Ionic currents of cultured horizontal cells isolated from white perch retina

Potassium channels are one of the most complex and wide-distributed channels in neurons, which have concentrated more and more attention, because it is an important signal pathway in the neuronal apoptosis and pathogenesis of Alzheimer's disease and neuron apoptosis. In this study whole-cell electrophysiological recording techniques were used to identify potassium currents in cultured rat hippocampal neurons (P7 to P14). The membrane potential was held at -65mV, before the activation of outward potassium currents, the membrane potential was pre-hyperpolarized to -110 mV, and then depolarized to +70 mV in 10 mV steps. We found that a kind of outward delayed potassium current was voltage dependent, and it could be significantly inhibited by 50 mM tetraethyl ammonium (TEA).

1. Horizontal cells of the cat retina were isolated by enzymatic dissociation. Two types of horizontal cells were identified: the axonless (A-type) horizontal cell having four to six thick, long (approximately 100 microns) dendrites, and the short-axon (B-type) horizontal cell having many (> 5) fine, short (approximately 30 microns) dendrites. 2. Membrane properties of isolated horizontal cells were analyzed under current-clamp and voltage-clamp conditions. In the A-type cell, the average resting potential was -55 mV and the mean membrane capacitance was 110 pF, whereas values in the B-type cell were -58 mV and 40 pF, respectively. The A-type cell showed long-lasting Ca spikes, but B-type cells had no Ca spikes. 3. Five types of voltage-dependent ionic currents were recorded: a sodium current (INa), a calcium current (ICa), and three types of potassium currents. Potassium currents consisted of potassium current through the inward rectifier (Ianomal), transient outward potassium current (IA), and potassium current through the delayed rectifier (IK(v)). INa was recorded only from A-type cells. Other currents were recorded from both types of cells. 4. INa activated when cells were depolarized from a holding potential (Vh) of -95 mV, and it was maximal at -25 mV. This current was blocked by tetrodotoxin. Approximately half of the A-type cells had INa, but no B-type cell had this current. 5. L-type ICa, an inward-going sustained current, was activated with depolarization more positive than -25 mV. Current amplitude reached a maximal value near 15 mV and became smaller with further depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of prolonged (greater than 2 hr) darkness and background illumination on the light responsiveness of cone horizontal cells were examined in isolated, superfused white perch retinas. In retinas from fish maintained in complete darkness for more than 2 hr, cone horizontal cells had a resting membrane potential of about -18 mV, and they generated only slow, low-amplitude (3-4 mV) responses even when stimulated with bright flashes. Following the presentation of dim background light, the cone horizontal cells slowly hyperpolarized and thereafter remained at a more hyperpolarized level (about -25 mV). Concurrently, their light responses were dramatically enhanced in size, and response amplitudes to bright flashes eventually increased to about 50 mV. This was accompanied by noticeable changes in response waveforms; following light exposure, the responses became faster and showed initial on-transients. The increase in cone horizontal cell responsiveness was graded with intensity of the background light. A similar enhancement in response amplitudes of cone horizontal cells occurred after presenting bright flashes repetitively at intervals of 9 sec. This background sensitization phenomenon was observed in both L- and C-type cone horizontal cells. When light-sensitized isolated retinas were maintained in darkness for long periods (greater than 30 min), the light responsiveness of cone horizontal cells gradually decreased. The changes in membrane potential and response waveform were opposite to those that occurred when prolonged dark-adapted retinas were exposed to background lights; the cells depolarized by 5-7 mV and light-evoked responses became slower. Effects of background illumination on rod-driven horizontal cells were examined as well. Rod horizontal cells were about 2 log units more sensitive to white light than were cone horizontal cells. When recorded in prolonged dark-adapted retinas, rod horizontal cells showed large responses (approximately 50 mV), which could not be further enhanced by background illumination. Cone horizontal cell responsiveness in the carp retina was also depressed by prolonged darkness and increased by illumination in a fashion similar to that observed in the white perch retina. The change in response amplitude was only about 2-fold in carp compared with 5- to 10-fold changes observed in white perch. These results indicate that the light responsiveness of cone horizontal cells in the teleost retina is suppressed in prolonged darkness and that background lights release the cells from suppression.(ABSTRACT TRUNCATED AT 400 WORDS)

