Local influence of mitochondrial calcium transport in retinal amacrine cells

Ca2+-dependent synaptic transmission from retinal amacrine cells is thought to be initiated locally at dendritic processes. Hence, understanding the spatial and temporal impact of Ca2+ transport is fundamental to understanding how amacrine cells operate. Here, we provide the first examination of the local effects of mitochondrial Ca2+ transport in neuronal processes. By combining mitochondrial localization with measurements of cytosolic Ca2+, the local impacts of mitochondrial Ca2+ transport for two types of Ca2+ signals were investigated. Disruption of mitochondrial Ca2+ uptake with carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) produces cytosolic Ca2+ elevations. The amplitudes of these elevations decline with distance from mitochondria suggesting that they are related to mitochondrial Ca2+ transport. The time course of the FCCP-dependent Ca2+ elevations depend on the availability of ER Ca2+ and we provide evidence that Ca2+ is released primarily via nearby ryanodine receptors. These results indicate that interactions between the ER and mitochondria influence cytosolic Ca2+ in amacrine cell processes and cell bodies. We also demonstrate that the durations of glutamate-dependent Ca2+ elevations are dependent on their proximity to mitochondria in amacrine cell processes. Consistent with this observation, disruption of mitochondrial Ca2+ transport alters the duration of glutamate-dependent Ca2+ elevations near mitochondria but not at sites more than 10 microm away. These results indicate that mitochondria influence local Ca2+-dependent signaling in amacrine cell processes.     

Second messenger pathways involved in up-regulation of an L-type calcium channel

Catfish cone horizontal cells contain a voltage-gated L-type calcium channel that is modulated by activation of metabotropic glutamate receptors (mGluRs). Activation of group I mGluRs with the mGluR I agonist, (S)-3,5-dihydroxyphenylglycine [(S) 3,5-DHPG], potentiated peak calcium current amplitude, shifted the membrane potential corresponding to peak current activity, and widened the calcium current's activation range. In this study, we have examined the mechanisms linking activation of the mGluRs with "up-regulation" of calcium current activity. Under whole-cell voltage-clamp conditions favoring expression of the L-type calcium current, we provide evidence that activation of mGluRs initiate the diacylglyceral (DG) second messenger pathway to activate protein kinase C (PKC) and up-regulate calcium channel activity. This evidence was based on results using a number of PKC activators and inhibitors. PKC activators mimicked the effect of (S) 3,5-DHPG on calcium current activity. Up-regulation of the calcium channel by PKC activators or (S) 3,5-DHPG was eliminated if PKC inhibitors were present. These results also demonstrated that activation of group I mGluRs were linked to a pertussis toxin sensitive G-protein. When the GTP analog, guanosine 5-0-(3-thiotriphosphate (GTPgammaS), was allowed to diffuse into voltage-clamp cells, up-regulation of the calcium channel occurred and mimicked the effect of (S) 3,5-DHPG. However, when pertussis toxin (PTX) was allowed to diffuse into the cell along with GTPgammaS, GTPgammaS failed to modulate calcium current activity. IP3 (inositol 1,4,5 triphosphate) is a second product produced by activation of group I mGluRs. Once formed, IP3 can trigger calcium release from IP3-sensitive intracellular stores. To determine if the IP3 second messenger system was involved in up-regulation of calcium channel, (S) 3,5-DHPG was applied to voltage-clamped cone horizontal cells containing different concentrations of the calcium buffer, EGTA. Low concentrations of EGTA failed to buffer calcium released from intracellular stores. In the presence of low EGTA concentrations, (S) 3,5-DHPG's enhancement of the calcium current amplitude was reduced. Inhibition of the calcium current amplitude in low concentrations of EGTA was eliminated in the presence of the intracellular calcium store blocker, heparin. These results suggest that both the DG and IP3 second messenger pathways are involved in modulation of the voltage-gated calcium channel in catfish cone horizontal cells. The DG pathway up-regulates the voltage-gated calcium channel activity whereas calcium released from IP3 intracellular stores inhibits peak current amplitude.     

