Caffeine-sensitive calcium stores regulate synaptic transmission from retinal rod photoreceptors.

We investigated the role of caffeine-sensitive intracellular stores in regulating intracellular calcium ([Ca(2+)](i)) and glutamatergic synaptic transmission from rod photoreceptors. Caffeine transiently elevated and then markedly depressed [Ca(2+)](i) to below prestimulus levels in rod inner segments and synaptic terminals. Concomitant with the depression was a reduction of glutamate release and a hyperpolarization of horizontal cells, neurons postsynaptic to rods. Caffeine did not affect the rods' membrane potentials indicating that caffeine likely acted via some mechanism(s) other than a voltage-dependent deactivation of the calcium channels. Most of caffeine's depressive action on [Ca(2+)](i), on glutamate release, and on I(Ca) in rods can be attributed to calcium release from stores: (1) caffeine's actions on [Ca(2+)](i) and I(Ca) were reduced by intracellular BAPTA and barium substitution for calcium, (2) other nonxanthine store-releasing compounds, such as thymol and chlorocresol, also depressed [Ca(2+)](i), and (3) the magnitude of [Ca(2+)](i) depression depended on basal [Ca(2+)](i) before caffeine. We propose that caffeine-released calcium reduces I(Ca) in rods by an as yet unidentified intracellular signaling mechanism. To account for the depression of [Ca(2+)](i) below rest levels and the increased fall rate of [Ca(2+)](i) with higher basal calcium, we also propose that caffeine-evoked calcium release from stores activates a calcium transporter that, via sequestration into stores or extrusion, lowers [Ca(2+)](i) and suppresses glutamate release. The effects of store-released calcium reported here operate at physiological calcium concentrations, supporting a role in regulating synaptic signaling in vivo.

Somatostatin modulates voltage-gated K(+) and Ca(2+) 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.

Rod sensitivity and visual pigment concentration in Xenopus.

Xenopus larvae were raised on a vitamin A-free diet under constant illumination until their visual pigment content had decreased to between 8% of normal and an undetectably low level. After the intramuscular injection of 2.1 X 10(13-2.1 X 10(16) molecules of [3H]vitamin A, ocular tissue showed a rapid rate of uptake of label which reached a maximum level of incorporation by 48 h. Light-microscopic autoradiography revealed that the retinal uptake of label was concentrated within the receptor outer segments. Spectral transmissivity measurements at various times after injection were made upon intact retinas and upon digitonin extracts. They showed that visual pigment with a lambdamax of 504 nm was formed in the retina and that the amount formed was a function of incubation time and the magnitude of the dose administered. Electrophysiological measures of photoreceptor light responses were obtained from the PIII component of the electroretinogram, isolated with aspartate. The quantal flux required to elicit a criterion response was determined and related to the fraction of visual pigment present. The results showed that rod sensitivity varied linearly with the probability of quantal absorption.

Transport and phosphorylation of 2-deoxy-D-glucose by amphibian retina. Effects of light and darkness.

We studied the uptake of 2-deoxy-D-glucose (2DG) and the synthesis of its phosphorylated product 2DG-6-phosphate (2DG-6P) by the retinas of the clawed frog (Xenopus laevis) and the bullfrog (Rana catesbeiana). Autoradiographs showed that most of the retinal 2DG uptake is by the photoreceptor layer. The 2DG accumulation by isolated Xenopus retinas was time and concentration dependent. The Kt for transport was 5.05 mM; Vmax was 6.99 X 10(-10) mol . mg-1 tissue wet weight min-1. The Km for 2DG-6P formation was estimated to be 2-3 mM and Vmax to be approximately 4 x 10(-9) mol . mg-1 min-1. 2DG uptake was inhibited competitively by glucose with a Ki of 2.29 mM. Exposure to light reduced 2DG uptake by no more than 10% as compared with dark uptake. Low sodium or ouabain (10(-4)-10(-7) M) treatment did not significantly alter 2DG uptake as compared with control retinas. In experiments upon intact, anesthetized bullfrogs, light reduced both the total amount of radioactivity acquired by the retina and the fraction of 2DG-6P present. The results are discussed in terms of the fraction of energy consumed by the retina required to maintain the photoreceptor dark current.

