Molecular analyses of circadian gene variants reveal sex-dependent links between depression and clocks.

An extensive literature links circadian irregularities and/or sleep abnormalities to mood disorders. Despite the strong genetic component underlying many mood disorders, however, previous genetic associations between circadian clock gene variants and major depressive disorder (MDD) have been weak. We applied a combined molecular/functional and genetic association approach to circadian gene polymorphisms in sex-stratified populations of control subjects and case subjects suffering from MDD. This approach identified significant sex-dependent associations of common variants of the circadian clock genes hClock, hPer3 and hNpas2 with major depression and demonstrated functional effects of these polymorphisms on the expression or activity of the hCLOCK and hPER3 proteins, respectively. In addition, hCLOCK expression is affected by glucocorticoids, consistent with the sex-dependency of the genetic associations and the modulation of glucocorticoid-mediated stress response, providing a mechanism by which the circadian clock controls outputs that may affect psychiatric disorders. We conclude that genetic polymorphisms in circadian genes (especially hClock and hPer3, where functional assays could be tested) influence risk of developing depression in a sex- and stress-dependent manner. These studies support a genetic connection between circadian disruption and mood disorders, and confirm a key connection between circadian gene variation and major depression.

Heterogeneous transgene expression in the retinas of the TH-RFP, TH-Cre, TH-BAC-Cre and DAT-Cre mouse lines.

Transgenic mouse lines are essential tools for understanding the connectivity, physiology and function of neuronal circuits, including those in the retina. This report compares transgene expression in the retina of a tyrosine hydroxylase (TH)-red fluorescent protein (RFP) mouse line with three catecholamine-related Cre recombinase mouse lines [TH-bacterial artificial chromosome (BAC)-, TH-, and dopamine transporter (DAT)-Cre] that were crossed with a ROSA26-tdTomato reporter line. Retinas were evaluated and immunostained with commonly used antibodies including those directed to TH, GABA and glycine to characterize the RFP or tdTomato fluorescent-labeled amacrine cells, and an antibody directed to RNA-binding protein with multiple splicing to identify ganglion cells. In TH-RFP retinas, types 1 and 2 dopamine (DA) amacrine cells were identified by their characteristic cellular morphology and type 1 DA cells by their expression of TH immunoreactivity. In the TH-BAC-, TH-, and DAT-tdTomato retinas, less than 1%, ∼ 6%, and 0%, respectively, of the fluorescent cells were the expected type 1 DA amacrine cells. Instead, in the TH-BAC-tdTomato retinas, fluorescently labeled AII amacrine cells were predominant, with some medium diameter ganglion cells. In TH-tdTomato retinas, fluorescence was in multiple neurochemical amacrine cell types, including four types of polyaxonal amacrine cells. In DAT-tdTomato retinas, fluorescence was in GABA immunoreactive amacrine cells, including two types of bistratified and two types of monostratified amacrine cells. Although each of the Cre lines was generated with the intent to specifically label DA cells, our findings show a cellular diversity in Cre expression in the adult retina and indicate the importance of careful characterization of transgene labeling patterns. These mouse lines with their distinctive cellular labeling patterns will be useful tools for future studies of retinal function and visual processing.

Interactions of the serotonin and circadian systems: nature and nurture in rhythms and blues.

The serotonin and circadian systems are principal regulatory networks of the brain. Each consists of a unique set of neurons that make widespread neural connections and a defined gene network of transcriptional regulators and signaling genes that subserve serotonergic and circadian function at the genetic level. These master regulatory networks of the brain are extensively intertwined, with reciprocal circuit connections, expression of key genetic elements for serotonin signaling in clock neurons and expression of key clock genes in serotonergic neurons. The reciprocal connections of the serotonin and circadian systems likely have importance for neurobehavioral disorders, as suggested by their convergent contribution to a similar range of mood disorders including seasonal affective disorder (SAD), bipolar disorder, and major depression, and as suggested by their overlapping relationship with the developmental disorder, autism spectrum disorder. Here we review the neuroanatomical and genetic basis for serotonin-circadian interactions in the brain, their potential relationship with neurobehavioral disorders, and recent work examining the effects on the circadian system of genetic perturbation of the serotonergic system as well as the molecular and behavioral effects of developmental imprinting of the circadian system with perinatal seasonal light cycles.

Gating of retinal horizontal cell hemi gap junction channels by voltage, Ca2+, and retinoic acid.