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 ).     

Influence of muscarinic agonists and tyrosine kinase inhibitors on L-type Ca(2+)Channels in human and bovine trabecular meshwork cells

Trabecularmeshwork (TM), a smooth muscle-like tissue with contractile properties, is involved in the regulation of aqueous humor outflow. However, little is known about the regulation of Ca(2+)influx in trabecular meshwork cells. We investigated the influence of acetylcholine and tyrosine kinases on Ca(2+)conductances of bovine TM (BTM) and human TM (HTM) cells using the perforated-patch configuration of the patch-clamp technique and measurements of intracellular free Ca(2+)([Ca(2+)](i)). Depolarization of the cells in the presence of 10 m m Ba(2+)or Ca(2+)led to an activation of inward currents at potentials positive to -30 mV with characteristics typical of L-type Ca(2+)currents: when using 10 m m Ba(2+), maximal inward current and inactivation time constant (tau) increased; the L-type Ca(2+)channel blocker nifedipine (1 microm) reduced and the L-type Ca(2+)channel agonist BayK8644 (5 microm) enhanced maximal inward current.Acetylcholine (100 microm) and carbachol (1 microm) led to an increase in inward Ba(2+)current whereas application of the tyrosine kinase inhibitors genistein (50 microm) and lavendustin A (20 microm) resulted in a decrease in inward current. The application of daidzein (10 microm), an inactive analog of genistein had no effect. Depolarization of the cells with 135 m m K(+)or direct stimulation of L-type channels by application of BayK 8644 led to an increase in [Ca(2+)](i). Carbachol (1 microm) induced an increase in [Ca(2+)](i)which was decreased by application of the tyrosine kinase inhibitor genistein (50 microm). We conclude that HTM and BTM cells express voltage-dependent L-type Ca(2+)channels that influence intracellular Ca(2+)concentration and thus may modulate TM contractility. The activity of L-type Ca(2+)currents is influenced by muscarinic agonists and tyrosine kinases.     

Activation of glutamate transporters in rods inhibits presynaptic calcium currents

We found that L-glutamate (L-Glu) inhibits L-type Ca2+ currents (ICa) in rod photoreceptors. This inhibition was studied in isolated rods or rods in retinal slices from tiger salamander using perforated patch whole cell recordings and Cl(-)-imaging techniques. Application of L-Glu inhibited ICa by approximately 20% at 0.1 mM and approximately 35% at 1 mM. L-Glu also produced an inward current that reversed around ECl. The metabotropic glutamate receptor (mGluR) agonists t-ADA (Group I), DCG-IV (Group II), and L-AP4 (Group III) had no effect on ICa. However, the glutamate transport inhibitor, TBOA (0.1 mM), prevented L-Glu from inhibiting ICa. D-aspartate (D-Asp), a glutamate transporter substrate, also inhibited ICa with significantly more inhibition at 1 mM than 0.1 mM. Using Cl imaging, L-Glu (0.1-1 mM) and D-Asp (0.1-1 mM) were found to stimulate a Cl- efflux from terminals of isolated rods whereas the ionotropic glutamate receptor agonists NMDA, AMPA, and kainate and the mGluR agonist, 1S,3R-ACPD, did not. Glutamate-evoked Cl- effluxes were blocked by the glutamate transport inhibitors TBOA and DHKA. Cl- efflux inhibits Ca2+ channel activity in rod terminals (Thoreson et al. (2000), Visual Neuroscience 17, 197). Consistent with the possibility that glutamate-evoked Cl- efflux may play a role in the inhibition, reducing intraterminal Cl- prevented L-Glu from inhibiting ICa. In summary, the results indicate that activation of glutamate transporters inhibits ICa in rods possibly as a consequence of Cl- efflux. The neurotransmitter L-Glu released from rod terminals might thus provide a negative feedback signal to inhibit further L-Glu release.     

