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REPORT
1Section of Neurobiology, Physiology, and Behavior and 2Department of Ophthalmology and Vision Science, University of California, Davis, California
Submitted 27 November 2006; accepted in final form 14 February 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Meister and Berry 1999; Wiesel 1959; Zaghloul et al. 2003), changes in the gating of several ion currents have been found and the kinetics of at least some of these changes are suitable for rendering spiking a malleable rather than static process. Among these changes are fast and slow inactivation of high-threshold current after depolarizations and priming of low-threshold currents by hyperpolarizations (Berry and Meister 1998; Eng et al. 1990; Hayashida and Ishida 2004; Hidaka and Ishida 1998; Kaneda and Kaneko 1991; Karschin and Lipton 1989; Kim and Rieke 2001; Lee et al. 2003). Other effects of hyperpolarization on ganglion cells are known, but descriptions of the ion channels gated by these voltage changes have been unclear in a number of ways. Initial recordings showed that moderate hyperpolarizations activate inward voltage rectification in axons (Eng et al. 1990) and a mixed-cation current (Ih) in somata (Tabata and Ishida 1996) of rat and goldfish retinal ganglion cells, respectively. Anatomical studies subsequently found that somata in the ganglion cell layer of rat retina bind antisera directed against Ih and inward rectifier K+ (Kir) channels (Chen et al. 2004; Ishii et al. 2003; Ivanova and Müller 2006; Müller et al. 2003; Tian et al. 2003). Although these studies suggest that more than one channel type may be activated by hyperpolarization, it is unknown whether individual ganglion cells possess both Ih and Kir channels or whether Ih and Kir occur only in different cells. Interestingly, no inward rectification was seen in large rat retinal ganglion cell somata (Reiff and Guenther 1999; see also Akamine et al. 2002; O'Brien et al. 2002) and the only inwardly rectifying current characterized in any mammalian retinal ganglion cell to date is a rapidly activating, Ba2+-sensitive Kir-like current observed during large hyperpolarizations or in elevated extracellular K+ levels (Chen et al. 2004).
Because these observations differ from the slowly activating, Cs+-sensitive mixed-cation current originally inferred on the basis of voltage recordings from rat optic nerve (Eng et al. 1990), we tested for the presence of Ih in adult rat retinal ganglion cells. We report here that 1) in perforated-patch whole cell recordings, Ih can be activated in some of these cells during moderate hyperpolarizations at a physiological extracellular K+ concentration (Ames and Nesbett 1981); 2) we found no evidence of Kir in cells that possessed Ih; 3) a Kir-like current can be activated in other cells under identical recording conditions; and 4) still other cells displayed neither Ih nor Kir. These results provide the first identification of different ion currents activated by hyperpolarization in retinal ganglion cells of a single species and the first measurement of Ih in mammalian retinal ganglion cells.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Adult rat retinas were used for the experiments reported here to compare our results with those of previous anatomical and electrophysiological studies (Chen et al. 2004; Eng et al. 1990; Ishii et al. 2003; Ivanova and Müller 2006; Müller et al. 2003; Reiff and Guenther 1999; Tian et al. 2003). Long–Evans rats (female, P60–P120, 150–250 g) were obtained from a commercial supplier (Harlan Bioproducts, San Diego, CA) and housed in standard cages at about 23°C on a 12-h/12-h light/dark cycle. All animal care and experimental protocols were approved by the Animal Use and Care Administrative Advisory Committee of the University of California, Davis.
Cell dissociation and panning
The retinal ganglion cells used in this study were dissociated by a combination of standard protocols and methods developed in our laboratory (e.g., Hayashida et al. 2004; Vaquero et al. 2001). Each rat used was killed by an overdose of pentobarbital. Sources of the reagents used are listed below. Briefly, retinas were isolated from two freshly enucleated eyes and cells on the distal side of these retinas (mostly photoreceptors) were manually sliced off with a razor. The remaining retinal tissue was incubated for 5–10 min at 30°C in a 5-ml plastic tube containing a papain solution [16 U/ml papain in low-Ca2+ solution mixed 1:1 with L-15 culture medium that was supplemented with 1 µM tetrodotoxin (TTX). The low-Ca2+ solution contained (in mM): 140 sucrose, 2.5 KCl, 70 CsOH, 20 NaOH, 1 NaH2PO4, 15 CaCl2, 20 EDTA, 11 D-glucose, 15 HEPES, 0.1 glutathione, 1 kynurenic acid, 0.001 TTX, and 0.025 mg/ml DNase I. The estimated free Ca2+ concentration was 100–200 nM]. To inhibit the papain activity, the enzyme solution was replaced with ovomucoid solution (0.5 mg/ml ovomucoid in low-Ca2+ solution mixed 1:1 with L-15 supplemented with 1 µM TTX and 0.025 mg/ml DNase I) for 5 min at room temperature. The retinal tissue was rinsed a few times with fresh L-15 medium (supplemented with 0.025 mg/ml DNase I; pH 7.2–7.3) and triturated. Supernatant was layered over fresh L-15 medium in a 5-ml plastic tube (10 mm ID) and allowed to sit for 20 min. The top 1–2 cm of this solution was then discarded and the remaining solution, except for undissociated retinal pieces at the tube bottom, was transferred to an empty 5-ml plastic tube.