PURPOSE: Hemi gap junction (HGJ) channels, precursors of gap junctional channels, are functionally expressed in retinal horizontal cells where they may play roles in osmoeregulation and ephaptic regulation of synaptic feedback to photoreceptors. In this study we examined mechanisms of gating of these channels by transmembrane voltage, Ca2+ and retinoic acid (RA). METHODS: Experiments were performed on cultured bass horizontal cells using the conventional whole cell patch clamp configuration. RESULTS: HGJ currents in isolated bass horizontal cells, revealed by perfusion with Ca2+ free media, were opened by positive holding potentials and inhibited by negative holding potentials. These currents were also inhibited by external application of either Ca2+ or RA. Using a rapid perfusion system, the latency of 2 mM Ca2+ to begin channel closure was unmeasurably brief, whereas the latency for 30 microM RA action was 177+/-9 ms (mean+/-standard error of the mean). The total inhibition of HGJ channel currents by coapplication of 0.3 microM RA and 100 microM Ca2+ was less than the sum of inhibition by RA alone and Ca2+ alone suggesting that the actions of RA and Ca2+ were not independent. In the presence of 0.3 microM RA, the half maximal concentration for Ca2+ inhibition was increased from a control value of 192 microM to 375 microM without affecting maximal inhibition. Similarly, the half maximal concentration for RA inhibition was increased from a control value of 0.44 microM to 1.1 microM without affecting maximal inhibition in the presence of 100 microM Ca2+. CONCLUSIONS: These results suggest that horizontal cell HGJ channels are closed by the normal negative resting potentials of these cells. Extracellular Ca2+ and the retinal neuromodulator RA also act to close HGJ channels through mechanisms or sites which are not independent.

Direct gating by retinoic acid of retinal electrical synapses.

Retinoic acid (RA), a signaling molecule derived from vitamin A, controls growth and differentiation of a variety of cell types through regulation of gene transcription. In the vertebrate retina, RA also regulates gap junction-mediated physiological coupling of retinal neurons through a nontranscriptional mechanism. Here we report that RA rapidly and specifically modulates synaptic transmission at electrical synapses of cultured retinal horizontal cells through an external RAR(beta)(/gamma)-like binding site, the action of which is independent of second messenger cascades. External application of all-trans retinoic acid (at-RA) reversibly reduced the amplitude of gap junctional conductance in a dose-dependent manner, but failed to affect non-gap-junctional channels, including glutamate receptors. In contrast, internal dialysis with at-RA was ineffective, indicating an external site of action. Selective RAR(beta)(/gamma) ligands, but not an RAR(alpha)-selective agonist, mimicked the action of at-RA, suggesting that gating of gap junctional channels is mediated through an RAR(beta)(/gamma)-like binding site. At-RA did not act on gap junctional conductance by lowering [pH](i) or by increasing [Ca(2+)](i). A G protein inhibitor and protein kinase inhibitors did not block at-RA uncoupling effects indicating no second messenger systems were involved. Direct action of at-RA on gap junction channels was further supported by its equivalent action on whole-cell hemi-gap-junctional currents and on cell-free excised patch hemichannel currents. At-RA significantly reduced single-channel open probability but did not change unitary conductance. Overall, the results indicate that RA modulates horizontal cell electrical synapses by activation of novel nonnuclear RAR(beta)(/gamma)-like sites either directly on, or intimately associated with, gap junction channels.

GFP fluorescence reports Period 1 circadian gene regulation in the mammalian biological clock.

Endogenous cyclic activation of a specific set of genes, including Period 1 (Per1), drive circadian rhythms in the suprachiasmatic nucleus (SCN), a biological clock nucleus of the brain. We have produced transgenic mice in which a degradable form of recombinant jellyfish green fluorescent protein (GFP) is driven by the mouse Period 1 (mPer1) gene promoter. GFP protein is expressed in the circadian neural structures of the retina and SCN. Fluorescent signals are resolved at the level of individual neurons. mPer1-driven GFP fluorescence intensity reports light-induction and circadian rhythmicity in SCN neurons. This circadian reporter transgene captures the gene expression dynamics of living biological clock neurons and ensembles, providing a novel view of this brain function.

Electrical coupling of retinal horizontal cells mediated by distinct voltage-independent junctions.

