Properties of depolarizing bipolar cell responses to central illumination in salamander retinal slices.

The voltage and current responses of depolarizing bipolar cells to central illumination were studied by means of whole-cell recording in retinal slices of the larval tiger salamander, Ambystoma tigrinum. To stabilize the responses, it was necessary to limit exchanges between the cytosol and the solution in the patch pipette by reducing the diameter of the pipette tip opening. The current-voltage relationship of the cell membrane in darkness displayed a strong outward rectification, and the inward current evoked by light could be consistently reversed by depolarization only when tetraethylammonium was added to the pipette solution. As a result of the membrane non-linearity, increases in the intensity of bright lights caused relatively smaller amplitude increases in the voltage than in the current responses and the latter had a proportionally smaller after-effect. With larger pipette tip openings, the cytosol equilibrated with the pipette filling solution. Under these conditions the light-evoked responses gradually became slower and acquired an on-off pattern, their final amplitude and polarity being determined by the ratio of the chloride concentrations on each side of the cell membrane. This finding is interpreted as revealing the existence of two response components: a chloride-dependent on-off increase in conductance and a faster depolarizing input that was lost through diffusional exchange. Addition of GTP and ATP to the electrode filling solution had a stabilizing effect on the labile component, whether or not cyclic GMP was also included. Observations on the magnitude of the conductance changes and on diphasic reversals indicate that the labile response component, presumably representing direct input from photoreceptors, is caused by an increase in conductance. The resulting inward current was still present at a low intracellular chloride concentration and may be assumed, therefore, to be carried by a cation influx.

Chemical synapses between turtle photoreceptors.

Rod photoreceptors of the snapping turtle retina wer Golgi impregnated and studied in the electron microscope. Telodendria arising from the synaptic bases ended at rods and cones as lateral or central elements of the ribbon synaptic complex, thus providing clear evidence of chemical synapses between turtle photoreceptors.

Synaptic action mediating cone responses to annular illumination in the retina of the larval tiger salamander.

1. Responses of salamander cones to steps of light on their surrounding area were intracellularly recorded through micropipettes filled with 2 M-potassium acetate or 2 M-potassium chloride. 2. Using 2 M-potassium acetate, cone step responses showed a larger relaxation for a 1100 micrometers diameter spot than for a 100 micrometers spot, but dim annular illumination failed to evoke any detectable response. With 2 M-potassium chloride the larger spot or an anulus evoked responses that were mainly or purely depolarizing, respectively, while the responses to the smaller spot were still hyperpolarizing. 3. The depolarizing response to annular illumination had an increased amplitude immediately after discontinuing the injection of inward current through a micropipette filled with 2 M-potassium chloride. 4. Depolarization by extrinsic current reversed the polarity of the depolarizing response to annular illumination. 5. It is suggested that the enhancement of the depolarizing influence of the surround is due to an increase in the intracellular concentration of chloride ions, which issue from the electrodes by passive diffusion or electrophoresis. Taking into consideration the effect of depolarizing current, it would follow that surround illumination induces an increase in the chloride conductance of the cone membrane. 6. Preliminary observations on the properties of the enhanced surround response reveal a slow time course and a receptive field of more than 270 micrometers radius.

Lateral contacts and interactions of horizontal cell dendrites in the retina of the larval tiger salamander.

1. The contacts of horizontal cell dendrites with processes of other second order neurones were studied at the level of the electron microscope in serial sections of the salamander retina. Intracellular recordings of the responses to light of horizontal and bipolar cells were used to investigate the possible significance of some of the morphological findings.2. Horizontal cell dendrites make close membrane appositions (gap junctions) with one another and are post-synaptic to bipolar cell dendrites at presumed chemical synapses. On the other hand, there is no clear evidence that horizontal cell dendrites are presynaptic to any other neuronal processes at the outer plexiform layer, so that the output connexions of horizontal cell bodies remain a matter of speculation.3. It is suggested that the bipolar cell input and the gap junctions between dendrites contribute, respectively, depolarizing and hyperpolarizing components to the responses of horizontal cell bodies to surround illumination. In addition, the facilitatory effect of central illumination on the surround response of horizontal cell bodies may result, although perhaps only partly, from observed properties of the surround response of bipolar cells.4. In the course of these observations, bipolar cells were found to be presynaptic at the outer plexiform layer not only to horizontal cell dendrites, but also to other bipolar cells, horizontal cell axon terminals and certain processes belonging to an as yet unidentified neurone.

