Dihydropyridine-sensitive calcium currents in bipolar cells of salamander retina are inhibited by reductions in extracellular chloride

Dihydropyridine-sensitive calcium currents (I_Ca) in photoreceptors are unusual in that they can be inhibited by reductions in extracellular chloride. The present study examined whether I_Ca in retinal bipolar cells, which as in photoreceptors mediates sustained neurotransmission, is also inhibited by reductions in chloride. Nystatin-perforated patch, whole cell recordings were obtained from bipolar cells in a retinal slice preparation of larval tiger salamander. In the presence of Ba²⁺, voltage steps above −40 mV evoked sustained inward currents, which were enhanced by the dihydropyridine, (−)BayK8644, and inhibited by nisoldipine. Similar to photoreceptors, replacing Cl⁻ with gluconate or CH₃SO₄ inhibited bipolar cell I_Ca and produced a negative shift in the current/voltage relationship. Thus, sensitivity to Cl⁻ may be a more general property of L-type Ca²⁺ channel subtypes that mediate sustained neurotransmission.     

Structure of the receptive fields of bipolar cells in the salamander retina

1. The receptive-field structure of bipolar cells in the salamander retina has been examined using isolated retinae from dark-adapted eyes. 2. Receptive-field mapping was carried out with a 25-microns diam spot of light whose wavelength and intensity was intended to stimulate rods rather than cones. 3. Both hyperpolarizing and depolarizing bipolar cells showed receptive fields having a single central point of maximum sensitivity from which sensitivity declined radially. Antagonistic surrounds could not be demonstrated using a small spot of light. 4. The diameter of receptive fields was found to vary between 374 and 662 micron, consistent with a single bipolar cell being effectively connected to 323-1,275 rods. 5. Lucifer yellow injections of bipolar cells revealed dendritic arbors whose greatest dimensions varied between 43 and 70 microns, consistent with a direct synaptic connection of between 10 and 24 rods to each bipolar cell. 6. We rule out signal spread within the rod network, extensive lateral ramification of rod process, nonlinearity of synaptic transmission, and light scatter, as possible explanations of large bipolar cell receptive fields. It seems likely, instead, that signals are extensively shared between bipolar cells.     

I4AA-Sensitive chloride current contributes to the center light responses of bipolar cells in the tiger salamander retina

Light-evoked currents in depolarizing and hyperpolarizing bipolar cells (DBCs and HBCs) were recorded under voltage-clamp conditions in living retinal slices of the larval tiger salamander. Responses to illumination at the center of the DBCs' and HBCs' receptive fields were mediated by two postsynaptic currents: DeltaI(C), a glutamate-gated cation current with a reversal potential near 0 mV, and DeltaI(Cl), a chloride current with a reversal potential near -60 mV. In DBCs DeltaI(C) was suppressed by L-2-amino-4-phosphonobutyric acid (L-AP4), and in HBCs it was suppressed by 6,7-dinitroquinoxaline-2,3-dione (DNQX). In both DBCs and HBCs DeltaI(Cl) was suppressed by imidazole-4-acetic acid (I4AA), a GABA receptor agonist and GABA(C) receptor antagonist. In all DBCs and HBCs examined, 10 microM I4AA eliminated DeltaI(Cl) and the light-evoked current became predominately mediated by DeltaI(C). The addition of 20 microM L-AP4 to the DBCs or 50 microM DNQX to HBCs completely abolished DeltaI(C). Focal application of glutamate at the inner plexiform layer elicited chloride currents in bipolar cells by depolarizing amacrine cells that release GABA at synapses on bipolar cell axon terminals, and such glutamate-induced chloride currents in DBCs and HBCs could be reversibly blocked by 10 microM I4AA. These experiments suggest that the light-evoked, I4AA-sensitive chloride currents (DeltaI(Cl)) in DBCs and HBCs are mediated by narrow field GABAergic amacrine cells that activate GABA(C) receptors on bipolar cell axon terminals. Picrotoxin (200 microM) or (1,2,5,6-tetrahydropyridine-4yl) methyphosphinic acid (TPMPA) (2 other GABA(C) receptor antagonists) did not block (but enhanced and broadened) the light-evoked DeltaI(Cl), although they decreased the chloride current induced by puff application of GABA or glutamate. The light response of narrow field amacrine cells were not affected by I4AA, but were substantially enhanced and broadened by picrotoxin. These results suggest that there are at least two types of GABA(C) receptors in bipolar cells: one exhibits stronger I4AA sensitivity than the other, but both can be partially blocked by picrotoxin. The GABA receptors in narrow field amacrine cells are I4AA insensitive and picrotoxin sensitive. The light-evoked DeltaI(Cl) in bipolar cells are mediated by the more strongly I4AA-sensitive GABA(C) receptors. Picrotoxin, although acting as a partial GABA(C) receptor antagonist in bipolar cells, does not suppress DeltaI(Cl) because its presynaptic effects on amacrine cell light responses override its antagonistic postsynaptic actions.     

