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D A Baylor

Publications and source records attributed to D A Baylor.

At least 37 records · Page 2Linked to original sources
PubMed1995

An alternative pathway for signal flow from rod photoreceptors to ganglion cells in mammalian retina.

Rod signals in the mammalian retina are thought to reach ganglion cells over the circuit rod-->rod depolarizing bipolar cell-->AII amacrine cell-->cone bipolar cells-->ganglion cells. A possible alternative pathway involves gap junctions linking the rods and cones, the circuit being rod-->cone-->cone bipolar cells-->ganglion cells. It is not clear whether this second pathway indeed relays rod signals to ganglion cells. We studied signal flow in the isolated rabbit retina with a multielectrode array, which allows the activity of many identified ganglion cells to be observed simultaneously while the preparation is stimulated with light and/or exposed to drugs. When transmission between rods and rod depolarizing bipolar cells was blocked by the glutamate agonist 2-amino-4-phosphonobutyric acid (APB), rod input to all On-center and briskly responding Off-center ganglion cells was dramatically reduced as expected. Off responses persisted, however, in Off-center sluggish and On-Off direction-selective ganglion cells. Presumably these responses were generated by the alternative pathway involving rod-cone junctions. This APB-resistant pathway may carry the major rod input to Off-center sluggish and On-Off direction-selective ganglion cells.

Aminobutyrates↗
PubMed1995

Conductance and kinetics of single cGMP-activated channels in salamander rod outer segments.

1. The conductance and kinetics of single 3',5'-cyclic guanosine monophosphate (cGMP)-activated channels of retinal rod outer segments were studied in inside-out membrane patches. The size of the single channel currents was increased by using low concentrations of divalent cations. 2. At saturating cGMP concentration, the current flickered at high frequency. Occasionally, the current was interrupted by closures lasting tens or hundreds of milliseconds. At +50 mV the maximum current during an opening was slightly more than 1 pA, but the open channel level was poorly resolved due to the speed of the gating transitions. 3. Amplitude histograms confirmed the presence of a sublevel of current, roughly a quarter the size of the peak current, at low cGMP concentrations. The fraction of time in the sublevel decreased with increasing cGMP concentration, suggesting that the sublevel may be due to opening by the partially liganded channel. 4. Consistent with previous macroscopic current recordings, single channel activation by cGMP had an apparent dissociation constant of 8.6 microM, and a Hill coefficient of 2.8. 5. At saturating cGMP concentrations, the channel was modelled as a two-state system with the following parameters. The open channel conductance was 25 pS. The opening rate constant, beta, was 1.5 x 10(4) s-1 at 0 mV, and had a voltage sensitivity equivalent to the movement of 0.23 electronic charges outward through the membrane electric field. The closing rate constant, alpha, was 2.1 x 10(4) s-1 and was voltage insensitive. Assuming that the open-state chord conductance was voltage independent, the inferred voltage dependence of beta largely accounted for the outward rectification in the steady-state macroscopic current-voltage relation of multichannel patches, at saturating cGMP concentration.

Animals↗
PubMed1995

Mechanisms of rhodopsin inactivation in vivo as revealed by a COOH-terminal truncation mutant.

Although biochemical experiments suggest that rhodopsin and other receptors coupled to heterotrimeric guanosine triphosphate-binding proteins (G proteins) are inactivated by phosphorylation near the carboxyl (COOH)-terminus and the subsequent binding of a capping protein, little is known about the quenching process in vivo. Flash responses were recorded from rods of transgenic mice in which a fraction of the rhodopsin molecules lacked the COOH-terminal phosphorylation sites. In the single photon regime, abnormally prolonged responses, attributed to activation of individual truncated rhodopsins, occurred interspersed with normal responses. The occurrence of the prolonged responses suggests that phosphorylation is required for normal shutoff. Comparison of normal and prolonged single photon responses indicated that rhodopsin begins to be quenched before the peak of the electrical response and that quenching limits the response amplitude.

Animals↗
PubMed1994

Calcium controls light-triggered formation of catalytically active rhodopsin.