The influences of metabotropic receptor activation on cellular signaling and synaptic function in amacrine cells

Amacrine cells receive glutamatergic input from bipolar cells and GABAergic, glycinergic, cholinergic, and dopaminergic input from other amacrine cells. Glutamate, GABA, glycine, and acetylcholine (ACh) interact with ionotropic receptors and it is these interactions that form much of the functional circuitry in the inner retina. However, glutamate, GABA, ACh, and dopamine also activate metabotropic receptors linked to second messenger pathways that have the potential to modify the function of individual cells as well as retinal circuitry. Here, the physiological effects of activating dopamine receptors, metabotropic glutamate receptors, GABAB receptors, and muscarinic ACh receptors on amacrine cells will be discussed. The retina also expresses metabotropic receptors and the biochemical machinery associated with the synthesis and degradation of endocannabinoids and sphingosine-1-phosphate (S1P). The effects of activating cannabinoid receptors and S1P receptors on amacrine cell function will also be addressed.     

Interaction between mGluR8 and calcium channels in photoreceptors is sensitive to pertussis toxin and occurs via G protein betagamma subunit signaling

PURPOSE: The most recently identified metabotropic glutamate receptor (mGluR), type 8 mGluR (mGluR8), has been identified functionally as a presynaptic autoreceptor in rod photoreceptors. This study analyzed the mechanism of action underlying mGluR8 activity and modulation of the cytosolic Ca2+ concentration in mouse photoreceptors. METHODS: The cytosolic Ca2+ concentration of acutely isolated rod photoreceptors was monitored optically with microspectrofluorimetry and in the presence of modulators of G protein activity. RESULTS: mGluR8 activation by the group III mGluR agonists l-2-amino-4-phosphonobutyrate and l-serine-O-phosphate or the physiological ligand l-glutamate produced a decrease in influx of extracellular Ca2+ into the cytosol. Pretreatment of isolated rod photoreceptors with the G protein uncoupler suramin or pertussis toxin, which inactivates Gi/o/z proteins and Gt protein/transducin, or a G protein betagamma subunit-inhibiting peptide abolished this activity. Preincubation of cells with cholera toxin (CTX), an activator of Gs protein, had no effect. CONCLUSIONS: These results suggest that the function of mGluR8 of modulating the cytosolic Ca2+ concentration and thereby potentially the release of neurotransmitter from rod spherules, the axon terminal systems of rod photoreceptors, is mediated by a pertussis toxin-sensitive G protein potentially via the betagamma subunit. The absence of Go and Gz proteins, as reported previously, implies a novel potential interaction between Gi2 and/or Gt protein/transducin and mGluR8 in photoreceptors. These results have potential implications for the regulatory function and pharmacologic targeting of mGluR8 in photoreceptors.     

cGMP modulates spike responses of retinal ganglion cells via a cGMP-gated current

Certain ganglion cells in the mammalian retina are known to express a cGMP-gated cation channel. We found that a cGMP-gated current modulates spike responses of the ganglion cells in mammalian retinal slice preparation. In such cells under current clamp, bath application of the membrane-permeant cGMP analog (8-bromo-cGMP, 8-p-chlorophenylthio-cGMP) or a nitric oxide donor (sodium nitroprusside, S-nitroso-N-acetyl-penicillamine) depolarized the membrane potential by 5-15 mV, and reduced the amount of current needed to evoke action potentials. Similar effects were observed when the membrane potential was simply depolarized by steady current. The responses to cGMP are unaffected by inhibitors of cGMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase. The response to cGMP persisted in Ca2+-free bath solution with Ca2+ buffers in the pipette. Under voltage clamp, cGMP analogs did not affect the response kinetics of voltage-gated currents. We conclude that cGMP modulates ganglion cell spiking simply by depolarizing the membrane potential via the inward current through the cGMP-gated channel. Modulation of this channel via the long-range NO-synthase amacrine cell may contribute to control of contrast gain by peripheral mechanisms.     

