 |
INTRODUCTION |
Cyclic guanosine 3
,5
monophosphate (cGMP)-gated channels, located in the outer segment of vertebrate photoreceptors, are open in darkness. The resulting dark current, carried largely by Na+ and to a lesser extent by Ca2+, flows into the cell through cGMP-gated channels along their electrochemical gradients (Yau and Baylor 1989
for review). The influx of Na+ depolarizes the inner segment and maintains the release of neurotransmitter from the synaptic terminal. Light leads to the closure of these cGMP-gated channels (Matthews 1987
; Matthews and Watanabe 1987
), decreases inward current in the outer segment, hyperpolarizes the inner segment, and reduces the neurotransmitter released (Cervetto and Piccolino 1974
). However, changes in the membrane potential of the inner segment do not follow the course of changes in the current flowing at the outer segment. The membrane potential is modulated by interactions with other photoreceptors (Barnes 1995
for review), by negative feedback from horizontal cells (Djamgoz et al. 1995
; Piccolino 1995
for reviews) as well as by voltage-sensitive ionic channels (Barnes 1994
for review).
Ionic currents have been studied with double-electrode voltage- and patch-clamp methods in the inner segments of isolated cones of tiger salamander (Attwell et al. 1982
; Barnes and Hille 1989
), lizard (Maricq and Korenbrot 1990a
,b
), chicken (Wilson and Gleason 1991
), and monkey (Yagi and MacLeish 1994
). Depolarization-elicited currents include an outward tetraethylammonium (TEA)-sensitive K+ current (IKx), an outward Ca2+-activated Cl
current (ICl(Ca)), an inward Ca2+ current (ICa). Also, a hyperpolarization-elicited nonselective inward cation current (Ih) (Attwell et al. 1982
), or an inward anion current in the case of chicken retina (Wilson and Gleason 1991
), has been documented. It has been proposed that Ih modulates the light-induced membrane potential changes in the inner segment by acting as a counter current to the hyperpolarizing light response (Barnes 1994
for review; Barnes and Hille 1989
; Fain et al. 1978
; Maricq and Korenbrot 1990a
,b
). All these prior reports were obtained from studies of isolated photoreceptors. Despite the complications introduced by intercellular coupling and a relatively intact circuitry compared with isolated photoreceptors, we have recorded whole cell currents from blue cone inner segments in living zebrafish retinal slices. We have identified voltage-activated currents described in the isolated cones and, in addition, have found an outward current carried by active transport of Cl
that is activated by hyperpolarizing-voltage steps. This novel current, which opposes Ih over the same voltage range, may be important in modulation of the photoresponse.
 |
METHODS |
Preparation of retinal slices
Adult zebrafish (Brachydanio rerio), of body length 27-29 mm, were stored in a water tank with salt (0.06 g/l Instant Ocean, Aquarium System) added on a 12 h on/12 h off light cycle. Dark adaptation before dissection was not necessary for making good slices. The animal was decapitated and the eye enucleated. Retinal slices were prepared using procedures similar to those described by Werblin (1978)
. Briefly, after the lens and iris were removed, the eyecup was cut into halves, placed photoreceptor-side up on a strip of Millipore filter (type HA, 0.45 µm pore size), after which the sclera and choroid were removed. The retina, together with the filter paper, was cut into 250-µm-thick slices using an apparatus consisting of a razor blade advanced by a micrometer. The slices were soaked in a solution containing 20% L-15 culture medium (GIBCO) and 80% NaCl-saline at 4°C for 1 h in the dark. A slice, still attached to the Millipore filter, was rotated 90° and embedded in petroleum gel tracks in the experimental chamber (Model RC-22C of Warner Instrument, Manden, CT). The slice was treated with hyaluronidase (Sigma Type V; 125 U/ml saline) for 90 s at 22°C to liquefy the vitreous, then thoroughly rinsed with saline. Hyaluronidase has no reported effect on a variety of physiological functions in isolated rat retina (Winkler and Cohn 1985
). Slices could stay in good condition for >5 h as judged by the whole cell currents from blue cones.
Electrophysiological methods
Slices were perfused with aerated saline at 22°C and viewed through a Zeiss 63X, long-working-distance (1.3 mm), water-immersion objective. The volume of solution in the chamber was ~0.3 ml. The ground Ag-AgCl lead was connected through a 0.15 M KCl agar-agar bridge near the outlet of the chamber. To avoid the photoelectric artifact produced by exposing the Ag-AgCl electrode to light, the electrode was covered with a black tubing. Whole cell patch-clamp techniques (Hamill et al. 1981
) were used to record current from the inner segment of blue cones in the retinal slices. Patch electrodes were pulled in two stages with a vertical electrode puller (Model PP-83, Narashigi, Tokyo) using thin wall microchematocrit capillary tubes (Fischer Scientific, Pittsburgh, PA) with an inner diameter of 1.1-1.2 mm. The tip resistance, measured in the bath, was between 3 and 10 M
; the seal resistance ranged from 1 to 20 G
. A model Axopatch 200A patch-clamp amplifier, pClamp software (version 6.0) and a model 1200 Digidata data acquisition instrument of Axon Instruments (Foster City, CA) were used. Junctional potentials were corrected; the low-pass Bessel filter of the amplifier was set at 2 kHz. Unless specified, leak subtraction was used. The series resistance was corrected with the series resistance correction on the Axopatch 200A amplifier. The digitized-current data were stored in an IBM PC 486 compatible computer. Unless specified otherwise, the current records were later plotted through a 1 kHz filter.