We isolated retinal ganglion cells from the final cell suspension by a "panning" method based on the expression of Thy1 (Barres et al. 1988) and nearly all cells were checked for the presence of voltage-gated Na+ current and/or large spikes to verify that they were ganglion cells (Barres et al. 1988; Boos et al. 1993). We prepared panning dishes by cutting a 13-mm hole in the bottom of 35-mm plastic tissue culture dishes and attaching a glass coverslip with Sylgard 184. The upper side of each coverslip was coated with goat anti-mouse IgM (diluted 1:200 in 0.1 M Tris, pH 9.5) for 2 h at 30°C. After three PBS rinses, the dishes were incubated with anti-Thy1 antibody for an additional 2 h at room temperature, rinsed again with PBS, followed by one final rinse with L-15.
Retinal ganglion cells were panned by placing several drops of the final cell suspension onto the prepared glass area of these culture dishes. After allowing cells to settle for 30 min at 30°C, nonadherent cells were removed from the dishes by rinsing each dish three times with L-15. The dishes were then filled with culture medium (L-15 medium; supplemented with 1% B-27; pH was adjusted to 7.2–7.3 with HCl). The cells were stored at 30°C for 12–16 h and the culture medium was replaced once more before electrophysiological recordings.
Recording configuration and solutions
The recordings presented here were performed in perforated-patch whole cell mode at room temperature (21–23°C) using methods we described previously (e.g., Hayashida and Ishida 2004; Hidaka and Ishida 1998; Lee et al. 2003; Tabata and Ishida 1996; Vaquero et al. 2001). Patch electrodes were pulled from borosilicate glass capillaries (#64–0786; Warner Instruments, Hamden, CT) to tip resistances of about 2–4 M
. Amphotericin B was included in the recording electrode-filling solution as the perforating agent (200 µg/ml, with 100 or 300 µg/ml Pluronic F-127) and data were collected after the series resistance fell to <100 M. The recording electrode solution contained (in mM): 115 K-D-gluconic acid, 15 KCl, 15 NaOH, 2.6 MgCl2, 0.34 CaCl2, 1 EGTA, and 10 HEPES; the pH of this solution was adjusted with methanesulfonic acid to 7.4. The extracellular solution contained (in mM): 145 NaCl, 3.5 KCl, 2.5 CaCl2, 1.0 MgCl2, 10 D-glucose, and 5 HEPES; the pH of this solution was adjusted with NaOH to 7.4. Osmolalities of the extracellular and recording electrode solutions were 290–310 and 260–280 mmol/kg, respectively. To apply reagents under uniform conditions, saline was continuously superfused over each cell recorded from, through a hole (ID 
500 µm) at the bottom of a U-shaped Teflon tube. By changing the solution reservoir feeding the top of this U-tube, effects of pharmacological agents or of altered extracellular ion concentrations could be measured at the control flow rate (200 µl/min). The extracellular solution was grounded by an agar bridge. The membrane potentials reported here were corrected for liquid junction potentials due to measured differences between the extracellular and patch electrode-filling solutions. For recordings in control bath solution, the correction was made by adding –13 mV to the voltages in the current- and voltage-clamp protocols. For those initiated in low-Na+ solutions, the correction was –20 mV. No correction was made for the 1-mV difference between the bath solutions containing different K+ concentrations.
An Axopatch-1D patch-clamp amplifier (Axon Instruments, Union City, CA) and pCLAMP software (v. 8.2.0.224 [EC] ; Axon Instruments) were used to generate voltage jumps, inject constant current, and acquire data. In voltage-clamp mode, the current monitor output of the amplifier was analog-filtered by the built-in four-pole Bessel filter (fc = 0.5 or 1 kHz) and digitally sampled (usually at 2 kHz). Current traces are displayed without leak subtraction, signal averaging, or post hoc filtering and series resistance compensation was used to check for voltage errors in current amplitudes. When recording spikes, the voltage monitor output was analog-filtered at either 2 or 5 kHz and digitally sampled at 10 kHz.