Sensory neuron N-type calcium currents are inhibited by both voltage-dependent and -independent mechanisms

The voltage dependence of -aminobutyric-acid- and norepinephrine-induced inhibition of N-type calcium current in cultured embryonic chick dorsal-root ganglion neurons was studied with whole-cell voltage-clamp recording. The inhibitory action of the neurotransmitters was comprised of at least two distinct modulatory components, which were separable on the basis of their differential voltage dependence. The first component, which we term kinetic slowing, is associated with a slowing of the activation kinetics — an effect that subsides during a test pulse. The kinetic-slowing component is largely reversed at depolarized voltages (i.e., it is voltage-dependent). The second component, which we term steady-state inhibition, is by definition not associated with a change in activation kinetics and is present throughout the duration of a test pulse. The steady-state inhibition is not reversed at depolarized voltages (i.e., it is voltage-independent). Although the two components can be separated on the basis of their voltage dependence, they appear to be indistinguishable in their time courses for onset and recovery as well as their rates of desensitization following multiple applications of transmitter. Furthermore, neither component requires cell dialysis, as both are observed using perforated-patch as well as whole-cell recording configurations. The co-existence in nerve terminals of both voltage-dependent and -independent mechanisms to modulate calcium channel function could offer a means of differentially controlling synaptic transmission under conditions of low- and high-frequency presynaptic discharge.     

Voltage- and transmitter-gated currents in isolated rod bipolar cells of rat retina

1. Bipolar cells were isolated from adult rat retinas after enzymatic and mechanical treatment. The cells could be unequivocally identified from their morphology because of high retention of their axon and dendritic processes after isolation. 2. Protein kinase C (PKC) immunoreactivity performed on sections of the rat retina labeled rod bipolar cells and a few amacrine cells. Virtually all bipolar cells in the dissociates expressed PKC immunoreactivity and were, therefore, rod bipolar cells. 3. Rod bipolar cells were examined with the tight-seal whole-cell and excised-patch recording techniques. Resting potentials of the isolated cells recorded under current-clamp conditions showed a broad unimodal distribution around -37 mV. 4. Membrane depolarization from a holding potential of -90 mV resulted in an outward current. A fast sodium inward current was not observed. Membrane hyperpolarization from a holding potential of -40 mV activated an inwardly rectifying current. 5. gamma-Aminobutyric acid (GABA) and glycine, the putative retinal neurotransmitters that mediate the bipolar cells' receptive field surround in vivo, activated chloride conductances in almost all isolated bipolar cells. GABA- and glycine-evoked currents were both desensitizing and could be antagonized by the classical blockers bicuculline, picrotoxin, and strychnine, respectively. 6. Pressure application of the drugs from fine microcapillaries to various parts of the isolated cells suggests a high GABA sensitivity at the axonal endings compared with either the somatic or dendritic region. A similar distribution was not found for glycine. On the contrary, glycine-induced single-channel events with main conductances of 52 and 34 pS were recorded from membrane patches excised from the cells' somata. 7. Conductances induced by glutamate and several excitatory amino acid agonists were observed in a number of the cells. Application of the glutamate agonist 2-amino-4-phosphonobutyric acid (APB) induced an inward current at negative holding potentials associated with the opening of ion channels. In only 5 of 93 cells, APB closed ion channels, leading to a decrease in membrane conductance.     