Fresh slices were used and the chamber was rinsed after any pharmacological manipulation to ensure that each cone recorded was not tainted by residual chemical effects from prior treatments. Records were taken immediately after patch rupture to determine the properties of the cell. However, the particular experimental data were not acquired until the cell produced stable control records for
5-10 min. Experiments could last for 1-2 h, during which time stability of recording was verified. The change of membrane conductances was monitored by passing a series of 50 Hz, 10 mV or 20 mV brief (2 ms) pulses. The threshold voltages of the hyperpolarization-elicited current were determined by the intersection of the I-V curve or the extrapolation of the I-V curve elicited by hyperpolarizing steps with the 0 current axis. Data in the graphswere fit by curves with a Spline smooth method using theTECH.GRAPH.PAD program. The statistical significance of the results was calculated with Student's t-test.
Solutions and chemicals
The NaCl saline bath solution contained (in mM) 140 NaCl, 3 KCl, 1.2 CaCl2, 1 MgSO4, 10 glucose, 5 N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid (HEPES), pH 7.4 adjusted with NaOH. In most experiments 5 mM CsCl (substituting equimolar NaCl) was added to suppress Ih, the Cs+-sensitive inward nonselective cation current induced by hyperpolarizing-voltage steps (Barnes and Hille 1989
). Other changes in the composition of the saline bath are described in the text or figure legends. The following pipette solutions were used: 1) K-pipette solution, (in mM) 140 KCl, 0.1 CaCl2, 0.6 ethylene glycol-bis(
-aminoethyl ether)-N,N,N
,N
-tetraacetic acid (EGTA), 0.5 MgCl2, 2 ATP (K-salt), 10 glucose, 5 HEPES, pH 7.4 adjusted with KOH. In some experiments, the concentration of CaCl2, EGTA, ATP, and MgSO4 was increased to 1, 5, 5, and 2 mM, respectively, with no significant difference in results. 2) Cs-TEA pipette solution, 140 mM KCl was substituted with 100 mM Cs-acetate, 20 mM CsCl, and 20 mM TEA-Cl; pH was adjusted with CsOH. This solution was used to suppress the outward K+ current elicited by depolarizing-voltage steps and to maintain the intracellular Cl
concentration close to the physiological value found in fish retinal neurons (Grant and Dowling 1995
).
Acetazolamide, ATP (K-salt), bumetanide, 4,4
-diisothiocynatostilbene-2,2
-disulfonic acid (DIDS), EGTA, ethacrynic acid, N-ethylmaleimide, furosemide, HEPES, Lucifer yellow CH, niflumic acid, 4-acetatamido-4
isothiocyanatostilbene-2,2
-disulfonic acid (SITS), and TEA-Cl were obtained from Sigma Chemical (St. Louis, MO); anthracene-9-carboxylic acid (A-9-C) and sodium orthovanadate from Aldrich Chemical (Milwaukee, WI.); [(2-nb u t y l - 6 , 7 - d i c h l o r o - 2 - c y c l o p e n t y l - 2 , 3 - d i h y d r o - 1 - o x o - 1 H - i n d e n - 5 yl)oxyl]acetic acid (DIOA), N-phenylanthranilic acid (DPC), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) from Research Biochemical International (Natick, MA). Stock solutions of the following chemicals were dissolved in dimethyl sulfoxide (DMSO) with concentrations of A-9-C, DPC, DIOA, NPPB, 2-octanol, and bumetanide equal to 500, 500, 200, 10, 100, and 20 mM, respectively. Stock solutions of niflumic acid and furosemide were dissolved in ethanol at concentrations of 100 and 20 mM, respectively. Ethacrynic acid, N-ethylmaleimide and acetazolamide were dissolved in physiological saline. When stocks with DMSO or ethanol were used as the solvent, an equivalent amount of the organic solvent was added into the bath solution before applying those chemicals.
Our first experiments were performed without regard to the intensity of background light once whole cell mode was achieved. We soon found that the photoreceptor nuclei became granular when subjected to prolonged intense light from the microscope lamp. The quality and duration of the recordings improved greatly when the illuminating light of the microscope was turned off once whole cell mode was achieved. All experiments to be described, except where noted, were performed under dim, indirect illumination provided by a GE 32 W daylight fluorescent bulb (10
10 W/mm2 as measured by a Tektronix J16 radiometer, placed in the position of the preparation). The membrane potential of the cone inner segment was usually held at
45 mV, which was close to the resting membrane potential under our experimental condition.
 |
RESULTS |
Visualization of the living slice and blue cone identification
The layers of the retina were visualized easily in a well-oriented slice of the living zebrafish retina (Fig. 1A). Zebrafish have three types of cones, which are organized in three layers. The most proximal layer contains short-single UV-sensitive cones, the middle layer contains long-single blue-sensitive cones, and the distal layer contains double cones that are long-wavelength sensitive (Branchek and Bremiller 1984
; Nawocki et al. 1985; Raymond et al. 1993
). The long-single blue cone was always the largest of the cone types with an inner segment diameter of 4-10 µm (depending on the fish). These blue cones were easy to visualize, and they could be identified by ionophoretical injection of Lucifer yellow through the patch pipette (Fig. 1B). The structure of the slice and the morphological features of the blue cone are the same as that described by others in fixed zebrafish retina (Branchek and Bremiller 1984
; Raymond et al. 1993
).

