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J Physiol (2003), 549.3, pp. 787-800
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.042051
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ABSTRACT |
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Glutamate either depolarizes or hyperpolarizes retinal neurons. Those are the initial and primary effects. Using a voltage probe (oxonol, DiBaC4 (5)) to study dissociated zebrafish retinal neurons, we find a secondary, longer-term effect: a post-excitatory restoration of membrane potential, termed after-hyperpolarization (AHP). AHP occurs only in neurons that are depolarized by glutamate and typically peaks about 5 min after glutamate application. AHP is seen in dissociated horizontal cells (HCs) and hyperpolarizing, or OFF type, bipolar cells (HBCs). These cells commonly respond with only an AHP component. AHP never occurs in depolarizing, or ON type, bipolar cells (DBCs), which are cell types hyperpolarized by glutamate. AHP is blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). It is evoked by kainate, AMPA and the AMPA-selective agonist (S)-5-fluorowillardiine, but not by NMDA, D-aspartate, the kainate-selective agonist SYM 2081 or by DL-2-amino-4-phosphonobutyric acid (DL-AP4). Cells with exclusively AHP responses are tonically depolarized. Resting potentials can be restored by nifedipine, suggesting a tonic, depolarizing action of L-type Ca2+ channels. However AHP is not blocked by nifedipine and is insensitive to [Cl-]o. AHP is blocked by Lisubstitution for Na
(Resubmitted 21 February 2003; accepted after revision 31 March 2003; first published online 2 May 2003)
Corresponding author R. Nelson: Basic Neurosciences Program, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 36 Room 2C02, 36 Convent Drive MSC 4066, Bethesda, MD 20892-4066, USA. Email: rnelson{at}codon.nih.gov
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INTRODUCTION |
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Na+,K+-ATPase is heavily expressed in the retinal outer plexiform layer (OPL) (McGrail & Sweadner, 1986; Yazulla & Studholme, 1987; Wetzel et al. 1999), and Na+,K+-ATPase activity is readily measured in distal retinal neurons (Shimura et al. 1998; Zushi et al. 1998). The role that Na+,K+-ATPase plays in the processing of visual information by retinal interneurons has been little studied. In this report, we examine the distribution of Na+,K+-ATPase in zebrafish retina, describe its activation in retinal neurons excited by glutamate, and argue that this activation provides a significant driving force for resting membrane potential in horizontal cells (HCs) and hyperpolarizing, or OFF centre, bipolar cells (HBCs).
We studied glutamatergic responses of acutely dissociated, adult, zebrafish retinal neurons (Connaughton & Dowling, 1998), using oxonol dye as a probe for neurotransmitter-induced changes in membrane potential (Waggoner, 1976; Walton et al. 1993; Nelson et al. 1999). The probe allows measurements of such changes without altering intracellular Na+, an activator of Na+,K+-ATPase. When glutamate responses were investigated with this method, we were surprised to find a group of cells in which the largest amplitude effect was a several minutes long loss of probe fluorescence (FL) following glutamate removal. This loss, indicating membrane hyperpolarization, we term after-hyperpolarization (AHP). The goals of this study are to examine the mechanism of the AHP response, which appears to be driven by Na+,K+-ATPase activation, and to identify the cell types with which it is associated.
Zebrafish retinal dissociations yield a mixture of type A (round stellate) and type B (elongate) HCs, long and short axon bipolar cells (BCs), as well as other types of retinal neurons (Connaughton & Dowling, 1998; Nelson et al. 2001). The ability to recognize several cell types in dissociation makes zebrafish retina a good tissue source for correlating physiological mechanisms with morphologically identified cell types. AHP responses were found in both types A and B HCs, in a subpopulation of HBCs, but not in depolarizing, or ON type, bipolar cells (DBCs). Results suggest a two-component model for retinal neurons excited by glutamate: a direct, membrane potential-sensitive component provided by ionotropic glutamate receptor (IgluR) channels gating Na+ and K+ permeabilities, and an indirect, long-term, hyperpolarizing, membrane-potential-insensitive component provided through stimulation of a ouabain and Na+-sensitive ATPase. While retinal Na+,K+-ATPase activity is usually associated with the high metabolic needs of photoreceptors in sustaining the dark current (Hagins et al. 1970), the present study provides a potential role for Na+,K+-ATPase in distal retinal interneurons excited by glutamate.
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METHODS |
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Retinal cell dissociations
Dark-adapted adult zebrafish (Danio rerio) were killed by decapitation and pithed by a rostrocaudal slice through the brain along the midline. Anterior portions of the eyes were cut away and retinas were removed. Animal care and tissue preparation protocols were approved by the Animal Care and Use Committee of the National Institute of Neurological Disorders and Stroke, NIH, in accordance with National Research Council Guidelines and Public Health Service Policy on Humane Care and Use of Laboratory Animals. Retinal cells were dissociated in 70 % Leibovitz L-15 medium (Life Technologies (Gibco BRL), Grand Island, NY, USA) using 33 U ml-1 papain (Worthington Biochemical Corp., Lakewood, NJ, USA) and 0.19 U ml-1 dispase (Sigma Chemical Co., St Louis, MO, USA), followed by gentle trituration (Connaughton & Dowling, 1998). The resultant cell suspension was allowed to settle and adhere to poly-D-lysine-coated plastic plates (2.1 µg cm-2, Collaborative Biomedical Products, Bedford, MA, USA; Nalge Nunc International, Rochester, NY, USA) and studied within 1-6 h of dissociation.
