Steven H. DeVries

Hemi-gap-junction channels in solitary horizontal cells of the catfish retina

1. Solitary horizontal cells were isolated from catfish retinas and their membrane current was recorded with a whole‐cell voltage clamp. Reducing the extracellular Ca2+ concentration produced a current that could be suppressed by dopamine. This Ca(2+)‐ and dopamine‐sensitive current is hereafter termed I gamma. The voltage dependence, cytoplasmic regulation, and permeability of the I gamma channel suggest that it is half of a gap‐junction channel. 2. I gamma was voltage and time dependent. In the steady state, the current‐voltage relation displayed outward rectification at voltages more depolarized than 0 mV and a negative resistance region at voltages more hyperpolarized than ‐15 mV. The reversal potential was 3.3 +/‐ 1.5 mV when NaCl was the predominant extracellular salt and potassium‐D‐aspartate was the predominant intracellular salt. 3. The size of I gamma depended on the extracellular Ca2+ concentration. I gamma was maximal at external Ca2+ concentrations below 10 microM, half‐maximal at 220 microM‐Ca2+, and reduced to less than 4% of its maximum amplitude at external Ca2+ concentrations above 1 mM. Increasing the extracellular Ca2+ concentration reduced the amplitude of I gamma without changing the shape of the current‐voltage relation or the kinetics of inactivation. Thus, rectification does not result from a voltage‐dependent block by extracellular Ca2+. 4. Patches of cell membrane were voltage clamped in both the cell‐attached and excised‐patch configurations. In the cell‐attached configuration, the addition of dopamine to the solution outside the patch pipette blocked the opening of channels within the membrane patch. Thus, dopamine closes I gamma channels by initiating an intracellular messenger cascade. In the excised‐patch configuration, a maximum conductance of 145 pS was measured while Cs+ and tetraethylammonium+ (TEA+) were the only monovalent cations on both sides of the membrane. 5. The ability of dopamine to suppress I gamma was blocked by introducing an inhibitor of the cyclic AMP‐dependent protein kinase, PKI5‐24, into the cytoplasm. Thus, the action of dopamine is mediated by a pathway that includes the activation of a cyclic AMP‐dependent kinase. 6. I gamma was suppressed by nitroprusside, an agent which activates guanylate cyclase and increases the intracellular cyclic GMP concentration. The effect of nitroprusside was not altered by the intracellular application of PKI5‐24. Thus, nitroprusside suppresses I gamma through a pathway that does not include the activation of a cyclic AMP‐dependent kinase.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of an electrical synapse between solitary pairs of catfish horizontal cells by dopamine and second messengers.

1. Retinas from channel catfish were dissociated and the cells maintained in culture. Horizontal cells that normally receive input from cone photoreceptors were identified. The conductance of the electrical junction formed between a pair of ‘cone’ horizontal cells was measured by controlling the membrane voltage of each cell with a voltage clamp maintained through either a micropipette or a patch pipette. The two techniques yielded similar results. 2. Transjunctional current was measured while transjunctional voltage was stepped to values between +/‐ 60 mV. The current (measured 5 ms after a step) was proportional to voltage over the range tested. For steps to voltages greater than +/‐ 45 mV, the current exhibited a slight time‐dependent decline. 3. Dopamine decreased junctional conductance in a dose‐dependent fashion. A 50% reduction was obtained with 10 nM‐dopamine. The D1 agonist fenoldopam (100 nM) also decreased junctional conductance. The uncoupling produced by either agent was rapid and reversible. 4. The introduction of 100 microM‐cyclic AMP into one cell of a pair decreased junctional conductance by, on average, 40%. Forskolin (1‐10 microM), an activator of adenylate cyclase, decreased junctional conductance 50‐90%. 5. The introduction of 80 microM‐cyclic GMP into one cell of a pair decreased junctional conductance by, on average, 40%. Nitroprusside (1‐10 microM), an activator of guanylate cyclase, reduced junctional conductance 40‐65%. 6. The introduction of a peptide inhibitor specific for the cyclic AMP‐dependent protein kinase reversed a decrease in junctional conductance produced by superfusion with either dopamine (1 microM), fenoldopam (100 nM) or forskolin (5‐10 microM). 7. Intracellular Ca2+ concentration was measured with the fluorescent indicator Fura‐2. The intracellular Ca2+ concentration was increased by activation of a Ca2+ current. Junctional conductance remained constant as the internal Ca2+ concentration changed from 100 to 700 nM. 8. Intracellular pH was measured with the fluorescent indicator bis‐carboxyethylcarboxyfluorescein. The application of acetate (2.5 mM) reduced intracellular pH by 0.2‐0.3 units and decreased junctional conductance by approximately 50%. A subsequent application of fenoldopam did not alter intracellular pH, but decreased junctional conductance by more than 50%. 9. The sensitivity of the junctional conductance between isolated horizontal cells to dopamine is consistent with dopamine having a direct effect on coupling in intact retina. Dopamine regulates the activity of a cyclic AMP‐dependent protein kinase which in turn modulates junctional conductance. Changes in intracellular pH and Ca2+ concentration are not involved in mediating the effect of dopamine on coupling. Cyclic GMP and intracellular pH may participate in regulatory pathways independent of that used by cyclic AMP.

