Structural basis for on-and off-center responses in retinal bipolar cells.

Electron microscopy of Golgi preparations of goldfish retina shows that dendrites of type a (hyperpolarizing, off-center) bipolar cells make wide cleft junctions unassociated with synaptic ribbons, while those of type b (depolarizing, on-center) bioplar cells make narrow cleft junctions and synaptic ribbon contacts, with rods and cones. This suggests that wide cleft junctions are the site of sign-conserving, and narrow cleft junctions or ribbon contacts (or both) are the site of sign-inverting synaptic transmission from photoreceptors to bipolars.

A dopamine- and protein kinase A-dependent mechanism for network adaptation in retinal ganglion cells.

Vertebrates can detect light intensity changes in vastly different photic environments, in part, because postreceptoral neurons undergo "network adaptation." Previous data implicated dopaminergic, cAMP-dependent inhibition of retinal ganglion cells in this process yet left unclear how this occurs and whether this occurs in darkness versus light. To test for light- and dopamine-dependent changes in ganglion cell cAMP levels in situ, we immunostained dark- and light-adapted retinas with anti-cAMP antisera in the presence and absence of various dopamine receptor ligands. To test for direct effects of dopamine receptor ligands and membrane-permeable protein kinase ligands on ganglion cell excitability, we recorded spikes from isolated ganglion cells in perforated-patch whole-cell mode before and during application of these agents by microperfusion. Our immunostainings show that light, endogenous dopamine, and exogenous dopamine elevate ganglion cell cAMP levels in situ by activating D1-type dopamine receptors. Our spike recordings show that D1-type agonists and 8-bromo cAMP reduce spike frequency and curtail sustained spike firing and that these effects entail protein kinase A activation. These effects resemble those of background light on ganglion cell responses to light flashes. Network adaptation could thus be produced, to some extent, by dopaminergic modulation of ganglion cell spike generation, a mechanism distinct from modulation of transmitter release onto ganglion cells or of transmitter-gated currents in ganglion cells. Combining these observations with results obtained in studies of photoreceptor, bipolar, and horizontal cells indicates that all three layers of neurons in the retina are equipped with mechanisms for adaptation to ambient light intensity.

Voltage-gated Na+ current availability after step- and spike-shaped conditioning depolarizations of retinal ganglion cells.

We used two conditioning voltage protocols to assess inactivation of voltage-gated Na+ current in retinal ganglion cells. The first protocol tested the possibility, raised by published activation and steady-state inactivation curves, that Na+ ions carry a "window" current in these cells. The second protocol was used, because these cells spike repetitively in situ, to measure the Na+ current available for activation following spikes. Na+ current activated at test potentials more positive than ­65 mV. At test potentials more positive than ­55 mV, Na+ current peaked and then declined along a time course that could be fit by the sum of a large, rapidly decaying component, a small, slowly decaying component and a non-decaying component. Both step- and spike-shaped conditioning depolarizations reduced the amount of current available for subsequent activation, sparing the non-decaying "persistent" component. Most of the Na+ current recovered from this inactivation along a rapid exponential time course (τ=3 ms). The remaining recovery was complete within at least 4 s (at ­70 mV). Our use of step depolarizations has identified a current component not anticipated from previous measurements of steady-state inactivation in retinal ganglion cells. Our use of spike-shaped depolarizations shows that Na+ current density at 1 ms after a single spike is roughly 25% of that activated by the conditioning spike, and that recovery from inactivation is 50­90% complete within 10 ms thereafter. Na+ current amplitude declines during spikes repeated at relatively low frequencies, consistent with a slow component of full recovery from inactivation.

Rod and cone inputs to bipolar cells in goldfish retina.

Five morphological types of bipolar cells which make synaptic contact with rods and cones are distinguished in the retina of adult goldfish (Carassius auratus) by characteristics readily observable in the light microscope. Cells were designated type a or type b according to whether their axons terminate in the distal part (sublamina a) or proximal part (sublamina b) of the inner plexoform layer, respectively. Analysis of serial semi-thin sections of Golgi-impregnated cells demonstrates that each subtype of bipolar contacts rods and a characteristic set of chromatic subtypes of cones: types a1 and b1 cells contact rods and red-sensitive cones, while types a2, b2 and b3 contact rods and red- and green-sensitive cones. Comparison with published descriptions of cells stained with Procion Yellow after intracellular recordings had been made suggests that type a cells should be off-center types and type b on-center. Furthermore, it is suggested that the receptive fields of cell types a1 and b1 should be non-color-coded, and those of a2, b2, and b3 color-coded.