Voltage-gated calcium and sodium currents of starburst amacrine cells in the rabbit retina

The voltage-gated calcium and sodium currents of starburst amacrine cells were examined in slices of the adult rabbit retina. ON-center starburst amacrine cells were targeted for whole-cell recording by prelabeling the retina with the nuclear dye 4'-6-diamidino-2-phenylindole hydrochloride (DAPI). Calcium currents were isolated using an external Ringer that contained tetrodotoxin to block sodium currents and barium to block potassium channels. When starburst amacrine cells were stepped to holding potentials positive to -50 mV, a series of voltage-dependent calcium currents were activated. The calcium current peaked at -10 mV. The calcium currents kinetics were mainly sustained in nature, showing only a small amount of slow inactivation. Nickel (100 microM), a T-type channel blocker, had no effect on the calcium current. Application of the L-type channel agonist BAY K8644 (1-2.5 microM) had small variable effects on the calcium current while the L-type channel antagonist nifedipine (10 microM) had no effect. However, addition of a reported N-type calcium channel antagonist, omega-conotoxin G6A (1 microM), blocked a large portion of the calcium current, as did a more nonselective antagonist, omega-conotoxin M7C (200 nM). Agatoxin 4A (500 nM) reduced a smaller sustained calcium current component, implying a P/Q-type calcium channel was present on these neurons. In addition to the calcium currents, a fast voltage-gated sodium current was observed in many starburst cells. This current could be blocked by tetrodotoxin (200-500 nM). The differing kinetics and durations of the sodium and calcium currents could play important roles in the regulation of synaptic release and in the coordination of spiking by starburst amacrine cell dendrites during retinal development and in the encoding of motion across the retinal surface.     

Retrograde endocannabinoid inhibition of goldfish retinal cones is mediated by 2-arachidonoyl glycerol

A functional role for retinal endocannabinoids has not been determined. We characterized retrograde suppression of membrane currents of goldfish cones in a retinal slice. Whole-cell recordings were obtained from cone inner segments under voltage clamp. I(K(V)) was elicited by a depolarizing pulse to +54 mV from a holding potential of -70 mV. A fifty-millisecond puff of saline with 70 mM KCl or Group I mGluR agonist DHPG was applied through a pipette directly at a mixed rod/cone (Mb) bipolar cell body. The amplitude of I(K(V)) decreased 25% compared to the pre-puff control. Retrograde suppression of I(K(V)) was blocked by CB1 receptor antagonist, SR141716A. The FAAH inhibitor URB597 had no effect on the suppression of I(K(V)), whereas nimesulide, a COX-2 inhibitor, prolonged the effects of the K+ puff 10-fold. Orlistat, a blocker of 2-AG synthesis, blocked the effect of the K+ puff. Group I mGluR activation of Gq/11 was demonstrated in that a puff with DHPG decreased I(K(V)) of cones by 32%, an effect blocked by SR141716A. The effect of DHPG was not blocked by the mGluR5 antagonist MPEP, indicating involvement of mGluR1. The suppressive effect of the K+ puff vanished in a Ca2+-free, 2 mM Co2+ saline. TMB-8 or ryanodine, blocked the effect of DHPG, but not that of the K+ puff, showing that calcium influx or release from intracellular stores could mediate retrograde release. We suggest that retrograde suppression of cone I(K(V)) is mediated by Ca2+-dependent release of 2-AG from Mb bipolar cell dendrites by separate mechanisms: (1) voltage-dependent, mimicked by the K+ puff, that may be activated by the depolarizing ON response to light; (2) voltage-independent, occurring under ambient illumination, mediated by tonic mGluR1 activation. The negative feedback of this latter mechanism could regulate tonic glutamate release from cones within narrow limits, regardless of ambient illumination.     

