| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1Department of Neuroscience, University of Pennsylvania School of Medicine; and 2Division of Neurology, Pediatric Regional Epilepsy Program, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
Submitted 3 June 2005; accepted in final form 19 December 2005
 |
ABSTRACT |
|---|
|
|
|
INTRODUCTION |
|---|
|
, 2002). GABA depolarizes neurons, activates voltage-gated calcium channels, and increases intracellular calcium concentration ([Ca2+]i) (Leinekugel et al. 1995; Owens et al. 1996; Yuste and Katz 1991). Through depolarization and calcium elevation, GABA regulates differentiation, survival, dendritic mobility, and spontaneous activity (Fischer et al. 1998; Liu et al. 1997; Marty et al. 1996; Redburn 1992; Wong and Wong 2001; Young and Cepko 2004). In retina, GABA and glycine also modulate retinal waves, spontaneous bursts of action potentials that propagate across amacrine and ganglion cells and are critical for refinement of the retinotopic and eye-specific maps in the brain (Firth et al. 2005; Penn et al. 1998; Shatz and Stryker 1988). GABA and glycine affect waves differently in different species: they increase the frequency of calcium transients (ferret: Fischer et al. 1998), control wave propagation rate (turtle: Sernagor et al. 2003), or generate the waves (rabbit: Zheng et al. 2004; Zhou 2001).
The excitatory action of GABA is mediated by the ionotropic GABA receptor, which permeates primarily chloride but may also permeate bicarbonate. Therefore depolarization by GABA may be due to efflux of chloride if the chloride equilibrium potential (ECl) is above the resting membrane potential (Erest) or to efflux of HCO3–, the typical reversal potential of which is 0 mV (Bormann et al. 1987; Kaila et al. 1989). In several brain regions, GABA's depolarizing effect correlates with measurements of ECl, lending support to the chloride hypothesis (Ehrlich et al. 1999; Eilers et al. 2001; Owens et al. 1996; Yamada et al. 2004). However, in retina, neither hypothesis was tested, and the evidence supporting the chloride hypothesis remained indirect.
In rat retina, the chloride extruder, KCC2, is undetectable early in development and becomes significantly greater at about the stage when GABA was predicted to switch from excitatory to inhibitory (Vu et al. 2000). This has been interpreted to mean that [Cl–]i is initially high and declines as the extruder appears. This interpretation, however reasonable, needed testing with actual measurements of the developmental changes in [Cl–]i, ECl, and the reversal potential of GABA (EGABA)—all in the same species, and only then comparing them to the appearance of KCC2. These measurements carry additional importance because the molecular mechanisms of chloride's accumulation in early development remain to be established in any brain region and to do so requires firm quantification of these various parameters. We performed these measurements in mouse where the availability of gene knockouts will facilitate studying the molecular mechanisms. Here we show, by optical measurements of [Cl–]i and [Ca2+]i and electrical measurements of ECl with perforated-patch recordings, that from postnatal day (P) 0 to P6, [Cl–]i is high enough for GABA to cause chloride efflux, calcium rise, and excitation. After P6, [Cl–]i declines abruptly and GABA becomes inhibitory.
|
|
METHODS |
|---|
|
Newborn mice were deeply anesthetized on postnatal days P0–P6 with halothane and killed by decapitation. Older mice were killed with ketamine (85 µg/g) and xylazine (13 µg/g ip) followed by anesthetic overdose. Animals were treated in compliance with federal regulations and University of Pennsylvania policy. After enucleation, eye cups were immersion-fixed for 1 h at room temperature in 4% paraformaldehyde and 0.01% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 and cryoprotected overnight with 30% sucrose in phosphate buffer. Eyes were frozen in a mixture of tissue-freezing medium (Electron Microscopy Sciences, Ft. Washington, PA) and 20% sucrose (1:2) and cryosectioned vertically at 10 µm. Sections were preblocked, then stained by incubation in guinea pig anti-GABA (Chemicon, Temecula, CA) diluted with 0.1 M phosphate buffer containing 10% normal goat serum, 5% sucrose, and 0.3% Triton X-100 (overnight at 4°C). Sections were then washed, incubated in donkey anti-guinea pig IgG conjugated to FITC (3 h; Jackson Immunoresearch, West Grove, PA), washed, and mounted in Vectashield (Vector Laboratories, Burlingame, CA). Sections were visualized with a confocal microscope (Leica, Nussloch, Germany). At each age group, omitting the primary antibody gave no staining at all.
Calcium imaging
A retina was cleaned of vitreous, detached from the pigment epithelium, and cut into two to three pieces. Retinal pieces were mounted on a filter paper and maintained in bicarbonate-based Ames medium saturated with carbogen (95% O2-5% CO2). For bicarbonate-free Ames medium, NaHCO3 was replaced with 20 mM HEPES, and the solution was saturated with oxygen (100% O2). In mice >P7, ganglion cells were exposed by gently scratching the vitreal surface with a scalpel tip or a brush.
