associated with these patterns at each cellular level in the retina and further dissected inhibitory components pharmacologically. Using perforated and loose patch recording, we measured the voltages, currents, or spiking at 91 lateral positions covering approximately 2 mm in response to a flashed 300-microm-wide bar. First, we showed how the effect of well known lateral inhibition at the outer retina, mediated by horizontal cells, evolved in time to compress the spatial representation of the stimulus bar at ON and OFF bipolar cell bodies as well as horizontal cells. Second, we showed, for the first time, how GABA(C) receptor mediated amacrine cell feedback to bipolar terminals compresses the spatial representation of the stimulus bar at ON bipolar terminals over time. Third, we showed that a third spatiotemporal compression exists at the ganglion cell layer that is mediated by feedforward amacrine cells via GABA(A) receptors. These three inhibitory mechanisms, via three different receptor types, appear to compensate for the effects of lateral diffusion of activity attributable to dendritic spread and electrical coupling between retinal neurons. As a consequence, the width of the final representation at the ganglion cell level approximates the dimensions of the original stimulus bar.

Voltage-dependent uptake is a major determinant of glutamate concentration at the cone synapse: an analytical study.

It was suggested that glutamate concentration at the synaptic terminal of the cones was controlled primarily by a voltage-dependent glutamate transporter and that diffusion played a less important role. The conclusion was based on the observation that the rate of glutamate concentration during the hyperpolarizing light response was dramatically slowed when the transporter was blocked with dihydrokainate although diffusion remained intact. To test the validity of this notion we constructed a model in which the balance among uptake, diffusion, and release determined the flow of glutamate into and out of the synaptic cleft. The control of glutamate concentration was assumed here to be determined by two relationships; 1) glutamate concentration is the integral over the synaptic volume of the rates of release, uptake, and diffusion, and 2) membrane potential is the integral over the membrane capacitance of the dark, leak, and transporter-gated chloride current. These relationships are interdependent because glutamate uptake via the transporter is voltage dependent and because the transporter-gated current is concentration dependent. The voltage and concentration dependence of release and uptake, as well as the light-elicited, transporter-gated, and leak currents were measured in other studies. All of these measurements were incorporated into our predictive model of glutamate uptake. Our results show a good quantitative fit between the predicted and the measured magnitudes and rates of change of glutamate concentration, derived from the two interdependent relationships. This close fit supports the validity of these two relationships as descriptors of the mechanisms underlying the control of glutamate concentration, it verifies the accuracy of the experimental data from which the functions used in these relationships were derived, and it lends further support to the notion that glutamate concentration is controlled primarily by uptake at the transporter.

Spatiotemporal patterns at the retinal output.

Edge enhancement in the retina is thought to be mediated by classical center-surround antagonism, first encountered as the interactions between horizontal cells and cones. But in the salamander retina these interactions do little to enhance edges. Instead, a robust dynamic interaction between amacrine and bipolar cells appears to be responsible for a sharp edge enhancement. To demonstrate this we recorded extracellularly from a single ganglion cell and moved a flashed square, 300 micro(m) on a side, over a 1.5 x 1.0 mm2 grid at 25-micro(m) increments. Playing back all of these recordings simultaneously simulated the pattern of responses that would have been measured from an array of ganglion cells. The emerging pattern of ganglion cell activity first faithfully represented the flashed square, but after approximately 60 ms the center of the representation collapsed, leaving a representation of only the edges. We inferred that the feedback synapse from amacrine to bipolar cells at gamma-aminobutyric acid-C (GABAC) receptors mediated this effect: bicuculline and strychnine were ineffective in altering the response pattern, but in picrotoxin the center of the representation did not collapse. The GABAergic amacrine cells thought to mediate this effect have quite narrow spread of processes, so the existence of this edge-enhancing effect suggests a mechanism quite different from classical lateral inhibition, namely the delayed inhibition of a spatially expanding input pattern.

Response to change is facilitated by a three-neuron disinhibitory pathway in the tiger salamander retina.