Background light reduces the gain of phototransduction in retinal rods so that the ability to register changes in light intensity is not prevented by saturation of the cell's response. The gain is reduced by a light-induced fall in the intracellular calcium concentration which results from blockage of Ca2+ entry through the channels closed by light and continued Ca2+ extrusion by the Na:Ca,K exchanger. Calcium seems to exert several coordinated effects on the cyclic GMP cascade: a fall in [Ca2+] stimulates cGMP synthesis, increases the affinity of the cGMP-gated channel for cGMP and accelerates rhodopsin deactivation by phosphorylation. We now report that lowering intracellular [Ca2+] reduces the catalytic rhodopsin activity produced by light. The effect is operationally equivalent to a fourfold reduction in the number of rhodopsin molecules available for activation. The reduction in gain is cooperative and half-maximal at about 35 nM Ca2+, suggesting that it is mediated by a specific Ca(2+)-binding protein. Reduced rhodopsin activity in low Ca2+ should contribute to adaptation in background light.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1994

Multi-neuronal signals from the retina: acquisition and analysis.

Throughout the central nervous system, information about the outside world is represented collectively by large groups of cells, often arranged in a series of 2-dimensional maps connected by tracts with many fibers. To understand how such a circuit encodes and processes information, one must simultaneously observe the signals carried by many of its cells. This article describes a new method for monitoring the simultaneous electrical activity of many neurons in a functioning piece of retina. Extracellular action potentials are recorded with a planar array of 61 microelectrodes, which provides a natural match to the flat mosaic of retinal ganglion cells. The voltage signals are processed in real time to extract the spike trains from up to 100 neurons. We also present a method of visual stimulation and data analysis that allows a rapid characterization of each neuron's visual response properties. A randomly flickering display is used to elicit spike trains from the ganglion cell population. Analysis of the correlations between each spike train and the flicker stimulus results in a simple description of each ganglion cell's functional properties. The combination of these tools will allow detailed study of how the population of optic nerve fibers encodes a visual scene.

Action Potentials↗
PubMed1993

Interactions between divalent cations and the gating machinery of cyclic GMP-activated channels in salamander retinal rods.

The effects of divalent cations on the gating of the cGMP-activated channel, and the effects of gating on the movement of divalent cations in and out of the channel's pore were studied by recording macroscopic currents in excised membrane patches from salamander retinal rods. The fractional block of cGMP-activated Na+ currents by internal and external Mg2+ as well as internal Ca2+ was nearly independent of cGMP concentration. This indicates that Mg2+ and Ca2+ bind with similar affinity to open and closed states of the channel. In contrast, the efficiency of block by internal Cd2+ or Zn2+ increased in proportion to the fraction of open channels, indicating that these ions preferentially occupy open channels. The kinetics of block by internal Ni2+, which competes with Mg2+ but blocks more slowly, were found to be unaffected by the fraction of channels open. External Ni2+, however, blocked and unblocked much more rapidly when channels were mostly open. This suggests that within the pore a gate is located between the binding site(s) for ions and the extracellular mouth of the channel. Micromolar concentrations of the transition metal divalent cations Ni2+, Cd2+, Zn2+, and Mn2+ applied to the cytoplasmic surface of a patch potentiated the response to subsaturating concentrations of cGMP without affecting the maximum current induced by saturating cGMP. The concentration of cGMP that opened half the channels was often lowered by a factor of three or more. Potentiation persisted after the experimental chamber was washed with divalent-free solution and fresh cGMP was applied, indicating that it does not result from an interaction between divalent cations and cGMP in solution; 1 mM EDTA or isotonic MgCl2 reversed potentiation. Voltage-jump experiments suggest that potentiation results from an increase in the rate of cGMP binding. Lowering the ionic strength of the bathing solution enhanced potentiation, suggesting that it involves electrostatic interactions. The strong electrostatic effect on cGMP binding and absence of effect on ion permeation through open channels implies that the cGMP binding sites on the channel are well separated from the permeation pathway.