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| FIG. 1.
Photomicrographs of a living slice of zebrafish retina. A: 3 layers of cone photoreceptors are visible: an inner layer of UV-sensitive, short-single cones (UV), a middle layer of blue-sensitive, single cones (BC), and an outer layer of red and green wavelength-sensitive, double cones (RG). Arrowheads indicate individual cones in each of 3 layers. Horizontal cells and other cells are seen clearly in inner nuclear layer (INL); outer plexiform layer, OPL. B: a Lucifer yellow-filled blue cone in retinal slice with synaptic terminal is indicated (arrowhead). Calibration bar = 10 µm.
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General aspects of the whole cell current
These data are based on recordings from 199 blue cones. Depolarizing-voltage steps from
45 mV elicited outward currents in the inner segment of all blue cones bathed in a Cs+-free NaCl-saline solution (Fig. 2A). These currents were carried mainly by K+ as they were suppressed 70-80% (n = 9) with bath application of 10 mM TEA-Cl or greatly reduced with substitution of KCl in the pipette solution with 120 mM CsCl and 20 mM TEA-Cl (n = 15). Other currents (n = 5) with a reversal potential at about 0 mV were elicited by depolarizing steps with 30 mM TEA-Cl in the bath and 140 mM CsCl in the pipette (Fig. 2B). This current had the same characteristics and the records appear virtually identical as the Ca2+-activated anion current reported in isolated cones of tiger salamander (for comparison see Fig. 9A of Barnes and Hille 1989
). No depolarization-elicited inward-calcium current was detected under our experimental condition ([Ca2+]o = 1.2 mM). No effort was made to detect the calcium currents either by increasing the [Ca2+]o or by substituting Ca2+ with Ba2+, as Ca2+ channels are more permeable to Ba2+ (Hagiwara and Byerly 1981
).
Hyperpolarizing-voltage steps elicited inward currents in 98 of the 199 blue cones bathed in the NaCl saline (Cs+-free; Fig. 2C). These inward currents were suppressed by bath application of 5 mM CsCl consistent with the properties of Ih (Attwell et al. 1982
; Barnes and Hille 1989
; Fain et al. 1978
).

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| FIG. 2.
IK, ICl(Ca), and Ih elicited in blue cones. A: outward IK elicited by depolarizing steps (left) with current-voltage relations displayed on right. Voltage steps were from 60 to +120 mV in 9 steps from a holding potential of 45 mV. Slice was bathed in a sodium saline and recorded with pipette filled with K-pipette solution. B: calcium-activated ICl (ICl(Ca)) elicited by depolarization steps. Voltages steps were to 20, +10, and +50 mV from a holding potential of 45 mV. Retinal slice was bathed in a solution containing 30 mM tetraethylammonium HCl (TEA-Cl), 60 mM NaCl, and 50 mM NaBr. CsCl was substituted completely for KCl in pipette solution. C: an inward current elicited by hyperpolarizing steps (left) with current-voltage relations displayed on right. Voltage steps ranged from 150 to 60 mV in 9 steps from a holding potential of 45 mV. Slice was bathed in a sodium saline and recorded with a pipette filled with K-pipette solution. This inward current was abolished by addition of 5 mM CsCl to bathing medium (not shown) and thus, was likely to be Ih.
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| FIG. 9.
Iout-h was blocked by 20 µM N-ethylmaleimide as indicated in records from 1 blue cone (A) and in I-V relation obtained from another blue cone (B). Currents in A were elicited by a 100 mV hyperpolarizing step from a holding potential of 45 mV and plotted through a 500-Hz filter. Slices were bathed in a NaCl saline with 5 mM CsCl added, and recording electrode was filled with K-pipette solution.
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Features of an outward current elicited by hyperpolarizing steps
As opposed to inward currents, hyperpolarizing-voltage steps elicited outward currents in 54 of the 199 blue cones in the NaCl Cs+-free saline solution (Fig. 3). The term "Iout-h" will be used to refer to this hyperpolarizing-induced outward current. Iout-h was observed for hyperpolarizing steps more negative than
70 ± 5.1 mV (n = 30) at a holding potential of
45 mV regardless of whether leak subtraction was applied or not (compare Fig. 3, A and B), demonstrating that the outward current was not an artifact. The I-V relation (Fig. 3C) showed a monotonic increase with hyperpolarizing steps with no sign of saturation even at
150 mV. More negative steps usually resulted in a loss of high resistance seal. No reversal potential was found after blockage of Ik with extracellular TEA (30 mM) and intracellular CsCl (140 mM). There were no changes in membrane conductance for outward currents (Iout-h) elicited by hyperpolarizing steps (n = 5), whereas there were increases in conductance for outward currents elicited by depolarizing steps (Fig. 4). The records shown in Fig. 4 were obtained without 0 subtraction in which the apparent currents include those passing through the leak resistance. The net outward currents were of equivalent amplitudes during the hyperpolarizing and depolarizing pulses. Iout-h also was recorded from blue cones in slices bathed in a Ca2+-free saline or in a solution containing 100 µM nifedipine and 2 mM CoCl2, suggesting that Iout-h was not activated synaptically through neuronal feedback circuits. This current also was recorded from four short single cones and one member of a double cone, identified by their position in the ONL. The analysis of Iout-h was carried on the blue-sensitive cones because they are the largest and most stable under our recording conditions.

