Immunocytochemical identification of proteins involved in dopamine release from the somatodendritic compartment of nigral dopaminergic neurons.

We examined the somatodendritic compartment of nigral dopaminergic neurons by immunocytochemistry and confocal microscopy, with the aim of identifying proteins that participate in dopamine packaging and release. Nigral dopaminergic neurons were identified by location, cellular features and tyrosine hydroxylase immunoreactivity. Immunoreactive puncta of vesicular monoamine transporter type 2 and proton ATPase, both involved in the packaging of dopamine for release, were located primarily in dopaminergic cell bodies, but were absent in distal dopaminergic dendrites. Many presynaptic proteins associated with transmitter release at fast synapses were absent in nigral dopaminergic neurons, including synaptotagmin 1, syntaxin1, synaptic vesicle proteins 2a and 2b, synaptophysin and synaptobrevin 1 (VAMP 1). On the other hand, syntaxin 3, synaptobrevin 2 (VAMP 2) and SNAP-25-immunoreactivities were found in dopaminergic somata and dendrites Our data imply that the storage and exocytosis of dopamine from the somatodendritic compartment of nigral dopaminergic neurons is mechanistically distinct from transmitter release at axon terminals utilizing amino acid neurotransmitters.

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.

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.

Contributions of AMPA- and kainate-sensitive receptors to the photopic electroretinogram of the Xenopus retina.

The effects of kainate receptor-preferring glutamate ligands were tested on the electroretinogram (ERG) of the Xenopus retina. Kainate, domoic acid, and 5-iodowillardiine (20-100 microM) acted similarly in every respect. They increased peak amplitudes of the ERG a-, b-, and d-waves significantly over controls. The AMPA-specific antagonist, GYKI 53655, prevented a kainate-induced increase in ERG a- and d-waves, but was without effect on an increase in the b-wave. Once the effect of agonist on the b-wave had peaked, the ERG began to subside, leading to its nearly complete disappearance within 20 min. Prior exposure to GYKI followed by a combination of GYKI + agonist did not significantly slow the rate of b-wave disappearance. Our results indicate that (1) AMPA receptors contribute to ERG a- and d-waves. (2) The kainate-evoked increase in ERG a-, b-, and d-waves probably results, in part, from an excitotoxic swelling of inner retinal processes. (3) The inner retina has a population of GYKI-resistant, kainate-sensitive receptors which may contribute to b-wave generation.

Intracellular calcium reduces light-induced excitatory post-synaptic responses in salamander retinal ganglion cells.

The whole-cell patch clamp technique was used to study the effect of intracellular Ca2+ on light-evoked EPSCs in on-off ganglion cells in salamander retinal slices. Both AMPA and NMDA receptors contributed to the light-evoked responses. In the presence of strychnine and picrotoxin, ganglion cells responded to light onset and offset with transient inward currents at -70 mV. These currents were reduced by 35 +/- 3 % when the light stimulus was preceded by a depolarizing step from -70 to 0 mV. The inhibitory effect of depolarization on light-evoked EPSCs was strongly reduced in the presence of 10 mM BAPTA. The degree of EPSC inhibition by the prepulse holding potential followed the current-voltage relationship of the Ca2+ current found in the ganglion cell. In the presence of the NMDA receptor antagonist AP-7, glutamate-dependent current was nearly abolished when high Ca2+ was substituted for high Na+ solution. The release of Ca2+ from internal stores by caffeine or inositol trisphosphate reduced the EPSCs by 36 +/- 5 and 38 +/- 11 %, respectively, and abolished the inhibitory effect of depolarization. The inhibitory effect of depolarization on EPSCs was reduced 5-fold in the presence of AP-7, but was not reduced by the AMPA receptor antagonist CNQX. Neither inhibition of Ca2+-calmodulin-dependent enzymes, nor inhibition of protein kinase A or C had any significant effect on the depolarization-induced inhibition of EPSCs. Our data suggest that elevation of [Ca2+]i, through voltage-gated channels or by release from intracellular stores, reduced primarily the NMDA component of the light-evoked EPSCs.

Influence of light and neural circuitry on tyrosine hydroxylase phosphorylation in the rat retina.

