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 ).

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.

Ionic mechanisms mediating oscillatory membrane potentials in wide-field retinal amacrine cells.

Particular types of amacrine cells of the vertebrate retina show oscillatory membrane potentials (OMPs) in response to light stimulation. Historically it has been thought the oscillations arose as a result of circuit properties. In a previous study we found that in some amacrine cells, the ability to oscillate was an intrinsic property of the cell. Here we characterized the ionic mechanisms responsible for the oscillations in wide-field amacrine cells (WFACs) in an effort to better understand the functional properties of the cell. The OMPs were found to be calcium (Ca2+) dependent; blocking voltage-gated Ca2+ channels eliminated the oscillations, whereas elevating extracellular Ca2+ enhanced them. Strong intracellular Ca2+ buffering (10 mM EGTA or bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid) eliminated any attenuation in the OMPs as well as a Ca2+-dependent inactivation of the voltage-gated Ca2+ channels. Pharmacological and immunohistochemical characterization revealed that WFACs express L- and N-type voltage-sensitive Ca2+ channels. Block of the L-type channels eliminated the OMPs, but omega-conotoxin GVIA did not, suggesting a different function for the N-type channels. The L-type channels in WFACs are functionally coupled to a set of calcium-dependent potassium (K(Ca)) channels to mediate OMPs. The initiation of OMPs depended on penitrem-A-sensitive (BK) K(Ca) channels, whereas their duration is under apamin-sensitive (SK) K(Ca) channel control. The Ca2+ current is essential to evoke the OMPs and triggering the K(Ca) currents, which here act as resonant currents, enhances the resonance as an amplifying current, influences the filtering characteristics of the cell membrane, and attenuates the OMPs via CDI of the L-type Ca2+ channel.

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.

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.

Regulation of voltage-sensitive Ca2+ channels in bipolar cells by divalent cations and polyamines.

Ca2+ plays a key role in intracellular signal transduction in neurons but in excess it can lead to cell death. Thus its entry into cells is highly regulated by both extrinsic and intrinsic mechanisms. Little is known of the regulation of Ca2+ entry into retinal neurons. Here we describe the role of divalent cations and polyamines as intrinsic modulators of Ca2+ entry into retinal bipolar cells. Cone-dominant (small) bipolar cells of the white bass retina were studied using whole cell patch clamp techniques. With biophysical and pharmacological tools it was determined that these cells expressed a Ca2+ current similar to an L-type current. This current was very susceptible to blockage by divalent cations including Ca2+. In addition, when tested with the polyamines, spermine, spermidine and putrescine, only spermine effectively inhibited the current. When the dose response curve was fit with the Hill function we found an EC50 of 28 microM and a Hill-coefficient of about 2. Our results indicate that divalent cations and the polyamine, spermine, are effective modulators of calcium entry into cone-dominated bipolar cells. The in vivo regulation of the concentrations of these molecules provides an exquisitely sensitive mechanism for regulating Ca2+ entry into bipolar cells under different conditions.