Electrical coupling between H2 horizontal cell pairs isolated from the hybrid bass retina was studied using dual whole-cell, voltage-clamp technique. Voltage-dependent inactivation of junctional currents in response to steps in transjunctional voltage (Vj) over a range of +/-100 mV was characterized for 89 cell pairs. Approximately one-quarter of the pairs exhibited strongly voltage-dependent junctions (>50% reduction in junctional current at +/-100 mV), another quarter of the pairs exhibited voltage-independent junctional current (<5% reduction at +/-100 mV), and the remainder of the pairs exhibited intermediate values for voltage inactivation. We focused on further characterizing the Vj-independent junctions of horizontal cells, which have not been described previously in detail. When Lucifer Yellow dye was included in one recording pipette, pairs exhibiting Vj-independent coupling showed no (9/12), or limited (3/12), passage of dye. Vj-independent coupling was markedly less sensitive to the modulators SNP (100-300 microM, -9% reduction in coupling) and dopamine (100-300 microM, -6%) than were Vj-dependent junctions (-45% and -44%). However, simultaneous application of both SNP and dopamine significantly reduced Vj-independent coupling (-56%). Both Vj-independent and Vj-dependent junctions were blocked by DMSO (1-2%), but Vj-independent junctions were not blocked by heptanol. Single-channel junctional conductances of Vj-independent junctions range from 112-180 pS, versus 50-60 pS for Vj-dependent junctions. The results reveal that Vj-independent coupling in a subpopulation of horizontal cells from the hybrid bass retina is mediated by cellular junctions with physiological and pharmacological characteristics distinct from those previously described in fish horizontal cells.

Serotonin modulates glutamate responses in isolated suprachiasmatic nucleus neurons.

Two input pathways to the suprachiasmatic nucleus (SCN) of the hypothalamus are the glutamatergic retinohypothalamic tract and the serotonergic afferent from the midbrain raphe nucleus. To determine whether these two temporal signaling pathways can converge at the cellular level, we have investigated the effects of serotonin on glutamate-induced calcium responses of individual SCN neurons isolated in cell culture. Dispersed cultures were formed from the SCN of neonatal rats. The calcium indicator Fura-2 acetoxymethyl ester was used to assess the changes in [Ca(2+)](i) by recording the 340-nm/380-nm excitation ratio. Application of glutamate (5 microM) to the culture caused a rapid (within 10 s) increase in the fluorescence ratio of neurons indicating a marked increase in the concentration of intracellular free calcium. However, when 5-hydroxytryptamine (5-HT; 5 microM) was coapplied with glutamate, 31% of neurons showed an overall 61% reduction in the peak of the glutamate-induced calcium increase. Application of the 5-HT(7/1A) receptor agonist, (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin [(+/-)-8-OH-DPAT] (1 microM), also reduced the calcium elevation this time by 80% in 18% of the neurons tested. When the 5-HT(7/2/1C) receptor antagonist, ritanserin (800 nM), was coapplied with serotonin, it blocked modulation of the glutamate responses. Further support for the involvement of the 5-HT(7) receptor was provided by the ability of the adenylate cyclase activator, forskolin (10 microM), and the cAMP analogue, 8-Br cAMP (0.5 mM), to mimic the suppressive effect of serotonin. Blocking spike-mediated cell communication with tetrodotoxin (1 microM) did not prevent the serotonergic suppression of glutamate-induced responses. These results support the hypothesis that the serotonergic modulation of photic entraining signals can occur in SCN neurons.

Horizontal cell glutamate receptor modulation by NO: mechanisms and functional implications for the first visual synapse.

Neurons of the horizontal cell retinal neural network are subject to modulation by the neurotransmitter nitric oxide (NO). We have examined the effects of NO on glutamate receptor function in isolated horizontal cells from the perch (Perca fluviatilis) using the concentration ramp technique to simultaneously record receptor current and agonist concentration. Dose-response curves for glutamate (0-1 mM) and kainate (0-200 microM) were measured in the presence and absence of 1-2 mM sodium nitroprusside (SNP), 1 mM 8-Br-cGMP, 100 microM cyclothiazide or 200 microM dopamine as modulators. SNP increased the EC50 (i.e. decreased affinity) for glutamate and increased Imax (i.e. increased efficacy), whereas 8-Br-cGMP increased EC50, but not Imax. In the presence of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor desensitization blocker cyclothiazide, the SNP-induced increase in EC50 persisted, but the increase in Imax was blocked. The increase in EC50, but not the increase in Imax was also observed when the non-desensitizing agonist kainate (100-200 microM) was applied in the presence of SNP. When 2 mM SNP and 200 microM dopamine were applied together, they increased Imax (740 vs. 2455 pA) and EC50 (422 vs. 682 microM). Our findings indicate that NO modulates horizontal cell glutamate responses by reducing the affinity of receptors for glutamate while simultaneously increasing the maximal current. The shift in affinity is cGMP-mediated and independent of desensitization. The action of NO on horizontal cell glutamate receptors is distinct from, but synergistic with. that of dopamine. Glutamate receptor modulation by NO qualitatively predicts the action of NO on horizontal cell light responses in situ and may alter transmission at visual synapses according to adaptational conditions.