Morphological identification
Prior to voltage probe recordings, dissociated cells were identified using Hoffman optics. BCs were identified by flask-shaped cell bodies with dendritic tufts emerging from the top and an axon extending from the bottom. The presence of a complete axon (including terminal) was a variable feature. BCs without axon could be readily identified by somal shape. HCs gave a large flattened appearance compared to BCs or other cells on the dish. Some HCs were round and stellate with short radiating processes (type A) while others were elongate with a few stubby extensions (type B), as described by Connaughton & Dowling (1998). Examples of a type A HC and a BC appear in Fig. 1A, labelled 1 and 2 respectively. Area-of-interest maps were generated from Hoffman images (Fig. 1A) and applied to fluorescence images as seen through the same objective (Fig. 1B).
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Figure 1. Glutamatergic responses of dissociated zebrafish retinal neurons A, Hoffman video image of (1) a type-A horizontal cell (HC) and (2) a bipolar cell (BC). B, oxonol fluorescence of these cells (false colour) prior to first glutamate treatment. C and D, oxonol fluorescence (FL) responses of the HC (C) and the BC (D) to L-glutamate (glu), DL-AP4 (APB), kainic acid (KA), gramicidin (grami) or Na | ||
Recording solutions
The recording medium (Connaughton & Nelson, 2000) was composed of (mM): 120 NaCl (120 LiCl or 120 N-methyl-D-glucamine for Na+ substitution, 120 sodium isethionate for Cl- substitution), 2 KCl, 1 MgCl2, 3 CaCl2, 3 D-glucose, 4 Hepes (pH adjusted to 7.4-7.5) and 80 nM oxonol, DiBaC4 (5) (Molecular Probes, Eugene, OR, USA), the voltage probe. A primary 2 mM oxonol stock was dissolved in ethanol; from this a secondary 20 µM aqueous stock was prepared each experimental day to be further diluted to 80 nM for experimental use (ethanol carrier was diluted by 25 000 in the process). Glutamate agonists, antagonists and other selective ligands were added to this recording medium in concentrations ranging from 2 to 250 µM. These included APB (DL-2-amino-4-phosphonobutyric acid; DL-AP4) obtained from Calbiochem, La Jolla, CA, USA; SYM 2081 ((2S-4R)-4-methylglutamic acid), (S)-5-fluorowillardiine and nifedipine obtained from Tocris Cookson, Ballwin, MO, USA; AMPA ((±-
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid), NMDA (N-methyl-D-aspartate), L-glutamate, CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and ouabain obtained from Research Biochemicals International, Natick, MA, USA; gramicidin and recording-medium salts obtained from Sigma.
Voltage probe acquisition
Cells were perfused at 140 µl min-1 under a cover-glass bridge mounted above the plated cells (150 µm gap, mean fluid velocity 2600 µm s-1). The chamber was scanned with a 
40 Hoffman objective to locate a suitable field, containing a total of approximately 102 cells including identifiable BCs and HCs. Voltage probe fluorescence was imaged through the same objective. Data were acquired in a darkened room using minimal light exposure as the probe is readily bleached. Cells were perfused 10-20 min for initial equilibration. Thereafter, under continuous perfusion with probe medium, or probe medium containing selected ligands, video images were obtained every 30 s using intensified video microscopy (V/ICCD GENIV, Princeton Instruments Inc., Trenton, NJ, USA), a PC frame grabber card (Matrox, PIP1024A, Dorval, Quebec, Canada) and software written in house (Walton et al. 1993). The excitation shutter (Texas red or rhodamine filter sets) was opened briefly (1 s) during acquisition. Total fluorescence within a cellular region was averaged and mean fluorescence of nearby cell-free background regions subtracted giving net probe fluorescence (FL). A log transformation of net probe fluorescence was made (log(FL)) (Walton et al. 1993).
Calibration
Oxonol is a negatively charged lipophilic dye that partitions across cell membranes according to membrane potential. The concentration ratio across the membrane follows, in principle, a Nernstian relationship with transmembrane potential, so that log of probe FL within the cell is a measure of membrane potential. Increases in FL correspond to depolarization; decreases correspond to hyperpolarization. Gramicidin makes cell membranes permeable to monovalent cations and sets transmembrane potential to '0' mV, providing a Y-axis '0' for voltage probe FL (Nelson et al. 1999; Maric et al. 2000). One log unit increase in FL corresponds to ~100 mV increase in membrane potential (±30 %) as determined from fluorescence changes with manipulation of [Na+]o in gramicidin-permeabilized cells (Dall'Asta et al. 1997; Langheinrich & Daut, 1997; Nelson et al. 1999). Response time constants of 1-4 min are limited by dye equilibration (Nelson et al. 1999; Maric et al. 2000).
Correction for optical noise
The microscopic field typically contained a number of objects that we interpreted as dead cells or cell debris. These accumulated oxonol and fluoresced, but did not respond to neurotransmitters or gramicidin. These objects provided information about drifts in optical efficiency over the course of an experiment: fluctuations in source emission, camera efficiency, or even minor focus drift. They also provided an index of the constancy of dye loading. We took the mean log(FL) of such debris objects as a function of time and subtracted this 'fluorescence efficiency index' from the raw log(FL) data of responsive cells. The process normalized raw log(FL) responses to a standard candle at each point in time. This noise subtraction is a subtle benefit of parallel recordings of many objects simultaneously within a microscope field.