Retinal pieces were loaded in 5–10 µM fura-2 AM (Molecular Probes, Eugene, OR) in oxygenated Ames medium for 1 h at 27–30°C. A retina was placed in an optical recording chamber mounted on a fixed-stage, upright microscope (Olympus BX51WI), and continuously superfused (3–4 ml/min) with Ames medium preheated to 30–33°C. Fura-2 was alternately excited at 340 and 380 nm with a xenon arc lamp in lambda DG4 (Sutter Instrument, Novato, CA) and attenuated with a neutral density filter (0.25). Fluorescent images were viewed with a x40 water-immersion lens (NA, 0.8, Olympus, Tokyo, Japan), filtered with a 510/40-nm emission filter (set 71000, Chroma, Rockingham, VT), and captured with Hamamatsu Orca ER digital CCD camera (Hamamatsu, Hamamatsu City, Japan). Wavelength switch and image capturing were controlled by Openlab software (Improvision, Lexington, MA) running under Mac G4. To minimize potential UV phototoxicity, the illuminated retinal area was restricted by closing the field aperture to match the CCD imaging field (250 x 190 µm), and pixels were binned (2 x 2). Exposure time was 25–200 ms, and sampling interval 3–4 s.
Chloride imaging
Fresh 6-methoxy-N-ethyl-1,2-dihydroquinoline (dihydro-MEQ) was synthesized from 5 mg 6-methoxy-N-ethylquinolinium iodide (MEQ) according to the protocol provided by Molecular Probe (Molecular Probes). The reduced product was resuspended in dimethyl sulfoxide and added to Ames medium to yield a loading concentration of 
300 µM dihydro-MEQ. Retinal pieces, prepared as described in the preceding text, were incubated in oxygenated dihydro-MEQ for 1 h at 27–30°C. To identify cells and monitor their volume, 1 µM calcein and 0.001- 0.02% pluronic acid were added for the last 15 min of loading. For dual calcium and chloride imaging, the loading solution contained 10 µM calcium indicator, Calcium Green-1 AM, and 0.001–0.02% pluronic acid. During loading, intracellular oxidation converted the membrane-permeable dihydro-MEQ into the charged and impermeable MEQ. After loading, retinas were transferred into fresh Ames medium at room temperature and then transferred to the recording chamber. The chamber was maintained at 27–29°C (rather than 33°C) to reduce MEQ leakage and increase MEQ sensitivity (Fukuda et al. 1998). The optical setup and acquisition system was the same as for calcium imaging except that excitation and emission was filtered for MEQ and calcein or for MEQ and Calcium Green-1 fluorescence. Excitation alternated between 345 nm (D345/10x, Chroma) and 485 nm (S485/25x, Chroma), and emission was collected with a dual band filter with peaks at 450 and 535 nm (set 91018, Chroma). Images were collected every 10–15 s to determine intracellular chloride, and every 3–4 s to record GABA-evoked chloride changes.
Optical signal analysis
Imaging and statistical analysis were done with Openlab and Excel (Microsoft, Seattle, WA) and curve fitting with Origin (Origin Lab, Northampton, MA). The time-lapse image stacks were first registered to correct for retinal movements, then a region of interest was drawn around a dye-loaded ganglion or amacrine cell in the ganglion cell layer, and the cell's fluorescence was measured at each time point. Background due to dark current, ambient light, and autofluorescence was estimated in control experiments with unloaded retinas and subtracted from the cell's fluorescence. For single wavelength indicators MEQ and Calcium Green-1, decline of baseline due to dye bleaching and leakage was fitted with a linear decay curve and subtracted from the cell's fluorescence (Fig. 1A). The index 
F/F, where F is the fluorescent intensity obtained at baseline (after baseline correction and background subtraction) and F is the deviation from F at a given time, was used as a measure of change in ionic concentration. Note that because MEQ fluorescence is quenched collisionally by Cl–, F/F goes down proportionally with an increase in Cl– (see APPENDIX). For calcium imaging with the ratiometric indicator fura-2, we computed the ratio of the cell's average fluorescence at 340 to that at 380 nm (F340/F380).
|
To measure intracellular chloride, we compared a cell's unperturbed MEQ fluorescence to its fluorescence when its intracellular chloride was set to a reference value. Internal chloride concentration was equilibrated by adding the K+/H+ exchanger nigericin (7 µM) and the Cl–/OH– exchanger tributyltin (10 µM) in the presence of high K + (Table 1). The Cl–/OH– exchanger equilibrates Cl– across the membrane, and the K+/H+ exchanger in presence of high K+ is used to clamp pH while Cl– changes (Krapf et al. 1988). As intracellular chloride equilibrated, if intracellular chloride concentration was reduced (i.e., initial intracellular chloride concentration was higher than reference), the baseline-corrected fluorescence increased. Conversely, if intracellular chloride increased, MEQ fluorescence decreased. Thus we first measured the cell's fluorescence in Ames or Ringer, then equilibrated with a single chloride solution and noted the direction and magnitude of fluorescent change. Noise level in constant chloride was estimated by measuring fluorescence over 15 min. Traces were baseline- corrected and normalized, and the fluorescence fluctuations were measured after additional 7 min. This gave a noise level of 2.5% of initial fluorescence; thus only changes >2.5% were considered significant (Fig. 1B).