Cloning and functional expression of a novel gap junction channel from the retina of Danio aquipinnatus.

Electrical synapses, or gap junctions, are widely distributed in the vertebrate retina and are thought to play critical roles in the transmission and coding of visual signals. To investigate the molecular basis of this form of neural communication in the retina, we have isolated, characterized, and functionally expressed a cDNA for a gap junction channel derived from the retina of the teleost fish Danio aquipinnatus (giant danio). The cDNA contained an open reading frame of 1146 nucleotides encoding a connexin with a predicted molecular mass of 43.3 kDa which shared extensive identity with Rattus norvegicus Cx43 (78%). This protein (DACX43) contained several consensus phosphorylation sequences in the c-terminal region, some of which are conserved among Cx43 orthologs. RNA blot hybridization revealed that DACX43 was expressed in the brain as well as in the retina. In addition, Southern analysis suggested that there are multiple copies of DACX43, or other closely related sequences, in the Danio aquipinnatus genome. When DACX43 was expressed by stable transfection in gap-junction-deficient mouse N2A neuroblastoma cells, functional gap junctions were formed as indicated by dual whole-cell recordings of electrical coupling. We conclude that DACX43 is a connexin43 ortholog, which is expressed in the retina of Danio aquipinnatus, and when translated is able to form functional gap junction channels.

Stimulus-secretion coupling in porcine adrenal chromaffin cells: effect of dexamethasone.

Recent studies from this laboratory established that dexamethasone (DEX) potentiates Ca2+ current via voltage-gated Ca2+ channels (VGCC), and as a consequence potentiates agonist-induced cytosolic Ca2+ transients in rat adrenal chromaffin cells. The present study examined whether DEX can also modulate VGCC activity and agonist-induced cytosolic Ca2+ transients in porcine adrenal medullary chromaffin (PAMC) cells, and if so whether this results in alterations in catecholamine secretion. Forty-eight-hr exposure to 1 microM DEX significantly increased peak Ca2+ current (delta + 138%; n = 6; P < 0.05) in PAMC cells. DEX treatment also significantly potentiated the increase in cytosolic Ca2+ in response to membrane depolarization with KCl (delta + 20%; n = 29; P < 0.05), but did not affect the amplitude of Ca2+ transients elicited by nicotine or acetylcholine. Despite the potentiation of intracellular Ca2+, DEX treatment had no effect on KCl-induced secretion of either norepinephrine or epinephrine. These data demonstrate that as in the rat chromaffin cell, DEX can also increase VGCC activity in PAMC cells. However, the subsequent potentiation of selected agonist-induced increases in intracellular Ca2+ does not appear to be sufficient to alter catecholamine secretion.

Modulation of hybrid bass retinal gap junctional channel gating by nitric oxide.

1. To elucidate the role of the nitric oxide (NO) transmitter system in the regulation of gap junctional channel gating, we have examined the effects of the NO donor sodium nitroprusside (SNP) on the electrical synapses of hybrid bass H2-type horizontal cells. 2. SNP reversibly reduced the macroscopic junctional conductance without significantly changing voltage sensitivity. 3. Kinetic analyses showed that SNP made the voltage-dependent decay of junctional currents more rapid. 4. Single-channel data showed that SNP reduced channel open probability by reducing channel open frequency. 5. The action of SNP can be prevented or largely reduced by the NO scavenger, haemoglobin. NO release by SNP solutions was detected directly by a NO sensor. 6. NO appears to modulate the gap junctional conductance by activating the cGMP-cGMP-dependent protein kinase G (PKG) pathway. A membrane-permeable cGMP analogue, 8-Br-cGMP, mimics the action of SNP. A soluble guanylate cyclase inhibitor (LY-83583) and a highly specific cGMP-dependent protein kinase inhibitor (RKRARKE) blocked the action of NO. 3-Isobutyl-1-methylxanthine (IBMX), a non-specific phosphodiesterase inhibitor, potentiated the effect of SNP. 7. [Ca2+]i image studies showed that NO donors did not change [Ca2+]i in horizontal cells, suggesting that the regulation of junctional channels by NO is [Ca2+]i independent.