Scoring of responses
Glutamate responses were separated into one of four categories: depolarizing, hyperpolarizing, depolarizing biphasic or AHP. Responses were hand scored based on the observed change in log(FL) immediately following glutamate application or removal. Rapid events occurring within the first 30 s of drug application, or other similarly rapid inflections, were not considered as the time constant of probe equilibration precludes full evaluation of rapid events. Depolarizing and hyperpolarizing responses displayed smooth asymptotic rises and declines throughout drug treatment and following removal. AHP responses began on agonist withdrawal, but the remaining time course was governed by a variable cell recovery period. For example, AHP responses typically peaked within 5 min of agonist withdrawal, but in a minority of cases they could last for as long as 20 min. Cells were judged to have an AHP component if the mean of the log(FL) values in the first 5 min after agonist application was less than pre-treatment baseline, and later recovery was observed. Cells with both depolarizing and AHP components were scored as depolarizing biphasic. Some very depolarized cells (resting potentials > +20 mV) exhibited a small hyperpolarization to glutamate agonists, followed by a much larger AHP response. These were grouped as AHP types and not separately scored. Membrane potentials were measured from the mean baseline log(FL) before the first drug application to the mean log(FL) achieved during gramicidin treatment and wash at the end of each recording.
Immunocytochemistry
Eyes were collected from zebrafish during either light or dark cycles. Retinas were removed, fixed in 4 % paraformaldehyde, equilibrated with 30 % sucrose, frozen, cryostat-sectioned at 14 µm and stored at -80 °C. Sections were warmed to room temperature prior to immunostaining (Connaughton et al. 1999). After two washes in PBS, sections were blocked 1 h (0.1 % goat serum, 1 % BSA, 0.05 % Triton X-100 in PBS), followed by two rinses. Sections were incubated overnight at 4 °C with the 5 monoclonal antibody, an antibody directed against the catalytic subunit of Na+,K+-ATPase. Dilutions of 1:10, 1:5 and 1:2 of 5 antibody supernatant were used. The antibody was obtained from the Developmental Studies Hybridoma Bank (DSHB) and was developed by D. M. Fambrough (Department of Biology, Johns Hopkins University, Baltimore, MD, USA). To determine background fluorescence, control sections were incubated in normal serum (NS-1; DSHB) instead of primary antiserum. Following 4 rinse in blocker, sections were incubated 40 min at room temperature in secondary antibody (Cy-3 conjugated donkey anti-mouse IgG) at a dilution of 1:100. Sections were washed 4 in blocker, 4 in PBS, overlaid with a mixture of 50 % glycerol in PBS, and a coverslip added. Each treatment was performed in three replicates. Sections were viewed with a 40 objective in both Hoffman and fluorescence modes. Images were photographed with a cooled CCD camera (Dage-MTI, Inc. CCD300TRC) and processed in Adobe Photoshop.
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RESULTS |
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Different responses to glutamate
In dissociated retinal cells, glutamate evoked four distinct fluorescence waveforms: depolarizing, depolarizing biphasic, AHP and hyperpolarizing. All these responses could be found in HCs and/or BCs. The first three waveforms are variants on the single theme of glutamate excitation (e-type responses, Table 1), while the fourth reflects inhibition by glutamate (i-type response, Table 1). AHP responses, unaccompanied by any depolarizing components, were the most common glutamate-driven responses observed in HCs (75 %) and HBCs (51 %) (Table 1).
In Fig. 1 a small stellate type A HC (cell 1) exhibits a depolarizing biphasic response. This cell appears adjacent to a BC (cell 2) in Hoffman image (Fig. 1A) and corresponding oxonol fluorescence image (Fig. 1B). Glutamate application depolarized the HC (log(FL) increase, Fig. 1C). Following glutamate removal a precipitous AHP component occurs. This AHP component reduced log(FL) to levels below the pretreatment baseline (Fig. 1C, left arrow). Kainate, an agonist selective for ionotropic AMPA-kainate glutamate receptors, also produced both these effects, while APB, a selective metabotropic glutamate agonist, had only a small effect, ~10 % of the AHP response. The response is termed a depolarizing biphasic type because both depolarizing and AHP components are combined in response to IgluR agonists.
Glutamate hyperpolarized the adjacent BC with a single-component waveform (FL decrease, Fig. 1D). This hyperpolarizing glutamate response was the defining property of DBCs. A low amplitude response to APB (~40 % of the glutamate response) was observed (Fig. 1D) suggesting that this DBC contained both APB- and Iglu-type receptors (Iglu is a glutamate-gated Cl- channel common to teleost DBCs (Grant & Dowling, 1995, 1996).) The response appeared primarily driven by Iglu; APB contributed only a small proportion of the overall glutamate response. In patch recordings of zebrafish DBCs (Connaughton & Nelson, 2000), 77 % responded to glutamate with an Iglu-only mechanism, 5 % with APB-only mechanism and 18 % with both mechanisms.
Relationship of resting fluorescence to glutamate response waveforms
A signature relationship was found between mean resting log(FL) and responses consisting of an AHP component without glutamate-evoked depolarization (Table 1). The mean resting level for HCs with only an AHP component was +0.25 ± 0.03 log(FL) units; that of HBCs was +0.29 ± 0.04 log(FL) units, both levels about +30 mV depolarized (Table 1). In contrast, the resting level of HCs with depolarizing biphasic responses was -0.34 ± 0.10 log(FL) units, and that of HBCs with the same response was -0.37 ± 0.02 log(FL) units; both levels were -30 to -40 mV hyperpolarized. HCs and HBCs with single component depolarizing responses were yet more hyperpolarized (-50 to -60 mV). The mean resting level of DBCs, cells hyperpolarized by glutamate, was -0.46 ± 0.01 log(FL) units (-50 mV).