Most retinal ganglion cells respond only transiently, for approximately 150 msec at the onset and termination of a light flash. The responses are transient because it has been shown that bipolar-to-ganglion cell transmission is truncated after 150 msec by a feedback inhibition to bipolar cell terminals. The feedback inhibition itself must be delayed by approximately 150 msec to allow the initial bipolar-ganglion cell transmission. This study identifies a three-component serial synaptic pathway from glycinergic amacrine cells to GABAergic amacrine cells to bipolar cell terminals as one source of this delay. We used perforated and whole-cell patch-clamp recordings to measure the timing of light responses in amacrine, bipolar, and ganglion cells under control and glycine and GABA receptor-blocked conditions. Our results suggest that, after a light flash, a population of glycinergic amacrine cells responds first, inhibiting a population of GABAergic amacrine cells for approximately 150 msec. The GABAergic amacrine cells feed back to bipolar terminals, but only after the 150 msec delay, allowing the bipolar terminals to excite ganglion cells for the first 150 msec. Blocking the glycinergic amacrine cell activity with strychnine allows the GABAergic system to become active earlier. GABAergic amacrine cells then inhibit release from bipolar cells earlier. Under these conditions, the ganglion cell response to change would be decreased.

Temporal contrast enhancement via GABAC feedback at bipolar terminals in the tiger salamander retina.

Most retinal amacrine (ACs) and ganglion cells (GCs) express temporal contrast by generating action potentials at only the onset and offset of the light stimulus. This study investigated the neural mechanisms that underlie this temporal contrast enhancement. Whole cell patch recordings were made from bipolar cells (BCs), ACs, and GCs in the retinal slice preparation. The cells were identified by the locations of their somas in the inner nuclear layer and ganglion cell layers, their characteristic light responses, and morphology revealed by Lucifer yellow staining. Depolarizing a single BC with a brief voltage pulse elicited a Cl- tail current that was completely abolished when Ca2+ entry to bipolar terminals was prevented, by either removing Ca2+ from the Ringer solution or blocking Ca2+ channels with Co2+. This suggests that the Cl- current is Ca2+-dependent. In those bipolar cells whose axon terminals were cutoff during slicing no Cl- current was observed, indicating that this current is generated at the synaptic terminals. The Cl- current consists of a predominant synaptic component that can be blocked by the non-N-methyl--aspartate (NMDA) glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or by the gamma-aminobutyric acid-C (GABAC) receptor antagonist picrotoxin. There also exists a relatively small nonsynaptic component. Thus both glutamatergic and GABAergic transmission were involved in the generation of this Cl- current, suggesting that it is mediated by a recurrent feedback to bipolar cells. Picrotoxin, which blocks both GABAC receptors at BC terminals and GABAA receptors on the dendrites of ACs and GCs, converted the light-elicited voltage response in most - ACs and GCs from transient to sustained. Bicuculline, which blocks only the GABAA receptors, did not prolong the transient response in - ACs and GCs. This suggests that a negative feedback mediated by the GABAC receptor on the bipolar terminals is responsible for making these responses transient. After the GABAergic feedback was blocked with picrotoxin the light-elicited voltage responses (recorded under current clamp) were more sustained than the current responses (recorded under voltage clamp) to the same light stimuli. This suggests that a voltage-dependent conductance converts the relatively transient current responses to more sustained voltage responses. Our results imply a synaptically driven local GABAergic feedback at bipolar terminals, mediated by GABAC receptors. This feedback appears to be a significant component of the mechanism underlying temporal contrast enhancement in - ACs and GCs.

Postsynaptic response kinetics are controlled by a glutamate transporter at cone photoreceptors.