The glossopharyngeal nerve contains the sensory fibers of the petrosal ganglion (PG) neurons that innervate the carotid body and sinus, through the carotid sinus nerve, and the tongue and the pharynx, through the glossopharyngeal branch. Based on the shape of the action potential, two populations of PG neurons can be recognized. However, little is known about the ionic currents underlying these action potentials. The application of ACh and ATP, transmitters putatively involved in the communication between the glomus cells and PG neuron terminals, to the isolated PG increase the frequency of discharge of the carotid sinus nerve, while only ATP increases discharges in the glossopharyngeal branch. The above suggests the presence of at least two neuronal populations. Petrosal ganglia were excised from cats anesthetized with sodium pentobarbitone (40 mg/kg, ip), dissociated, and the neurons cultured for 3–16 days. Using whole-cell patch-clamp technique, we recorded from isolated PG neurons both action potentials and ionic currents evoked by electrical stimulation, and by application of ACh and ATP. Neurons presented either fast action potentials (F-type) or slower spikes with an inflexion in the repolarizing phase (H-type). The F-type neurons had fast inward and a sustained outward current. The spike and inward currents were reversibly blocked when choline replaced Na+ or in the presence of 3 μM tetrodotoxin (TTX). The outward current was partly blocked by 5 mM tetraethylammonium (TEA). The H-type neurons presented an inward current with at least two components, and an outward current with a fast -partly inactivating- and a slower sustained component. When external Na+ was replaced by choline, or when 3 μM TTX was added, the amplitude of the spike and the inward current were reversibly decreased. Similarly, when intracellular K+ was replaced by Cs+, or when 5 mM TEA was added, the amplitude and duration of the spike increased and the outward current was reduced. Under voltage-clamp, ATP induced a dose-dependent inward current that was partly desensitized during 20 s application pulses. The ATP-induced current had a threshold of 100 nM, and saturated between 20–50 μM. ACh also induced a fast, inactivating inward current, with a threshold between 10–50 μM, and saturated about 1–5 mM. From 34 neurons tested for ACh and ATP, 9% responded only to ACh, 24% to ATP only, 50% responded to both agents, and the remaining 17% were unresponsive. In current-clamp, ATP and ACh depolarized the neurons and may induce action potentials. Our results show that H- and F-type PG neurons express different voltage-gated ionic and receptor-gated currents, which are activated either by ACh or ATP, with a half population responding to both putative transmitters.

The functional and morphological connectivity between various horizontal cell (HC) types (H1, H2, H3, and H4) and photoreceptors was studied in zebrafish retina. Since HCs are strongly coupled by gap junctions and feedback from HCs to photoreceptors depends strongly on connexin (Cx) hemichannels, we characterized the various HC Cxs (Cx52.6, Cx52.7, Cx52.9, and Cx55.5) in Xenopus oocytes. All Cxs formed hemichannels that were conducting at physiological membrane potentials. The Cx hemichannels differed in kinetic properties and voltage dependence, allowing for specific tuning of the coupling of HCs and the feedback signal from HCs to cones. The morphological connectivity between HC layers and cones was determined next. We used zebrafish expressing green fluorescent protein under the control of Cx promoters. We found that all HCs showed Cx55.5 promoter activity. Cx52.7 promoter activity was exclusively present in H4 cells, while Cx52.9 promoter activity occurred only in H1 cells. Cx52.6 promoter activity was present in H4 cells and in the ventral quadrant of the retina also in H1 cells. Finally, we determined the spectral sensitivities of the HC layers. Three response types were found. Monophasic responses were generated by HCs that contacted all cones (H1 cells), biphasic responses were generated by HCs that contacted M, S, and UV cones (H2 cells), and triphasic responses were generated by HCs that contacted either S and UV cones (H3 cells) or rods and UV cones (H4 cells). Electron microscopy confirms that H4 cells innervate cones. This indicates that rod-driven HCs process spectral information during photopic and luminance information during scotopic conditions.