Animals↗
PubMed1992

Cyclic GMP-activated channels of salamander retinal rods: spatial distribution and variation of responsiveness.

1. Patch-clamp methods were used to investigate the areal density and spatial location of cyclic GMP-activated channels in the surface membrane of salamander rod outer segments. 2. The density of active channels (i.e. channels able to respond to cyclic GMP) in patches excised from outer segments was determined from the number of active channels, N, and the membrane area, A. N was estimated from the current induced by a saturating concentration of cyclic GMP, while A was estimated from the electrical capacitance of the patch. 3. In patches excised from forty-one isolated outer segments prepared in the light the active channel density varied over a remarkable range: 0.34-629 microns-2, with a mean of 166 microns-2. Density was not correlated with patch area in this or any of the conditions studied. 4. The spatial distribution of open channels on the outer segment of a transducing rod was measured by recording the local dark current at various positions with a loose-patch electrode. The apparent density of open channels varied by only about +/- 50% around the circumference of the outer segment and up and down its length. This indicates that the wide range of densities in excised patches did not result from sampling a non-uniform spatial distribution of channels. 5. Patches excised from sixteen dark-adapted whole cells with healthy appearances and saturating light responses of normal size had active channel densities of 1.1-200 microns-2, with a mean of 60 microns-2. Patches from twenty light-adapted whole cells had similar densities. Many densities from the whole cells were much lower than expected. This, and the wide variation in densities, suggests that obtaining a patch often lowered the density of active channels. The number of channels in a patch was quite stable from 1 s to 30 min after excision, ruling out progressive denaturation or adsorption of channels to the glass as a cause for this effect. 6. The mean active channel density in patches excised from whole cells was lower with calcium present in the external solution than with calcium absent (80 vs. 152 microns-2, n = 36 and 30 respectively). 7. We conclude that copies of the channel protein were present at a density of at least 650 microns-2 in the surface membrane of the outer segment and that the distribution of channels was fairly uniform on a 1 micron scale.(ABSTRACT TRUNCATED AT 400 WORDS)

Adaptation, Ocular↗
PubMed1992

Cation interactions within the cyclic GMP-activated channel of retinal rods from the tiger salamander.

1. The ionic dependence of current through the 3',5'-cyclic guanosine monophosphate (cyclic GMP)-activated channels of salamander rods was studied in excised inside-out membrane patches from isolated outer segments. Voltage-clamp experiments on transducing rods were performed so that the channels in intact cells could be compared with those in excised patches. 2. The reversal potential of the cyclic GMP-induced patch current was close to the Na+ equilibrium potential when the concentration of NaCl on the cytoplasmic surface of a patch was varied at constant external NaCl concentration. Fitting the Goldman-Hodgkin-Katz equation indicated that the apparent ratio of permeabilities for Na+ and Cl- was at least 50. This confirms a previous report that the channel's Na+ permeability is much larger than its Cl- permeability. 3. Na+ currents through the channel did not obey the independence principle. The outward patch current at large positive potential began to saturate with increasing concentrations of internal Na+, as if permeation required Na+ to bind to a site with an apparent dissociation constant around 180 mM. 4. In symmetrical NaCl solutions containing very low concentrations of divalent cations the current-voltage relation measured from excised patches 50 microseconds after switching the voltage showed mild outward rectification. By 1 ms the rectification was more pronounced. The rectification at 50 microseconds is attributed to voltage dependence of Na+ permeation. The additional rectification at later times is attributed to voltage dependence of the channel's probability of being open, depolarization favouring the open state. 5. In symmetrical Mg2+ solutions the cyclic GMP-induced patch currents were smaller and the outward rectification was more pronounced. 6. Addition of Mg2+ or Ca2+ to an internal Na+ solution blocked the cyclic GMP-induced Na+ current through the channels, as if by occupying a single binding site with an affinity in the 0.1-2 mM range. Block by Mg2+ was voltage dependent, suggesting that the binding site was within the channel's transmembrane electric field. Raising the Mg2+ concentration on the external surface of the patch increased the apparent dissociation constant of block by internal Mg2+, as expected if external and internal Mg2+ compete for the same binding site. 7. Block by internal Ca2+ had an opposite and weaker voltage dependence than block by internal Mg2+. 8. In symmetrical solutions containing both Na+ and Mg2+ the outward rectification was more pronounced than in solutions containing Na+ alone. In solutions thought to be close to physiological the outward patch current increased e-fold for a depolarization of 24-30 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Synchronous bursts of action potentials in ganglion cells of the developing mammalian retina.