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| FIG. 3.
Hyperpolarizing steps elicited outward currents. NaCl saline bath contained 5 mM CsCl to block Ih; recording pipette was filled with K-pipette solution. A: an outward current elicited by a 105 mV hyperpolarizing voltage step from a holding potential of 45 mV. No leak subtraction was used. B: outward currents elicited by hyperpolarizing steps. Voltage steps ranged from 150 to 66 mV in 7 steps from a holding potential of 45 mV. Leak subtraction was used. C: current-voltage relations.
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| FIG. 4.
Hyperpolarizing steps elicited outward currents with no changes in membrane conductance. Slices were bathed in NaCl saline with 5 mM CsCl added and recorded with electrodes filled with K-pipette solution. A: sample current records during depolarizing (top) and hyperpolarizing (bottom) voltage steps with markers displayed below current traces. Leak subtraction was not used. Conductance was measured with a series of 2-ms, 20-mV pulses. Insets are currents elicited by brief pulses displayed at a 5 times faster sweep than used for displaying whole cell current. A comparison of pulse amplitudes obtained before, during, and after voltage steps (a-c) showed that there was no change in relative conductance during hyperpolarizing voltage step ( 105 mV, A, bottom) while relative conductance increased during depolarizing voltage step (+60 mV, top). Holding potential was 45 mV in both cases. B: there were no changes in relative conductance (mean ± SD, n = 5) during outward current elicited by a hyperpolarizing voltage step ( 105 mV) from a holding potential of 45 mV. For comparison, an increase in conductance of one blue cone during a depolarizing voltage step (to +60 mV) also is shown.
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Because both inward (Ih) and outward (Iout-h) currents were elicited by hyperpolarizing steps, there was the possibility that both currents coexisted in the cells and that the observed current was the algebraic sum of the two. This was determined to be the case as indicated by the following results. Recall that Ih was recorded in 98 blue cones in the NaCl Cs+-free saline. First, addition of 5 mM Cs+ to the bath resulted in a reversal of the hyperpolarizing-elicited Ih in 24 of the 98 blue cones (Fig. 5, A-C). Note the similarity in the time courses of the inward and outward currents. Second, the amplitude of Iout-h, elicited by a
100 mV hyperpolarizing step from a holding potential of
45 mV, was increased 2.5-fold by the addition of 5 mm CsCl (68.0 ± 21.3 pA to 169.2 ± 61.1 pA; means ± SD; n = 30), an effect due presumably to inhibition of Ih.

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| FIG. 5.
Manipulation of Ih and Iout-h with Cs+ and acetate. Currents were elicited by a voltage step of 150 mV from a holding potential of 45 mV. First step evoked an inward current Ih (A). Both Ih and Iout-h were expressed in this blue cone. Bath application of 5 mM Cs+ suppressed Ih and unmasked Iout-h (B). After washout of Cs+ and return to control (C), substituting Cl in bath with acetate, resulted in an increase in Ih (D), presumably by suppression of Iout-h. This effect also was reversible after washout (E). Slice was bathed in a NaCl saline, and recording electrode was filled with K-pipette solution.
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Iout-h was independent of Na+, K+, and the Na+-K+ ATPase
Iout-h could be carried by cation efflux, anion influx or the reverse, a decrease in cation influx or anion efflux. Our observation that the membrane conductance was unchanged (Fig. 4) and that the current flow was against the electrochemical gradients for K+, Na+, Ca2+, and Cl
, indicated that Iout-h was carried by active transport of ions. A common electrogenic-ion transporter in neurons is the Na+-K+ATPase. The effect of Na+-K+-ATPase inhibitors, ouabain and dihydroouabain, on Iout-h was examined. A 2-min application of 200 µM dihydroouabain suppressed Iout-h, followed by a partial recovery after a 20-min washout (Fig. 6A). The effect of ouabain octahydrate was similar to that of the dihydroouabain (data not shown). Although these results indicated that Iout-h was related to the activity of Na+-K+-ATPase, the following results do not support this notion. First, the Na+-K+-ATPase-transporter current is increased with voltage steps to a less negative value (Gadsby and Nakao 1989
), whereas Iout-h was decreased. Second, the activity of the Na+-K+-ATPase is dependent on [Na+]i and [K+]o and is affected by [Na+]o and [K+]i (Bielen et al. 1991
; Nakao and Gadsby 1989
; Rakowski et al. 1991
). However, Iout-h was not affected by alterations in any of these conditions.

