Eric M. Lasater

Membrane properties of an unusual intrinsically oscillating, wide‐field teleost retinal amacrine cell

In the retina, amacrine cells modulate the transfer of information from bipolar to ganglion cells. The nature of the modulation depends on the synaptic input and the membrane properties of the cells. In the retina of white bass, we identified a class of bistratified, wide‐field amacrine cell characterized by immunopositive labelling for GABA and calmodulin. In isolation, the cells presented resting membrane potentials averaging ‐69 mV although some cells settled at more depolarized values (‐30 mV). Injection of depolarizing current pulses induced oscillatory membrane responses. When elicited from depolarized cells, the oscillations were short‐lived (< 40 ms). For the most part, the oscillatory potentials of hyperpolarized cells remained unattenuated throughout the depolarizing pulse. The frequency of the oscillations increased logarithmically with mean membrane potential, ranging from 74 to 140 Hz. Cells exhibiting depolarized membrane potentials oscillated at twice that rate. When the membrane potential of these cells was hyperpolarized to ‐70 mV, the oscillations became unattenuated and slowed. We found the cells expressed voltage‐gated sodium, potassium and calcium currents and calcium‐dependent potassium currents. We demonstrate that the oscillatory potentials arose as a result of the interplay between calcium and potassium currents. The cells responded to local application of GABA and glycine, both of which modulate the oscillatory potentials. Glutamate and its analogues depolarized the cell and induced oscillatory potentials. Our results indicate that oscillatory responses of a type of wide‐field amacrine cell are an intrinsic feature of the cell and not due to circuit properties.

Membrane currents of spiking cells isolated from turtle retina.

SummaryWe examined the membrane properties of spiking neurons isolated from the turtle (Pseudemys scripta) retina. The cells were maintained in culture for 1–7 days and were studied with the whole cell patch clamp technique. We utilized cells whose perikaryal diameters were >15 μm since Kolb (1982) reported that ganglion cell perikarya in Pseudemys retina are 13–25 μm, whereas amacrine perikarya are less than 14 μm in diameter.We identified 5 currents in the studied cells: (1) a transient sodium current (INa) blocked by TTX, (2) a sustained calcium current (ICa) blocked by cobalt and enhanced by Bay-K 8644, (3) a calcium-dependent potassium current (IK(Ca)) (4) an A-type transient potassium current (IA) somewhat more sensitive to 4-AP than TEA, (5) a sustained potassium current (IK) more sensitive to TEA than 4-AP.The estimated average input resistance of the cells at — 70 mV was 720 ± 440 MΩ. When all active currents were blocked, the membrane resistance between — 130 and +20 mV was 2.5 GΩ.When examined under current clamp, some cells produced multiple spikes to depolarizing steps of 0.1–0.3 nA, whereas other cells produced only a single spike irrespective of the strength of the current pulse. Most single spikers had an outward current that rose to a peak relatively slowly, whereas multiple spikers tend to have a more rapidly activating outward current.Under current clamp, 4-AP slowed the repolarization phase of the spike thus broadening it, but did not always abolish the ability to produce multiple spikes. TEA induced a depolarized plateau following the initial spike which precluded further spikes. It thus appears that the spiking patterns of the retinal cells are shaped primarily by the kinetics of INa, IK and IA and to a lesser extent by IK(Ca).

Intracellular calcium release resulting from mGluR1 receptor activation modulates GABAA currents in wide-field retinal amacrine cells: a study with caffeine.

The modulatory action of calcium (Ca2+) released from intracellular stores on GABAA receptor‐mediated current was investigated in wide‐field amacrine cells isolated from the teleost, Morone chrysops, retina. Caffeine, ryanodine or inositol 1,4,5‐triphosphate (IP3) markedly inhibited the GABAA current by elevating [Ca2+]i. The inhibition resulted from the activation of a Ca2+→ Ca2+/calmodulin → calcineurin cascade. Long (>12 s) exposure to glutamate mimicked the caffeine effect, i.e. it inhibited the GABAA current by elevating [Ca2+]i through mGluR1 receptor activation and consequent IP3 generation. This pathway provides a ‘timed’ disinhibitory mechanism to potentiate excitatory postsynaptic potentials in wide‐field amacrine cells. It occurs as a result of the suppression of GABA‐mediated conductances as a function of the duration of presynaptic excitatory input activity. This is much like some forms of long‐term potentiation in the central nervous system. In a local retinal circuit this will selectively accentuate particular excitatory inputs to the wide‐field amacrine cell. Similar to other neural systems, we suggest that activity‐dependent postsynaptic disinhibition is an important feature of the signal processing in the inner retina.