The development of orderly connections in the mammalian visual system depends on action potentials in the optic nerve fibers, even before the retina receives visual input. In particular, it has been suggested that correlated firing of retinal ganglion cells in the same eye directs the segregation of their synaptic terminals into eye-specific layers within the lateral geniculate nucleus. Such correlations in electrical activity were found by simultaneous recording of the extracellular action potentials of up to 100 ganglion cells in the isolated retina of the newborn ferret and the fetal cat. These neurons fired spikes in nearly synchronous bursts lasting a few seconds and separated by 1 to 2 minutes of silence. Individual bursts consisted of a wave of excitation, several hundred micrometers wide, sweeping across the retina at about 100 micrometers per second. These concerted firing patterns have the appropriate spatial and temporal properties to guide the refinement of connections between the retina and the lateral geniculate nucleus.

Action Potentials↗
PubMed1991

Rapid charge movements and photosensitivity of visual pigments in salamander rods and cones.

1. Photosensitivities of visual pigments were determined by measuring early receptor currents (ERCs) in voltage-clamped photoreceptors from larval salamanders. 2. As expected from previous work of others, the ERC elicited by a brief flash consisted of a rapid inward component followed by a larger and slower outward component. The magnitude of the outward component corresponded to the movement of about 0.18 electronic charge across the membrane per photoisomerization. 3. The time course of the ERC was independent of the flash intensity, the flash wavelength and the magnitude of the response. The outward component of the cone ERC declined about twice as rapidly as the outward component of the rod ERC.. 4. The amplitude of the ERC decreased as successive flashes bleached the cell's pigment. Using the proportional relation between the size of the ERC and the number of pigment molecules photoisomerized, photosensitivities of the native A2 pigments in rods, red-sensitive cones, blue-sensitive cones and UV-sensitive cones were determined. Calculated solution photosensitivities for rhodopsin, red-sensitive and blue-sensitive cone pigments were not significantly different and the average value for all three pigments at their respective absorption maxima was (7.3 +/- 1.6) x 10(-9) micron 2 molecule-1. A value of 44.0 x 10(-9) micron 2 molecule-1 was obtained in a single UV-sensitive cone. 5. Substitution of the native dehydroretinal chromophore in the red-sensitive cone pigment with 11-cis-retinal increased the solution photosensitivity to (9.6 +/- 0.62) x 10(-9) micron 2 molecule-1. 6. We conclude that cone pigments have large molecular absorption cross-sections and high quantum efficiencies of photoisomerization. These properties seem well suited for the receptive molecules of a highly sensitive, miniaturized transducer.

Animals↗
PubMed1990

Effects of modified chromophores on the spectral sensitivity of salamander, squirrel and macaque cones.