Data were analyzed in pCLAMP. Three standard equations were routinely used in these analyses. First, the sum of two exponential time functions for fitting current traces
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x is the time constant for the change in Ax, and C is an offset constant. Second, a Boltzmann equation
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Reagents
Reagents were obtained from the following sources: Abbott Laboratories (North Chicago, IL): sodium pentobarbital (#0074-378-05); Aldrich Chemical (Milwaukee, WI): D-gluconic acid, potassium salt (#86037–9), methanesulfonic acid (#47135–6); BDH Laboratory Supplies (Poole, UK): CaCl2; Calbiochem (San Diego, CA): tetrodotoxin (#584411); Dow Corning (Midland, MI): Sylgard 184; Fluka (Milwaukee, WI): EGTA (#03777); Invitrogen (Carlsbad, CA): B-27 (#99-0254DG), L-15 (#41300–039); ICN (Aurora, OH): CsCl (#813061); Jackson ImmunoResearch (West Grove, PA): goat anti-mouse IgM (#115-005-020); Molecular Probes (Eugene, OR): Pluronic F-127 (#P6867); Sigma Chemical (St. Louis, MO): amphotericin B (#A-4888), bovine serum albumin (#A7284), DMSO (#317275), DNase I (#D4527), HEPES (#H-4034), ovomucoid trypsin inhibitor type 3–0 (#T2011); Tocris Bioscience (Ellisville, MO): 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride (ZD7288, #1000); Worthington, (Freehold, NJ): papain (#3126). The salts (NaCl, etc.) used for electrophysiological recordings and buffers were all reagent grade and obtained from Sigma unless otherwise specified.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Müller et al. 2003) because inwardly rectifying current types and densities were previously found to change with age in other preparations (e.g., Bayliss et al. 1994; Hogg et al. 2001; Tanaka et al. 2003). Second, the control external K+ concentration was set to 3.5 mM, to match the K+ concentration in solutions used to perfuse retinas during electrophysiological studies (Ames and Nesbett 1981; Newman and Bartosch 1999; Weinstein et al. 1967) and because the gating kinetics of Ih (and the amplitude of Ih and Kir) were found to differ at higher K+ concentrations (Halliwell and Adams 1982). Third, all recordings were made in perforated-patch mode (see METHODS) because second messengers modulate inwardly rectifying currents in various preparations including retinal photoreceptor, horizontal, and Müller cells (Akopian and Witkovsky 1996; Demontis et al. 2002; Dixon and Copenhagen 1997; Kusaka and Puro 1997; Puro et al. 1996; Solessio et al. 2001) and we sought to minimize perturbation of the intracellular milieu. Fourth, currents were recorded at room temperature to compare with results of previous studies (Chen et al. 2004; Ma et al. 2003; Tabata and Ishida 1996). Different conductances were detectable
Under these conditions, we observed three conductances when cells were hyperpolarized from a holding value of –73 mV to test potentials between –83 and –123 mV. In several cells, the clamp current changed in amplitude without delay after the voltage steps; the amplitude did not change during voltage steps as long as 4 s; and the amplitude increased linearly with test potential (Fig. 1A1). These resemble time-independent currents in previous recordings from postnatal rat retinal ganglion cells (Akamine et al. 2002; Reiff and Guenther 1999) and were not routinely examined here. In a very small number of other cells, the current amplitude also showed no time dependence after either the onset or termination of voltage steps, although the slope conductance increased considerably as the test potential was made more negative than –93 mV (Fig. 1B1). Low concentrations of Ba2+ (50–100 µM) blocked this rectification (Fig. 1B2), indicating it is likely to be Kir (Hagiwara et al. 1978). Aside from finding only an ohmic current (that is, a voltage- and time-independent conductance) during this block, we could not study this conductance further because we encountered it in only two of the cells we recorded from over the course of several months. In the remainder of the cells we recorded from, the whole cell current presented a slowly gating component (Fig. 1C) due to Ih, as described below.
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Ih can be reduced to negligibly small amplitudes by Cs+ (1–5 mM) and ZD7288 (1–100 µM) (Dickson et al. 2000; Santoro and Tibbs 1999) and it resists blockade by 2 mM Ba2+ (Ludwig et al. 1998). Because even smaller amounts of Ba2+ block Kir (see above), because some neurons possess both Ih and Kir (Dickson et al. 2000; Hogg et al. 2001; Ma et al. 2003; Scroggs et al. 1994), and given the results of previous ganglion cell studies (Chen et al. 2004; Eng et al. 1990; O'Brien et al. 2002; Tabata and Ishida 1996), we first characterized the slowly gating current by sequentially applying Ba2+ and Cs+ to individual cells. Figure 1C shows the whole cell current activated by voltage steps from a holding potential of –73 mV to test potentials ranging from –83 to –123 mV in 10-mV decrements, during superfusion of control saline (Fig. 1C1), saline supplemented with 1 mM Ba2+ (Fig. 1C2), saline supplemented with 1 mM Ba2+ plus 2 mM Cs+ (Fig. 1C3), and thereafter, control saline again (Fig. 1C4). In control saline, after the onset of each hyperpolarizing step, the whole cell current increased in amplitude instantaneously and then continued to increase gradually. During small hyperpolarizations (i.e., to test potentials less negative than about –100 mV), the current did not stop increasing in amplitude even if the test pulse duration was as long as 4 s. During larger hyperpolarizations (e.g., to –113 and –123 mV), the current increased instantaneously, rose to a peak amplitude within 1 s, and remained at that amplitude for the remainder of the hyperpolarization. The current in Fig. 1, C1, C2, and C4 thus activates more slowly than Kir currents seen in other retinal neurons (Chen et al. 2004; Ma et al. 2003; Tachibana 1983). After repolarizing to the holding potential, the whole cell current partially decreased instantaneously and then continued to decrease over the next several seconds. In most somata, and as subsequently described in more detail, the current returned to the holding level within 5–10 s. In a few cells, this deactivation appeared to be incomplete even after 10 s; in these cases, 
25 s were allowed to elapse between successive test pulses. Slowly gating, hyperpolarization-activated currents with these general properties were observed in roughly 75% of the cells from which we recorded (n 
120).