Functional differentiation of ganglion cells from multipotent progenitor cells in sliced retina of adult goldfish.

Multipotent progenitor cells at the retinal margin of adult goldfish give rise to all cell types in the rest of the retina. We took advantage of this spatial arrangement of progenitor and mature cells in slices of peripheral retina, to investigate the appearance and maturation of voltage-activated Na(+) current. We divided the peripheral retina into three broad regions (marginal, intermediate, and mature) on the basis of their morphological development. Whole-cell patch-clamp recordings were performed in ruptured-patch mode, so that cells from which currents were recorded could be identified by Lucifer Yellow fills. No voltage-activated Na(+) current was detected in the slender, peripherally located marginal cells. Voltage-activated Na(+) currents were detected in rounded cells found alongside or near marginal cells, facing the vitreal side of the retina. Some of these "intermediate cells" had a long axon-like process which ran along the vitreal surface. Intermediate cells adjacent to the marginal region tended to have smaller Na(+) currents than intermediate cells closer to the mature region. On average, the maximum Na(+) current amplitude recorded from intermediate cells was roughly 6-fold smaller than that of mature ganglion cells. In addition, the activation threshold of the Na(+) current in intermediate cells was nearly 14 mV more positive than that of mature ganglion cells. The results indicate that voltage-activated Na(+) current, as a possible marker of retinal ganglion cells, begins to develop well before these cells migrate to their adult position within the retina.

Omega-conotoxin-MVIID blocks an omega-conotoxin-GVIA-sensitive, high-threshold Ca2+ current in fish retinal ganglion cells.

Reduction of Ca2+ current amplitude by the Conus peptide omega-conotoxin-MVIID (omega-CTx-MVIID) was measured in voltage-clamped, goldfish retinal ganglion cells. Effects of depolarizing shifts in holding potential, and sequential applications of omega-CTx-MVIID, omega-CTx-GVIA, and BAY-K-8644, together with effects of Ni2+ and omega-Aga-IIIA, indicated that omega-CTx-MVIID may target Ca2+ channels differing from those termed T, L, N, P < Q and R.

Responses of solitary retinal horizontal cells to L-glutamate and kainic acid are antagonized by D-aspartate.

Solitary horizontal cells dissociated from goldfish retinas depolarized when exposed to micromolar doses of either L-glutamate or kainic acid. The responses to both of these agonists were antagonized by D-aspartate, and unaffected by L-aspartate, L-glutamic acid diethyl ester and folic acid. the results of the present study thus suggest that L-glutamate and kainic acid may produce depolarizations of horizontal cells by interacting with pharmacologically similar membrane receptors.

Regenerative sodium and calcium currents in goldfish retinal ganglion cell somata.

Whole-cell patch-clamp recordings were made from isolated goldfish retinal ganglion cell somata. Sodium- and calcium-dependent action potentials--blocked with tetrodotoxin (TTX) and Co2+ ions, respectively--were evoked under current-clamp. Depolarization of these somata under voltage clamp activated distinguishable Na+ and Ca2+ conductances: the former was permeable to Na+, blocked by TTX, impermeable to N-methyl-D-glucamine and tris(hydroxymethyl)aminomethane, and began to activate around -45 mV; the latter were permeable to Ca2+, but not Co2+, and began to activate around -55 mV. These results are consistent with recordings from carp, frog, salamander and rat retinal ganglion cells.

Ratfish retina--intracellular recordings and HRP injections in an isolated, superfused all-rod retina.

Hyperpolarizing responses to light were studied by intracellular recording in the isolated, superfused retina of the ratfish (Hydrolagus colliei). Two of these hyperpolarizing units were identified as horizontal cells by iontophoretic injection of horseradish peroxidase (HRP). These cells had rather large cell bodies (15 x 30 micrometer), elliptical dendritic arborizations measuring 150 x 300 micrometers and no axons. Since their physiological receptive fields were found to be at least 2.15 mm in diameter, it appears likely that either the photoreceptors or the horizontal cells are electrically coupled. Electron microscopy of HRP-injected horizontal cells showed their dendrites to end laterally in ribbon synapses of rods only and revealed dendro-dendritic contacts resembling gap junctions between injected and uninjected horizontal cells. The spectral sensitivity function of a dark-adapted horizontal cell can be described by a Dartnall nomogram based on a retinene pigment with lambda max = 473 nm. These findings are consistent with the histological observation that the ratfish retina appears to contain only rod photoreceptors.