Responses consisting of only an AHP component were always found in depolarized cells; nonetheless, this same component was also detected in depolarizing biphasic cells, which have hyperpolarized resting levels. Thus the AHP component appears insensitive to membrane potential. It is the depolarizing glutamate response component that is membrane potential sensitive, as it occurs only in cells with hyperpolarized resting levels, and is abolished or reversed in depolarized cells. Together AHP and depolarizing components combine to produce composite, membrane potential-dependent, e-type waveforms, with the depolarizing component being highly dependent on resting membrane potential, and the AHP component being insensitive.
AHP sensitivity to selective agonists
To identify the glutamate receptor types involved in the generation of the AHP responses seen in HCs and HBCs, agonists selective for AMPA, kainate and NMDA receptors, the three major IgluR classes, were tested. The results (Fig. 2A) indicate that glutamate and kainate evoked the AHP response, as did the selective AMPA agonist (S)-5-fluorowillardiine (Patneau et al. 1992; Jane et al. 1997), but not the IgluR agonist SYM 2081, an agonist selective for kainate-type receptors (Jones et al. 1997; Bleakman et al. 1999). The observed hyperpolarizing response of a DBC on the same plate (Fig. 2B) was stimulated by glutamate alone. Agonists selective for IgluR receptors did not elicit responses. In an HBC studied in a different preparation (Fig. 2C) AMPA effectively elicited an AHP response (Fig. 2C, arrow), but NMDA did not. The AMPA-evoked AHP was prolonged. During this event, glutamate evoked a depolarizing response (Fig. 2C, asterisk), without any AHP component. This suggests that further AHP components cannot be evoked during an ongoing AHP event; only depolarizing components occur.
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Figure 2. Actions of selective IgluR agonists on dissociated retinal neurons A, horizontal cell (HC) with AHP responses (arrows), observed following treatments with glutamate (glu), kainic acid (KA) or (S)-5-fluorowillardiine (Fwill, an AMPA receptor agonist), but not SYM 2081 (SYM, a kainate receptor agonist). B, in the same plate, glutamate hyperpolarized a depolarizing bipolar cell (DBC), but kainic acid SYM 2081, and (S)-5-fluorowillardiine were ineffective. C, on a different plate, a hyperpolarizing bipolar cell (HBC) produced an AHP response to AMPA, but not NMDA. During the long-lasting AMPA-induced AHP, glutamate (glu) evoked a depolarizing response (asterisk) without any AHP component. | ||
IgluR (e-type) responses in glutamate-sensitive HCs and HBCs were readily evoked by AMPA (17/17, 50 µM), kainate (79/81, 10 µM) and (S)-5-fluorowillardiine (5/6, 10 µM). Responses were not seen with SYM 2081 (0/6, 10 µM), NMDA (0/6, 100 µM) or APB (0/24, 250 µM). D-Aspartate was infrequently effective in glutamate-sensitive HCs and HBCs (5/24, 50 µM), but both HCs and BCs were represented in the sensitive group. This scoring overall suggests that AMPA-like receptors are important in the generation of IgluR responses in zebrafish HCs and HBCs, and that the AHP response component also results from stimulation of AMPA-like receptors. Metabotropic, NMDA and kainate receptors appear much less significant. NMDA failed to stimulate even depolarized units with AHP-type responses, conditions expected to relieve block on the NMDA channel.
Concentration dependence of AHP responses
The glutamate concentration dependence of an AHP response is illustrated in Fig. 3A. The threshold for the AHP response in this HBC was about 8 µM. At this dose there was a separation between a small hyperpolarizing event occurring during glutamate application (Fig. 3, asterisk) and the large AHP event occurring after (Fig. 3, arrow). The hyperpolarization during glutamate application in highly depolarized AHP responding cells appeared to be a direct action of IgluR channels on membrane potential, similar to depolarizing responses, except that in depolarized cells, a net outward flow of cations hyperpolarized the unit back towards the 0 mV IgluR reversal potential. The evidence suggests that the AHP component of e-type responses is more delayed, and less glutamate sensitive than the direct action of glutamate on IgluR channels. Half-saturation for AHPs in these fields was 17.4 ± 1.9 µM (n = 5, mean ± S.E.M.). The glutamate concentration dependence of an HBC with depolarizing responses is illustrated in Fig. 3B. The amplitude of this response appears about half-saturated by the 3 µM dose. Half-saturation for depolarizing responses of cells in these fields was 5.2 ± 0.8 µM (n = 27).