L-type calcium channels mediate transmitter release in isolated, wide-field retinal amacrine cells.

Transmitter release in neurons is triggered by intracellular Ca2+ increase via the opening of voltage-gated Ca2+ channels. Here we investigated the voltage-gated Ca2+ channels in wide-field amacrine cells (WFACs) isolated from the white-bass retina that are functionally coupled to transmitter release. We monitored transmitter release through the measurement of the membrane capacitance (Cm). We found that 500-ms long depolarizations of WFACs from -70 mV to 0 mV elicited about a 6% transient increase in the Cm or membrane surface area. This Cm jump could be eliminated either by intracellular perfusion with 10 mM BAPTA or by extracellular application of 4 mM cobalt. WFACs possess N-type and L-type voltage-gated Ca2+ channels. Depolarization-evoked Cm increases were unaffected by the specific N-type channel blocker omega-conotoxin GVIA, but they were markedly reduced by the L-type blocker diltiazem, suggesting a role for the L-type channel in synaptic transmission. Further supporting this notion, in WFACs the synaptic protein syntaxin always colocalized with the pore-forming subunit of the retinal specific L-type channels (Cav1.4 or alpha1F), but never with that of the N-type channels (Cav2.2 or alpha1B ).

The role of potassium conductance in the generation of light responses in Müller cells of the turtle retina

Müller cells are highly permeable to potassium ions and play a major role in maintaining potassium homeostasis in the vertebrate retina during light-evoked neuronal activity. Potassium fluxes across the Müller cells membrane are believed to underlie the light-evoked responses of these cells. We studied the potassium currents of turtle Müller cells in the retinal slice and in dissociated cell preparations and their role in the genesis of the light-evoked responses of these cells. In either preparation, the I-V curve, measured under voltage-clamp conditions, consisted of inward and outward currents. A mixture of cesium ions, TEA, and 4-AP blocked the inward current but had no effect on the outward current. Extracellular cesium ions alone blocked the inward current but exerted no effect on the photoresponses. Extracellular barium ions blocked both inward and outward currents, induced substantial depolarization, and augmented the light-evoked responses, especially the OFF component. Exposing isolated Müller cells to a high potassium concentration did not cause any current or voltage responses when barium ions were present. In contrast, application of glutamate in the presence of barium ions induced a small inward current that was associated with a substantially augmented depolarizing wave relative to that observed under control conditions. This observation suggests a role for an electrogenic glutamate transporter in generating the OFF component of the turtle Müller cell photoresponse.

Properties of non-NMDA excitatory amino acid-activated channels in isolated retinal horizontal cells

The excitatory amino acid glutamate is believed to be the neurotransmitter used by some photoreceptors in the teleost retina. Past studies have shown that exogenous glutamate, and its analogs, are capable of affecting second-order retinal neurons in a manner consistent with the action of a photoreceptor transmitter. In an effort to characterize the properties of retinal glutamate channels on second- order neurons, non-NMDA excitatory amino acid-activated channels were studied in single horizontal cells isolated from the retina of the white bass. Using patch-clamp techniques single glutamate, kainate, and quisqualate channels were recorded. Two categories of channels were observed. The first was labeled slow-channels. Single-channel conductances and open times for this channel showed a range of values, but the average for channels activated by glutamate was 12 pS and 5.6 msec; quisqualate, 8.5 pS and 8.8 msec; and kainate, 8.5 pS and 4.5 msec. Openings of slow-channels elicited by the agonists tended to occur in bursts with a mean burst length of 38 msec. The bursts were punctuated by numerous, brief closings. The second channel category was termed fast channels. The agents glutamate, quisqualate, and kainate all activated channels in this category with open times of 1–2 msec and 2 prominent conductances in the range of about 10 and 20–30 pS. Activity of the fast channels tended to be noisy and no bursting behavior was observed.

Calcium-induced calcium release and calcium buffering in retinal horizontal cells.