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| FIG. 6.
Iout-h is independent of the Na+-K+ ATPase. In all cases, the slices were bathed in a NaCl saline with 5 mM CsCl added, and recording electrode was filled with K-pipette solution. Voltage steps ranged from 145 to 55 mV in 3 steps from a holding potential of 145 mV. A:Iout-h was suppressed by 200 µM dihydroouabain (DHO) and recovered partially after return to control bath for 20 min. Equimolar substitution of Na+ in the bath solution with choline (B) or TEA (C) had no effect on Iout-h.
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Iout-h was recorded, using pipettes filled with a KCl solution that contained no Na+, for >1 h, a time more than sufficient to equilibrate the cell's interior with the pipette solution. Because Iout-h was elicited by hyperpolarizing voltage steps, extracellular Na+ could not have entered the cell via voltage-sensitive Na+ channels, thereby eliminating extracellular Na+ as source for the activation of the sodium pump. Also, equimolar substitution of NaCl with either choline chloride (Fig. 6B) or TEA-Cl did not affect Iout-h (Fig. 6C). Iout-h was not affected when [K+]o was changed from 0 to 30 mM (substituting equimolar [Na+]o; records not shown) nor when both Na+ and K+ were substituted with equimolar TEA (n = 5). Also, Iout-h was not affected by substituting CsCl for KCl (n = 24) in the pipette solution (see also Table 1). Thus Iout-h was independent of intracellular and extracellular Na+ and K+, further supporting the contention that Iout-h was not a Na+-K+-ATPase transporter current.
Iout-h was Cl
dependent
Iout-h was sensitive to equimolar substitution of Cl
in the bath with other anions. Total substitution of Cl
with acetate (n = 8) completely abolished Iout-h (Fig. 7A). The effect of acetate substitution on the currents of a blue cone in a Cs2+-free solution (Fig. 5, C-E) was to increase the amplitude of Ih, presumably by inhibiting Iout-h. Under the control conditions, the intracellular and extracellular Cl
concentrations were comparable at ~141 and 146 mM, respectively. Iout-h was not affected by Cl
substitution in the bath with acetate until there was a 50% reduction of Cl
(73 mM in the bath). The half-inhibition of Iout-h occurred with a 78% substitution of Cl
with acetate (Fig. 7B), a reduction of [Cl
]o to 33 mM. This persistence of Iout-h indicates a substantial movement of Cl
into the blue cone against its chemical concentration gradient, considering that the recording pipette contained ~141 mM Cl
. Total substitution of bath Cl
with Br
(n = 4) had less of an effect than acetate; the maximum suppression observed was ~50%. Reversal of the effects of both acetate and Br
was very rapid, occurring within the time course of bath replacement. Gluconate substitution also suppressed Iout-h. However, reversal of the inhibition took >30 min to recover after NaCl saline was reintroduced. The mechanism of the long-lasting after effect of gluconate is not known.

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| FIG. 7.
Iout-h is Cl dependent. Substitution of Cl in bath solution with acetate completely abolished Iout-h. Slice was bathed in a NaCl saline with 5 mM CsCl added, and recording electrode was filled with K-pipette solution. A: complete substitution of NaCl with sodium acetate. Voltage steps ranged from 130 to 70 mV in 6 steps from a holding potential of 45 mV. B: relative amplitude of the steady value of Iout-h, plotted against percentage of Cl substitution, shows a 50% reduction at a 78% Cl substitution. Holding potential was 45 mV with a 55-mV hyperpolarizing pulse to 100 mV.
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Agents that are known to affect the Cl
movement across the cell membrane were tested because Iout-h was sensitive to [Cl
]o. None of the Cl
-channel blockers: A-9-C (2 mM), DIDS (1 mM), DPC (1 mM), NPPB (30 µM), niflumic acid (500 µM), and SITS (200 µM) had any effect on Iout-h.
Bumetanide and furosemide inhibit the Na+-K+-Cl
cotransporter with high affinity (Aull 1981
; Crook et al. 1992
; Vigne et al. 1994
; von Brauchitsch and Crook 1993
) and the Na+-Cl
cotransporter with lower potency (Johanson et al. 1990
). Both bumetanide and furosemide suppressedIout-h. The effect of bumetanide (Fig. 8; n = 10) developed slowly, taking 10-30 min for Iout-h to reach a new stable value and a further 20-30 min to recover after bumetanide was washed out. The half-inhibition concentration (IC50) of bumetanide was 50 µM as against 1 µM for inhibition of the Na+-K+-Cl
cotransporter. Furosemide (n = 7) was less potent than bumetanide. It had no effect at a concentration <30 µM and a variable effect above this concentration. For example, Iout-h was suppressed completely by 200 µM furosemide in one cell, whereas in another cell Iout-h was suppressed only 60% by 300 µM furosemide.

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| FIG. 8.