TMEM16B induces chloride currents activated by calcium in mammalian cells

Ca²⁺-activated Cl^? channels play important physiological roles in various cell types, but their molecular identity is still unclear. Recently, members of the protein family named transmembrane 16 (TMEM16) have been suggested to function as Ca²⁺-activated Cl^? channels. Here, we report the functional properties of mouse TMEM16B (mTMEM16B) expressed in human embryonic kidney (HEK) 293T cells, measured both in the whole-cell configuration and in inside-out excised patches. In whole cell, a current induced by mTMEM16B was activated by intracellular Ca²⁺ diffusing from the patch pipette, released from intracellular stores through activation of a G-protein-coupled receptor, or photoreleased from caged Ca²⁺ inside the cell. In inside-out membrane patches, a current was rapidly activated by bath application of controlled Ca²⁺ concentrations, indicating that mTMEM16B is directly gated by Ca²⁺. Both in the whole-cell and in the inside-out configurations, the Ca²⁺-induced current was anion selective, blocked by the Cl^? channel blocker niflumic acid, and displayed a Ca²⁺-dependent rectification. In inside-out patches, Ca²⁺ concentration for half-maximal current activation decreased from 4.9 µM at ?50 mV to 3.3 µM at +50 mV, while the Hill coefficient was >2. In inside-out patches, currents showed a reversible current decrease at ?50 mV in the presence of a constant high Ca²⁺ concentration and, moreover, an irreversible rundown, not observed in whole-cell recordings, indicating that some unknown modulator was lost upon patch excision. Our results demonstrate that mTMEM16B functions as a Ca²⁺-activated Cl^? channel when expressed in HEK 293T cells.     

Intracellular chloride concentration is higher in rod bipolar cells than in cone bipolar cells of the mouse retina

Bipolar cells (BCs) have antagonistic center-surround receptive field. Surround illumination evokes depolarization in the OFF-type cone BC, and hyperpolarization in the rod BC and the ON-type cone BC. Surround illumination reduces gamma-aminobutyric acid (GABA) release from horizontal cells. If GABA hyperpolarize BCs, the polarity of the GABA-induced effect agrees with the light-evoked surround response in the OFF-type BC, but contradicts in the rod BC and the ON-type cone BC. Immunohistochemical study of the Cl⁻ transporter of BCs has suggested that the intracellular Cl⁻ concentration is different among BC subtypes. We examined the reversal potential of GABA-induced current of BCs using gramicidin-perforated patch clamp technique in the mouse retina, and found that GABA depolarizes rod BC and hyperpolarizes cone BCs. Our results are consistent with the GABAergic input to rod BC dendrite.     

Differential expression of high- and two types of low-voltage-activated calcium currents in rod and cone bipolar cells of the rat retina

Whole cell voltage-clamp recordings were performed to investigate voltage-activated Ca(2+) currents in acutely isolated retinal bipolar cells of rats. Two groups of morphologically different bipolar cells were observed. Bipolar cells of the first group, which represent the majority of isolated bipolar cells, were immunoreactive to protein kinase C (PKC) and, therefore likely to be rod bipolar cells. Bipolar cells of the second group, which represent only a small population of isolated bipolar cells, did not show PKC immunoreactivity and were likely to be cone bipolar cells. The validity of morphological identification of bipolar cells was further confirmed by the presence of GABA(C) responses in these cells. Bipolar cells of both groups displayed low-voltage-activated (LVA) Ca(2+) currents with similar voltage dependence of activation and steady-state inactivation. However, the activation, inactivation, and deactivation kinetics of the LVA Ca(2+) currents between rod and cone bipolar cells differed. Particularly, the LVA Ca(2+) currents of rod bipolar cells displayed both transient and sustained components. In contrast, the LVA Ca(2+) currents of cone bipolar cells were mainly transient. In addition, the LVA Ca(2+) channels of rod bipolar cells were more permeable to Ba(2+) than to Ca(2+), whereas those of cone bipolar cells were equally or less permeable to Ba(2+) than to Ca(2+). The LVA Ca(2+) currents of both rod and cone bipolar cells were antagonized by high concentrations of nimodipine with IC(50) of 17 and 23 microM, respectively, but largely resistant to Cd(2+) and Ni(2+). Bipolar cells of both groups also displayed high-voltage-activated (HVA) Ca(2+) currents. The HVA Ca(2+) currents were, at least in part, to be L-type that were potentiated by BayK-8644 (1 microM) and largely antagonized by low concentrations of nimodipine (5 microM). The L-type Ca(2+) channels were almost exclusively located at the axon terminals of rod bipolar cells but expressed at least in the cell soma of cone bipolar cells. Results of this study indicate that rod and cone bipolar cells of the mammalian retina differentially express at least two types of LVA Ca(2+) channels. Rod and cone bipolar cells also show different spatial distribution of L-type Ca(2+) channels.     