Stimulation of L-type Ca(2+) channels by increase of intracellular InsP3 in rat retinal pigment epithelial cells

The purpose of this study was to investigate the role of voltage-dependent L-type Ca(2+)channels in intracellular Ca(2+)signaling of the retinal pigment epithelium (RPE). Patch-clamp techniques in conjunction with measurements of the intracellular free Ca(2+)using the Ca(2+)-sensitive fluorescence dye fura-2 were performed using cultured rat RPE cells. Intracellular application of inositol-1,4,5-trisphosphate (InsP3; 10 microM) via the patch-pipette during the whole-cell configuration led to an increase in the intracellular free Ca(2+)([Ca(2+)](i)). This effect could be reduced by the L-type Ca(2+)channel blocker nifedipine (2 microM). At the moment of the maximal rise in [Ca(2+)](i)L-type currents displayed an increase in the current density and shifts in the activation curve and of the steady-state inactivation. Comparable changes of L-type channel activity could be observed by induction of capacitative Ca(2+)entry, a maneuver to release Ca(2+)from intracellular Ca(2+)stores independently from InsP3. The increase in L-type Ca(2+)channel activity and [Ca(2+)](i)by intracellular application of InsP3 or induction of capacitative Ca(2+)entry could be inhibited by blocking tyrosine kinase activity using genistein (5 microM) or tyrphostin 51 (10 microM). It is concluded that L-type Ca(2+)channels are involved in the Ca(2+)/InsP3 second messenger system by generating an influx of extracellular Ca(2+)into the cell. This is enabled by depletion of cytosolic Ca(2+)stores and tyrosine kinase-dependent activation of L-type channels.     

Spontaneous regenerative activity in mammalian retinal bipolar cells: roles of multiple subtypes of voltage-dependent Ca2+ channels

Patch-clamp recordings were used to investigate the properties of the regenerative activity in acutely isolated bipolar cells from the rat retina. Spontaneous, pacemaker-like membrane potential oscillations were observed in all rod bipolar cells and the majority of cone bipolar cells. The waveform of the regenerative potential was stereotypical but distinct among different bipolar cell groups, especially between rod and cone bipolar cells. The spontaneous activity was completely blocked by Co2+, suggesting that Ca2+ influx through voltage-dependent Ca2+ channels was required for initiating such activity. Ca2+-induced Ca2+ release, however, was not found to be involved. The spontaneous activity was also blocked by mibefradil, a T-type Ca2+ channel antagonist. In contrast, application of nimodipine, an L-type Ca2+ current antagonist, affected mainly the waveform of the regenerative potential. This study shows that mammalian retinal bipolar cells in isolation are also capable of generating Ca2+-dependent spontaneous regenerative potential. However, T-type Ca2+ channels appear to be essential for the initiation of the spontaneous activity in mammalian bipolar cells.     

Immunolocalization of metabotropic glutamate receptors 1 and 5 in the synaptic layers of the chicken retina

We have examined the distribution of metabotropic glutamate receptors (mGluRs) 1 and 5 in the adult chicken retina using preembedding immuno-electronmicroscopy. Immunoreactivity for mGluRs 1 and 5 was found in both the outer plexiform layer (OPL) and the inner plexiform layer (IPL). For mGluR1, OPL labeling was observed at cone pedicles and horizontal and bipolar cell processes. In the IPL, mGluR1 labeling could be found on bipolar cell terminals, as well as postsynaptic processes, including amacrine cell processes. Neither presynaptic nor postsynaptic elements were labeled at rod synapses. For mGluR5, OPL labeling was associated with cone pedicles as well as bipolar and horizontal cell processes. As for mGluR1, rod synapses were unlabeled. In the IPL, labeling for mGluR5 was found on bipolar cell terminals and amacrine cell processes. The presynaptic expression of these receptors in the OPL was confirmed at the light level by double-labeling experiments with SV2. The distributions of mGluRs 1 and 5 indicate that they have the potential to regulate function in both synaptic layers. Furthermore, the similar expression patterns for these two receptors indicate that they might be co-expressed and thus have the potential to interact functionally.     