Suppression of Iout-h by a Cl transporter blocker, bumetanide. Slices were bathed in a NaCl saline with 5 mM CsCl added, and recording electrode was filled with K-pipette solution. A: application of 60 µM bumetanide reduced Iout-h by ~65%. Effect was reversed completely on washout. Voltage steps ranged from 110 to 50 mV in 6 steps from a holding potential of 45 mV. B: dose-response relations of bumetanide were obtained from 10 blue cones. Two concentrations were tested in 1 cone; 10 µM bumetanide was tested 1st. After washout and return of Iout-h to its control value, 100 µM bumetanide was applied. Iout-h was inhibited 50% at 50 µM bumetanide. Currents in B were elicited by a 100 mV hyperpolarizing step from a holding potential of 45 mV.
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N-ethylmaleimide, acetazolamide, vanadate, and ethacrynic acid inhibit an ATP-driven Cl
pump in various tissues including membrane vesicles from rat brain (Inagaki et al. 1985
; Shiroya et al. 1989
) vertebrate smooth muscle (Chipperfield et al. 1993
; Inoue et al. 1991
), and hippocampal neurons (Hara et al. 1982
). Iout-h was blocked completely and reversibly by N-ethylmaleimide (n = 8) at concentrations as low as 25 µM (Fig. 9) and sodium orthovanadate (1 mM; n = 3). Ethacrynic acid had no effect
1 mM (n = 2). The inhibitory effect of acetazolamide at 1 mM (n = 3) was complete but irreversible. However, we confirmed the viability of the blue cones by observing Ih after a return to a Cs2+-free medium.
At a membrane potential of
45 mV, application of bumetanide, furosamide, acetazolamide, etc., had no effect onthe steady current, indicating that there was no bumetanide-,furosemide-, acetazolamide-sensitive (etc.) steady current around the resting membrane potential.
The dependence of Iout-h on intracellular K+ and Cl
was investigated comparing the average amplitude of Iout-h as the recording pipettes were filled with different solutions (Table 1). Iout-h was not affected by low Cl
, K+-free (regardless of Cl
), or high Na+ in the electrode.
Iout-h was inhibited by increased background light intensity
All of the preceding data were obtained under dim background daylight fluorescence (GE 32 W bulb,
10
10W/mm2). However, in some later preliminary experiments, we investigated the effect of increased background light on Iout-h. This was done crudely by increasing the intensity of the quartz-halogen microscope illuminator, being well aware of the alterations in the spectral composition of the light with the increases in intensity. Iout-h became smaller (n = 9) with increased light intensity and reversed as the light intensity reached 10
7 W/mm2 (Fig. 10). It took <3 min forIout-h to reverse and reach a new steady value with increased light intensity. This effect of light was not due to a deterioration of the preparation because it was reversible but over a longer time course. When the microscope lamp was turned off, the inward current decreased, an outward current appeared and increased >10-15 min to reach a steady state level. It was possible that the effect of light was to increase the amplitude of Ih rather than inhibiting Iout-h. This was found not to be the case in cells in which an outward current, recorded in dim light, reversed to an inward current in the light. However, this inward current was abolished by extracellular cesium; no outward current was detected in the light in the presence of cesium. This reversal of direction of current flow by brighter light indicated that Ih was masked by Iout-h under the dimmer light conditions.

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| FIG. 10.
Iout-h was inhibited by increased background light. Iout-h was recorded under dim daylight fluroescence (10 10 W/mm2), was suppressed with a reversal of current within 3 min by bright light (quartz-halogen illuminator 10 7 W/mm2) and recovered to control within 10-15 min when the illuminator was turned off. Slice was bathed in a Cs-free, NaCl saline, and recording electrode was filled with K-pipette solution. Voltage steps ranged from 130 to 70 mV in 6 steps from a holding potential of 45 mV.
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 |
DISCUSSION |
Currents from the inner segment of zebrafish blue cones in retinal slices are similar to those recorded from isolated cones in other species (Attwell et al. 1982
; Barnes and Hille 1989
; Maricq and Korenbrot 1990a
,b
; Yagi andMacLeish 1994): a TEA-sensitive outward K+ current, a Ca2+-activated anion current, and Ih. We did not observe an inward Ca2+ current; this cannot be explained by a run down that is often observed in neurons under whole cell patch mode because the first record usually was taken within 30 s after achieving whole cell mode. The maximum magnitude of ICa from salamander cone inner segment is ~60 pA in the presence of 3 mM Ca2+ (Barnes and Hille 1989
). The [Ca2+]0 we used was lower (1.2 mM), and as the diameter of zebrafish cones is ~50% smaller than those of tiger salamander, it is likely that ICa was too small to be detected directly. However, we obtained indirect evidence for ICa in zebrafish cones because the presence of Ca2+-activated anion currents implies a Ca2+ influx during depolarization needed to activate the anion channels. In addition to those currents already documented, we observed a hyperpolarizing-elicited outward current, Iout-h in all cone types, although the data presented here were derived from the largest type, the long-single blue-sensitive cone.
Nature of Iout-h
An apparent outward current could be due to either an initiation of an outward current or a decrease of a standing inward current. The latter option seems improbable because the blue cones would have to have had a standing-inward, bumetanide-/acetazolamide-sensitive current
250 pA at a membrane potential of
45 mV. No evidence for such a current was found.