At 1–2 mM, Ba2+ reduced the total current when the test hyperpolarizations activated a slow component. However, the Ba2+-sensitive portion did not have the same fast onset and voltage dependence as Kir (not shown) and the reduction was slight (Fig. 1C2), unlike the suppression of rapidly gating, inward rectifier current noted earlier (Fig. 1B2). For example, during hyperpolarizations from –83 to –123 mV, 1 mM Ba2+ reduced the total current amplitude by 13 ± 3% (mean ± SE, n = 4) and the remaining (Ba2+-resistant) current gated slowly and rectified inwardly like the control current (Fig. 1C2). By contrast, subsequent addition of 2 mM Cs+ markedly reduced the remaining current (Fig. 1C3), especially by suppressing the slowly activating and deactivating current (see following text). The ability of Cs+ to block the slowly gating current did not require previous or concurrent exposure to Ba2+, and Ba2+ produced little or no reduction of the total current when its application started only after current was blocked by Cs+ (not shown), as if Ba2+ was affecting a small portion of the Cs+-sensitive current (see Ludwig et al. 1998). We address this possibility further, by use of a Kir-sparing Ih antagonist, after describing the effects of Cs+.
Cs+ reduced the whole cell current in four ways. First, it reduced the holding current (compare Fig. 1, C1, C2, and C3). Digital subtraction of the current recorded before and during the Cs+ application (i.e., subtraction of the current recorded in Ba2+ and Cs+ from that recorded in Ba2+) therefore yielded a net inward "Cs+-blocked" current at the holding potential (see current trace at –73 mV, Fig. 2A). Second, Cs+ reduced the amplitude of the instantaneous current steps seen at the onset and termination of the test hyperpolarizations (compare Fig. 1, C2 and C3). This is shown by the instantaneous current steps at the onset and termination of the test hyperpolarizations in the Cs+-blocked current traces (Fig. 1C5). Third, it abolished the slowly activating current during the test hyperpolarizations, leaving only a time-independent current (Fig. 1C3). (Consistent with this, we found no effect of 1 mM Cs+ on currents that were ohmic in control solution; see Fig. 1A2.) Fourth, Cs+ abolished the slowly deactivating "tail" current seen after terminating each test hyperpolarization, leaving a time-independent tail current (Fig. 1C3). Thus the Cs+-blocked current consisted of a standing inward current at the holding potential, an instantaneous component at the onset of the test hyperpolarization, a slowly activating component during the test hyperpolarization, and a slowly deactivating tail after shifting the voltage back to the holding potential (Fig. 1C5).
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Current activation was gauged from the rate at which Cs+-blocked current rose in amplitude when cells were hyperpolarized. The gradual increases in current (after the instantaneous increases) at test potentials between –93 and –123 mV were fitted by sums of two exponential time functions (see METHODS; Fig. 2A). These fits were not significantly improved by using the sum of three exponentials, but they were clearly poorer with one exponential (Fig. 2, C1 and C2). Both the faster and slower time constants of the fitted functions decreased exponentially with voltage (closed symbols, Fig. 2E), ranging from 459 ± 82 and 2,718 ± 783 ms at –93 mV to 105 ± 10 and 726 ± 122 ms at –123 mV, respectively (mean ± SE, n = 5); the faster component comprised 70 ± 3% of these fits at –123 mV and less at more positive test potentials (e.g., 50 ± 13% at –93 mV; mean ± SE, n = 5). The decrease in the faster time constant is readily apparent from the increased rate of current rise as cells were hyperpolarized to more negative test potentials (Fig. 2A). The rate at which Cs+-blocked current deactivated at the holding potential could also be fitted better by the sum of two exponential time functions in three of the cells from which we recorded; monoexponential fits were adequate in the other two cells (Maricq and Korenbrot 1990; Mayer and Westbrook 1983). In either case, we observed no dependence of the tail current time constants on the membrane potential at which the current was initially activated (open symbols, Fig. 2E). For example, after repolarizing to the holding potential (–73 mV) from the test jump to –93 mV, the faster time constant of the biexponential fits was 317 ± 23 ms (mean ± SE, n = 3); after repolarizing from –123 mV, it was 328 ± 6 ms. The slower time constant of these fits was 2,122 ± 228 ms after the repolarizations from –93 mV and 2,113 ±385 ms after the repolarizations from –123 mV. The fast deactivating component was 51 ± 4% of the biexponential fits and did not appreciably differ over the voltage range we examined. The average monoexponential decay constant (in the two cells noted above) was 1,163 ± 114 ms.
The range of membrane potentials that activated current (i.e., the "activation range") was estimated from the amplitude of Cs+-blocked current immediately after each repolarization to the holding potential (i.e., after terminating each test hyperpolarization). These "tail" current amplitudes were back-extrapolated to the moment of repolarization, based on the time functions fitted to them and provided that the currents did not noticeably diverge from these functions near the moment of repolarization (compare Fig. 2, B and D). The tail current amplitude (IV, in pA) from each cell was then plotted against test potential and fitted with a Boltzmann equation (see METHODS). The means of the test potential that activates half of the maximum conductance (V1/2) and the slope factor values obtained this way were –86 ± 3 mV (mean ± SE, n = 5) and 7.9 ± 0.9, respectively. Figure 2F displays these measurements, from all of the cells recorded under this condition, after normalizing the current amplitudes for each cell to its respective Imax (measured during the repolarization from –123 mV).