Contacts between receptors and electrophysiologically identified neurones in the retina of the larval tiger salamander.

1. Following the intracellular recording of bipolar and horizontal cell responses, each unit was injected with horseradish peroxidase and a histochemical staining used to identify it at the level of the light and electron microscopes.2. Centre-depolarizing bipolar cells made contact with rods and cones at basal and ribbon junctions, the latter being fewer. Centre-hyperpolarizing bipolar cells made the same types of contacts with the receptors, but ribbon junctions predominated.3. It appears, therefore, that there is no fixed relationship between the sign of synaptic transmission from receptors to bipolar cells and the junctional features revealed by present methods for electron microscopy of tissue sections. Accordingly, the reason for the existence of more than one type of contact between receptors and bipolar cells remains to be determined.4. Light microscopy of the peroxidase-injected horizontal cells gave further support to the notion that type B responses are recorded from the cell body, and type A responses from the axon terminal, of a single type of horizontal cell. Electron microscopy showed that the processes (dendrites) originating from the cell body make ribbon and distal junctions with rods and cones, just as shown before for the axon terminals.5. As a result of these observations, it is possible to exclude one of two alternative hypotheses previously proposed to account for the properties of the surround responses recorded from the horizontal cell bodies.

Horizontal cell responses in the retina of the larval tiger salamander.

The responses to light of horizontal cells were recorded intracellularly in the retina of the larval tiger salamander. 2. All the units studied had a large summation area and were hyperpolarized by circles of light of any wave-length centred on the recording electrode, but two types could be distinguished according to the properties of their receptive fields. Type A units were hyperpolarized following illumination of any portion of their receptive field, while type B units were not hyperpolarized by illumination of their surround unless the centre was simultaneously illuminated, stimulation of the surround alone resulting in either a small depolarization or virtually no response. 3. Procion yellow injections showed that type A responses are recorded from thick and long processes not directly continuous with an identifiable cell body, while type B responses originate from the cell body of cells that send very fine and tortuous processes towards the receptors. The histological observations also suggested that the type A units represent expansions or swellings of one or more of the fine processes originating from the type B units. Therefore, it seems possible that both types of units are just different parts of a single kind of horizontal cell, and that a majority of the dye injections failed to stain them simultaneously because of the small diameter of the connecting process. 4. The large summation area of type A units can be explained, just as for horizontal cells in other retinae, by supposing that they are electrically coupled to other units of the same type. The receptive field properties of type B units, however, can only be partly explained by electrical coupling, and then only if the existence of voltage-dependent junctions is postulated. Instead, the reversal of the polarity of responses to an annulus of light during steady illumination of the centre, plus the available electron microscopic evidence, suggest that the effect of the surround on the type B units is due to a chemical synaptic impingement from the type A units.

Light-induced resistance changes in retinal rods and cones of the tiger salamander.

1. The electrical properties of retinal rods and cones of the larval tiger salamander were investigated with intracellular electrodes, and the cells identified by means of dye injections.2. Both types of photoreceptors are hyperpolarized by illumination. Following stimulation with brief flashes of dim light, rod responses show a slower time course than cone responses; with bright flashes, rod responses can be recognized because of their long recovery time.3. Values of input resistance were derived from the voltage displacement induced by constant current pulses in darkness or at the peak of the photoresponse. The input resistance following illumination was also calculated from the effect of steady polarizing currents on the amplitude of the photoresponse.4. In darkness, the input resistance of the rod cells is time- and voltage-dependent, but the voltage-current relations of most cells have a linear region which includes the physiological limits of membrane potential. At the peak of the photoresponse, the input resistance (slope of the linear region of the v-i relations) is decreased.5. Cone cells show approximately linear v-i relations. As reported by previous authors, illumination increases the input resistance.6. These results support the current view that the cone photoresponse is the consequence of a reduction in the permeability of channels which in darkness shunt the membrane. In rods, however, it appears that the main effect of illumination is to increase the permeability of the membrane to ions for which the equilibrium potential is more negative than the membrane potential in darkness.