Action spectra and adaptation properties of carp photoreceptors.

The mass photoreceptor response of the isolated carp retina was studied after immersing the tissue in aspartate-Ringer solution. Two electro-retinogram components were isolated by differential depth recording: a fast cornea-negative wave, arising in the receptor layer, and a slow, cornea-negative wave arising at some level proximal to the photoreceptors. Only the fast component was investigated further. In complete dark adaptation, its action spectrum peaked near 540 nm and indicated input from both porphyropsin-containing rods (lambda(max) approximately 525 nm) and cones with longer wavelength sensitivity. Under photopic conditions a broad action spectrum, lambda(max) approximately 580 nm was seen. In the presence of chromatic backgrounds, the photopic curve could be fractionated into three components whose action spectra agreed reasonably well with the spectral characteristics of blue, green, and red cone pigments of the goldfish. In parallel studies, the carp rod pigment was studied in situ by transmission densitometry. The reduction in optical density after a full bleach averaged 0.28 at its lambda(max) 525 nm. In the isolated retina no regeneration of rod pigment occurred within 2 h after bleaching. The bleaching power of background fields used in adaptation experiments was determined directly. Both rods and cones generated increment threshold functions with slopes of +1 on log-log coordinates over a 3-4 log range of background intensities. Background fields which bleached less than 0.5% rod pigment nevertheless diminished photoreceptor sensitivity. The degree and rate of recovery of receptor sensitivity after exposure to a background field was a function of the total flux (I x t) of the field. Rod saturation, i.e. the abolition of rod voltages, occurred after approximately 12% of rod pigment was bleached. In light-adapted retinas bathed in normal Ringer solution, a small test flash elicited a larger response in the presence of an annular background field than when it fell upon a dark retina. The enhancement was not observed in aspartate-treated retinas.

Formation, conversion, and utilization of isorhodopsin, rhodopsin, and porphyropsin by rod photoreceptors in the Xenopus retina.

The visual pigment content of rod photoreceptors in Xenopus larvae was reduced greater than 90% through a combination of vitamin A-deficient diet and constant light. Thereafter, a dose of either all-trans-retinol or 9-cis-retinal was injected intramuscularly, leading to the formation of a rhodopsin (lambdamax 504 nm) or isorhodopsin (lambdamax 487-493 nm) pigment, respectively. Electrophysiological measurements were made of the threshold and spectral sensitivity of the aspartate-isolated PIII (photoreceptoral) component of the electroretinogram. These measures established that either rhodopsin or isorhodopsin subserved visual transduction with the same efficiency as the 519 nm porphyropsin pigment encountered normally. When animals with rhodopsin or isorhodopsin were kept in darkness or placed on a cyclical lighting regimen for 8 days, retinal densitometry showed that either pigment was being converted to porphyropsin; significantly more porphyropsin was formed as a result of cyclical lighting than after complete darkness.

Slow PIII component of the carp electroretinogram.