An outward current could be carried either by an outflow of cations or an inflow of anions. With slices bathed in a NaCl saline and pipettes filled with 140 mM KCl, the candidate-current carrier could only be K+ or Cl
. However, the direction of current flow at voltage steps more negative than the equilibrium potential of K+ or Cl
is against the electrochemical gradients for both K+ and Cl
. The following results indicate that Iout-h is carried by transport of Cl
. First, Iout-h was independent of intracellular and extracellular Na+ and K+, eliminating any involvement of the Na+-K+-ATPase, or the Na+-K+-Cl
, Na+-Cl
, and K+-Cl
symporters, as well as any Na+-dependent electrogenic amino acid transport, as described for L-glutamate in salamander cones (Eliasof and Werblin 1993). Second, Iout-h was Cl
dependent in the bath. Third, Iout-h was recorded in the absence of HCO
3 in the bathing medium, a condition in which the Cl
/HCO
3 anion antiporter does not operate (Davis 1992
). Fourth, Iout-h was not affected by the Cl
channel blockers A-9-C, DIDS, DPC, NPPB, niflumic acid, and SITS. Fifth, Iout-h was blocked by inhibitors of Cl
transport, including bumetanide, furosemide, orthovanadate, acetazolamide, and N-ethylmaleimide but not ethacrynic acid. This pharmacology of the inhibition of Iout-h indicates a Cl
transporter of a different character than previously reported.
The most commonly recognized Cl
transporters in cells are K+-Cl
, Na+-Cl
, and Na+-K+-Cl
symporters and HCO3-Cl
antiporter that are all electroneutral (Haas 1994
; Lauf et al. 1992
for review). The Cl
transporter reported in this paper is electrogenic, transporting Cl
into the cell and requiring neither Na+ nor K+ for cotransport. Anion-stimulated ATPase was first suggested in the leaf of a saltmarsh plant (Hill and Hill 1973
) and subsequently reported in a wide variety of invertebrate and vertebrate preparations (Gerencser and Lee 1983
for review). The conclusion as to whether these reports of Cl
transport represented a primary Cl
pump was debatable (Gerencser et al. 1988
). However, more recent studies have demonstrated the presence of an electrogenic Cl
pump (i.e., Cl
-ATPase) in three quite different preparations: an alga Acetabularia acetabulum (Gradmann et al. 1982
; Ikeda and Oesterhelt 1990
), Aplysia intestine (Gerencser 1988
; Gerencser and Purushotham 1995
), and rat brain (Zeng et al. 1994
). Studies in Aplysia intestine indicate that the Cl
pump is a P-type ATPase (Gerencser and Zelzna 1993) that has been associated with electrogenic transport of cations rather than anions.
The Cl
pumps found in various preparations have different pharmacological properties, direction of Cl
translocation and presumably, functions. The ATP-driven Cl
pump reduces intraneural Cl
concentration in rat hippocampal neurons (Inoue et al. 1991
) while increasing Cl
concentration in alga and rat vascular smooth muscle (Chipperfield et al. 1993
; Davis 1996
; Ikeda and Oesterfelt 1990). The ATP-dependent Cl
transport tends to be inhibited by acetazolamide, N-ethylmaleimide, ethacrynic acid, and vanadate and is rather insensitive to bumetanide and furosemide (Chipperfield et al. 1993
; Davis 1996
; Gerencser 1983
, 1988
; Inagaki et al. 1985
; Shiroya et al. 1989
). Iout-h found in this paper has the following characteristics: 1) it is electrogenic; 2) it is increased by hyperpolarization similar to the property of electrogenic Cl
pump reported by Gerencser (1988)
in Aplysia intestine; 3) the outward direction of current flow indicates that Cl
is transported into the cell; 4) Iout-h is not affected by Na+ and K+ in the medium similar to the properties of Cl
pump reported for rat brain (Inagaki and Shiroya 1988
; Shiroya et al. 1989
) and algae (Ikeda et al. 1990
); and 5) similar to most other preparations, Iout-h is inhibited by vanadate, acetazolamide and N-ethylmaleimide. Unlike other preparations, it is not inhibited by ethacrynic acid but it is inhibited by bumetanide and furosemide. We conclude that Iout-h is caused by an ATP-dependent Cl
pump that is electrogenic, inwardly directed, with a unique pharmacological profile that has similarities and differences with other ATP-dependent Cl
transporters.