The site of origin of electrical responses in visual cells of the leech, Hirudo medicinalis.

In leech visual cells the presumed light-absorbing structures are microvilli arising from the membrane of what would seem to be a large intracellular vacuole. This vacuole, however, is an extracellular compartment, since it communicates with the intercellular spaces through narrow channels. Therefore, the membrane of the microvilli is-as in other invertebrate visual cells-a part of the cell membrane. Visual responses recorded with an electrode within the vacuole were compared with the intracellular recordings. Following illumination the vacuole becomes negative with respect to the outside fluid, while the cells are depolarized. This finding indicates that inward current penetrates the cell through the microvillar membrane. It is concluded, therefore, that the electrical response (receptor potential) originates as a result of changes in the properties of the light-absorbing membrane.

The pathway between hyaloid blood and retinal neurons in the toad. Structural observations and permeability to tracer substances.

The hyaloid vessels form a capillary network on the inner surface of the retina. These capillaries are embedded in the vitreous humor, and they lack a glial investment. The intercellular spaces of the retina communicate with the ocular cavity, as can be evidenced by following the penetration of tracer substances. Hence, there is an extracellular diffusion pathway between hyaloid capillaries and retinal neurons, without interposition of glial cells. Trypan blue and ferrocyanide were not detected within the vitreous humor nor the retina after systemic injection. To this extent, at least, the hyaloid capillaries functionally resemble central nervous system capillaries. Intravascular injections of horseradish peroxidase established the absence of vesicular transfer across the endothelium of the hyaloid capillaries. In addition, quintuple-layered junctions between endothelial cells prevented the intercellular passage of the enzyme. It is likely, therefore, that the only pathway across the endothelium of the hyaloid capillaries is through the plasmalemma of the endothelial cells.

Cell junctions in ommatidia of Limulus.

The intercellular relationships in the ommatidia of the lateral eye of Limulus have been investigated. The distal process of the eccentric cell gives origin to microvilli which interdigitate with the microvilli of the retinular cells. Therefore, both types of visual cells contribute to form the rhabdom and may have an analogous photoreceptor function. Quintuple-layered junctions are found within the rhabdom at the lines of demarcation between adjoining microvilli, whether the microvilli originate from a single retinular cell, from two adjacent retinular cells, or from a retinular cell and the eccentric cell. Furthermore, quintuple-layered junctions between the eccentric cell and the tips of the microvilli of the retinular cells occur at the boundary between the distal process and the rhabdom. These findings are interpreted to indicate that the rhabdom provides an extensive electrotonic junction relating retinular cells to one another and to the eccentric cell. Quintuple-layered junctions between glial and visual cells, as well as other structural features of the ommatidial cells, are also described.

Potential, current, and ionic fluxes across the isolated retinal pigment epithelium and choriod.

A flux chamber was utilized for in vitro studies of a membrane formed by the retinal pigment epithelium and choroid of the eye of the toad (Bufo arenarum and Bufo marinus). A transmembrane potential of 20 to 30 mv was found, the pigment epithelium surface positive with respect to the choroidal surface. Unidirectional fluxes of chloride, sodium, potassium, and calcium were determined in the absence of an electrochemical potential difference. A net transfer of chloride from pigment epithelium to choroid accounted for a major fraction of the mean short-circuit current. A small net flux of sodium from choroid to pigment epithelium was detected in Bufo marinus. In both species of toads, however, about one-third of the mean short-circuit current remained unaccounted for. Manometric determinations of bicarbonate suggested an uptake of this ion at the epithelial surface of the membrane but did not provide evidence of a relationship between this process and the short-circuit current.