1. Chemically modified retinal chromophores were used to investigate the mechanisms that produce the characteristic spectral absorptions of cone pigments. Spectral sensitivities of single cones from the salamander, squirrel and macaque retina were determined by electrical recording. The chromophore was then replaced by bleaching the pigment and regenerating it with a retinal analogue. 2. Exposing a bleached cone to 9-cis-retinal for a brief period (less than 20 min) caused its flash sensitivity to recover to about 0.2 of the pre-bleach value. Similar exposure to a locked 6-s-cis, 9-cis analogue gave a recovery to about 0.03 of the pre-bleach value. 3. Unlike the flash sensitivity, the saturating photocurrent amplitude often recovered completely after bleaching and regenerating the pigment. 4. When the 3-dehydroretinal chromophore in the salamander long-wavelength-sensitive (red) cone was replaced with 11-cis-retinal, shortening the conjugated chain in the chromophore, the spectral sensitivity underwent a blue shift of 67 nm. 5. Pigments containing the planar-locked 6-s-cis.9-cis-retinal analogue absorbed at substantially longer wavelength than those containing unmodified 9-cis-retinal. The opsin shift, a measure of the protein's ability to modify the chromophore's absorption was larger for the locked analogue than for 9-cis-retinal. This suggests that the native chromophore assumes a twisted 6-s-cis conformation in these pigments. 6. The spectral sensitivities of red and green macaque cones containing 9-cis-retinal or planar-locked 6-s-cis.9-cis-retinal retained the 30 nm separation characteristic of the native pigments. This suggests that the different absorptions of of the 6-7 carbon bond in the retinal chromophore.

Animals↗
PubMed1990

Visual transduction in cones of the monkey Macaca fascicularis.

1. Visual transduction in macaque cones was studied by measuring the membrane current of single outer segments projecting from small pieces of retina. 2. The response to a brief flash of light was diphasic and resembled the output of a bandpass filter with a peak frequency near 5 Hz. After the initial reduction in dark current there was a rebound increase which resulted from an increase in the number of open light-sensitive channels. The response to a step of light consisted of a prominent initial peak followed by a steady phase of smaller amplitude. 3. Responses to dim light were linear and time-invariant, suggesting that responses to single photons were linearly additive. From the flash sensitivity and the effective collecting area the peak amplitude of the single photon response was estimated as about 30 fA. 4. With flashes of increasing strength the photocurrent amplitude usually saturated along a curve that was gentler than an exponential but steeper than a Michaelis relation. The response reached the half-saturating amplitude at roughly 650 photoisomerizations. 5. The response-intensity relation was flatter in the steady state than shortly after a light step was turned on, indicating that bright light desensitized the transduction with a delay. This desensitization was not due to a reduction in pigment content. In the steady state, a background of intensity I lowered the sensitivity to a weak incremental test flash by a factor 1/(1 + I/IO), where IO was about 2.6 x 10(4) photoisomerizations s-1, or about 3.3 log trolands for the red- and green-sensitive cones. 6. Bleaching exposures produced permanent reductions in flash sensitivity but had little effect on the kinetics or saturating amplitude of subsequent flash responses. The sensitivity reductions were consistent with the expected reductions in visual pigment content and gave photosensitivities of about 8 x 10(-9) microns2 (free solution value) for the red- and green-sensitive pigments. During a steady bleaching exposure the final exponential decline of the photocurrent had a rate constant given by the product of the light intensity and the photosensitivity. 7. In some cells it was possible to measure a light-induced increase in current noise. The power spectrum of the noise resembled the spectrum of the dim flash response and the magnitude of the noise was consistent with a single photon response roughly 20 fA in size. 8. The membrane current recorded in darkness was noisy, with a variance near 0.12 pA2 in the band 0-20 Hz.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Hindered diffusion in excised membrane patches from retinal rod outer segments.

Excised inside-out membrane patches are useful for studying the cGMP-activated ion channels that generate the electrical response to light in retinal rod cells. We show that strong ionic current across a patch changes the driving force on the current by altering the ionic concentration near the surface membrane, an effect somewhat like that first described by Frankenhaeuser and Hodgkin (1956) in squid axons. The dominant concentration change occurs in the solution adjacent to the cytoplasmic (inner) surface of the membrane, where diffusion is impaired by intracellular material that adheres to the patch during excision. The magnitude and time course of the ionic changes are consistent with the expected volume of this material and with an effective diffusion coefficient about an order of magnitude less than that in free solution. Methods are described for correcting current transients observed in voltage clamp experiments, so that channel gating kinetics can be obtained without contamination by changes in driving force. We suggest that restricted diffusion may occur in patches excised from other types of cells and influence rapid kinetic measurements.

Ambystoma↗