Neural reprogramming in retinal degeneration

PURPOSE: Early visual defects in degenerative diseases such as retinitis pigmentosa (RP) may arise from phased remodeling of the neural retina. The authors sought to explore the functional expression of ionotropic (iGluR) and group 3, type 6 metabotropic (mGluR6) glutamate receptors in late-stage photoreceptor degeneration. METHODS: Excitation mapping with organic cations and computational molecular phenotyping were used to determine whether retinal neurons displayed functional glutamate receptor signaling in rodent models of retinal degeneration and a sample of human RP. RESULTS: After photoreceptor loss in rodent models of RP, bipolar cells lose mGluR6 and iGluR glutamate-activated currents, whereas amacrine and ganglion cells retain iGluR-mediated responsivity. Paradoxically, amacrine and ganglion cells show spontaneous iGluR signals in vivo even though bipolar cells lack glutamate-coupled depolarization mechanisms. Cone survival can rescue iGluR expression by OFF bipolar cells. In a case of human RP with cone sparing, iGluR signaling appeared intact, but the number of bipolar cells expressing functional iGluRs was double that of normal retina. CONCLUSIONS: RP triggers permanent loss of bipolar cell glutamate receptor expression, though spontaneous iGluR-mediated signaling by amacrine and ganglion cells implies that such truncated bipolar cells still release glutamate in response to some nonglutamatergic depolarization. Focal cone-sparing can preserve iGluR display by nearby bipolar cells, which may facilitate late RP photoreceptor transplantation attempts. An instance of human RP provides evidence that rod bipolar cell dendrite switching likely triggers new gene expression patterns and may impair cone pathway function.     

Voltage-dependent calcium channels in the rat retina: involvement in NMDA-stimulated influx of calcium

Rises in intracellular Ca2+ induced by activation of glutamate receptors are of ultimate importance for neuronal excitability and pathophysiological processes. In the present study, we aimed to elucidate the types of voltage-dependent Ca2+ channels involved in the NMDA-stimulated influx of Ca2+ into the isolated rat retina by using selective blockers. Additionally, the number of binding sites for radioligands labelling L- ([3H]nitrendipine), N- ([125I]omega-conotoxin MVIIA) and P/Q-type ([125I]omega-conotoxin MVIIC) Ca2+ channels was assessed in the rat retina and, for further comparison, in the rat cortex. Incubation of isolated rat retinas with 100 microM NMDA produced a three-fold increase in the influx of 45Ca2+ that was completely blunted by MK-801, a NMDA receptor antagonist, and partially attenuated (approximately 20%) by tetrodotoxin, a Na+ channel blocker. The L-type Ca2+ channel blocker nifedipine reduced NMDA-stimulated Ca2+ influx in a dose-related fashion, with a maximum reduction of approximately 50%. Similar effects were observed with verapamil and diltiazem. Blockers of N- and P/Q-type Ca2+ channels had no significant effect on the influx of Ca2+ evoked by NMDA. Co2+, a non-specific Ca2+ channel blocker, caused an inhibition of NMDA-stimulated Ca2+ influx similar to that of nifedipine. Therefore, of all voltage-dependent Ca2+ channels, L-type channels appear to make the greatest contribution (up to 50%) to the NMDA-stimulated influx of Ca2+ into the isolated rat retina. This finding contrasts with evidence obtained in brain neurones supporting a role for L-, N- and P/Q-type channels in NMDA-evoked Ca2+ signals. A comparison of the number of radioligand binding sites associated with L-, N- or P/Q-type Ca2+ channels in the rat cortex and retina revealed that such a difference cannot be ascribed to a distinct expression pattern of these channels in both tissues, although some variations were found. Interestingly, a different affinity of [3H]nitrendipine for L-type Ca2+ channels in the rat retina and cortex was observed which may reflect the expression of different classes of L-type channels in these tissues. The ability of L-type Ca2+ channel blockers to attenuate NMDA-stimulated Ca2+ influx may underlie their neuroprotective effects in the retina.     

