Chapter 5 Membrane properties of solitary retinal cells

Although isolated retinal cell preparations have been used widely to study retinal function in lower vertebrates, dissociated cells from primate retina have not been developed for routine physiological experiments. In this study, we demonstrated the feasibility of obtaining viable and identifiable dissociated cells from the primate retina. In addition, we characterized voltage-dependent membrane currents in each type of primate retinal cell with the whole-cell patch-clamp technique. Multiple types of ionic conductance with distinctive current profiles were recorded in various types of primate retinal neurons. Photoreceptors exhibited an inward I_H activated by membrane hyperpolarization and an outward current activated at depolarized potentials. Two types of potassium currents (transient potassium current, I_K(A), and delayed rectifier potassium current, I_K(V)) were recorded from bipolar cells. I_K(A) dominated the current response in putative midget bipolar cells, and I_K(V) was mainly associated with putative rod bipolar cells. L-type calcium currents (I_Ca) were observed in primate bipolar cells with axon terminals, but not in axotomized bipolar cells. Large voltage-dependent sodium currents (I_Na) were only recorded from ganglion cells. Müller cells exhibited I_K(V) and large potassium inward rectifier current (I_K(IR)), and occasionally a small I_Na. Neurons with electrophysiological signatures of amacrine cells and horizontal cells were also studied even though their morphological features were lost during cell dissociation. By using both morphological and physiological criteria outlined in this report, it is possible to use the dissociated retinal cell preparation as an in vitro system for physiological, biochemical and pharmacological studies of the primate visual system.

A model system for syncytial integration is the outer vertebrate retina, where graded signals or electrotonic potentials interact laterally via gap junctions to form an integrated response that is relayed by chemical synapses to the next layer of interconnected cells. Morphological and physiological experiments confirm that bipolar cells form quasisyncytial lattices, and so this review will aim to address two important issues: the function of coupling in visual information processing and the construction of a robust mathematical model that can adequately simulate signal spread in the bipolar cell syncytium. It is shown that the role of coupling in bipolar cells differs from that associated in the presynaptic networks, namely, loss in spatial resolution in order to increase the signal-to-noise ratio. The intrinsic membrane properties of bipolar cells which give rise to voltage-dependent currents are inactive over the normal in vivo operating range of membrane potential and may be shunted as a direct result of electrotonic coupling, suppressing any possibility of action potential propagation in the bipolar cell syncytium. It is therefore speculated that the mechanisms underlying processing of information in bipolar networks are dependent on the structure of bipolar cells and in particular, on the presence of gap junctions. It is proposed that a three-dimensional model which incorporates the spatial properties of each bipolar cell in the network in the form of a leaky cable is the most likely model to simulate signal spread in the bipolar cell syncytium in vivo. This is because discrete network models represent each bipolar cell in the syncytium as isopotential units without any spatial structure, and thus are unable to reproduce the temporal characteristics of electrotonic potential spread within the central receptive field of bipolar cells.

We propose an ionic current model of bipolar cells based on the published experimental data. Five types of ionic currents identified in bipolar cell bodies,I_h,I_Kv,I_A,I_Ca andI_K(Ca) were described by a mathematical formulation similar to the Hodgkin and Huxley (Journal of Physiology,117, 500–544, 1952) equations.. The model parameters were estimated from the voltage clamp data. In simulation, we demonstrate that the present model reproduces not only the voltage clamp responses but also the current clamp responses of the bipolar cells. As a result, the model provides a better understanding of the functional role of the ionic currents in bipolar cells in generating the electrical responses.

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°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 33000 to 204000 μm². Finally, although these neurites grew as fast as 35 μm hr⁻¹ at 23°C and individually reached lengths of up to 735 μm, they showed essentially no growth at 13°C. Neurite outgrowth at 23°C was vigorous even in cells whose growth had previously been suppressed for as long as 8 hr at 13°C.

Retinal horizontal cells (HCs) are second-order neurons that integrate information from photoreceptors over large retinal areas, mediating the lateral spread of visual signals in the distal retina. The ‘glial’ vs. ‘neuronal’ nature of the HC has been widely debated. For example, carbonic anhydrase (CA), glutamine synthetase (GS), and glial fibrillary acidic protein (GFAP) are considered ‘glial’ markers, yet both CA and GFAP have been previously reported in HCs of the teleost retina in species-specific patterns^6,15. In contrast, the neurofilament triplet (NFT) proteins are considered ‘neuronal’ markers; these proteins have been immunolocalized to a mammalian HC³, but are absent from teleost HCs¹⁵. We have studied these cytochemical characteristics in HCs from the white bass, by immunolabeling both cryosections of intact retina and freshly isolated, identified cells attached to coverslips. We found that both HCs (neurons) and Müller cells (MCs; glia) immunolabeled with antisera to CA. Both type 1 (external) HCs and MCs immunolabeled with an antibody to vimentin. Only MCs immunolabeled with antisera to GS and GFAP. Neither HC perikarya (and their major dendrites) nor MCs immunolabeled with an antibody to the 160-kDa subunit of NFT protein. Thus, bass HCs and MCs share the presence of CA and vimentin epitopes and absence of the NFT 160-kDa epitope. Moreover, retinal cell isolation, by itself, does not affect cell-type specific immunolabeling patterns in identified cells, except for what may be lost with the finer processes of the various cells. Isolated cell studies can aid in interpreting immunolabeling patterns observed in the intact retina, especially in retinal layers where several cell types may be present.