We evaluated the role of the sodium/glutamate transporter at the synaptic terminals of cone photoreceptors in controlling postsynaptic response kinetics. The strategy was to measure the changes in horizontal cell response rate induced by blocking transporter uptake in cones with dihydrokainate (DHK). DHK was chosen as the uptake blocker because, as we show through autoradiographic uptake measurements, DHK specifically blocked uptake in cones without affecting uptake in Mueller cells. Horizontal cells depolarized from about -70 to -20 mV as the exogenous glutamate concentration was increased from approximately 1 to 40 microM, so horizontal cells can serve as "glutamate electrodes" during the light response. DHK slowed the rate of hyperpolarization of the horizontal cells in a dose-dependent way, but didn't affect the kinetics of the cone responses. At 300 microM DHK, the rate of the horizontal cell hyperpolarization was slowed to only 17 +/- 8.5% (mean +/- SD) of control. Translating this to changes in glutamate concentration using the slice dose response curve as calibration in Fig. 2, DHK reduced the rate of removal of glutamate from approximately 0.12 to 0.031 microM/s. The voltage dependence of uptake rate in the transporter alone was capable of modulating glutamate concentration: we blocked vesicular released glutamate with bathed 20 mM Mg2+ and then added 30 microM glutamate to the bath to reestablish a physiological glutamate concentration level at the synapse and thereby depolarize the horizontal cells. Under these conditions, a light flash elicited a 17-mV hyperpolarization in the horizontal cells. When we substituted kainate, which is not transported, for glutamate, horizontal cells were depolarized but light did not elicit any response, indicating that the transporter alone was responsible for the removal of glutamate under these conditions. This suggests that the transporter was both voltage dependent and robust enough to modulate glutamate concentration. The transporter must be at least as effective as diffusion in removing glutamate from the synapse because there is only a very small light response once the transporter is blocked. The transporter, via its voltage dependence on cone membrane potential, appears to contribute significantly to the control of postsynaptic response kinetics.

Use-dependent and use-independent blocking actions of picrotoxin and zinc at the GABAC receptor in retinal horizontal cells.

The inhibitory actions of picrotoxin (PTX) and zinc on the GABAC receptor in acutely isolated catfish cone horizontal cells were studied and compared using the whole-cell patch clamp technique. PTX blocked the GABAC current elicited by 30 microM GABA with IC50 = 0.64 microM. Over a PTX concentration range of 1-100 microM, simultaneous application of PTX with GABA (30 microM) produced current transients at both the onset and offset of the drug pulse. When the PTX concentration was maintained before, during, and after GABA application, the current transients at the onset and offset of GABA application disappeared. Thus, these transients seem to reflect a slower initial action of PTX at, and faster washout of PTX from, the GABAC receptor than GABA when they were co-applied. The full recovery from PTX inhibition required a second GABA application. Recovery could not be achieved by a prolonged wash in the absence of GABA. These results suggest that PTX effect is use-dependent. Zinc also potently blocked the GABAC current elicited by 30 microM GABA with an IC50 about an order of magnitude higher than that of PTX (IC50 = 8.2 microM). However, only the onset, but not the offset current transient was observed when zinc was simultaneously applied with GABA. The full recovery of the GABAC current from zinc inhibition was obtained after washing for 20 sec and did not require a subsequent GABA application. This indicates that the zinc effect is use-independent. Our findings suggest that: (1) the zinc binding site is on the surface of the GABAC receptor molecule; (2) there is a PTX binding site that is probably inside the receptor and its access requires GABA binding to the receptor.

Requirement for cholinergic synaptic transmission in the propagation of spontaneous retinal waves.

Highly correlated neural activity in the form of spontaneous waves of action potentials is present in the developing retina weeks before vision. Optical imaging revealed that these waves consist of spatially restricted domains of activity that form a mosaic pattern over the entire retinal ganglion cell layer. Whole-cell recordings indicate that wave generation requires synaptic activation of neuronal nicotinic acetylcholine receptors on ganglion cells. The only cholinergic cells in these immature retinas are a uniformly distributed bistratified population of amacrine cells, as assessed by antibodies to choline acetyltransferase. The results indicate that the major source of synaptic input to retinal ganglion cells is a system of cholinergic amacrine cells, whose activity is required for wave propagation in the developing retina.

Noise analysis of the glutamate-activated current in photoreceptors.