Exocytosed protons feedback to suppress the Ca2+ current in mammalian cone photoreceptors.

A proton pump acidifies synaptic vesicles and provides the electrochemical gradient for transmitter uptake. Although external protons can modulate membrane voltage- and ligand-gated conductances, the fate of the protons released when vesicles fuse with the plasma membrane is unclear. In the dark, the glutamate-laden vesicles of cone photoreceptors fuse continuously with the plasma membrane. I now show that vesicular protons feed back to block the nearby calcium channels that mediate release. This local proton-mediated feedback is a novel mechanism through which neurons may regulate the release of transmitter.

Kainate receptors mediate synaptic transmission between cones and ‘Off’ bipolar cells in a mammalian retina

Light produces a graded hyperpolarization in retinal photoreceptors, that decreases their release of synaptic neurotransmitter,. Cone photoreceptors use glutamate, as a neurotransmitter with which to communicate with two types of bipolar cell. Activation of metabotropic glutamate receptors in ‘On’ bipolar cells, initiates a second-messenger cascade that can amplify small synaptic inputs from cones. In contrast, it is not known how the ionotropic glutamate receptors that are activated in ‘Off’ bipolar cells, are optimized for transmitting small, graded signals. Here we show, by recording from a cone and a synaptically connected ‘Off’ bipolar cell in slices of retina from the ground squirrel, that transmission is mediated by glutamate receptors of the kainate-preferring subtype. In the dark, a cone releases sufficient neurotransmitter to desensitize most postsynaptic kainate receptors. The small postsynaptic current that persists (<5% of maximum) is quickly modulated by changes in presynaptic voltage. Since recovery from desensitization is slow (the decay time constant is roughly 500 milliseconds), little recovery can occur during the brief (roughly 100-millisecond) hyperpolarization that is produced in cones by a flash of light. By limiting the postsynaptic current, receptor desensitization prevents saturation of the ‘Off’ bipolar cells voltage response and allows the synapse to operate over the cones entire physiological voltage range.

Parallel Processing in Two Transmitter Microenvironments at the Cone Photoreceptor Synapse

A cone photoreceptor releases glutamate at ribbons located atop narrow membrane invaginations that empty onto a terminal base. The unique shape of the cone terminal suggests that there are two transmitter microenvironments: within invaginations, where concentrations are high and exposures are brief; and at the base, where concentrations are low and exposure is smoothed by diffusion. Using multicell voltage-clamp recording, we show that different subtypes of Off bipolar cells sample transmitter in two microenvironments. The dendrites of an AMPA receptor-containing cell insert into invaginations and sense rapid fluctuations in glutamate concentration that can lead to transient responses. The dendrites of kainate receptor-containing cells make basal contacts and respond to a smoothed flow of glutamate that produces sustained responses. Signaling at the cone to Off bipolar cell synapse illustrates how transmitter spillover and synapse architecture can combine to produce distinct signals in postsynaptic neurons.

Electrical Coupling between Mammalian Cones

BACKGROUND Cone photoreceptors are noisy because of random fluctuations of photon absorption, signaling molecules, and ion channels. However, each cones noise is independent of the others, whereas their signals are partially shared. Therefore, electrically coupling the synaptic terminals prior to forward transmission and subsequent nonlinear processing can appreciably reduce noise relative to the signal. This signal-processing strategy has been demonstrated in lower vertebrates with rather coarse vision, but its occurrence in mammals with fine acuity has been doubted (even though gap junctions are present) because coupling would blur the neural image. RESULTS In ground squirrel retina, whose triangular cone lattice resembles the human fovea, paired electrical recordings from adjacent cones demonstrated electrical coupling with an average conductance of approximately 320 pS. Blur caused by this degree of coupling had a space constant of approximately 0.5 cone diameters. Psychophysical measurements employing laser interferometry to bypass the eyes optics suggest that human foveal cones experience a similar degree of neural blur and that it is invariant with light intensity. This neural blur is narrower than the eyes optical blur, and we calculate that it should improve the signal-to-noise ratio at the cone terminal by about 77%. CONCLUSIONS We conclude that the gap junctions observed between mammalian cones, including those in the human fovea, represent genuine electrical coupling. Because the space constant of the resulting neural blur is less than that of the optical blur, the signal-to-noise ratio can be markedly improved before the nonlinear stages with little compromise to visual acuity.