The slow PIII component of the electroretinogram (ERG) was studied in the isolated, aspartate-treated carp retina. Although the latter is richly populated with cones, slow PIII appeared to reflect almost exclusively the activity of rods; e.g. the spectral sensitivity of the potential paralleled closely the rod pigment curve, its operating range (i.e. the V-log I curve) was limited to 3 log units above absolute threshold, and raising background intensities to photopic levels produced saturation of the increment threshold function without evidence of a cone-mediated segment. Only after bleaching away a significant fraction of the porphyropsin was it possible to unmask a small photopic contribution to slow PIII, as evidenced by a displacement in the action spectrum to longer wavelengths. The spatial distribution of the slow PIII voltage within the retina (Faber, D.S. 1969. Ph.D. Thesis. State University of New York. Buffalo, N.Y.; Witkovsky, P.J. Nelson, and H. Ripps. 1973. J. Gen Physiol. 61:401) and its ability to survive aspartate treatment indicate that this potential arises in the Müller (glial) fiber. Additional support for this conclusion is provided by the slow rise time (several seconds) and long temporal integration (up to 40s) of the response. In many respects the properties of slow PIII resemble those of the c-wave, a pigment epithelial response also subserved by rod activity. On the other hand, the receptoral (fast PIII) and the b-wave components of the ERG behave quite differently. Unlike slow PIII, response saturation could not be induced, since both potentials are subserved by cones when the stimulus conditions exceed the limits of the scotopic range. Receptors appear to govern light adaptation at photopic background levels; both fast PIII and b-wave manifest identical incremental threshold values over this range of intensities. However, under scotopic conditions, the sensitivity of the b-wave is affected by luminous backgrounds too weak to alter fast PIII threshold, indicating a postreceptoral stage of adaptation.

Glutamate receptors and circuits in the vertebrate retina.

We survey the evidence for L-glutamate's role as the primary excitatory neurotransmitter of vertebrate retinas. The physiological and molecular properties of glutamate receptors in the retina are reviewed in relation to what has been learned from studies of glutamate function in other brain areas and in expression systems. We have focused on (a) the evidence for the presence of L-glutamate in retinal neurons, (b) the processes by which glutamate is released, (c) the presence and function of ionotropic receptors for L-glutamate in retinal neurons, (d) the presence and function of metabotropic receptors for L-glutamate in retinal neurons, and (e) the variety and distribution of glutamate transporters in the vertebrate retina. Modulatory pathways which influence glutamate release and the behavior of its receptors are described. Emphasis has been placed on the cellular mechanisms of glutamate-mediated neurotransmission in relation to the encoding of visual information by retinal circuits.

Involvement of D1 and D2 Dopamine Receptors in the Control of Horizontal Cell Electrical Coupling in the Turtle Retina.

We studied the actions of D1 and D2 dopamine agonists and antagonists on the coupling of horizontal cell axons in the turtle retina by a combination of pharmacological and electrophysiological methods. Both D1 and D2 receptors were identified in membrane fractions by radioligand binding using [3H]-SCH 23390 and [3H]-spiperone, respectively. The KD of both receptor classes were identical (0.21 nM) but D1 receptor density exceeded that of D2 receptors by more than four-fold. D1 agonists increased the activity of adenylate cyclase in a dose-dependent manner, whereas D2 agonists were without significant effect by themselves, nor did D2 antagonists block the D1-mediated increase in adenylate cyclase activity. Intracellular recordings and Lucifer Yellow dye injections were used to characterize the modifications of the receptive field profile of horizontal cell axons (H1AT) exposed to different pharmacological agents. Dopamine or D1 agonists (0.05 - 10 microM) induced a marked constriction of the H1AT receptive field, whereas D2 agonists elicited a small expansion of the receptive field. However, in the presence of a D1 antagonist, as well as IBMX to inhibit phosphodiesterase, D2 agonists (10 - 70 microM) induced a marked increase in the receptive field profile. These results indicate that both D1 and D2 dopamine receptors play a role in shaping the receptive field profile of the horizontal cell axon terminal in the turtle retina.

Uptake and localization of 3H-2 deoxy-D-glucose by retinal photoreceptors.