The glutamate-activated current in photoreceptors has been attributed both to a sodium/glutamate transporter and to a glutamate-activated chloride channel. We have further studied the glutamate-activated current in single, isolated photoreceptors from the tiger salamander using noise analysis on whole-cell patch-clamp recordings. In cones, the current is generated by chloride channels with a single-channel conductance of 0.7 pS and an open lifetime of 2.4 ms. The number of channels per cell is in the range of 10,000-20,000. Activation of the channels requires the presence of both glutamate and sodium. The single-channel conductance and the open lifetime of the channel are independent of the external concentration of glutamate and sodium. External glutamate and sodium affect only the opening rate of the channels. D,L-Threo-3-hydroxyaspartate (THA), a glutamate-transport blocker, is shown to be a partial agonist for the channel. The single-channel conductance is the same regardless of whether glutamate or THA is the ligand, but the open lifetime of the channel is only 0.8 ms with THA as ligand. The glutamate-activated current in rods has a similar single-channel conductance (0.74 pS) and open lifetime (3 ms). We propose a kinetic model, consistent with these results, to explain how a transporter can simultaneously act both as a sodium/glutamate-gated chloride channel and a glutamate/sodium cotransporter.

A glutamate-elicited chloride current with transporter-like properties in rod photoreceptors of the tiger salamander.

Glutamate, when puffed near the synaptic terminals, elicits a current in rod photoreceptors. The current is strongly dependent upon both the intracellular and extracellular chloride concentration: its reversal potential follows the predicted Nernst potential for a chloride permeable channel. The glutamate-elicited current also requires the presence of extracellular sodium. This glutamate-elicited current is pharmacologically like a glutamate transporter: it is elicited, in order of efficacy, by L-glutamate, L-aspartate, L-cysteate, D-aspartate, and D-glutamate, all shown to activate glutamate transport in other systems. Furthermore, it is reduced by the glutamate transport antagonists dihydrokainate (DHKA) and D,L-threo-3-hydroxyaspartate (THA). THA, when applied alone, elicits a current similar to that elicited by glutamate. The current cannot be activated by the glutamate receptor agonists kainate, quisqualate, NMDA and APB, nor can it be blocked by the glutamate receptor antagonists CNQX and APV. Thus, the current does not appear to be mediated by a conventional glutamate receptor. Taken together, the ionic dependence and pharmacology of this current suggest that it is generated by glutamate transporter coupled to a chloride channel.

Inwardly rectifying potassium conductance can accelerate the hyperpolarizing response in retinal horizontal cells.

1. We studied the activation properties and assessed the functional role of the inwardly rectifying potassium conductance (GK.IR) in acutely isolated retinal horizontal cells (HCs) with the use of the whole cell patch-clamp technique. 2. The potassium current mediated by GK.IR was isolated by the use of Cs+ or Ba2+ ions. This current was outward, although relatively small in amplitude, in the voltage range between the potassium equilibrium potential (EK) and 50-60 mV more positive. The current reversed its polarity at EK and became inward at potentials more negative than EK. When HCs were bathed in normal Ringer (EK = -90 mV), GK.IR began to active at about -30 mV, was 30-40% activated at the resting potential (-70 to -80 mV) and about fully activated at -130 mV. Thus a significant portion of the activation range of GK.IR overlaps the HC physiological response range (-20 to -80 mV). 3. GK.IR has a dramatic effect on the kinetics of membrane polarization. Blocking GK.IR with Cs+ or Ba2+ significantly slowed the rate of membrane hyperpolarization in response to a hyperpolarizing current ramp over the HC physiological response range. Blocking GK.IR also dramatically slowed the onset rate of a simulated light response generated by a brief break in a sustained glutamate puff. 4. These results suggest that GK.IR can enhance the temporal resolution of the HC by accelerating the onset rate of the hyperpolarizing light response.

Glutamate-gated chloride channel with glutamate-transporter-like properties in cone photoreceptors of the tiger salamander.