Mechanisms of synaptic transmission in the retina.

This paper reviews the mechanisms of transmitter release, the kinetics of synaptic transfer, the mechanisms for the production of conductance changes by transmitters, and the nature of the conductance changes at synapses in vertebrate retina. A method for the culturing of adult retinal cells is described, together with preliminary experiments on the identification of cells in culture.

Solitary horizontal cells in culture--II. A new tool for examining effects of photoreceptor neurotransmitter candidates.

Membrane potentials of solitary horizontal cells dissociated from goldfish retinas were intracellularly measured while applying various acidic amino acids. L-glutamate and kainic acid depolarized solitary horizontal cells at micromolar doses. Neither L- nor D-aspartate produced any change in solitary horizontal cell resting potentials. Low-amplitude responses to L-glutamate showed no sign of desensitization. A steady, plateau-like, dose-dependent component of solitary horizontal cell responses to either L-glutamate or kainic acid, though obscured during its rising phase by action potentials, was always recorded during maintained agonist applications. Responses to either L-glutamate or kainic acid reversed in polarity at membrane potentials between 0 and -20 mV. Responses to L-glutamate collapsed reversibly when extracellular sodium ions were replaced by choline ions. Responses to either L-glutamate or kainic acid were antagonized by relatively high doses of D-aspartate. These results demonstrate that retinal horizontal cell chemosensitivity to acidic amino acids persists after dissociation, and in several respects resembles that found in several other preparations, including retinas in situ.

Selective potentiation of retinal horizontal cell responses to L-glutamate by D-aspartate.

1. L-Glutamate and L-aspartate depolarize type H1 horizontal cells in the isolated retina of goldfish, but only at millimolar concentrations. 2. When applied in the presence of D-aspartate, L-glutamate depolarizes H1 cells at concentrations nearly 15-fold lower than when it is applied alone. The effects of L-aspartate were not potentiated by either D-aspartate or D-glutamate. 3. Since D-aspartate seems also to enhance the effect of the transmitter released by cone photoreceptors, these results are consistent with the possibility that L-glutamate is a neurotransmitter of cones.

Deactivation, recovery from inactivation, and modulation of extra-synaptic ion currents in fish retinal ganglion cells.

As is shown magnificently by Heron Island's reef, the visual environment of many fishes includes various light intensities, hues and shapes that can change on large and small scales in space and time. Several articles in this issue address why fishes are sensitive to some of these properties, and how fishes and other aquatic species have acquired or fostered these sensitivities. This article discusses contributions of extrasynaptic ion currents, in a specific population of neurons, to the detection of ambient light levels, the appearance of certain visual stimuli and the disappearance of others.

(-)-baclofen and gamma-aminobutyric acid inhibit calcium currents in isolated retinal ganglion cells.

Of the various synaptic inputs known to converge upon retinal ganglion cells, the major inhibitory inputs are thought to be GABAergic. Although gamma-aminobutyric acid (GABA) is known to activate anion-selective ion channels in retinal ganglion cells, we have tested the possibility that GABA can also modulate cationic conductances in these cells, as seen in other central and peripheral neurons. Specifically, we have made whole-cell patch-clamp recordings to test whether voltage-gated calcium currents in isolated goldfish retinal ganglion cells are sensitive to GABAB receptor ligands. (-)-Baclofen and GABA inhibited calcium currents activated by moderately long depolarizations and, during large depolarizations (e.g., to 0 mV), also appeared to accelerate the rate of current decay. The calcium current inhibition induced by (-)-baclofen and GABA was not prevented by 2-hydroxysaclofen, phaclofen, or bicuculline, even though bicuculline suppressed a GABA-activated conductance in these cells. These results demonstrate the presence of baclofen- and GABA-sensitive calcium currents in vertebrate retinal ganglion cells as well as the coexistence of GABAA and GABAB receptors in individual retinal ganglion cells.

Cold inhibits neurite outgrowth from single retinal ganglion cells isolated from adult goldfish.