Gap junction channel gating at bass retinal electrical synapses.

To further characterize the properties of retinal horizontal cell electrical synapses, we have studied the gating characteristics of gap junctions between cone-driven horizontal cells from the hybrid striped bass retina using double whole-cell voltage-clamp techniques. In a total of 105 cell pairs, the macroscopic conductance ranged from 0.4-100 nS with most cell pairs exhibiting junctional conductances between 10 and 30 nS. The junctional current-voltage relationship showed that peak or instantaneous currents (Iinst) were linear within the Vj range of +/- 100 mV and that steady-state junctional currents (Iss) exhibited rectification with increasing voltage beginning around +/- 30-40 mV Vj. The normalized junctional current-voltage relationship was well fit by a two-state Boltzmann distribution, with an effective gating charge of 1.9 charges/channel, a half-maximal voltage of approximately +/- 55 mV, and a normalized residual conductance of 0.28. The decay of junctional current followed a single exponential time course with the time constant decreasing with increasing Vj. Recovery of junctional current from voltage-dependent inactivation takes about 1 s following a pulse of 80 mV, and is about five times slower than the inactivation time course at the same Vj. Single-channel analysis showed that the unitary conductance of junctional channels is 50-70 pS. The overall open probability decreased in a voltage-dependent manner. Both the mean channel open time and the frequency of channel opening decreased, while the channel closure time increased. The ratio of closed time/total recording time significantly increased as Vj increased. Increased Vj reduced the number of events at all levels and shifted the unitary conductance to a lower level. Kinetic analysis of channel open duration showed that the distribution of channel open times was best fit by two exponentials and increased Vj significantly reduced the slower time constant. These results indicate that bass retina horizontal cells exhibit voltage-dependent inactivation of macroscopic junctional current. The inactivation occurs at the single-channel level mainly by increasing the rate of closure of voltage-sensitive channels.

Nitric oxide and cGMP modulate retinal glutamate receptors.

1. In the retina, as in other regions of the vertebrate central nervous system, glutamate receptors mediate excitatory chemical synaptic transmission and are a critical site for the regulation of cellular communication. In this study, retinal horizontal cells from the hybrid less were dissociated in cell culture, voltage clamped by the whole cell recording technique, and the currents evoked by application of excitatory amino acids recorded. 2. Responses to glutamate and its agonist kainate were reduced by approximately 50% in the presence of the nitric oxide (NO) donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine. The effect of these compounds was blocked by the NO scavenger hemoglobin. 3. This effect of NO donors on kainate currents could be mimicked by the application of a membrane permeable guanosine 3',5'-cyclic monophosphate (cGMP) analogue, 8-Br-cGMP. The NO effect was also blocked by application of the guanylate cyclase inhibitor LY-83583, and by a protein kinase G inhibitor peptide. 4. In H1-type horizontal cells, stimulation of endogenous nitric oxide synthase with L-arginine reduced kainate responses, whereas application of D-arginine had no effect. 5. This receptor modulation mechanism may act in concert with other pre- and postsynaptic mechanisms to modify horizontal cell synaptic function according to the adaptational state of the retina and also may protect horizontal cells from glutamate excitotoxicity.

Horizontal cell electrical coupling in the giant danio: synaptic modulation by dopamine and synaptic maintenance by calcium.

Electrical synapses, and their structural manifestation, gap junctions, are critical elements of retinal circuitry. These synapses are subject to both rapid modulation and slower structural changes by physiological signals which mediate changes in the adaptational state of the retina. The electrical synapses of fish retinal horizontal cells are an excellent preparation for in vitro studies of electrical synapses. We have examined the rapid modulation of electrical coupling by dopamine and effects on the expression and maintenance of electrical synapses by cell calcium in pairs of horizontal cells isolated from retinas of the giant danio (Danio aquipinnatus). We report that rapid modulation by dopamine reduces junctional conductance by modifying gap junction channel gating, while maintaining cells in reduced calcium medium, and lowering intracellular calcium concentration, results in the loss of electrical coupling. The effects of calcium on synaptic maintenance may be related to structural changes observed in horizontal cell electrical synapses during light adaptation.

Modulation of gap-junction channel gating at zebrafish retinal electrical synapses.