1. Using the patch-clamp technique, we investigated whether the glutamate-elicited current in mechanically isolated cone photoreceptors from the salamander retina is generated by a Cl- channel or a glutamate transporter. 2. The current reversed near the equilibrium potential for Cl-, was decreased by three Cl- channel blockers, 5-nitro-2-(3-phenyl-propylamino) benzoic acid, 4,4'-diisothiocyanostilbene-2,2'-disulfonate, and diphenylamine 2,2'-dicarboxylic acid, and was eliminated when gluconate was substituted for both internal and external Cl-, features consistent with the current being mediated by a Cl- channel. 3. The single-channel conductance of the Cl- channel was estimated by noise analysis of the glutamate-elicited current fluctuations to be 0.7 pS with an open time of 2 ms. 4. The magnitude of the current was dependent on both internal and external Na+ and K+, features consistent with the current being related to the activation of a glutamate transporter. Yet changes in their concentrations did not affect the reversal potential of the current. 5. Taken together with earlier reports on this current showing that it has a glutamate-transporter-like pharmacology, our results suggest that the glutamate-elicited current is carried by a Cl- channel but gated by a glutamate receptor whose pharmacology and ionic requirement resemble those previously described for glutamate transporters.

Dopamine modulates GABAc receptors mediating inhibition of calcium entry into and transmitter release from bipolar cell terminals in tiger salamander retina.

Using optical recording techniques, we directly monitored pre- and postsynaptic calcium dynamics at bipolar cell terminals while inhibiting synaptic release with applied GABA and modulating inhibition with dopamine. To monitor pre-synaptic activity, individual bipolar cells in the retinal slice were filled with either fura-2 or fluo-3 through a patch electrode. Calcium entry into bipolar terminals, elicited by depolarization from -60 mV to 0 mV, was reduced to 36% of control in the presence of 200 microM bath-applied GABA. Further addition of 100 microM dopamine to the bath relieved the GABAergic inhibition and nearly doubled the calcium entry. Yet dopamine alone had no apparent direct effect upon calcium entry. The relief from GABAergic inhibition could be reproduced with SKF-38393, a dopamine D1 receptor agonist, and with forskolin, an adenylyl cyclase activator, suggesting that dopamine acts through a cAMP second-messenger pathway. To monitor transmitter release from bipolar cells, slices were loaded with fura-2AM, a membrane permeable form of the dye. Puffs of 110 mM KCl at bipolar dendrites depolarized bipolar cells and elicited calcium signals that could be monitored both at bipolar terminals and in postsynaptic cells. Consistent with the results above, GABA inhibited calcium entry at bipolar terminals and also reduced transmitter release, measured as a decrease in calcium entry in amacrine and ganglion cells. The addition of dopamine relieved this inhibition and increased transmitter release. Our results show the spatiotemporal correlation between the GABAergic inhibition of calcium entry at bipolar terminals, the resulting reduction in postsynaptic activity, and the relief of this inhibition with dopamine.

Zinc downmodulates thf GABAc receptor current in cone horizontal cells acutely isolated from the catfish retina.

1. We studied the effect of zinc on the gamma-aminobutyric acid-C (GABAC) receptor in acutely isolated catfish cone horizontal cells using the whole cell patch-clamp technique. 2. GABA activates the GABAC receptor with a half-activation concentration (EC50) of 2.99 microM. The Hill coefficient is 1.32. Desensitization of the receptor is evident when GABA concentration is > 3 microM. 3. Zinc downmodulates the GABAc receptor current, elicited by 30 microM GABA, with a half-inhibition concentration (IC50) of 8.20 microM. 4. The inhibition of zinc is both competitive and noncompetitive. In the presence of 10 microM zinc, the maximum GABA response was reduced to approximately 60 percent of control and the EC50 increased to 17.32 microM, whereas the Hill coefficient (1.39) was not significantly altered. 5. The steady-state block by zinc is virtually voltage independent. 6. These results suggest that the GABAC receptor of horizontal cells can be modulated by endogenous zinc found in photoreceptors.

Low-cost data acquisition and analysis programs for electrophysiology.

In the laboratory, powerful personal computers are invaluable tools for data acquisition and analysis. The commercially available software for these purposes is often very expensive and does not always conform to the experimenter's needs. In our laboratory, we have developed two programs for acquisition and analysis of electrophysiological data from retinal neurons, although they can be used for many other types of data acquisition and analysis as well. These programs were written for IBM PC-compatible computer systems and are being distributed as shareware. Thus they are available for users to try out free of charge; if they are found to be of use, they can be purchased for a minimal cost. PATCHIT, the data-acquisition program can use one of several popular data-acquisition boards to simultaneously stimulate and record from an experimental preparation. TACK, the data analysis program was developed to analyze data recorded by PATCHIT and other data-acquisition programs. PATCHIT and TACK are powerful programs which are competitive with similar commercially available programs, but are sold at a much lower cost. Details on the development and operation of these programs are presented in this paper.

