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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.
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.
When humans respond to sensory stimulation, their reaction times tend to be long and variable relative to neural transduction and transmission times. The neural processes responsible for the duration and variability of reaction times are not understood. Single-cell recordings in a motor area of the cerebral cortex in behaving rhesus monkeys (Macaca mulatta) were used to evaluate two alternative mathematical models of the processes that underlie reaction times. Movements were initiated if and only if the neural activity reached a specific and constant threshold activation level. Stochastic variability in the rate at which neural activity grew toward that threshold resulted in the distribution of reaction times. This finding elucidates a specific link between motor behavior and activation of neurons in the cerebral cortex.
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.
Transport of norepinephrine (NE+) by cocaine- and antidepressant-sensitive transporters in presynaptic terminals is predicted to involve the cotransport of Na+ and Cl-, resulting in a net movement of charge per transport cycle. To explore the relationship between catecholamine transport and ion permeation through the NE transporter, we established a human norepinephrine transporter (hNET) cell line suitable for biochemical analysis and patch-clamp recording. Stable transfection of hNET cDNA into HEK-293 (human embryonic kidney) cells results in lines exhibiting (1) a high number of transporter copies per cell (10(6)), as detected by radioligand binding and hNET-specific antibodies, (2) high-affinity, Na(+)-dependent transport of NE, and (3) inhibitor sensitivities similar to those of native membranes. Whole-cell voltage-clamp of hNET-293 cells reveals NE-induced, Na(+)-dependent currents blocked by antidepressants and cocaine that are absent in parental cells. In addition to NE-dependent currents, transfected cells posses an NE-independent mode of charge movement mediated by hNET. hNET antagonists without effect in non-transfected cells abolish both NE-dependent and NE-independent modes of charge movement in transfected cells. The magnitude of NE-dependent currents in these cells exceeds the expectations of simple carrier models using previous estimates of transport rates. To explain our observations, we propose that hNETs function as ion-gated ligand channels with an indefinite stoichiometry relating ion flux to NE transport. In this view, external Na+ and NE bind to the transporter with finite affinities in a cooperative fashion. However, coupled transport may not predict the magnitude or the kinetics of the total current through the transporter. We propose instead that Na+ gates NE transport and also the parallel inward flux of an indeterminate number of ions through a channel-like pore.