Calcium-permeable glutamate receptors in horizontal cells of the mammalian retina

Mechanisms that mediate the calcium influx in mammalian horizontal cells were studied. Horizontal cells (HCs) enzymatically dissociated from the rabbit retina were recorded by the whole-cell configuration of the patch-clamp technique and by calcium image ratioespectrophotometry of Fura-2 loaded cells. AMPA-preferring glutamate receptors were shown to permeate Ca2+ in mammalian HCs by ionic substitution experiments. Furthermore, after blocking the L-type calcium current with nifedipine (100 microM), calcium current through the AMPA-preferring glutamate receptors was measured. Calcium image ratioespectrophotometry was performed on the dissociated HCs in order to determine the changes in the intracellular calcium ([Ca2+]i). Fura-2 microspectrophotometry showed that in HCs, K+-induced cell depolarization promoted an increase in [Ca2+]i, mediated by the L-type calcium channels, since it was abolished in the presence of nifedipine. The increase in [Ca2+], upon cell depolarization was observed throughout each cell: however, it was maximal at the cell soma. Activation of glutamate receptors in dissociated HCs by glutamate, AMPA or kainate promoted an increase in [Ca2+]i. This increase in [Ca2+]i, was abolished in nominally Ca2+-free solution (0 mM Ca2+); in contrast, nifedipine decreased the glutamate-induced influx of calcium in ca. 50%. The present study demonstrates that calcium may permeate through glutamate receptors expressed in HCs of the rabbit retina.     

AsialoGM1-mediated IL-8 release by human corneal epithelial cells requires coexpression of TLR5

PURPOSE: In this study, it was determined that human corneal epithelial cells (HCECs) express asialoganglioside ganliotetraosylceramide (asialoGM1) and toll-like receptor (TLR)-5, and their interaction induces interleukin (IL)-8 release through Ca(2+) transient activation and mitogen-activated protein kinase (MAPK) stimulation. METHODS: Expression of asialoGM1 and TLR5 was detected in SV40 HCECs by Western blot and flow cytometry analyses and their association by coimmunoprecipitation. Single-cell fluorescence imaging was used to measure intracellular free Ca(2+) transients in fura-2-loaded cells. The enzyme-linked immunosorbent assay (ELISA) was used to quantify IL-8 production in both cultured and primary HCECs. RESULTS: The HCECs expressed both asialoGM1 and TLR5 receptors. Ligation of asialoGM1 resulted in protein-protein interaction with TLR5, followed by transient increases in Ca(2+) influx through L-type voltage-dependent Ca(2+) channels. This led to P2Y receptor stimulation along with membrane depolarization, resulting from increases in ATP release into the medium. Intracellular Ca(2+) transients led to time-dependent extracellular signal-regulated kinase (ERK) MAPK pathway stimulation, followed by a 9.5-fold increase in IL-8 release. Similarly, in primary HCECs, asialoGM1 receptor stimulation resulted in an 8.1-fold increase. With a TLR5 neutralizing antibody, no asialoGM1-induced increases in IL-8 release occurred, and this response was not suppressed in the presence of a TLR2 neutralizing antibody. CONCLUSIONS: IL-8 release by HCECs is mediated through ligand-induced asialoGM1 protein-protein interactions with TLR5. This response is dependent on ATP efflux into the medium, followed by P2Y receptor stimulation. Such activation, in turn, results in increases in Ca(2+) influx through L-type voltage-dependent Ca(2+) channels, as well as stimulation of the ERK pathway.     

Voltage-clamp analysis and computational model of dopaminergic neurons from mouse retina