Following dark incubation of isolated retinas of Xenopus laevis in Ringer solution supplemented with 3H-2-Deoxy-D-glucose (2DG), virtually all of the uptake of the label was by the inner segments and synaptic bases of the photoreceptor cells. Autoradiographs prepared from conventionally fixed tissue showed the same cellular distribution of label as those prepared from identically incubated, unfixed, freeze-dried retinas. However, fixation removed about 77% of the total counts. This fixation-labile, soluble fraction was identified as being primarily 2DG-6 phosphate by thin-layer chromatography. The remaining insoluble fraction corresponded in distribution to glycogen grains. In cones, glycogen is stored primarily in the paraboloid, whereas in rods it is distributed throughout the inner segment and synaptic base. EM autoradiographs illustrated that these were the sites over which fixation-resistant 2DG label was localized. Measurements of radioactivity associated with extracts of retinal glycogen following 2DG incubation demonstrated that a disproportionately high fraction of total counts were associated with the glycogen fraction. We conclude that in the amphibian retina 2DG may be incorporated into glycogen.

Synaptic connections linking cones and horizontal cells in the retina of the pikeperch (Stizostedion vitreum).

Cones and horizontal cells of the pikeperch retina were studied with morphological and physiological techniques. Gap junctions were observed between cone pedicles and basal processes emitted by neighboring cones. Intracellular recordings showed that the light-evoked hyperpolarizing cone response was enhanced by light falling upon neighboring receptors within a radius of 50 microns. We suggest that the network of gap junctions between cones mediates the summative lateral interaction described. Three sub-classes of horizontal cells (H1, H2, H3) send dendrites to cones; H1 and H2 cells appear to contact twin cones, exclusively or preferentially, whereas H3 cells appear to synapse only with single cones. Horizontal cells of the same sub-class are joined by gap junctions between dendrites or at the lateral faces of perikarya. These unions extend over several micron 2 and as seen in transmission electron microscopy consist of patches of close apposition alternating with areas of membrane separation, folding and occasional zonulae adherents. Freeze-fracture profiles of horizontal gap junctions show localized areas of dense particle aggregation on the P-face and pits on the E-face flanked by regions of unspecialized membrane. These morphological findings provide support for the known spatial and color-coding properties of pikeperch horizontal cells.

Retinal neurochemistry of three elasmobranch species: an immunohistochemical approach.

We surveyed retinas of Raja erinacea, Mustelus canis, and Squalus acanthias for neurotransmitter substances by using antisera directed against the substances themselves or against their synthesizing enzymes. Both the peroxidase-antiperoxidase (PAP) and indirect fluorescent techniques were employed to visualize the primary antisera. In all three species positive results were obtained with antisera directed against tyrosine hydroxylase (TOH), glutamic acid decarboxylase (GAD), serotonin (5-HT), and leucine enkephalin (Lenk). Antisera directed against glucagon, neurotensin, beta-endorphin, vasoactive intestinal peptide, or bombesin failed to show any specific staining. Immunoreactivity was located in amacrine, interplexiform, and horizontal cells as well as in axons of the optic fiber layer. The four antisera labelled different amacrine cell classes, distinguished on the bases of perikaryal morphology and the distribution of cell processes in the inner plexiform layer (IPL). Amacrine cells that labelled with the same marker were seen to have different morphologies in the species studied. Thus, TOH-like immunoreactivity was distributed in layers 1, 3, and 5 of the IPL in Mustelus but only in layers 1 and 3 in Raja retina. GAD-like immunoreactivity was found diffusely over all layers of the IPL in Raja, but in Mustelus it was confined primarily to layers 1, 3, and 5 of the IPL. Lenk- and 5-HT-like immunoreactivities showed similar species variations. Two neurochemical classes of interplexiform cell were identified in this study. In Mustelus GAD-like and Lenk-like immunoreactive interplexiform cells were seen whereas in Raja only GAD-positive interplexiform cells were detected. In squalus no unequivocal demonstration of any interplexiform cell was made with these antisera. The GAD antiserum also labelled a subset of the horizontal cells in the dorsal retina of Raja. TOH and 5-HT-antisera labelled axons in the optic fiber layer of all three species but reactive ganglion cell perikarya were not identified.

Gap junctions among the perikarya, dendrites, and axon terminals of the luminosity-type horizontal cell of the turtle retina.