Competitive inhibition of NMDA receptor-mediated currents by extracellular calcium chelators

Calcium chelators have been widely used in electrophysiological recordings of N-methyl-D-aspartate (NMDA) receptor-mediated currents, as well as in studies of excitotoxicity. Intracellularly applied calcium chelators are known to inhibit, at least in part, such calcium-dependent processes as calmodulin-dependent inactivation, calcineurin-dependent desensitization, and rundown of NMDA receptors. On the other hand, the functional consequences and potential nonspecific effects of extracellularly applied chelators have not been extensively investigated. In whole-cell patch-clamp recordings from human embryonic kidney (HEK) 293 cells transiently transfected with recombinant NMDA receptors, we found that addition of calcium chelators such as EGTA shifted the glutamate dose-response curve to the right, from an EC(50) for NR1A/NR2A of 8 microM in 1.8 mM Ca(2+) to approximately 24 microM in a solution containing nominal 0 Ca(2+)/5 mM EGTA and further to approximately 80 microM in 20 mM EGTA. A similar shift in glutamate dose-response was observed for NR1A/NR2B currents. This dose-response shift was not due to a decrease in extracellular Ca(2+) concentration because there was no change in the glutamate EC(50) at Ca(2+) concentrations ranging from 10 mM to nominal 0/200 microM EGTA. Moreover, addition of 5 mM EGTA fully chelated with 6.8 mM Ca(2+) did not produce any shift in the glutamate dose-response curve. We propose that calcium chelators, containing four free carboxyl moieties, competitively inhibit glutamate binding to NMDA receptors.     

Functional organization of ganglion cells in the salamander retina

Recently, we reported a novel technique for recording all of the ganglion cells in a retinal patch and showed that their receptive fields cover visual space roughly 60 times over in the tiger salamander. Here, we carry this analysis further and divide the population of ganglion cells into functional classes using quantitative clustering algorithms that combine several response characteristics. Using only the receptive field to classify ganglion cells revealed six cell types, in agreement with anatomical studies. Adding other response measures served to blur the distinctions between these cell types rather than resolve further classes. Only the biphasic off type had receptive fields that tiled the retina. Even when we attempted to split these classes more finely, ganglion cells with almost identical functional properties were found to have strongly overlapping spatial receptive fields. A territorial spatial organization, where ganglion cell receptive fields tend to avoid those of other cells of the same type, was only found for the biphasic off cell. We further studied the functional segregation of the ganglion cell population by computing the amount of visual information shared between pairs of cells under natural movie stimulation. This analysis revealed an extensive mixing of visual information among cells of different functional type. Together, our results indicate that the salamander retina uses a population code in which every point in visual space is represented by multiple neurons with subtly different visual sensitivities.     