1. The electrical properties of horizontal cells in the mudpuppy in light and dark were measured with a pair of micropipettes separated by about 1 mum with low coupling resistance so that no bridge circuitry was required. 2. All horizontal cells studied showed significant anomalous rectification: the current-voltage characteristic for about 60 per cent of the cells studied had a slope resistance of about 20-30 M omega at the dark potential level; the slope resistance increased by about 15% for each 10 mV depolarization and decreased by about 15% for each 10 mV hyperpolarization. The remaining 40% of the horizontal cells showed a higher input resistance at corresponding potential levels but had similar rectifying properties. 3. The increase in resistance with depolrization developed with a time course of about 1/2 sec when steady steps of outward current were passed across the membrane, but the time course for resistance decrease with hyperpolarization was much shorter for steady inward current steps. In about half the horizontal cells there was a transient decrease in resistance lasting about 100 msec immediately following the outward current steps superimposed upon the slower sustained resistance increase. 4. The normal 20-30 mV hyperpolarizing light response was associated with little or no change in input resistance. However, if the membrane potential was held at the dark potential level with extrinsic current, thereby eliminating the potential-dependent resistance change, a light-elicited resistance increase of about 10 M omega was measured. 5. The time-dependent change in membrane resistance elicited by polarizing steps of current obscured the reversal potential for the response. However, when the reversal potential was measured at short times following polarization of the membrane, before the time-dependent resistance change developed, it was estimated at between +15 and +50 m V. 6. The results suggest that the horizontal cell response is mediated by a light-elicited resistance increase at the synaptic membrane which is obscured by a potential- and time-dependent resistance decrease at another part of the membrane.

White perch horizontal cells in culture: Methods, morphology and process growth

Interplexiform cells are centrifugal neurons in the retina carrying information from the inner to the outer plexiform layers. In teleost fish, interplexiform cells appear to release dopamine in the outer plexiform layer after prolonged darkness that modulates the receptive-field size and light responsiveness of horizontal cells (Mangel & Dowling, 1985; Yang et al., 1988a, b). It has been proposed that interplexiform cells may also release dopamine upon steady illumination because horizontal cells' receptive fields shrink in the light (Shigematsu & Yamada, 1988). Here, we report the shrinkage of the receptive fields of horizontal cells seen in the presence of background illumination is not blocked by dopamine antagonists, indicating that dopamine does not underlie the receptive-field size changes observed during steady illumination. Flickering light, however, does appear to stimulate the release of dopamine from the interplexiform cells, resulting in a marked reduction of horizontal cell receptive-field size. Taken together, experiments on horizontal cells indicate that dopamine is released from interplexiform cells in the teleost retina after prolonged darkness and during flickering light, but that dopamine release from interplexiform cells during steady retinal illumination is minimal.

The peptide omega-agatoxin-IIIA (omega-Aga-IIIA) blocks ionic current through L-type Ca channels in guinea pig atrial cells without affecting the associated gating currents. omega-Aga-IIIA permits the study of L- type Ca channel ionic and gating currents under nearly identical ionic conditions. Under conditions that isolate L-type Ca channel currents, omega-Aga-IIIA blocks all ionic current during a test pulse and after repolarization. This block reveals intramembrane charge movements of equal magnitude and opposite sign at the beginning of the pulse (Q(on)) and after repolarization (Q(off)). Q(on) and Q(off) are suppressed by 1 microM felodipine, saturate with increasing test potential, and are insensitive to Cd. The decay of the transient current associated with Q(on) is composed of fast and slow exponential components. The slow component has a time constant similar to that for activation of L-type Ca channel ionic current, over a broad voltage range. The current associated with Q(off) decays monoexponentially and more slowly than ionic current. Similar charge movements are found in guinea pig tracheal myocytes, which lack Na channels and T-type Ca channels. The kinetic and pharmacological properties of Q(on) and Q(off) indicate that they reflect gating currents associated with L-type Ca channels. omega-Aga-IIIA has no effect on gating currents when ionic current is eliminated by stepping to the reversal potential for Ca or by Cd block. Gating currents constitute a significant component of total current when physiological concentrations of Ca are present and they obscure the activation and deactivation of L-type Ca channels. By using omega- Aga-IIIA, we resolve the entire time course of L-type Ca channel ionic and gating currents. We also show that L- and T-type Ca channel ionic currents can be accurately quantified by tail current analysis once gating currents are taken into account.