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Figure 3. Concentration dependence of AHP and depolarizing responses from hyperpolarizing bipolar cells (HBC) A, at AHP threshold (8 µM, above), a direct glutamate hyperpolarization occurred during glutamate treatment (asterisk) and was followed by a delayed AHP response (arrow). In depolarized cells with AHP responses, a small hyperpolarizing component was commonly seen during glutamate application (asterisk), and is attributed to a net outflow of cations through IgluR channels. The 20 µM glutamate dose evoked fast onset AHP kinetics. B, the depolarizing components of HBC responses are more sensitive than the AHP components. The response to 3 µM glutamate was about half-saturating for this cell. This response was recorded on a different plate than that for A. | ||
CNQX antagonism of AHP responses
Both AHP and depolarizing e-type components were blocked by CNQX, an antagonist of AMPA-kainate receptors (Fig. 4). AHP responses of a tonically depolarized HC were evoked by glutamate in control and recovery treatments (Fig. 4A), but not during CNQX application (circled). This is an example of a complete block. In another HC glutamate evoked depolarizing responses in control and recovery treatments, but not in the presence of CNQX (Fig. 4B). In eight HBCs and six HCs, 50 µM CNQX fully blocked responses to 50 µM glutamate. This group included six depolarizing, one depolarizing biphasic and seven AHP response types. In further trials 20 µM CNQX was tested on a group of 11 HCs. In combination with the 50 µM glutamate test, this dose provided complete block in six cases, partial block in two cases, and was ineffective in three cases. These results suggest some variability in the CNQX sensitivity of HCs near the threshold agonist/antagonist ratio for this competitive inhibitor. All but one of these responses was an AHP type.
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Figure 4. AHP and depolarizing responses to glutamate (glu) are blocked by CNQX A, the AHP responses of a horizontal cell (HC) were not evoked in the presence of CNQX (dashed circle). B, depolarizing responses of an HC recorded on a different plate were similarly prevented. The depolarizing inflection at control glutamate onset in A is not a part of the AHP response and is considered to be a recording artifact. | ||
L-type Ca2+ channels tonically depolarize cells with AHP responses
In other species stimulated HCs produce Ca2+ action potentials, which are maintained for many seconds, or in some cases, indefinitely due to the activation of L-type Ca2+ channels (Tachibana, 1981; Shingai & Christensen, 1986; Sullivan & Lasater, 1990, 1992). To test the idea that the persistent depolarized state characteristic of AHP responses (as seen in zebrafish HCs) is maintained through the action of long-lasting Ca2+ currents, and that glutamate-induced modulation of such currents might be the mechanism of AHP responses, HCs were bathed in nifedipine, an L-type Ca2+ channel blocker. In Fig. 5A an HC with glutamate-elicited AHP responses hyperpolarized dramatically during nifedipine application (open arrowhead). A depolarizing biphasic glutamate response (asterisk), with a prominent AHP component (arrow), was then evoked by glutamate on this hyperpolarized background. The AHP component persisted in the presence of nifedipine (Fig. 5A). Of 11 HCs treated with nifedipine, all were hyperpolarized. Similar to the HC in Fig. 5A, depolarizing biphasic responses were seen on these hyperpolarized backgrounds in 8 of the 11 HCs. The remainder exhibited depolarizing responses. Activated L-type Ca2+ channels appear to hold HCs in a depolarized state. Glutamatergic suppression of these L-type Ca2+ currents, however, does not appear to be the mechanism by which AHP response components are generated. These components are frequently observed even in the presence of a visibly effective Ca2+ channel blocker.
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Figure 5. Neither L-type Ca2+ channels nor extracellular Cl- ions are required for AHP responses A, nifedipine (nifed), an L-type Ca2+ channel blocker, restored membrane potential (open arrowhead) to a depolarized horizontal cell (HC). During the hyperpolarizing nifedipine treatment, a depolarizing biphasic glutamate (glu) response occurred (asterisk). This response has an AHP component (arrow), which persists in nifedipine. B, reduction of [Cl-]o from 130 to 10 mM was achieved by substituting sodium isethionate for NaCl in the recording medium. The AHP response of this HC persisted in the low [Cl-]o medium (arrow). | ||
AHP responses require Na but not Cl
Entry of Cl is a common mechanism that hyperpolarizes neurons and might be involved in generation of AHP responses. Zebrafish photoreceptors contain an electrogenic Cl- pump (Fan & Yazulla, 1997). In DBCs, glutamate activates a Cl- current (Grant & Dowling, 1995; Connaughton & Nelson, 2000). However, transmembrane movement of Cl- does not appear to be the mechanism generating the AHP response. This response persists in medium with low [Cl-]o (Fig. 5B, arrow). Fifteen HCs and four HBCs with AHP responses to glutamate in standard media were bathed in media with low [Cl-]o. Of these 19 cells, 13 retained AHP response components in the substituted media, giving either AHP or depolarizing biphasic responses. Thus, [Cl-]o-dependent mechanisms appear not to play a major role in generating AHP responses. Further, in the HC (Fig. 5B) there was an initial hyperpolarization during glutamate treatment. This response also persisted in low [Cl-]o, providing evidence against [Cl-]o dependence of this component, and agreeing with the previous suggestion of generation by cation efflux through IgluR channels.
Sodium ions, however, are essential for glutamate-induced AHPs. Substitution of Na by Li reversibly blocked AHP responses. Representative data are presented in Fig. 6A from an HC that gave AHP responses to glutamate in control and recovery treatments, but in which no significant AHP components were seen during Li substitution (circled). In the substituted medium, glutamate depolarized this HC by about +0.3 log(FL) units (30 mV, Fig. 6A, asterisk). Following glutamate treatment, rather than an AHP-accelerated repolarization, a delayed repolarization was seen (circled). In the recovery glutamate treatment, however, a depolarizing biphasic response was seen, with a large and rapid AHP component (Fig. 6A). In 11/11 HCs and 3/3 HBCs with AHP or depolarizing biphasic control responses, no AHP components were seen during Li+ substitution for Na+. Depolarizing responses to glutamate were seen instead in all 14 of these cases, in each example with a delayed repolarization after glutamate withdrawal. The results suggest that: (1) Li+ permeates most AMPA-like IgluR channels in zebrafish HCs and HBCs, (2) an AHP component cannot be stimulated in Li+ substituted medium, and (3) AHP components normally participate in repolarization after IgluR-induced depolarizations.