Gap junctions of the H1 horizontal cell of the turtle retina (Leeper, '78) were studied in thin-sectioned material and in freeze-fracture replicas. Perikaryal gap junctions were extremely restricted, 0.02-0.07 micron2 in in area, whereas those of axon terminals were much larger, most being 0.1-1.0 micron2. Both varieties, however, had the usual seven-layered appearance in thin section and measured 15 +/- 1 nm in overall width between cytoplasmic faces. Freeze-fractured views of the perikaryal junctions revealed roughly circular patches of P-face 9-nm particles and E-face pits. The axon terminal gap junctions were seen as large areas of P-face particles and E-face pits containing occasional islands of unspecialized membrane. Particle densities varied from 1,455 to 2,448 microns-2. A serial reconstruction was made of a portion of the axon terminal network in order to measure the surface areas of the axons contained therein and the fraction occupied by gap junctions. These data demonstrated that the fractional area occupied by gap junctions was roughly in inverse proportion to the area of the axon region (tuberous core vs. terminal process). It is argued that this constitutes an impedance matching device to ensure adequate current flow through the axon processes. Assuming that each P-face particle represents a connection having a conductance of 10(-10) S and given the P-face particle density and gap junctional areas determined in this report, we calculated that the gap junction distribution is adequate to account for the spatial properties of the horizontal cell axon network (Lamb, '76).

Morphology and synaptic connections of HRP-filled, axon-bearing horizontal cells in the Xenopus retina.

Axon-bearing horizontal cells of the Xenopus retina were studied by intracellular injection of HRP following physiological characterization. The profile of the cell viewed in whole mount consisted of a round or oval perikaryon about 50 microns in diameter and an axon about 1 mm long which lacked a prominent terminal expansion. The axonal diameter was 0.5-1.0 microns in its proximal third but 2-4 microns in its distal portion. Along its course the axon emitted 25-40 branchlets each 0.2 micron in diameter, up to 10 micron long and terminating in a cluster of two to six synaptic knobs. Electron microscopic examination revealed that both perikaryal dendrites and axon branchlets ended in both rod and cone synaptic bases; cone contacts outnumbered rod contacts by two- to threefold. We were unable to document synapses of presumed interplexiform cells onto identified horizontal cells. Horizontal cell axons are joined in their distal portions by numerous, small (0.2 micron long) gap junctions. Other gap junctions were noted between horizontal cell processes within the synaptic endings of photoreceptors. An hypothesis is advanced whereby the cluster of axon branchlet synaptic knobs permits dynamic interaction of rod and cone synaptic inputs to the horizontal cell.

Catecholamine- and indoleamine-containing neurons in the turtle retina.

We identified a population of presumed dopaminergic amacrine cells and populations of presumed serotonergic bipolar and amacrine cells in the retina of the turtle Pseudemys scripta elegans by a combination of autoradiographic, fluorescence, and immunocytochemical techniques. Antisera directed against the dopamine-synthesizing enzyme, tyrosine hydroxylase (TOH), stained perikarya located at the border of inner nuclear (INL) and inner plexiform (IPL) layers. Processes emitted by these cells arborized in sublaminae 1, 3, and 5 of the IPL. Incubation of retinas in 10(-6) M 3H-dopamine yielded a labeling pattern identical to the staining pattern achieved with TOH antisera, but when the concentration of 3H-dopamine was increased 25-fold, both amacrine and bipolar cells are labeled. Following intraocular injection of dopamine, fluorescence micrography revealed both stained amacrine and bipolar cells. The bipolar cells had Landolt's clubs, pyriform perikarya located in the distal portion of the INL, and axons that coursed horizontally in the INL, then entered the IPL, and ramified in both its superficial and deeper layers. Although no fluorescent neuronal profiles were revealed following injection of serotonin (5HT), bipolar cells identical to those described were visualized with 5HT antisera. The intensity of bipolar cell staining with 5HT antisera was improved by preinjection of the eye with exogenous 5HT. We suggest that the bipolar cell is serotonergic, but that it also can actively accumulate dopamine. The 5HT antisera also stained a population of large amacrine cells whose processes ramified in IPL sublaminae 1, 4, and 5. The same populations of presumed serotonergic bipolar and amacrine cells were labeled following incubation of the eyecup in 10(-6) M 3H-5HT.