Isolated dopaminergic amacrine (DA) cells in mouse retina fire rhythmic, spontaneous action potentials and respond to depolarizing current with trains of low-frequency action potentials. To investigate the roles of voltage-gated ion channels in these processes, the transient A-type K+ current (I(K,A)) and Ca2+ current (I(Ca)) in isolated mouse DA cells were analyzed by voltage clamp. The I(K,A) activated at -60 mV and inactivated rapidly. I(Ca) activated at around -30 mV and reached a peak at 10 mV without apparent inactivation. We also extended our previous computational model of the mouse DA cell to include the new electrophysiological data. The model consisted of a membrane capacitance in parallel with eight currents: Na+ transient (I(Na,T)), Na+ persistent (I(Na,P)), delayed rectifier potassium (I(Kdr)), I(K,A), calcium-dependent potassium (I(K,Ca)), L-type Ca2+ I(Ca), hyperpolarization-activated cation current (I(h)), and a leak current (I(L)). Hodgkin-Huxley type equations were used to define the voltage- and time-dependent activation and inactivation. The simulations were implemented using the neurosimulator SNNAP. The model DA cell was spontaneously active from a wide range of initial membrane potentials. The spontaneous action potentials reached 35 mV at the peak and hyperpolarized to -76 mV between spikes. The spontaneous firing frequency in the model was 6 Hz. The model DA cell responded to prolonged depolarizing current injection by increasing its spiking frequency and eventually reaching a depolarization block at membrane potentials greater than -10 mV. The most important current for determining the firing rate was I(K,A). When the amplitude of I(K,A) was decreased, the firing rate increased. I(Ca) and I(K,Ca) also affected the width of action potentials but had only minor effects on the firing rate. Ih affected the firing rate slightly but did not change the waveform of the action potentials.     

Transmitter-gated currents of GABAergic amacrine-like cells in chick retinal cultures.

A subpopulation of cells developing in dissociated neuronal cultures prepared from 8-day-old embryonic chick retinae can be identified as putative in vitro counterparts of GABAergic amacrine cells by immunocytochemical and autoradiographic markers and by their electrophysiological responses to transmitter agonists. In the present study, transmitter-gated conductances expressed by these neurons were examined using the whole-cell patch-clamp technique. At negative holding potentials, the excitatory amino acid agonists N-methyl-D-aspartate (NMDA), kainate quisqualate, and glutamate induced inward currents with reversal potentials close to 0 mV in most of the cells selected for recording. NMDA-evoked responses were selectively blocked by the noncompetitive inhibitor MK 801 and by Mg2+ (in a voltage-dependent manner) and were potentiated in the presence of submicromolar concentrations of glycine. Glutamate apparently interacted with both NMDA and non-NMDA type receptors. All cells tested responded to the inhibitory transmitters GABA and glycine. Both inhibitory agonists could be shown to activate chloride conductances. Responses to GABA and glycine were specifically inhibited in the presence of bicuculline and strychnine, respectively. Thus, GABAergic neurons in retinal cultures express at least two different excitatory amino acid receptors--NMDA and non-NMDA--and two different inhibitory amino acid receptors--the GABAA and the glycine receptor. The results demonstrate the ability of the cultured neurons to develop an apparently mature phenotype and contribute to the understanding of the functional properties of GABAergic amacrine cells in the vertebrate retina.     

Rabbit cone bipolar cells: correlation of their morphologies with whole-cell recordings

The superfused retinal slice preparation was used to examine the morphology and glutamate-activated whole-cell currents of rabbit bipolar cells. There were six morphologically distinct types of cone bipolar cells and a rod bipolar cell that had axon terminals stratifying in stratum 3 to 5 of sublamina-b. All of these bipolar cell types exhibited an outward current in response to the application of the metabotropic glutamate receptor, mGluR6, agonist AP-4 (APB), and had I/V curves indicative of membrane channel closure. Conversely, there were no currents activated during the application of kainate, the AMPA/kainate receptor agonist. These data demonstrate they were on-bipolar cells. In addition, there were six morphologically distinct cone bipolar cells that stratified in sublamina-a. Every cell with axonal arborizations in stratum 1 and 2 exhibited an inward current when the ionotropic glutamate receptor agonist kainate was applied. This current was blocked by application of the AMPA/kainate receptor antagonist CNQX. These cells also decreased their membrane resistance in response to kainate, a characteristic of the opening of channels within the plasma membrane. Without exception, no cells stratifying in sublamina-a responded to the mGluR6 agonist AP-4, further identifying them as off-bipolar cells.