Light has been shown to increase dopamine synthesis and release in vertebrate retinas, but the retinal circuits mediating the light signal are unknown. We utilized three antibodies which recognize phosphorylated forms of tyrosine hydroxylase (TH) at serines 19, 31, and 40 to study the effects of light and neuroactive drugs on TH phosphorylation in the rat retina. Phosphorylated TH and total TH immunoreactivities were co-localized exclusively in retinal neurons whose shape and location are characteristic of dopaminergic interplexiform cells. Phosphorylated TH was weak to absent in darkness, but light strongly stimulated phosphorylation in all the three serine residues. Light-induced phosphorylation of TH induction by light was uniformly blocked by a combination of NMDA and AMPA glutamate receptor antagonists. In darkness, the combination of NMDA+AMPA induced phosphosphorylation of TH at serines 19 and 40 but it was weak at serine 31. A GABA(A) antagonist had the same effect. An agonist of depolarizing (ON) bipolar cells, L-(+)-2-amino-4-phosphonobutyric acid, did not prevent light-induced phosphorylated TH formation. Carbachol, a non-specific cholinergic agonist, selectively induced phosphorylation of TH at serine 31 in darkness, an effect which was blocked by the nicotinic antagonist, d-tubocurarine. These results show that retinal circuits involving glutamatergic, GABAergic and cholinergic synapses influence phospho-TH formation at different serine residues in this enzyme. Gamma amino butyric acid (GABA) and glutamate influence TH phosphorylation at serines 19 and 40, whereas cholinergic inputs affect its phosphorylation at serine 31.

Photoreceptor classes and transmission at the photoreceptor synapse in the retina of the clawed frog, Xenopus laevis.

The photoreceptor population in Xenopus consists of a green-sensitive rod (lambda(max) = 523 nm), a blue-sensitive rod (lambda(max) = 445 nm) and three classes of cone. The largest cone is red-sensitive (lambda(max) = 611 nm). The intermediate cone is presumed to be blue-sensitive based on physiological criteria, whereas the miniature cone may be UV-sensitive. Horizontal cells (HC) are of two sorts: axon-bearing and axonless. The axon-bearing HC is of the luminosity type and probably contacts all types of photoreceptor. The axonless HC is of the chromaticity type and contacts only intermediate (blue) cones and at least one type of rod. During development dendrites of HCs and bipolar neurons penetrate photoreceptor bases. A progressive maturation of HC and bipolar synapses with rods and cones occurs between tadpoles stages 37/8 and 46. Neighboring rods and cones are joined by gap junctions. During this same period, the outer segments are laid down and photopigments synthesized. A linear relation was found between the quantum capturing ability of the rod and its absolute threshold. Mature rods of the Xenopus retina release glutamate in a calcium-dependent manner. Glutamate release was found to be a linear function of calcium influx through L-type calcium channels. Both types of HC possess ionotropic glutamate receptors of the AMPA subtype.

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.

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.

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.

Sub-millimolar cobalt selectively inhibits the receptive field surround of retinal neurons.

Recent work has indicated that cobalt, at sub-millimolar concentrations, blocks horizontal cell (HC) to cone feedback, without attenuating direct cone to second-order cell synaptic transmission. We utilized low concentrations (0.25-0.5 mM) of cobalt to test the contribution of the feedback circuit, and other possible cobalt-sensitive mechanisms, to the receptive-field surrounds of retinal neurons. In the great majority of cases, low cobalt blocked ganglion cell surrounds, and it invariably blocked driving the ganglion cell by extrinsic current injected into the HC network. Although low cobalt reduced the integrating area of the HC network, dopamine, which similarly constricted the HC receptive area, did not block ganglion cell surrounds. Low cobalt reduced a late depolarizing wave in the HC light-evoked waveform and selectively suppressed the depolarizing component of chromatic HCs, both signs of HC to cone feedback. Low cobalt also reduced or blocked completely the receptive-field surrounds of a small sample of bipolar and amacrine cells. These results implicate the HC to cone feedback synapse in the formation of the receptive-field surround of retinal neurons.

Calcium released from intracellular stores inhibits GABAA-mediated currents in ganglion cells of the turtle retina.