Diurnal and circadian variation of protein kinase C immunoreactivity in the rat retina.

We studied the dependence of the expression of protein kinase C immunoreactivity (PKC-IR) in the rat retina on the light:dark (LD) cycle and on circadian rhythmicity in complete darkness (DD). Two anti-PKC alpha antibodies were employed: One, which we call PKCalphabeta recognized the hinge region; the other, here termed PKCalpha, recognized the regulatory region of the molecule. Western blots showed that both anti-PKC antibodies stained an identical single band at approximately 80 kD. The retinal neurons showing PKC-IR were rod bipolar cells and a variety of amacrine neurons. After 3 weeks on an LD cycle, PKCalphabeta-IR in both rod bipolar and certain amacrine cells manifested a clear rhythm with a peak at zeitgeber time (ZT) of 06-10 hours and a minimum at ZT 18. No rhythm in total PKC-IR was observed when using the PKCalpha antibody, but, at ZT 06-10 hours, rod bipolar axon terminals showed increased immunostaining. After 48 hours in DD, with either antibody, rod bipolar cells showed increased PKC-IR. The PKCalpha antibody alone revealed that, after 48 hours, AII amacrine neurons, which lacked PKC-IR in an LD cycle, manifested marked PKC-IR, which became stronger after 72 hours. Light administered early in the dark period greatly increased PKCalphabeta-IR in rod bipolar and some amacrine neurons. Our data indicate that light and darkness exert a strong regulatory influence on PKC synthesis, activation, and transport in retinal neurons.

Dopamine D2 receptor-mediated modulation of rod-cone coupling in the Xenopus retina.

We studied the responses of rod photoreceptors that were elicited with light flashes or sinusoidally modulated light by using intracellular recording. Dark-adapted Xenopus rod photoreceptors responded to sinusoidally modulated green lights at temporal frequencies between 1 Hz and 4 Hz. In normal Ringer's solution, 57% of the rods tested could follow red lights that were matched for equal rod absorbance to frequencies >5 Hz, indicating an input from red-sensitive cones. Quinpirole (10 microM), a D2 dopamine agonist, increased rod-cone coupling, whereas spiperone (5 microM), a selective D2 antagonist, completely suppressed it. D1 dopamine ligands were without effect. Neurobiotin that was injected into single rods diffused into neighboring rods and cones in quinpirole-treated retinas but only diffused into rods in spiperone-treated retinas. A subpopulation of rods (ca. 10% total rods) received a very strong cone input, which quickened the kinetics of their responses to red flashes and greatly increased the bandpass of their responses to sinusoidally modulated light. Based on electron microscopic examination, which showed that rod-rod and cone-cone gap junctions are common, whereas rod-cone junctions are relatively rare, we postulate that cone signals enter the rod network through a minority of rods with strong cone connections, from which the cone signal is further distributed in the rod network. A semiquantitative model of coupling, based on measures of gap-junction size and distribution and estimates of their conductance and open times, provides support for this assumption. The same network would permit rod signals to reach cones.

Dependence of photoreceptor glutamate release on a dihydropyridine-sensitive calcium channel.