We studied the properties of voltage-activated outward currents in two types of spider cuticular mechanoreceptor neurons to learn if these currents contribute to the differences in their adaptation properties. Both types of neurons adapt rapidly to sustained stimuli, but type A neurons usually only fire one or two action potentials, whereas type B neurons can fire bursts lasting several hundred milliseconds. We found that both neurons had two outward current components, 1) a transient current that activated rapidly when stimulated from resting potential and inactivated with maintained stimuli and 2) a noninactivating outward current. The transient outward current could be blocked by 5 mM tetraethylammonium chloride, 5 mM 4-aminopyridine, or 100 microM quinidine, but these blockers also reduced the amplitude of the noninactivating outward current. Charybdotoxin or apamin did not have any effect on the outward currents, indicating that Ca2+-activated K+ currents were not present or not inhibited by these toxins. The only significant differences between type A and type B neurons were found in the half-maximal activation (V50) values of both currents. The transient current had a V50 value of 9. 6 mV in type A neurons and -13.1 mV in type B neurons, whereas the V50 values of noninactivating outward currents were -48.9 mV for type A neurons and -56.7 mV for type B neurons. We conclude that, although differences in the activation kinetics of the voltage-activated K+ currents could contribute to the difference in the adaptation behavior of type A and type B neurons, they are not major factors.

There are two types of horizontal cells, the luminosity and the chromaticity cells, in the retina of the kissing gourami, Helostoma rudolfi. Luminosity cells occupy the outermost layer proximal to the receptor terminals, whereas chromaticity cells form a layer proximal to the layer of luminosity cells. Neither type of cell has axons. Responses were evoked by light from red and green light-emitting diodes. The two stimuli were modulated either by a pulsatile or a white-noise signal. The luminosity cell always produced a hyperpolarizing response. The chromaticity cell produced a hyperpolarizing response when stimulated by only one color. However, in the presence of a steady or modulated green input, a red stimulus produced a depolarizing response. Such chromaticity cells were similar to the (spectral) biphasic chromaticity horizontal cells observed in other retinae. The depolarizing phase of the red response was produced by the balance of intensity of the two inputs, red and green. We used white-noise methodology to identify the dynamics of the horizontal cell's modulation response by taking advantage of the fact that a Wiener kernel is a measure of a cell's incremental sensitivity, which includes its response dynamics. Under all conditions, a steady state modulation response by both luminosity and chromaticity cells always was related linearly to the input modulation. The average mean square error (MSE) of the model predicted by the first-order kernel was approximately 8% for both luminosity (n = 116) and chromaticity (n = 23) cells. In some cases, the MSE was a few percent even when the peak-to-peak response amplitude was nearly 30 mV. The ratio of inputs from red and green cones to both types of horizontal cells was variable; the major input for luminosity cells came from red cones, whereas the major input for chromaticity cells came from green cones. First-order kernels generated by the major input were robust in terms of waveform in the sense that the waveform remained unchanged whether or not there was a steady or modulated illumination by the opposing color. The results reported here do not address the question of the neural circuitry that generates horizontal cell responses, in particular, the depolarizing response. However, whatever that circuitry might be, the high degree of linearity of the modulation response by both types of cell under various stimulus conditions imposes restrictions on the performance of any proposed model as well as on mechanisms that underlie the generation of the horizontal cell response.