ZD7288 and high [K+]o effects are consistent with Ih
ZD7288 is a bradycardiac agent that can block current through all of the cloned Ih channel types studied to date (Santoro and Tibbs 1999; Stieber et al. 2005) and abolish native Ih without blocking Kir (e.g., Dickson et al. 2000; Hogg et al. 2001; Ma et al. 2003). Given this specificity, we tested whether ZD7288 blocked the slowly gating and inwardly rectifying type of current shown in Figs. 1C and 2. We did so in control saline (Fig. 3A) to assay for Ih in physiological concentrations of Na+ and K+, and after raising the extracellular K+ concentration from 3.5 to 38 mM (Fig. 3B) to double-check for the presence of Kir (by using conditions that increase its amplitude; Chen et al. 2004; Dixon and Copenhagen 1997; Hagiwara et al. 1978; Tachibana 1983). Figure 3A shows the current activated by voltage steps from a holding potential of –73 mV to test potentials between –78 and –98 mV during superfusion of control saline (Fig. 3A1) and then control saline supplemented with 100 µM ZD7288 (Fig. 3A2); the ZD7288-blocked current obtained by digital subtraction of these currents is shown at the right (Fig. 3A3).
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Because we were able to measure current kinetics and voltage sensitivities in saline containing a physiological K+ concentration, we did not analyze the currents recorded in high-K+ medium. However, the tail currents in both normal and high-K+ medium were inward at the holding potential (–73 mV), even though the K+ equilibrium potential (calculated from the bath and electrode solutions) moved from –91 mV in normal saline (Fig. 3B1) to –31 mV when the bath contained 38 mM K+ (Fig. 3B2). This is consistent with previous findings that the activation range of Ih is unaltered by increases in extracellular K+ concentration (Hogg et al. 2001; Tabata and Ishida 1996; Wollmuth and Hille 1992) and this differs from the change in tail current polarity that would be expected for a K+-selective current under the same conditions.
As in other tissues (Harris and Constanti 1995; Stieber et al. 2005), current amplitude declined over the course of 5–10 min of continuous 100 µM ZD7288 application in all of the cells we tested (n = 23, i.e., n = 14 in control saline; n = 5 in high-K+ saline; n = 4 in low-Na+ saline, as shown below). The fraction of total current suppressed by 10 µM ZD7288 was nearly equivalent (n = 7), with slower onset; 2 µM ZD7288 had only a slight effect (n = 3) and 5 µM was intermediate (n = 3; e.g., Fig. 4). We did not routinely test for reversibility of this block because other studies found the block to reverse slowly (>1 h), if at all (e.g., Berger et al. 1994; Harris and Constanti 1995), and we found only very slight reversal of 100 µM ZD7288 after washing continuously with control saline for as long as 25 min.
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The pharmacological properties, kinetics, and activation range described earlier suggest that Ih can be activated in rat retinal ganglion cell somata. To test further whether our protocols activated Ih, we measured the reversal potential of ZD7288-sensitive current. Because Ih is carried slightly better by K+ than by Na+ in solutions containing physiological concentrations of Na+ and K+, the reversal potential in control saline would be expected to be more negative than 0 mV, but more positive than our holding potential. However, various K+ currents activate in ganglion cells at voltages more positive than –70 mV (unpublished observations; see also Lipton and Tauck 1987). Because part of this current defies suppression by K+ channel blockers (Lukasiewicz and Werblin 1988; Sucher and Lipton 1992), we looked for reversal of Ih tail currents at voltages more negative than –70 mV. To do so, we measured ZD7288-blocked current after replacing 95% of the Na+ in the superfusate solution with an impermeant organic cation (N-methyl-D-glucamine; cf. Tabata and Ishida 1996; Wollmuth and Hille 1992).