Miniature excitatory postsynaptic currents in bipolar cells of the tiger salamander retina.

The synapse between photoreceptor and bipolar cell is important for at least three reasons: (1) it is the first synapse in the visual pathway; (2) it is the best-known tonic chemical synapse; and (3) it has perhaps the most complex and highly organized synaptic morphology in the entire brain. Yet little is known about how neurotransmitter is released from this synapse. We present in this report evidence which suggests that the release of photoreceptor neurotransmitter, presumably glutamate, is probably mediated by clusters of synaptic vesicles which give rise to discrete miniature excitatory postsynaptic currents (MEPSCs) in bipolar cells. The MEPSCs are Ca(2+)-, osmotic- and CNQX-sensitive, and they share the same reversal potential (near -3 mV) as the glutamate-induced postsynaptic current. The frequency of MEPSCs increases upon presynaptic depolarization, and the mean peak conductance is about 54 pS. MEPSCs exhibit wide variations of amplitudes and durations, probably resulting from random variations of number of synaptic vesicles and the degree of synchronization in individual release clusters.

Spike initiation and propagation in wide field transient amacrine cells of the salamander retina.

Our results suggest that the prominent spike, commonly recorded in wide field amacrine cells, is actively propagated along its processes. Current was passed through a patch pipette at the soma to elicit spike activity in the cell. The field potentials accompanying this spike activity were then measured with an extracellular electrode positioned at different sites along the cell and its processes, which had been made visible with Lucifer yellow. Different extracellular waveforms were measured at the soma, stalk, and cell processes: A monophasic negative-going extracellular voltage waveform, typically found at the site of action potential initiation, was recorded along the stalk between the soma and the radial processes. A biphasic, positive-negative waveform, typically associated with the truncated propagation of an action potential, was measured at the soma. A triphasic, positive-negative-positive extracellular waveform, typically associated with a fully propagated action potential, was recorded along the peripheral processes. The time to peak of this triphasic waveform increased with distance from the soma such that the calculated propagation velocity ranged from 0.5 to 2.5 cm/sec. The membrane regions carrying potassium, sodium, and calcium currents were examined by depolarizing the soma and eliminating different ionic currents in the cell. With only sodium present, extracellular potentials were measured at the stalk and processes, but rarely at the soma. When only potassium was present, extracellular potentials were measured at the soma and processes, but not the stalk. When only calcium channels carried the membrane current, extracellular potentials were measured only at the processes. The sites of different ligand-gated receptors were identified by puffing various transmitter substances at different positions radially along the processes and measuring their effects at the soma. In all cells tested, glutamate puffs elicited currents only when applied at processes within 200 microns of the soma. In some cells, GABA and glycine elicited currents up to 300 microns from the soma. As a control for the measurement of electrotonic spread, potassium puffs elicited depolarizations along a broader region of the processes. These results suggest that the excitatory glutamate-elicited synaptic input to these cells is confined to a narrow area of the processes near the soma. The spike then appears to be initiated at the stalk and propagated along the processes. Calcium currents at these processes suggest the presence of possible transmitter release sites. Thus, each wide field amacrine cell seems to be functionally and concentrically polarized, receiving input centrally near the soma and broadcasting its output by propagating spikes along its extensive processes.

GABA transporters and GABAC-like receptors on catfish cone- but not rod-driven horizontal cells.