A "reduced retina" preparation, consisting of the photoreceptor layer attached to the pigment epithelium in the eyecup, was used to study the pharmacology of the calcium channels controlling glutamate release by photoreceptors in Xenopus. Glutamate release was evoked either by dark adaptation or by superfusion with elevated (20 mM) potassium medium. Both darkness- and potassium-induced release were blocked by cadmium (200 microM). The N-type calcium channel blocker, omega-conotoxin GVIA (500 nM), the P-type calcium channel blocker, omega-agatoxin IVA (20 nM), and the P- and Q-type channel blocker omega-conotoxin MVIIC (1 microM) had no effect on glutamate release. In contrast, the dihydropyridines, nifedipine (10 microM) and nitrendipine (10 microM), which affect L-type calcium channels, blocked both darkness- and potassium-induced release. Bay K 8644 (10 microM), which promotes the open state of L-type calcium channels, enhanced glutamate release. These results indicate that photoreceptor glutamate release is controlled mainly by dihydropyridine-sensitive calcium channels. A dependence of glutamate release on L-type calcium channels also has been reported for depolarizing bipolar cells of a fish retina. Thus, it appears that non-inactivating L-type calcium channels are appropriate to mediate transmitter release in neurons whose physiological responses are sustained, graded potentials.

Kinetics of synaptic transfer from rods and cones to horizontal cells in the salamander retina.

We examined synaptic transmission between rods or cones and horizontal cells, using perforated patch recording techniques in salamander retinal slices. Experimental conditions were established under which horizontal cells received nearly pure rod or pure cone input. The response-intensity relation for both photoreceptors and horizontal cells was described by a Michaelis-Menten function with an exponent close to 1. A dynamic model was developed for the transduction from photoreceptor voltage to postsynaptic current. The basic model assumes that: (i) photoreceptor light-evoked voltage controls Ca2+ entry according to a Boltzmann relation; (ii) the rate of glutamate release depends linearly on the voltage-gated Ca2+ current (ICa) in the synaptic terminal; (iii) glutamate concentration in the synaptic cleft reflects the balance of release and reuptake in which reuptake obeys first order kinetics; (iv) the binding of glutamate to its receptor and channel gating are fast compared with glutamate kinetics in the synaptic cleft. The good fit to the model confirms that these are the key features of synaptic transmission from rods and cones. The model accommodated changes in kinetics induced by the glutamate uptake blocker, dihydrokainate. The match between model and response was not improved by including an estimate of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor desensitization or by making glutamate uptake voltage dependent.

GABA release from Xenopus retina does not correlate with horizontal cell membrane potential.

The relationship between horizontal cell membrane potential and the release of GABA was explored in the retina of Xenopus laevis. The intracellularly recorded membrane potential of horizontal cells was monitored while the retina was exposed to different concentrations of depolarizing agents. The dose-response curves obtained revealed a rise from 5 to 95% maximum depolarization in 0.5-1.5 log unit concentration change. The molar concentrations that elicited a 20 mV depolarization were 40 mM (potassium), 0.8 mM (glutamate), 0.8 mM (glycine), 5 microM (kainate) and 1.3 microM (quisqualate). Autoradiography revealed that radiolabel was accumulated almost exclusively by horizontal cells when isolated retinas were incubated in medium containing 1 microM [3H]GABA. Thus, retinal release of radioactivity was used as a measure of [3H]GABA release from horizontal cells. Endogenous GABA released from retinas was measured using high performance liquid chromatography and was taken to reflect both amacrine and horizontal cell GABA pools. The release of both [3H]GABA and endogenous GABA was stimulated by glutamate, kainate and potassium, but not by glycine or quisqualate. Similar dose-response curves for GABA release and for depolarization were obtained in the case of potassium and kainate but not for glutamate. Potassium-evoked release either of endogenous GABA or [3H]GABA was both calcium- and sodium-dependent, whereas kainate- or glutamate-evoked GABA release was sodium-dependent but calcium-independent. The results indicate that depolarization per se is not necessarily associated with transmitter release in Xenopus retinal horizontal cells. It is suggested that the action of a given neurotransmitter upon the efflux of GABA from horizontal cells may depend on the degree to which it modifies the sodium conductance of the horizontal cell.