Figure 3C shows the whole cell current activated by 3-s hyperpolarizations from a holding potential of –80 mV to a conditioning potential of –120 mV, followed by depolarizations to test potentials of –90, –80, and –70 mV, during superfusion of low-Na+ saline (Fig. 3C1) and low-Na+ saline supplemented with 100 µM ZD7288 (Fig. 3C2). The difference between the currents recorded before and during application of ZD7288 (Fig. 3, C3 and C4) shows that the tail current was inward at –90 mV, null near –80 mV, and outward at –70 mV. The "shoulder" on the outward current (immediately after the voltage shift from –120 to –70 mV; see bracket in Fig. 3C4) resembles that recorded after long conditioning pulses in other preparations (Maruoka et al. 1994). The outward current subsequently declines to a steady plateau, as expected from partial deactivation of current at this voltage and the activation range in Fig. 2F. Similar results were obtained from all three cells tested and linear regressions on plots of the currents measured at these test potentials yielded a reversal potential of –78 ± 0.6 mV (mean ± SE, n = 3; Fig. 3C5). Assuming that only Na+ and K+ carried these currents and that the relative permeabilities to Na+ and K+ ions are described by the GHK equation (see METHODS), the ratio of Na+ permeability to K+ permeability ("PNa/PK") is estimated from these data to be 0.37 ± 0.02. In extracellular solution containing 10% of the normal Na+, the ZD7288-sensitive tail current was inward at –90 and –80 mV and nulled at around –70 mV (n = 2; result not illustrated). In normal saline, the ZD7288-sensitive tail current was inward at –83, –73, and –63 mV (n = 2; result not illustrated). Thus the null potential was more positive when the extracellular solution contained 10% of the control Na+ than when it contained 5% of the control Na+ and, in normal saline, it appears to have shifted even more in the positive direction. From this result and because the K+ equilibrium potential was unchanged (at –91 mV) during all of these recordings, the ZD7288-sensitive current appeared to be carried by a mixture of Na+ and K+ ions, and not by K+ alone.
Activation of Ih can elicit rebound spiking
After activation, the slow deactivation of Ih produces depolarizations that drive various cells to spike threshold. To determine whether Ih could serve a similar role in mammalian retinal ganglion cells, we used a combination of voltage and current clamp on individual cells. Two examples of this are shown in Fig. 4 (A–C and D–F). Figure 4, A1 and D1 shows the total current during voltage steps like those used in Figs. 1 and 2. Switching to current clamp, we injected a small amount of current to hold the membrane potential at about the same level as we used in voltage clamp and verified we had a cell capable of firing action potentials by injecting a series of depolarizing current steps (Fig. 4, C1 and F1). We then injected constant-current steps that hyperpolarized this cell, at some point, to the voltages used in Fig. 4, A1 and D1. As is typical of Ih, prolonged injection of hyperpolarizing current led to activation of inward current and a depolarizing sag of the membrane potential (Fig. 4, B1 and E1). When the hyperpolarizing current was turned off, the membrane potential rebounded and elicited a short series of spikes.
To demonstrate that the membrane potential sag and rebound spikes were a consequence of Ih activation, we applied ZD7288, at 100 µM to the first cell (Fig. 4, A–C) and 5 µM to the second (Fig. 4, D–F). This antagonist of Ih produced four characteristic changes: 1) it caused the cell to hyperpolarize, suggesting that there is a small component of Ih active at our holding potential; 2) it removed the membrane potential sag observed during injections of hyperpolarizing current; 3) it increased the size of the membrane potential excursion elicited by the same hyperpolarizing current, suggesting that the membrane resistance is increased in this voltage range; and 4) it eliminated the rebound spikes (Fig. 4, B2 and E2). All of this occurred without significant effect on spikes elicited by depolarizing current steps (Fig. 4, C2 and F2). We then switched back to voltage clamp to show directly the block of Ih (Fig. 4, A2 and D2). Both voltage rectification and rebound spikes were blocked when ZD7288 blocked the slowly activating and deactivating current, as seen here (n = 8). Although block of Ih by 5 µM ZD7288 was not complete (note current activating during strong hyperpolarization in Fig. 4D2 and persisting voltage sag in Fig. 4E2), cells hyperpolarized 10 ± 1 mV and membrane resistance to hyperpolarizing current increased 25 ± 5% (n = 4) during application of 5 or 10 µM ZD7288. With 100 µM ZD7288, cells hyperpolarized 14 ± 4 mV and membrane resistance increased 52 ± 19% (n = 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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General features of Ih
Our finding of Ih fundamentally agrees with the inference that an Ih-like conductance produces voltage rectification in recordings from rat optic nerve (Eng et al. 1990) and with the finding that Cs+ blocks the slowly developing voltage rectification and hyperpolarization-activated, Ba2+-resistant, inwardly rectifying current in goldfish retinal ganglion cell somata (Tabata and Ishida 1996). The properties we found—block by 2 mM Cs+ (Fig. 1), dose-dependent block by 2–100 µM ZD7288 (Figs. 3, A–C and 4), and a PNa/PK estimate of 0.37 in low-Na+ solution (Fig. 3C)—resemble those of Ih in other preparations (e.g., photoreceptors: Wollmuth and Hille 1992; brain: Dickson et al. 2000; Harris and Constanti 1995; peripheral neurons: Hogg et al. 2001; HCN1–4: Santoro and Tibbs 1999; Steiber et al. 2005). Furthermore, the activation kinetics and activation range of Ih did not differ significantly from those found in other retinal cells under similar conditions (e.g., Fig. 1 of Tabata and Ishida 1996; Fig. 3 of Demontis et al. 2002; Table 3 of Ivanova and Müller 2006).