Voltage-sensitive calcium currents are acutely increased by nerve growth factor in PC12 cells

Whole cell calcium currents were recorded from PC12 cells with the perforated patch technique. Currents were evoked by step depolarization from a holding potential of -90 mV. Nerve growth factor (NGF) increased calcium currents through L-type calcium channels by >75% within 3-5 min. This increase was inhibited by K-252a, by nifedipine, and by inhibition or down-regulation of kinase C. Brain-derived neurotrophic factor (BDNF) also increased calcium current, but to a smaller extent. Thus increases in calcium current can be linked to activation of either the high- or the low-affinity nerve growth factor receptor. Increases in presynaptic calcium uptake appear to be a crucial element in the short-term actions of the neurotrophins on neurotransmitter release leading to long-term potentiation. Also, the control of calcium uptake is likely to be an important factor in the long-term actions of the neurotrophins on neuronal survival and neuronal protection. The present data indicate that the PC12 cell may be a useful model for studying the effect of the neurotrophins on calcium uptake.     

顺铂激活的低分化鼻咽癌细胞氯电流为非钙激活氯电流*

 目的：探讨顺铂激活的低分化鼻咽癌细胞（CNE-2Z）氯通道电流是否为钙激活的氯电流。方法：采用膜片钳全细胞记录技术记录细胞内/外无Ca2+及钙通道阻断剂对顺铂激活氯电流的影响,并用高渗灌流液观察顺铂激活氯电流的容积敏感性。结果：去除细胞外液的Ca2+后,5 μmol/L顺铂能诱发氯电流,且电流大小与细胞外有Ca2+ 时无明显差异,但潜伏期与达峰时间延长。细胞内外均无Ca2+ 对顺铂激活氯电流未产生影响。钙通道阻断剂nifedipine未能抑制顺铂诱发的氯电流。但细胞外灌流高渗液几乎可完全抑制顺铂激活的氯电流。结论: 顺铂激活的氯通道开放不依赖于细胞内/外的Ca2+,该通道不是钙激活氯通道而很可能是容积敏感性氯通道。     

Intracellular and extracellular chloride ions in the horizontal cells of the stingray retina.

Intracellular and extracellular concentrations of chloride ([Cl-]i, [Cl-]o) ions in the horizontal cells of the stingray retina were studied by means of ion-selective microelectrodes. The electrodes used were of the double-barreled type and Corning's #477315 resin was used as the exchanger. The average [Cl-]o and [Cl-]i in the L-type cells were 320 and 130 mM, respectively (n = 37), and the equilibrium potential (ECl) was calculated as -23.3 mV. In some cases, dark membrane potentials were more positive than the ECl. With white light stimulus, the membrane hyperpolarized to a more negative level than ECl. Based on the experiments described above, the hypothesis that the hyperpolarizing response of horizontal cells is due to the permeability change of the membrane to chloride ion was excluded in the stingray retina.     

Membrane currents of retinal bipolar cells in culture

1. Retinal bipolar cells were isolated from white bass retinas and maintained in a cell culture preparation. Two morphological types of bipolar cells were observed in cell culture. These were labeled large- and small-bipolar cells based mainly on the size of their somata and primary dendrites. Two types of small-bipolar cells were observed. Isolated bass bipolar cells are very similar to those described in the intact retina. 2. Under current clamp, to depolarizing current injection, small-bipolar cells produced a spike followed by a plateau. Large-bipolar cells showed a slow depolarization to a plateau level. 3. Voltage-gated membrane currents were studied using whole-cell patch-clamp techniques. Channel blocking agents were used to define the ion channels found in the membranes of these cells. 4. The large-bipolar cells were found to possess an A-current, a calcium current, and a calcium-dependent potassium current. 5. Large bipolar cells also possessed an inward rectifier that did not correspond to any previously described. 6. The two types of small-bipolar cells were found to have very similar membrane properties to one another. They lacked a large A-current but possessed a slowly activating, outward rectifying potassium current. Similar to the large-bipolar cells, they showed a calcium current and a calcium-activated potassium current. 7. The inward rectifier of small-bipolar cells was characterized as an H-current. 8. The results suggest that the membrane currents of bipolar cells set a narrow operating range about which the cells function in the intact retina. In addition these currents help shape the responses of bipolar cells to light stimuli but do not confer ON or OFF properties.