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Figure 6. AHP responses require Na A, the glutamate-evoked AHP responses of a horizontal cell (HC), seen after control and recovery glutamate treatments (glu, arrows), disappeared when Na | ||
Ouabain sensitivity of AHP responses
Na
is a requirement for activation of Na+,K+-ATPase, the sodium pump. Since the pump is electrogenic and hyperpolarizes neurons (Baylor & Nicholls, 1969; Thomas, 1969), the pump is a candidate to generate the AHP response. Ouabain is a Na+,K+-ATPase inhibitor. The effects of ouabain on an AHP response appear in Fig. 6B. Glutamate removal elicited an initial sustained AHP in this HC (left arrow). While ouabain itself did not immediately depolarize this cell (Fig. 6B), depolarization occurred rapidly in the presence of the combination ouabain plus glutamate (asterisk). In the presence of ouabain the AHP component of this response was blocked (circled), and the cell remained in a depolarized state. There was a partial recovery and return of a small AHP (right arrow) after 10 min of wash. Of seven HCs with either AHP or depolarizing biphasic responses, the AHP component was blocked by ouabain in five cases, and partially blocked in the other two. Full recovery was not seen in any of these instances. In five HBCs treated with ouabain, all with depolarizing responses, glutamate-evoked depolarization was not blocked. The combination of a requirement for Na+ and sensitivity to ouabain suggests that AHP responses are Na+,K+-ATPase mediated.
Localization of Na+,K+-ATPase in zebrafish retina
The monoclonal antibody 5 is raised against chicken kidney (Fambrough & Bayne, 1983). It is reactive with all Na+,K+-ATPase -subunit isoforms in a variety of species including avian, mammalian, amphibian and insect (DSHB database). A total of eight -subunit genes have been identified in zebrafish with up to 92 % amino acid identity with rat. Six of these are expressed in ocular tissue including orthologs of rat 1, 2 and 3 subunits (Rajarao et al. 2001).
Retinal sections incubated with 5 were immunoreactive, while sections incubated with NS-1 control were not. Both nuclear and plexiform layers were labelled. The outer nuclear layer (ONL), inner nuclear layer (INL) and the ganglion cell layer (GCL), were typically brighter than the inner plexiform layer (IPL). Bright rims of cytoplasm bordered dark cell nuclei in the nuclear layers (Fig. 7). In Fig. 7, BC somata are brightest; HCs, amacrine cell bodies and ganglion cell bodies are moderately labelled. Rod and cone cell bodies are labelled, as are cone inner segments. The single line of cone pedicles (CP) in the outer plexiform layer (OPL) was always bright. This layer, of course, also contains dendritic terminations of HCs and BCs. In Fig. 7, several prominent HCs (asterisks) appear just vitread to the bright CP band. By contrast the zone of rod spherules (RS), just sclerad to this band, is free of label.
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Figure 7. Patterns of Na+,K+-ATPase antibody staining in zebrafish retina A, Fambrough | ||
In some preparations the INL was uniformly labelled; however more frequently nuclear labelling was differentiated within the INL, as in Fig. 7. Axons descending into the IPL identified brightly labelled distal INL cell bodies as BCs. In some sections patterns of terminal ramification identified distinct populations. In Fig. 7, a prominent band is found in stratum 1 of sublamina a, and a weaker band in stratum 4 of sublamina b (arrows). In previous studies expression of GAD67, an enzyme found in HCs, varied between light- and dark-adapted retinas (Connaughton et al. 2001). No light-dark difference in the 5 Na+,K+-ATPase labelling pattern was found, however.
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DISCUSSION |
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Voltage probe recordings of dissociated zebrafish retinal neurons reveal four waveforms in response to glutamate stimulation. A hyperpolarizing, inhibitory (i-type) waveform is monophasic and occurs in DBCs. Three excitatory (e-type) waveforms were identified: depolarizing, depolarizing biphasic and AHP. These responses represent summations of two mechanisms: a direct, membrane depolarization by activated IgluR channels and an indirect, long-term hyperpolarization caused by activation of an electrogenic Na+,K+-ATPase. The long-term effect typically peaked within 5 min of the termination of IgluR stimulation, while the direct polarization terminated immediately. The difference between depolarizing biphasic and AHP responses is membrane potential. Cells with only AHP response components rest in a depolarized state, at or above the IgluR reversal potential of 0 mV. Therefore no IgluR-evoked depolarization can occur, only a null response or small hyperpolarization. The AHP component appears in isolation, but the hidden IgluR depolarization reappears if membrane potential is restored. Cells with depolarizing biphasic responses rest in a hyperpolarized state. IgluR stimulation directly depolarizes them. The depolarization is followed by an AHP component. This component does not reverse even at high membrane potential. While HCs and HBCs exhibited all three e-type waveforms, isolated AHP responses were the most frequent.
Mechanism of Na+ entry
AHP requires Na+ in the bathing medium and is blocked by ouabain. Glutamate apparently induces Na+ entry into the cytoplasm stimulating the electrogenic Na+,K+-ATPase, hyperpolarizing the cell. There are at least four mechanisms by which glutamate might stimulate Na+ entry: (1) by opening IgluR channels, (2) by stimulation of Na-dependent glutamate transporters (Brines & Robbins, 1993), (3) by stimulating Na+ entry through voltage-dependent Na+ channels, (4) by increasing Ca2+ entry, directly or indirectly, and stimulating subsequent removal by Na-dependent antiporters.