Our current measurements show that the activation and deactivation time constants are larger than the membrane time constants reported for mammalian retinal ganglion cells (<50 ms in nearly all cell types; O'Brien et al. 2002; Robinson and Chalupa 1997). This suggests that Ih gates slowly enough to contribute to the delayed sag during inward voltage rectification seen in mammalian retinal ganglion cells and also to rebound depolarizations seen at the termination of even moderate hyperpolarizations (Eng et al. 1990; O'Brien et al. 2002). Our measurements from individual cells under both voltage and current clamp confirmed these possibilities (and our preliminary measurements of the temperature dependency of Ih gating indicates that this could also occur at body temperature). Ih differs from Kir in this respect because the activation threshold of Ih is less negative and Kir gates so rapidly that the voltage rectification it produces would not include either a slowly developing or decaying component (e.g., Dickson et al. 2000; Hogg et al. 2001; Ma et al. 2003). However, because marked inactivation has not yet been found in either conductance, both Ih and Kir are likely to contribute to membrane resistance in the cells that express them. Our current and voltage measurements show that Ih could do so at voltages near typical resting potentials and at more negative voltages; Kir would probably do so around the K+ equilibrium potential and at more negative voltages.
Ih in some, but not all, ganglion cells
All retinal ganglion cells are known to have high-threshold Na+, K+, and Ca2+ currents. Our results suggest that Ih differs from these depolarization-activated currents, in mammalian retinal ganglion cells, because we detected Ih in roughly 75% of the cells from which we recorded. We do not yet know whether only specific subtypes of rat retinal ganglion cells possess Ih—and, if so, which ones—because our dissociation protocol, like those used in all previous studies of which we are aware, shears the dendrites and axons off of ganglion cells (see Hayashida et al. 2004). Although it was consequently impossible for us to identify anatomical types of ganglion cells in our recordings, three electrophysiologically distinct cell types could account for the results we have presented here along with those of other laboratories.
One of these possesses Ih. This would be consistent with the presence of Ih channel-like immunoreactivity in some somata in the ganglion cell layer of rat (Müller et al. 2003) and rabbit (Kim et al. 2003) retinas. A consistent feature of the cells possessing Ih is that, both in normal and high-K+ extracellular solutions, the whole cell current did not noticeably rectify at the voltages we tested in the presence of ZD7288. Also, the reversal potential of this current was sensitive to extracellular Na+ and, after reducing the total current by Cs+, we found no further effect of Ba2+. We conclude that we found no evidence of Kir in any of the cells that displayed Ih. Although comparison of cells with Ih versus cells that are stained by antibodies directed against Ih channels might reveal further properties of Ih, we refrained from attempting this with the data at hand for two reasons. First, we have no way of knowing whether the cells we recorded from included all morphological cell types in the rat retina. Second, the immunostaining data published to date do not show whether the cells stained are ganglion cells as opposed to displaced amacrine cells (cf. Perry 1981).
A second group of cells possesses Kir. The absence of slow gating at the voltages that activate inwardly rectifying current (Fig. 1B), the agreement between the whole cell current reversal potential and the calculated K+ equilibrium potential at various K+ concentrations (see Fig. 5F of Chen et al. 2004), and the nonselective suppression of Ba2+-resistant outwardly rectifying and inwardly rectifying current by Cs+ (see Fig. 5D of Chen et al. 2004) are all consistent with the possibility that these cells lack Ih. The absence of cells with detectable Ih in the latter study and the near absence of cells with Kir in the present study are consistent with the possibility that we recorded from subtypes of ganglion cells that, in almost all cases, differ from those studied by Chen et al. (2004). Whereas Thy1 panning as used here could introduce a selection bias in some circumstances (Barres et al. 1988), it is unlikely to be the sole explanation of the dichotomy of our results, given that we observed a broader range of current types than Chen et al. (2004). Although our results do not exclude the possibility that Ih and Kir colocalize in some ganglion cells (Chen et al. 2004), we also do not have results that support this possibility. Moreover, the expression of Ih and Kir in different cell populations might explain why Ih isoforms were found in only some somata in the ganglion cell layer of rat and rabbit retina (Kim et al. 2003; Müller et al. 2003) and why several adjacent, or nearly adjacent, ganglion cell layer somata were stained by an anti-Kir3.2 antibody in one study (see Fig. 3C of Chen et al. 2004), whereas no ganglion cell somata were stained in another study (see Fig. 1D of Tian et al. 2003).
A third set of cells lacks Ih and, more generally, inward rectification altogether. This would be consistent with the absence of time-dependent voltage rectification in intact cat retinal ganglion cells morphologically identified as the "
" type (O'Brien et al. 2002) and the absence of hyperpolarization-activated current in large-diameter, type "1" rat retinal ganglion cells (Reiff and Guenther 1999). At the same time, it remains to be seen to what extent this explains the exclusively ohmic currents we found in some cells (Fig. 1A). Although rat "I" cells may be anatomical homologs of cat cells (e.g., Huxlin and Goodchild 1997), the largest ganglion cells in rat are thought to be comprised of at least two distinct subtypes (Peichl 1989). Because no methods are available to unequivocally distinguish these cell subtypes after cell isolations, we could not test whether rat ganglion cells possess Ih. Recording in situ was not a viable alternative because ganglion cells are coupled by gap junctions to amacrine and other ganglion cells (e.g., Hidaka et al. 2004; Vaney 1991) and this would obscure the origin and detailed properties of inward rectification.