Bipolar cell pathways for color and luminance vision in a dichromatic mammalian retina

The mammalian retina is fundamentally dichromatic, with trichromacy only recently emerging in some primates. In dichromats, an array of short wavelength–sensitive (S, blue) and middle wavelength–sensitive (M, green) cones is sampled by approximately ten bipolar cell types, and the sampling pattern determines how retinal ganglion cells and ultimately higher visual centers encode color and luminance. By recording from cone–bipolar cell pairs in the retina of the ground squirrel, we show that the bipolar cell types sample cone signals in three ways: one type receives input exclusively from S-cones, two types receive mixed S/M-cone input and the remaining types receive an almost pure M-cone signal. Bipolar cells that carry S- or M-cone signals can have a role in color discrimination and may contact color-opponent ganglion cells. Bipolar cells that sum signals from S- and M-cones may signal to ganglion cells that encode luminance.

Separate blue and green cone networks in the mammalian retina

The distinct absorbance spectra of the cone photopigments form the basis of color vision, but ultrastructural and physiological evidence shows that mammalian cones are electrically coupled. Coupling between cones of the same spectral type should average voltage noise in adjacent photoreceptors and improve the ability to resolve low-contrast spatial patterns. However, indiscriminate coupling between spectral types could compromise color vision by smearing chromatic information across channels. Here we show, by measuring the junctional conductance between green-green and blue-green cone pairs in slices from the dichromatic ground-squirrel retina, that green-green cone pairs are routinely coupled with an average conductance of 220 pS, whereas coupling is undetectable in blue-green cone pairs. Together with a lack of tracer coupling and the selective localization of connexin proteins, our results show that signals in blue and green cones are processed separately in the photoreceptor layer.

A Mammalian Retinal Bipolar Cell Uses Both Graded Changes in Membrane Voltage and All-or-Nothing Na+ Spikes to Encode Light

Barlow (1953) studied summation in ganglion cell receptive fields and observed a fine discrimination of spatial information from which he inferred that retinal interneurons use analog signals to process images. Subsequent intracellular recordings confirmed that the interneurons of the outer retina, including photoreceptors, horizontal cells, and bipolar cells, respond to light with slow, graded changes in membrane potential. Analog processing may enable interneurons to discriminate fine gradations in light intensity and spatiotemporal pattern, but at the expense of the speed, temporal precision, and threshold discrimination that are characteristic of all-or-nothing Na+ spikes. We show that one type of mammalian On bipolar cell, the ground squirrel cb5b, has a large tetrodotoxin (TTX)-sensitive Na+ current. When recorded from in the perforated patch configuration, cb5b cells can signal the onset of a light step with 1–3 all-or-nothing action potentials that attain a peak amplitude of −10 to −20 mV (peak width at half-height equals 2–3 ms). When exposed to a continuous, temporally fluctuating stimulus, cb5b cells generate both graded and spiking responses. Cb5b cells spike with millisecond precision, selecting for stimulus sequences in which transitions to light are preceded by a period of darkness. The axon terminals of cb5b bipolar cells costratify with the dendrites of amacrine and ganglion cells that encode light onset with a short latency burst of spikes. The results support the idea that a spiking On bipolar cell is part of a dedicated retinal pathway for rapidly and reliably signaling dark to light transitions.

A non-canonical pathway for mammalian blue-green color vision

The dynamic range of visual coding is extended by having separate ganglion cell types that respond to light increments and decrements. Although the primordial color vision system in mammals contains a well-characterized ganglion cell that responds to blue light increments (a blue On center cell), less is known about ganglion cells that respond to blue light decrements (blue Off center cells). We identified a regular mosaic of blue Off center ganglion cells in the ground squirrel. Contrary to the standard scheme, blue Off responses came from a blue On bipolar and inverting amacrine cell.