1. Transmission at electrical synapses is modulated by a variety of physiological signals, and this modulation is a potentially general mechanism for regulating signal integration in neural circuits and networks. In the outer plexiform layer of the retina, modulation of horizontal-cell electrical coupling by dopamine alters the extent of spatial integration in the horizontal-cell network. By analyzing the activity of individual gap-junction channels in low-conductance electrical synapses of zebrafish retinal horizontal cells, we have defined the properties of these synaptic ion channels and characterized the functional changes in them during modulation of horizontal-cell electrical synapses. 2. Zebrafish horizontal-cell gap-junction channels have a unitary conductance of 50-60 pS and exhibit open times of several tens of milliseconds. The kinetic process of channel closure is best described by the sum of two rate constants. 3. Dopamine, and its agonist, (+/-)-6,7-dihydroxy-2-amino-tetralin (ADTN), modulates electrical synaptic transmission between horizontal cells predominantly by affecting channel-gating kinetics. These agents reduced the open probability of gap-junction channels two- to threefold by reducing both the duration and frequency of channel openings. Both time constants for channel open duration were reduced, whereas the duration of shut periods was increased. Similar changes in open-time kinetics were observed in power spectra of higher conductance gap junctions. 4. These results provide a description of rapid electrical synaptic modulation at the single channel level. The description should be useful in understanding the mechanisms of plasticity at these synapses throughout the vertebrate central nervous system.

Protein content and cAMP-dependent phosphorylation of fractionated white perch retina.

In the retinas of teleost fish dopamine, released from interplexiform cells, modulates synaptic transmission at both the chemical and electrical synapses of retinal horizontal cells. This modulation is due to activation of adenylate cyclase and phosphorylation by protein kinase A, perhaps of the synaptic ion channel proteins themselves. In this study we have fractionated the white perch retina by Percoll density gradient centrifugation in order to identify proteins which coenrich with horizontal cells. In addition we have tested retinal fractions for phosphorylation by native cAMP-dependent kinase. Our findings indicate that there are at least 3 proteins of molecular weights 28, 43/44 and 50 kDa which coenrich with horizontal cells and 3 proteins of 30/31 kDa, 35 kDa (putative rhodopsin) and 48 kDa (putative arrestin) which coenrich with photoreceptor fractions. The 43/44 kDa phosphoprotein is a target for cAMP-dependent protein phosphorylation and thus is apparently an element of the dopaminergic modulatory pathway in perch horizontal cells.

Modulation of electrical synaptic transmission in zebrafish retinal horizontal cells.

Electrical synaptic transmission is widespread in the vertebrate CNS and its modulation plays a critical role in altering the properties of coupled neural networks. In order to define further the mechanisms of electrical synaptic plasticity in the vertebrate retina, the electrophysiological characteristics of solitary horizontal cells and horizontal cell pairs from the zebrafish (Brachydanio rerio) were examined by whole-cell patch-clamp recordings from cells in primary cell culture. In solitary cells, the current-voltage relation exhibited inward current at potentials negative to -60 mV, a linear region of high resistance from -50 mV to 0 mV, and outward current positive to +20 mV. The inward current at negative potentials was blocked by substituting Cs+ for K+ in the extracellular medium, while the outward current at positive potentials was blocked by substitution of Cs+ for K+ in the pipette solution. Measurements of gap junctional conductance from electrically coupled cell pairs revealed that zebrafish horizontal cells expressed a mean junctional conductance of considerably smaller magnitude than other teleost retinal horizontal cells. Junctional conductance was found to be voltage dependent, exhibiting time-dependent closure with increasing transjunctional voltage. Voltage sensitivity was symmetrical about 0 mV junctional potential. At +/- 90 mV the ratio of steady state to peak current was approximately 0.5 and the time constant for inactivation of the junctional current was approximately 120 msec. Junctional conductance was also modulated by dopamine and cAMP. Pairs of horizontal cells responded to puff application of dopamine with a two- to threefold reduction in junctional conductance, but there was no discernible effect on extrajunctional conductances. The action of dopamine on coupling was mimicked by application of the dopamine agonist (+/-)-6,7-dihydroxy-2-amino-tetralin (ADTN) and the membrane permeable cAMP analog 8-bromo-cAMP. The selective D1 dopamine receptor antagonist SCH23390 blocked uncoupling by dopamine. These data provide a primary description of the electrophysiological characteristics of solitary horizontal cells and the electrical coupling between pairs of horizontal cells dissociated from the zebrafish retina. They indicate that zebrafish horizontal cells are distinct from the horizontal cells of other teleosts in their coupling characteristics. The results suggest that zebrafish horizontal cells exhibit differences in the regulation of synaptic assembly and maintenance that have important implications for the function of the zebrafish horizontal cell network in vivo.