We studied spiking neurons isolated from turtle retina by the whole cell version of the patch clamp. The studied cells had perikaryal diameters > 15 microns and fired multiple spikes in response to depolarizing current steps, indicating they were ganglion cells. In symmetrical [Cl-], currents elicited by puffs of 100 microM gamma-aminobutyric acid (GABA) were inward at a holding potential of -80 mV. All of the GABA-evoked current was blocked by SR95331 (20 microM), indicating that it was mediated by a GABAA receptor. The GABA-evoked currents were unaltered by eliciting a transmembrane calcium current either just before or during the response to GABA. On the other hand caffeine (10 mM), which induces Ca2+ release from intracellular stores, inhibited the GABA-evoked current on average by 30%. The caffeine effect was blocked by introducing the calcium buffer bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) into the cell but was unaffected by replacing [Ca2+]o with equimolar cobalt. Thapsigargin (10 microM), an inhibitor of intracellular calcium pumps, and ryanodine (20 microM), which depletes intracellular calcium stores, both markedly reduced a caffeine-induced inhibition of the GABA-evoked current. Another activator of intracellular calcium release, inositol trisphosphate (IP3; 50 microM), also progressively reduced the GABA-induced current when introduced into the cell. Dibutyryl adenosine 3'5'-cyclic monophosphate (cAMP; 0.5 mM), a membrane-permeable analogue of cAMP, did not reduce GABA-evoked currents, suggesting that cAMP-dependent kinases are not involved in suppressing GABAA currents, whereas calmidazolium (30 microM) and cyclosporin A (20 microM), which inhibit Ca/calmodulin-dependent phosphatases, did reduce the caffeine-induced inhibition of the GABA-evoked current. Alkaline phosphatase (150 micrograms/ml) and calcineurin (300 micrograms/ml) had a similar action to caffeine or IP3. Antibodies directed against the ryanodine receptor or the IP3 receptor reacted with the great majority of neurons in the ganglion cell layer. We found that these two antibodies colocalized in large ganglion cells. In summary, intracellular calcium plays a role in reducing the currents elicited by GABA, acting through GABAA receptors. The modulatory action of calcium on GABA responses appears to work through one or more Ca-dependent phosphatases.

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.

Cholinergic, but not the rod pathway-related glycinergic (All), amacrine cells contain calretinin in the rat retina.

Double-label immunocytochemistry was carried out on cryostat sections of rat retina to test for the presence of calretinin in cholinergic starburst and the rod pathway-related glycinergic (All) amacrine cells. All cholinergic cells contained calretinin, but calretinin-immunoreactive cells were much more numerous in both the inner nuclear and ganglion cell layers than the cholinergic cells. Glycinergic All amacrine cells have been found to contain calretinin in cat, monkey and rabbit retinas. Since All amacrine cells in rat can be selectively labeled with antibodies against parvalbumin, in a second experiment we attempted to colocalize these proteins. We found that calretinin- and parvalbumin-immunoreactive neurons belonged to distinct amacrine cell populations permitting the conclusion that, in the rat retina, All amacrine cells do not contain calretinin. The results indicate that even those amacrine cells of the mammalian retina that are highly conserved with respect to morphology and transmitter content, may differ with respect to other neurochemical characteristics, such as their calcium-binding proteins.

Gain of rod to horizontal cell synaptic transfer: relation to glutamate release and a dihydropyridine-sensitive calcium current.

We related rod to horizontal cell synaptic transfer to glutamate release by rods. Simultaneous intracellular records were obtained from dark-adapted rod-horizontal cell pairs. Steady-state synaptic gain (defined as the ratio of horizontal cell voltage to rod voltage evoked by the same light stimulus) was 3.35 +/- 0.60 for dim flashes and 1.50 +/- 0.03 for bright flashes. Under conditions of maintained illumination, there was a measurable increment of horizontal cell hyperpolarization for each light-induced increment of rod hyperpolarization over the full range of rod voltages. In separate experiments we studied glutamate release from an intact, light-responsive photoreceptor layer, from which inner retinal layers were removed. Steady light reduced glutamate release as a monotonic function of intensity; spectral sensitivity measures indicated that we monitored glutamate release from rods. The dependence of glutamate release on rod voltage was well fit by the activation function for a high-voltage-activated, dihydropyridine-sensitive L-type calcium current, suggesting a linear dependence of glutamate release on [Ca]i in the synaptic terminal. A simple model incorporating this assumption accounts for the steady-state gain of the rod to horizontal cell synapse.

Both high- and low voltage-activated calcium currents contribute to the light-evoked responses of luminosity horizontal cells in the Xenopus retina.