Calcium plays an integral role in intracellular signaling and process control in neurons. In the outer retina, it is a key component to the phototransduction cycle and neurotransmitter release in photoreceptor and bipolar cell terminals. It also contributes to the responses of horizontal and bipolar cells. In the dark, horizontal cells are depolarized and calcium enters via calcium permeant AMPA receptors and voltage-activated calcium channels. As a result, horizontal cells must be capable of handling high calcium loads without sustaining damage. The aim of this study was to examine the components determining the intracellular calcium levels in H2 horizontal cells in the retina of white bass. Calcium responses were evoked in isolated cells by depolarizing voltage steps and monitored by conventional imaging techniques. The responses consisted of two components: calcium entry through voltage-gated calcium channels and subsequent release from intracellular stores by calcium-induced calcium release (CICR). Under control conditions, changes in calcium levels reached 541 nM on average from a basal level of 60 nM. When release from CICR stores was blocked with ryanodine or dantrolene, calcium levels barely reached 180 nM. The threshold level needed to trigger CICR was dependent on the duration of the applied depolarization and increased in response to shorter pulses. In studies of temporal integration, cells were depolarized to 0 mVs for increasing periods of time. In the absence of CICR, the responses grew exponentially with time and saturated at approximately 200 nM in response to pulses of 8 s or longer. CICR extended the range of temporal integration to 20 s and the saturating maximum rose to 600 nM. Our results indicate that the slow time-course of the responses, the relatively small changes in intracellular calcium, and the contribution of CICR are shaped by the activity of strong calcium-removal mechanisms and an unusually large calcium-buffering ratio estimated to be over 2,500.

Dopamine modulates unitary conductance of single PL-type calcium channels in Roccus chrysops retinal horizontal cells.

1. Dopamine modulation of the PL‐type calcium channel of white bass retinal horizontal cells was studied in isolated, cultured neurons. Single‐channel recordings were made of calcium channels in outside‐out patches, under conditions which favoured the expression of calcium channel activity. 2. Analysis of single‐channel properties revealed that dopamine potentiated the activity of the sustained calcium channel in three ways. First, it increased unitary conductance through individual channels. Under the influence of dopamine, single‐channel conductance doubled. 3. Dopamine also increased the probability of channel opening and increased channel mean open time. The probability of opening increased 4‐fold while mean open time doubled. 4. The mean closed time was also affected. The time between individual openings was not affected but the closed time between bursts of openings was shortened by over 50%. 5. The effects of dopamine were mediated via the activation of a D1‐type receptor and the resulting activation of a cAMP‐mediated second messenger system. 6. The combination of the effects of dopamine significantly increased the net calcium influx into the cell.

Signal integration at the pedicle of turtle cone photoreceptors: an anatomical and electrophysiological study

The morphology of the axon which connects the cell body and pedicle of turtle cone photoreceptors was studied by light and electron microscopy. The axon which contains numerous synaptic vesicles, some endoplasmic reticulum, and a few cisternae is basically filled with cytoplasm. The length of the axon is related to the class of cone and varies slightly with retinal location, with axons as short as 3-6 microns found in red cones, and as long as 60 microns in cones containing colorless oil droplets. By simultaneously voltage clamping the cell body and pedicle regions of single isolated cones, we measured the longitudinal axonal resistance and the cell body and pedicle membrane resistances. For each cell studied, the axonal resistance of cones with short axons was lower than the cell and pedicle membrane resistances. Thus, the cell can be considered to be an isopotential structure. However, in some cones with long axons, the axonal, cell body, and pedicle resistances were comparable. The pedicles of these cones, therefore, could act like summing points and may provide a locus for spatial signal integration. Electrical coupling between the principal and accessory members of double cones was also studied. Electron-microscopic observation of the membrane junction between the apposed inner segments of the double cones in the intact retina show narrow segments which resemble gap junctions. However, in every double cone studied in culture, passing currents into one member of the double cone did not result in measurable current flow in the adjacent cell. Thus, the two members of the double cone, isolated from the turtle retina, are not electrically coupled.

An unusually small potassium current that is well-suited to a retinal neuron which is chronically depolarized

Here we describe a sustained outward potassium current (IK) in retinal horizontal cells (HCs). IK is unusually small over the range of membrane potentials normally experienced by these cells, which are chronically depolarized. We hypothesize that this unique IK will reduce the amount of neurotransmitter required to shift the cells membrane potential over a wide range, and will minimize the redistribution of potassium ions across the post-synaptic membrane when the cell is depolarized.