The effects of GABA and related agents were studied in solitary rod- and cone-driven horizontal cells, acutely isolated from the catfish retina using enzymatic and mechanical treatment. Both types of horizontal cells, which normally receive glutamatergic input from photoreceptors, responded to pressure ejection of the glutamate analog kainate (50 microM) with an inward current of 300-800 pA when voltage-clamped at -70 mV using the whole-cell patch-clamp technique. But pressure ejection of GABA (500 microM) elicited an inward current only in cone-driven horizontal cells. This current, ranging between 100 and 400 pA, consisted of two components: (1) a GABA receptor-gated chloride current that reversed near the chloride equilibrium potential and was blocked by bath application of picrotoxin (100-500 microM), and (2) a GABA transporter-mediated current that was picrotoxin resistant but was blocked by NO-711 (1 microM) and cis-4-hydroxynipecotic acid (250 microM), two potent GABA transporter blockers. The GABA transporter current could also be eliminated when sodium was replaced by either choline or lithium in the bathing medium. The picrotoxin-sensitive receptor-gated current could not be elicited by the GABAB receptor agonist baclofen, nor could it be blocked by the potent GABAB receptor antagonist 2-hydroxysaclofen. The picrotoxin-sensitive current could be divided into two components based on their sensitivity to the specific GABAA receptor antagonist bicuculline methiodide. The bicuculline-sensitive component was found only in some cells, whereas the bicuculline-resistant, picrotoxin-sensitive component was found in all cells tested. The bicuculline-resistant current was insensitive to pentobarbital, an allosteric modulator of GABAA receptor. To confirm the effectiveness of the specific batch of bicuculline methiodide and pentobarbital, we tested both drugs in ganglion cells in the salamander retinal slice preparation, where the GABA-elicited current is almost exclusively mediated by GABAA receptors. Bicuculline methiodide almost completely blocked, while pentobarbital significantly enhanced, the GABA current recorded in ganglion cells. Thus, in catfish cone horizontal cells the bicuculline-resistant GABA receptor current is most likely mediated by the GABAC receptor based on the above pharmacological profile. The relative effectiveness of GABA, muscimol, trans- and cis-4-aminocrotonic acid (TACA and CACA) was determined at this GABAC receptor site after cells were bathed in choline Ringer to eliminate the transporter current and in the presence of 100 microM bicuculline methiodide to block GABAA receptor current.(ABSTRACT TRUNCATED AT 400 WORDS)

A novel GABA receptor on bipolar cell terminals in the tiger salamander retina.

We studied the pharmacology of the GABA receptors on bipolar cell terminals in the retinal slice preparation. Whole-cell patch-clamp recordings were made from the somas of bipolar cells and GABA was puffed near their terminals, after synaptic transmission was blocked. GABA puffs evoked a large chloride current that was reduced by picrotoxin, but in many cells this current was insensitive to blockade by the competitive GABAA receptor antagonists bicuculline and SR95531. Pentobarbital, an enhancer of GABAA receptor-mediated responses, did not significantly increase the magnitude of the current responses to GABA puffed at the bipolar cell terminals. To confirm the effectiveness of GABAA antagonists and pentobarbital in the slice preparation, we measured GABA currents in ganglion cells. In contrast to bipolar cells, the ganglion cell GABA responses were strongly reduced by both bicuculline and SR95531. In addition, pentobarbital strongly enhanced the action of GABA at the ganglion cells. The isomeric GABA agonists cis- and transaminocrotonic acid (CACA and TACA), elicited picrotoxin-sensitive currents in both bipolar and ganglion cells. TACA was more effective than CACA at both cell types. In bipolar cells, TACA and CACA currents were relatively resistant to bicuculline blockade, but in ganglion cells both currents were reduced by bicuculline. GABA receptors on bipolar terminals appear to be pharmacologically different from the GABA receptors found on ganglion cell dendrites. The bipolar cell terminal GABA receptor pharmacology is similar to the pharmacology reported for the rho 1 GABA receptor subunit that was isolated from retina and expressed in Xenopus oocytes (Cutting et al., 1991; Polenzani et al., 1991; Shimada et al., 1992). This receptor, which is both bicuculline and pentobarbital insensitive, has been called the GABAC receptor (Johnston, 1986; Shimada et al., 1992). However, some bipolar cells were somewhat sensitive to blockade by bicuculline, suggesting that these cells had both GABAA and GABAC receptors on their bipolar terminals.