Recent recordings from intact cat retina show a similarly small number of substantial electrophysiological differences among a large number of anatomically distinct ganglion cell subtypes (O'Brien et al. 2002). These recordings resemble ours in that constant-current injections elicited no inward rectification in some retinal ganglion cells and a slowly developing, inward rectification as other cells were hyperpolarized to voltages more negative than –70 mV. However, a similarly delayed inward rectification was found in still other cells only if they were hyperpolarized beyond –90 mV. This differs from our results in suggesting that a slowly activating, inwardly rectifying current may have appreciably different activation thresholds in different cell types. Identifying the channel isoforms and properties that contribute to these differences and understanding why inward rectification is detectable in some cell types but not others remain to be determined by future studies.
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Address for reprint requests and other correspondence: A. Ishida, Section of Neurobiology, Physiology and Behavior, University of California, One Shields Avenue, Davis, CA 95616-8519 (E-mail: atishida{at}ucdavis.edu)
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Akopian A, Witkovsky P. D2 dopamine receptor-mediated inhibition of a hyperpolarization-activated current in rod photoreceptors. J Neurophysiol 76: 1828–1835, 1996.
Ames A 3rd, Nesbett FB. In vitro retina as an experimental model of the central nervous system. J Neurochem 37: 867–877, 1981.[CrossRef][ISI][Medline]
Barres BA, Silverstein BE, Corey DP, Chun LL. Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning. Neuron 1: 791–803, 1988.[CrossRef][ISI][Medline]
Bayliss DA, Viana F, Bellingham MC, Berger AJ. Characteristics and postnatal development of a hyperpolarisation-activated inward current in rat hypoglossal motoneurones in vitro. J Neurophysiol 71: 119–128, 1994.
Baylor DA, Fettiplace R. Transmission from photoreceptors to ganglion cells in turtle retina. J Physiol 271: 391–424, 1977.
Berger F, Borchard U, Gelhaar R, Hafner D, Weis T. Effects of the bradycardiac agent ZD 7288 on membrane voltage and pacemaker current in sheep cardiac Purkinje fibres. Naunyn-Schmiedeberg's Arch Pharmacol 350: 677–684, 1994.[ISI][Medline]
Berry MJ 2nd, Meister M. Refractoriness and neural precision. J Neurosci 18: 2200–2211, 1998.
Boos R, Schneider H, Wässle H. Voltage- and transmitter-gated currents of AII-amacrine cells in a slice preparation of the rat retina. J Neurosci 13: 2874–2888, 1993.[Abstract]
Chen L, Yu YC, Zhao JW, Yang XL. Inwardly rectifying potassium channels in rat retinal ganglion cells. Eur J Neurosci 20: 956–964, 2004.[CrossRef][ISI][Medline]
Demontis GC, Moroni A, Gravante B, Altomare C, Longoni B, Cervetto L, DiFrancesco D. Functional characterisation and subcellular localisation of HCN1 channels in rabbit retinal rod photoreceptors. J Physiol 542: 89–97, 2002.
Dickson CT, Magistretti J, Shalinsky MH, Fransen E, Hasselmo ME, Alonso A. Properties and role of Ih in the pacing of subthreshold oscillations in entorhinal cortex layer II neurons. J Neurophysiol 83: 2562–2579, 2000.
Dixon DB, Copenhagen DR. Metabotropic glutamate receptor-mediated suppression of an inward rectifier current is linked via a cGMP cascade. J Neurosci 17: 8945–8954, 1997.
Eng DL, Gordon TR, Kocsis JD, Waxman SG. Current-clamp analysis of a time-dependent rectification in rat optic nerve. J Physiol 421: 185–202, 1990.
Hagiwara S, Miyazaki S, Moody W, Patlak J. Blocking effects of barium and hydrogen ions on the potassium current during anomalous rectification in the starfish egg. J Physiol 279: 167–185, 1978.
Halliwell JV, Adams PR. Voltage-clamp analysis of muscarinic excitation in hippocampal neurons. Brain Res 250: 71–92, 1982.[CrossRef][ISI][Medline]
Harris NC, Constanti A. Mechanism of block by ZD 7288 of the hyperpolarization-activated inward rectifying current in guinea pig substantia nigra neurons in vitro. J Neurophysiol 74: 2366–2378, 1995.
Hayashida Y, Ishida AT. Dopamine receptor activation can reduce voltage-gated Na+ current by modulating both entry into and recovery from inactivation. J Neurophysiol 92: 3134–3141, 2004.
Hayashida Y, Partida GJ, Ishida AT. Dissociation of retinal ganglion cells without enzymes. J Neurosci Methods 137: 25–35, 2004.
) and slow (
) activation time constants change exponentially with voltage. Fast (
) and slow (
) deactivation time constants are roughly constant over the same voltage range. F: the mean amplitudes of the tail component of Cs+-blocked current from each cell in E, when they were repolarized from each test potential (abscissa) to the holding potential (–73 mV). Amplitudes back-extrapolated to the moment of repolarization by best fit of mono- or biexponential function to deactivation (see text), and normalized to the largest amplitude recorded from each cell. Error bars not visible in E and F are within the symbols.