As AHP is blocked by CNQX, induced by non-transported ligands such as kainate, AMPA and (S)-5-fluorowillardiine, and poorly stimulated by D-aspartate, a well-transported analogue, glutamate transport does not appear to be a significant source of Na+ entry in present experiments. Similarly AHP does not appear to require Ca2+ entry. AHP persists under conditions of Ca2+ channel block by nifedipine. Furthermore even the persistent state of Ca2+ channel activation found in cells with AHP responses, such as HCs and HBCs, does not trigger sufficient pump activity to repolarize the cells. In these depolarized cells it is possible that Ca2+ extrusion is accomplished by a Ca2+-ATPase (Hayashida et al. 1998; Krizaj et al. 2002) and Na+ entry is avoided. While teleost HCs can express voltage-sensitive Na+ channels (Lasater, 1986), these are only transiently active and require repolarization to reactivate. Our results do not suggest Na+ entry in tonically depolarized zebrafish HCs is maintained by this route. If it were, pump activity would repolarize the cells and depolarized cells would not be encountered. Only entry of Na+ through activation of IgluR channels appears a likely candidate for pump activation.
Survival of cells with persistent Ca2+ activation
A peculiarity of distal retinal neurons including HCs, HBCs and DBCs is prolonged activation of L-type Ca2+ channels resulting in long duration spikes and persistent depolarized states. Ca2+ channels are major contributors to signal processing in retinal BCs (Burrone & Lagnado, 1997; Zenisek & Matthews, 1998; Protti et al. 2000). Ca2+ currents contribute to transient oscillations in the HC light response (Akopian et al. 1997). They are also a prominent feature of the physiology of isolated HCs (Tachibana, 1981; Shingai & Christensen, 1986; Ueda et al. 1992). In isolated zebrafish HCs and HBCs, Ca2+ channels dominate membrane potential over long intervals. One might wonder how cells persistently depolarized by Ca2+ survive for extended periods without undergoing apoptosis and cell death. Our tissue culture preparations are studied within 1-6 h of dissociation, so the survival time is not extended. The further question, however, is how distal retinal neurons, excited continuously by photoreceptor glutamate, avoid apoptosis through excitotoxicity. Special mechanisms may have developed. One of these, robust activity of Na+,K+-ATPase is suggested by present results and would serve to remove excess Na resulting from IgluR stimulation. A mechanism to limit [Ca2+]i is suggested by the results of Sullivan & Lasater (1990). They found that K+ currents in HCs were unusually weak. This weakness causes cells to remain in depolarized states, but also limits Ca2+ entry. Continued influx of Ca2+ requires continued efflux of K+. In the limiting case of open Ca2+ channels in a cell with no countervailing K+ conductance, membrane potential could be maintained at Ca2+ equilibrium indefinitely without net Ca2+ flux. In many zebrafish BCs it is known that only K+ A-currents are activated by depolarization (Connaughton & Maguire, 1998). Similarly, the transient nature of this current may limit long-term Ca2+ flux in the depolarized state. Because of weak K+ currents, it is possible that only limited Ca2+ entry occurs in depolarized distal retinal neurons, and that such entry does not overwhelm extrusion mechanisms.
Significance of Na+,K+-ATPase for light responses in distal retina
Na+,K+-ATPase activation may affect the light responses of HCs and HBCs. In the present studies, these cells attained maximal hyperpolarization when glutamate was removed. The increased polarization was caused by Na-induced pump activation. Synaptic glutamate is naturally removed during light stimulation; as photoreceptors hyperpolarize, release ceases. Membrane potentials of skate HCs have been recorded for up to 30 min during experiments on light adaptation (Dowling & Ripps, 1971). While steady background stimuli initially caused a saturating hyperpolarization, after about 5 min, these HCs began to repolarize. We speculate some of this slow repolarization might reflect a reduction in electrogenic Na+,K+-ATPase activity as a buildup of [Na+]i from the previous dark period is removed. Similarly natural stimuli which increase [Na+]i in HCs and HBCs should activate Na+,K+-ATPase and increase currents flowing through post-synaptic IgluR channels. AMPA receptors found on these cells rapidly inactivate and are most conductive during transient stimulation. A light stimulus containing many transients, such as flicker, might tend to increase [Na+]i, activate the pump, and produce greater light-evoked voltage fluctuations across glutamate channels because of this increased current flow. Indeed the peak-to-peak flicker responses in cat HCs are observed to grow in amplitude as the stimulus is continued (Nelson, 1985; Pflug et al. 1990). In the previous two examples of natural stimulation, it is difficult to distinguish mechanisms occurring in photoreceptors from those occurring in HCs. In a more direct test, Na+ was injected into HCs in Necturus retina. After injection, the amplitude of light responses increased (Nelson, 1973), consistent with a mechanism where Na+,K+-ATPase activation increases the amplitude of light responses.
Na+,K+-ATPase-induced hyperpolarizations following electrical stimulation have been reported in snail neurons (Baylor & Nicholls, 1969), unmyelinated nerve fibres (Rang & Ritchie, 1968), and barnacle photoreceptors (Koike et al. 1971). We suggest that electrogenic Na+,K+-ATPase activation should be included among the mechanisms that may, in addition to restoring ionic gradients, hyperpolarize membranes and increase the amplitudes of light responses in distal retina. Currents generated by Na+,K+-ATPase activation will flow through postsynaptic IgluR channels increasing voltage fluctuations across them.