We have studied the growth of neurites from single retinal ganglion cells isolated from adult goldfish and maintained under various primary cell culture conditions. In 10% Leibovitz's L-15 medium at 23 degrees C, these ganglion cells remained viable for up to 10 days and generated extensive fields of neurites. We found two patterns of neuritic fields. In one, a pair of neurites exited from opposite sides of the cell soma, forming a bipolar pattern. In the second pattern, three to five neurites exited from several points around the soma, forming a multipolar pattern. Characteristically, each neurite of this latter type tapered and branched two to seven times, whereas neurites forming bipolar patterns showed less branching and little or no taper. The fields subtended by the neurites in multipolar patterns ranged in size from 33,000 to 204,000 microns 2. Finally, although these neurites grew as fast as 35 microns hr-1 at 23 degrees C and individually reached lengths of up to 735 microns, they showed essentially no growth at 13 degrees C. Neurite outgrowth at 23 degrees C was vigorous even in cells whose growth had previously been suppressed for as long as 8 hr at 13 degrees C.

Synchronous neurite branchings in single goldfish retinal ganglion cells.

We have examined the time course of branch formation in neurites of retinal ganglion cells isolated from adult goldfish (Carassius auratus). These neurites elongate at approximately 13 microns/h, and usually branch by bifurcation of growth cones at their tips. The times elapsed between branchings in different neurites of single cells can be described by a Poisson distribution with a mean interval of approximately 2 h. As predicted by this distribution, a relatively large number of branchings occur simultaneously in different neurites of individual cells. Simultaneous branchings of neurites elongating at a common rate generate branch points that lay equidistant from their soma. Since similar branching patterns can be seen in dendrites of retinal ganglion and amacrine cells in situ, these results are consistent with the possibility that dendrites of individual neurons branch synchronously and grow at common rates during development.

D-aspartate potentiates the effects of L-glutamate on horizontal cells in goldfish retina.

The amino acids L-glutamate and L-aspartate depolarize H1 horizontal cells in the perfused goldfish retina but only at millimolar concentrations. The effects of L-glutamate (but not of L-aspartate) are potentiated approximately 15-fold by exposure to D-aspartate. D-Aspartate blocks acidic amino acid uptake in goldfish retina, so that the potentiation of L-glutamate may be produced by an increase in its effective concentration at the horizontal cell membrane. Because D-aspartate also augments the light responses of horizontal cells, our results are consistent with the possibility that L-glutamate is a neurotransmitter of cone photoreceptors in goldfish.

Conotoxin-sensitive and conotoxin-resistant Ca2+ currents in fish retinal ganglion cells.

Using whole-cell patch-clamp methods, we tested whether omega-toxins from Conus block voltage-gated Ca2+ currents in teleost central neurons. The fractions omega-CTx-GVIA and omega-CTx-MVIIC, together with omega-toxins from Agelenopsis, the dihydropyridine BAY-K-8644, and voltage steps, produced effects indicating three types of Ca2+ current in dissociated goldfish retinal ganglion cells. One was activated by depolarization of most cells beyond -65 mV, primed at -95 mV but not at -45 mV, reduced by Ni2+, and unchanged by conotoxins, agatoxins, or BAY-K-8644. The second type constituted more than three-quarters of the total Ca2+ current in all cells, and at test potentials more positive than -30 mV, was reduced consistently by omega-CTx-GVIA, omega-CTx-MVIIC, and omega-Aga-IA, but not omega-Aga-IVA. The third Ca2+ current type was augmented by BAY-K-8644 at test potentials as negative as -45 mV, even in the presence of omega-CTx-GVIA. Replacement of extracellular Ca2+ by Ba2+ augmented current amplitude and slowed current decay. Conditioning depolarizations reduced Ca2+ current amplitude less than did omega-CTx-GVIA, and slowed current decay to imperceptible rates. These results provide the first description of conotoxin-sensitive, voltage-gated Ca2+ current recorded from teleost central neurons. Although most of the high-threshold Ca2+ current in these cells is blocked by omega-CTx-GVIA, it is also Ni(2+)-sensitive, and relatively resistant to omega-Aga-IIIA. The voltage sensitivities of low-and high-threshold Ca2+ current may suit current recruitment in situ after light-evoked hyperpolarizations end, and after light-evoked depolarizations begin, respectively.