Lack of Iout-h in isolated cones
Iout-h has not been described in isolated cone photoreceptors (Attwell et al. 1982
; Barnes and Hille 1989
; Maricq and Korenbrot 1990a
,b
; Wilson and Gleason 1991
; Yagi and MacLeish 1994
). First, Iout-h may be unique to cones of zebrafish. This seems unlikely given the description of Cl
pumps throughout the plant and animal kingdoms (see above). Also, in preliminary experiments, currents similar to Iout-h have been recorded from bipolar cells in a goldfish retinal slice (unpublished observations). Second, our data were recorded in a retinal slice preparation, whereas all other reports were obtained from isolated cones. Proteases used for cell dissociation affect membrane proteins (i.e., cGMP-activated channels of retinal rods) (Shen et al. 1995
) and could affect Iout-h, unlike our use of hyaluronidase to liquefy the vitreous humour. Hyaluronidase has no effect on the electroretinogram (ERG), oxygen consumption, glutathione content or ATPase activities in the isolated rat retina (Winkler and Cohn 1985
). Mechanical means were used to dissociate the retina in one study, but Iout-h was not recorded in that study either (Attwell et al. 1982
). Third, cones in a slice preparation are in an environment in which circuitry and supporting glial cells are somewhat intact compared with the total absence of such an environment for dissociated cells. Iout-h was not affected by a gap junction decoupler, 2-octanol (250 µM), nor in a Ca2+-free saline or in a solution containing 100 µM nifedipine and 2 mM CoCl2. These data suggest that Iout-h does not depend on electrical coupling between photoreceptors (reviewed in Barnes 1995
; Wu 1994
) nor on the consequences of calcium-dependent synaptic output of cones and subsequent feedback from horizontal cells (Djamgoz et al. 1995
; Piccolino 1995
). However, potential effects of calcium-independent transmitter release from cones (Schwartz 1986
) may be ruled out because such release is Na+-dependent and Iout-h was recorded in Na+-free medium. Fourth, Iout-h is suppressed by bright light. Isolated cones tend to be recorded under brighter illumination (Barnes and Hille 1989
).
Ouabain inhibits Iout-h without blocking the Na+-K+ ATPase
Ouabains (dihydroouabain and ouabain octahydrate) generally are regarded as specific Na+-K+-ATPase blockers (Glynn and Karlish 1975
), and as such the inhibition ofIout-h by the ouabains indicated an involvement of the Na+-K+-ATPase. However, Iout-h was independent of [K+]o and [Na+]i, both of which are required to activate the Na+-K+-ATPase (Bielen et al. 1991
; Nakao and Gadsby 1989
). Ouabain reportedly has effects other than blocking the Na+-K+-ATPase, for example in rabbit kidney, erythrocytes, and cat cardiac muscle (Fujino et al. 1989
; Kudo et al. 1991
; Ozaki et al. 1985
). Thus the present data add to those studies indicating that ouabain has effects other than inhibition of Na+-K+-ATPase.
Possible physiological role of Iout-h
The magnitude of Iout-h reported here depended on extracellular [Cl
] (Fig. 7B) but not on intracellular [Cl
] (Table 1), indicating that Iout-h is not particularly affected by ECl. Considering that the extracellular [Cl
] used in this study was close to body fluid, the magnitude of Iout-h reported here should be comparable with that elicited under in vivo conditions. Iout-h is activated by a 25-mV hyperpolarizing step, but only in the dark-adapted state. Potential roles for Iout-h include modulation of neurotransmitter release from photoreceptors and shaping both the onset and offset of the photoresponse.
Removal of extracellular Cl
is reported to suppress transmitter release from photoreceptor terminals in mudpuppy; the possibility was raised that regulation of Cl
levels in and around photoreceptors may in turn regulate glutamate release (Thoreson and Miller 1996
). Suggested regulatory mechanisms included Cl
/HCO
3 exchange,
-aminobutyric acid (GABA)-activated Cl
channels and Ca2+-activated Cl
channels. The electrogenic Cl
pump described in this study is another possible mechanism. Considering that Iout-h can be elicited only under dim-light conditions, its role would depend on whether the retina were light- or dark-adapted.
Iout-h could shape the photoresponse to both onset and offset of light in the following manners. At the onset of an intense light flash, Iout-h would oppose Ih. Cone photoreceptors hyperpolarize to a flash of light and if the flash is intense enough, the photo-response relaxes from its peak to a less hyperpolarized value (Baylor et al. 1974
). A similar relaxation phenomenon in rods is suppressed by Cs+ (Beech and Barnes 1989
; Fain et al. 1978
). It is suggested that under a dark-adapted state an intense flash would activate Ih andIout-h at similar voltage ranges and with comparable time courses (cf. Fig. 5). The two currents would oppose one another, resulting in a tonic voltage response to bright flashes. With light adaptation, an intense flash would not be able to activate Iout-h, relieving Ih from antagonism. The result should be an enhanced depolarizing inflection on the response to bright flashes. Second, Iout-h may affect the Ca2+-activated Cl
current, which is involved in the regenerative depolarization of cone photoreceptors of lower vertebrates (Barnes and Deschênes 1992
; Lasansky 1981
; Piccolino and Gerschenfeld 1978; Thoreson and Burkhardt 1991
). Iout-h increases Cl
influx and, in turn, increases the intracellular Cl
concentration and the Cl
equilibrium potential during bright flashes under scotopic conditions. This action could be reflected in an increase in ICl(Ca) induced at light offset by the depolarizing off response.
Conclusions
The photoresponse of rods and cones is determined by complex interactions of voltage-activated currents. Here, evidence has been provided for an additional current, Iout-h, which is unique in that it is the first demonstration of an electrogenic Cl
transporter in any tissue by use of whole cell recording techniques. It is suggested that the more tonic response of cones to intense flashes under scotopic, compared with photopic conditions, is due to the complexity of interacting Iout-h with Ih rather than simply an absence of hyperpolarizing-activated currents. Furthermore, it seem likely that Iout-h is not unique to zebrafish, but will be found to be a common feature in cone photoreceptors and perhaps in other retinal neuron types.