An electrogenic Na+,K+-ATPase has been physiologically characterized in acutely dissociated carp HCs. A K+-, Na+-, ouabain-sensitive, but voltage-insensitive current of about 13 pA was observed, with a suggested Na+/K+ ratio of 3/2. Carp HCs exhibited a steady inward leak of Na+, making the pump continuously active. Ouabain sensitivity had two limbs, suggesting the presence of high affinity and low affinity isoforms. The former had an IC50 of 20 nM, while the latter had an IC50 of 10.4 µM (Shimura et al. 1998). Assuming similar affinities in zebrafish, both isoforms would have been inhibited in present experiments. Isolated carp BCs also manifested an ouabain-sensitive pump (Zushi et al. 1998).
Patterns of ATPase immunoreactivity
ATPase immunoreactivity in zebrafish was found prominently in the distal retina, in HCs, BCs and the band of cone pedicles. The S1, S4 branching pattern of labelled BC axons suggests that at least some HBCs heavily express ATPase epitope. Candidates fitting this banding pattern include bistratified OFF type HBCs, or some mixture of monostratified HBCs, monostratified DBCs and bistratified HBCs (Connaughton & Nelson, 2000). The lack of label within rod spherules argues against a prominent labelling of rod BC dendrites, such as arising from mb1 rod BCs. Correspondingly, the large mb-type synaptic terminals, readily observed in glutamate-labelled retinas (Connaughton et al. 1999), were not identified in sections labelled with the 5 antibody. In goldfish OPL the cone pedicle band stained densely for ouabain and K+ sensitive p-nitrophenylphosphatase (K+-pNPPase) activity, a marker for Na+,K+-ATPase. Electron microscopy of goldfish OPL revealed staining of HC lateral elements and cone presynaptic structures (Yazulla & Studholme, 1987). Rod spherules remained unstained. The pattern of dense labelling of cone pedicles and absent label in rod spherules is also observed in the present study.
In rat retina, the OPL was densely immunofluorescent for Na+,K+-ATPase, with participation of HCs and BCs in the staining (McGrail & Sweadner, 1986). In rat as in zebrafish, cytoplasmic rims were observed on INL cell bodies, and there was a suggestion of IPL banding. Unlike zebrafish, the rat IPL is typically more brightly labelled than the INL. In rat and mouse, retinal neurons express multiple Na+,K+-ATPase -subunits (McGrail & Sweadner, 1986; Wetzel et al. 1999). Orthologues of rat 1, 2 and 3 subunits are expressed in zebrafish ocular tissue (Rajarao et al. 2001).
The overall high ATPase expression in OPL neurons may reflect the need to counter the increase in [Na+]i resulting from persistent AMPA receptor activation by photoreceptor glutamate (Brines & Robbins, 1993). On this argument, HCs and HBCs may require greater Na+,K+-ATPase expression than DBCs. In zebrafish retina DBCs utilize predominantly Iglu, a glutamate-gated Cl- conductance mechanism (Connaughton & Nelson, 2000), and consequently experience no Na+ entry related to synaptic activity.
AMPA channels in distal retina
AMPA-type glutamate responses are common in retinal HCs (Yang et al. 1998; Blanco & de la Villa, 1999; Shen et al. 1999) and HBCs (Maple et al. 1999; DeVries, 2000). Both AMPA and kainate type glutamate receptor subunits can be found by antibody staining of retinal HCs and BCs (Peng et al. 1995; Brandstatter et al. 1997; Vardi et al. 1998; Vandenbranden et al. 2000; Nelson et al. 2001; Pourcho et al. 2002). Nonetheless, it is the AMPA type responses that dominate the physiology of HCs and HBCs in zebrafish. The multiple components of e-type glutamate responses, seen by voltage probe techniques in dissociated zebrafish retinal neurons, all appear to be AMPA-receptor driven. Half-saturation of AHP components occurred at ~20 µM. This is similar to the EC50 found for the AMPA-type inward currents activated by glutamate in rabbit A-type HCs (Blanco & de la Villa, 1999), suggesting that AHPs are likely to occur in concentration ranges that normally activate AMPA receptor currents.
AHP responses may continue to grow in magnitude even beyond voltage saturation for direct AMPA channel responses. In the voltage-saturated state, Na+-K+ exchange through IgluR channels continues to increase in proportion to the number of channels open, even though membrane potential does not. A signal to cell physiology could be provided through increasing [Na+]i and stimulation of the electrogenic Na+,K+-ATPase even in cells depolarized to IgluR reversal potential. It is important to recall that while the net current through IgluR channels reverses at 0 mV, the Na+ component of this current does not. Na+ reversal is +60 to +90 mV (Johnston & Wu, 1995). Thus the opening of further AMPA channels even during voltage saturation can have long-term effects on cell physiology through stimulation of Na+,K+-ATPase activity; one of these effects is to hyperpolarize the membranes of distal retinal neurons.
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Acknowledgements
The 5 antibody developed by D. M. Fambrough was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, USA.
Supplementary material
The online version of this paper can be found at:
http://www.jphysiol.org/cgi/content/full/549/3/787 DOI: 10.1113/jphysiol.2003.042051
and contains material entitled:
Acquisition of voltage probe signals from dissociated zebrafish retinal neurons.
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