Spatial Distribution of Potential in a Flat Cell: Application to the Catfish Horizontal Cell Layers

Effect of TEA on light emission from aequorin-injected Aplysia central neurons

​ByBy the time visual information leaves the retina for the brain, it has undergone several rounds of processing. Most prominently, that information has been filtered to amplify differences in contrast, between either two points in space or two points in time. The initial step in that amplification is carried out by horizontal cells, whose job is not only to receive signals from a group of adjacent photoreceptor cells (rods or cones) but also to send inhibitory feedback signals back to the photoreceptors, turning down the weakest outputs and allowing only the strongest to get through. Schematic drawing of the vertebrate retina combined with the immunolocalization of the ecto-ATPase NDTPdase1 (green). This much has been known for several decades, but the cell physiological details of the feedback mechanism have been controversial. Part of the puzzle has been the paradoxical requirement for both very fast feedback, needed to highlight spatial differences, and slower feedback, needed to process temporal ones. In this issue of PLOS Biology, Rozan Vroman, Maarten Kamermans, and colleagues resolve this apparent paradox: they show that horizontal cells in the goldfish retina employ two simultaneous feedback mechanisms, the slower of which relies on a newly described mechanism, creating an extracellular buffer by secreting and hydrolyzing ATP. Horizontal cells affect the activity of cone cells by changing the rate of flow of positively charged calcium ions from the extracellular space through calcium channels into the cone cell. The authors measured that current within individual cone–horizontal cell synapses, and found that the curve reflecting the current change was best described as the sum of two exponential functions, one representing an initial fast increase and the other a slower one. The fast component was very fast indeed, inconsistent with the inevitable delay imposed when cells are separated by a synaptic gap. Instead, the authors show that connexin hemichannels on the horizontal cell membrane were involved. These channels allowed current to flow into the horizontal cell, increasing the negative charge of the intercellular space and increasing the flow of calcium into the cone cell, locally depolarizing it. This direct (“ephaptic”) connection makes the synapse among the fastest of all known inhibitory synapses. But it was the slow component that held the most surprises. The authors noted that light-induced feedback responses of horizontal cells can be completely blocked by carbenoxolone, which antagonizes not only connexin but also another channel, called pannexin 1 (Panx1). Panx1 is also present in the synapse and is known to mediate release of ATP. The authors showed that depolarized horizontal cells released ATP, an effect that could be blocked by addition of a specific Panx1 antagonist, probenecid. When hydrolyzed, ATP produces phosphates and H+ ions (protons), which could acidify the intercellular space as well as buffer it against pH changes. The authors used immunolabeling to show that an ATP hydrolase was present on the exterior surface of the horizontal cell, and that blocking this enzyme reduced the slow component of the feedback response. Artificial alkalization of the synaptic space had the same effect. The ATP that is released via Panx1 is thus hydrolyzed and leads to slight acidification of the synaptic cleft, which inhibits presynaptic calcium channels and inhibits the neurotransmitter release of photoreceptors. The combination of two feedback components with different speeds allows the horizontal cell–cone system to respond to both spatial and temporal variation, the authors argue. Spatially, the role of the horizontal cell is, in effect, to subtract the average level of activity in multiple adjacent cones from the output of each, allowing only the most prominent signals through. That subtraction must happen very fast for a moving signal to be processed correctly—and the ephaptic component is about as fast as it gets. Temporally, the horizontal cell must subtract out long-lasting stimulation from the cone output, which by definition requires a slower mechanism, provided by the Panx1–ATPase system. Although these experiments were done in fish, mice also express Panx1 at their horizontal cell synapses, suggesting the mechanism likely operates in mammals as well. The authors speculate that variations in the relative activity of the fast and slow mechanisms may contribute to interspecies differences in responsiveness to spatial versus temporal stimuli. They also point out that Panx1 is widely distributed in the brain, suggesting that an ATP pH effect may contribute to feedback systems in other types of neurons as well. Vroman R, Klaassen LJ, Howlett MHC, Cenedese V, Klooster J, et al. (2014) Extracellular ATP Hydrolysis Inhibits Synaptic Transmission by Increasing pH Buffering in the Synaptic Cleft. doi:10.1371/journal.pbio.1001864

Ionic currents in isolated vestibular hair cells from the guinea-pig crista ampullaris