We examined the contribution of two intrinsic voltage-dependent calcium channels to the light-evoked responses of a non-spiking retinal neuron, the horizontal cell (HC). HC's isolated from the Xenopus retina were studied by the whole cell version of the patch clamp. In a mixture of agents which suppressed Na- and K-dependent currents, we identified a transient, low voltage-activated Ca current suppressed by Ba2+ and blocked by Ni2+ (T-type) and a sustained, high voltage-activated, dihydropyridine-sensitive Ca current that was enhanced by Ba2+ (L-type). We made simultaneous intracellular recordings from rods and HC's in the intact, dark-adapted Xenopus retina. Under certain stimulus conditions, transient oscillations appeared in HC responses but were absent in rod light-evoked waveforms. One type of transient was seen at relatively hyperpolarized potentials (< -45 mV), was enhanced by Sr2+ and inhibited by Ni2+. It thus appears to depend on a T-type Ca-current. A second type of oscillation was seen to be superimposed on a prolonged depolarizing wave following light off in the HC and as spike-like depolarizations in rods. These oscillations were enhanced by Ba2+ and Sr2+, but blocked by the dihydropyridine, nifedipine, indicating their dependence on an L-type calcium conductance. All calcium-dependent oscillations were suppressed by 0.05-0.5 mM Co2+. Suppression of glutamate neurotransmission with CNQX or kynurenate, or glycine neurotransmission with strychnine, enhanced the HC oscillations.

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.

Glutamate release by the intact light-responsive photoreceptor layer of the Xenopus retina.

In order to study glutamate release from light responsive photoreceptors, we used an eyecup preparation treated with detergent and distilled water, which permitted removal of the inner retina. The remaining 'reduced' retina consists mainly of photoreceptors attached to the pigment epithelium. The viability of the preparation was established by exclusion of trypan blue, light and electron microscopic examination of the photoreceptor layer and by intracellular recordings from rods. The 'reduced' retina was superfused at 1 ml/h and overflow samples were analyzed for their glutamate content by a fluorimetric enzyme assay. We tested the response to dark and light adaptation and to treatment with 100 microM CdCl2. We found a baseline glutamate level in light-adapted preparation which was not affected by cadmium. Dark adaptation induced a 2-fold increase of glutamate release, which was completely blocked by cadmium.

D2 dopamine receptor-mediated inhibition of a hyperpolarization-activated current in rod photoreceptors.

1. Using the whole cell patch clamp method, we investigated the effect of dopamine on a hyperpolarization-activated current (Ih) in the inner segments of rod photoreceptors of the Xenopus retina. 2. Ih was elicited by hyperpolarizing voltage steps to -120 mV from a holding potential of -40 mV. Dopamine reversibly reduced Ih in a dose-dependent manner. Dopamine-mediated inhibition of Ih was blocked by the D2 dopamine antagonist sulpiride. 3. The D2 dopamine agonist quinpirole (0.1-20 microM) inhibited Ih whereas the D1 agonist SKF-38393 (100 microM) had no effect on Ih. Quinpirole-induced inhibition of Ih was blocked by sulpiride, but not by the D4 antagonist, clozapine. The D3 agonists (+/-)-7-hydroxy-2-dipropylaminotetralin hydrochloride and trans-7-hydroxy-2[N-propyl-N-(3'-iodo-2'-propenyl)amino]-tetralin maleate were, respectively, 5 and 100 times less effective than quinpirole in inhibiting Ih. 4. Quinpirole failed to reduce Ih when the internal solution contained GDP beta S (500 microM). Internal application GTP gamma S (300 microM) progressively and irreversibly reduced Ih and blocked a further reduction by quinpirole, indicating that the inhibition of Ih by quinpirole involves a G protein. 5. The inhibition of Ih by quinpirole was not affected by intracellularly applied adenosine 3',5'-cyclic monophosphate (cAMP) or by the protein kinase inhibitor H-7, indicating that a cAMP-mediated second messenger cascade does not participate in the dopamine-mediated inhibition. 6. Ih was not altered when the patch pipette contained a nominally Ca(2+)-free internal solution, but the inhibition of Ih by quinpirole was abolished, suggesting an involvement of Ca(2+) in the quinpirole-induced effect. 7. We conclude that a D2 dopamine receptor modulates Ih through the activation of a G protein and that intracellular Ca2+, but not cAMP, plays a key role in this process. 8. The reduction of Ih by dopamine may reduce the ability of rods to signal time-modulated light stimuli.