|
 |
where F is the fluorescence in Ames or Ringer, Fref is the difference between equilibrated fluorescence and the initial fluorescence, K50 is the half quenching constant obtained from the Stern-Volmer plot as described in the following text, and [Cl–]ref is the equilibrated chloride concentration (see APPENDIX).
We noted that following the switch of solution and before approaching the new steady state value, fluorescence transiently dropped (see Fig. 7A). Fukuda et al. (1998) had described a similar phenomenon and attributed it to the optical interference of nigericin and tributyltin. We found that nigericin and tributyltin did not absorb in the spectrum of MEQ excitation and emission and that the initial drop remained after omitting these ionophores. A similar drop was also obtained with a high K+ solution with normal chloride or with Ames medium containing glutamate, suggesting it is a secondary effect of the depolarization. Similar excitation-induced chloride influx has also been reported by Inglefield and Schwartz-Bloom (1998). We concluded that the initial drop reflects a transient chloride influx due to the high K+ solution; afterward the ionophores integrate into the membrane and the new steady-state fluorescence reflects the equilibrated chloride level.
|
We first measured a cell's unperturbed MEQ fluorescence, then calibrated MEQ fluorescence by introducing a series of internal chloride concentrations of known values (Table 1). At the end of this series, MEQ fluorescence was completely quenched by adding 150 µM potassium thiocyanate (KSCN) and the K+ ionophore valinomycin (5 µM); the remaining fluorescence was the un-quenchable component Fnq. A cell's fluorescence was corrected for declining baseline as described in the preceding text and corrected for quenching by subtracting Fnq (Fig. 1, C and D). Each solution exchange caused an exponential decrease or increase in fluorescence. The decreases were best fitted with the equation
 |
and the increases with
 |
where FClhigh is the initial fluorescent, FCllow is the steady-state fluorescent after solution change, and t1/2 is the time at which the fluorescence reached the middle value between FClhigh and FCllow. We then used the asymptotes of these equations to extrapolate the steady state fluorescence in each solution (FCl). By calculating the ratio of F at 0 chloride (F0) over FCl for each cell, we constructed a Stern-Volmer plot and fit a straight line. The half quenching constant K50 was taken as the reciprocal of the line's slope (Marandi et al. 2002; Verkman 1990) (Fig. 1E). Although intracellular chloride can be estimated from these plots, this method was not used for many retinas because the procedure was long with low success rate.
Perforated-patch recording
A retina was mounted in a recording chamber and superfused with either bicarbonate-based or bicarbonate-free Ames medium. Before experiments started, the retina was maintained at room temperature. Cells in the retinal ganglion cell layer were visualized with DIC-infrared optics. Glass electrodes (tip resistance of 2–5 M
) were front-filled with gramicidin-free pipette solution (150 mM KCl, 10 mM HEPES, pH 7.25) and backfilled with the pipette solution containing gramicidin D (100–200 µg/ml). Membrane potential was amplified (Axopatch 200B, Axon Instruments, Foster City, CA), sampled at 5 kHz, and stored on a computer (PClamp8 software, Axon Instruments). Data were analyzed with Clampfit (Axon Instruments) and Excel. Gramicidin integrated into the intrapipette membrane 20–60 min after establishing a gigaohm seal, and access resistance stabilized shortly thereafter at values between 60 and 500 M. In general, access resistance was higher for cells in the first postnatal week than those in the second postnatal week (average: 268 vs. 182 M), possibly due to different membrane properties and its ability to incorporate gramicidin. However, EGABA measured with high access resistance did not differ from those with low access resistance, so all the data with a stable access resistance were included. Access resistance dropped to 20–70 M when the patch was ruptured by negative pressure to form the whole cell configuration. This was conducted in each recording to ensure that no occult break-ins occurred, and the perforated-patch recording mode was maintained in all experiments.
During recording, a retina was incubated in a recycled Ames medium containing O-(CNB-caged) GABA (1 mM; Molecular Probes), the Na+ channel blocker tetrodotoxin (TTX, 300 nM), the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM), and the N-methyl-D-aspartate (NMDA)-receptor antagonist, 2-amino-7-phosphonoheptanoic acid (AP-7, 100 µM). For retinas before P6, the nicotinic receptor antagonist curare (50 µM) was also added. GABA was focally uncaged at the soma by an ultraviolet laser pulse (5- or 10-ms laser pulse, the uncaging radius was 5 µm in the focal plane) every 20 s (Enterprise II, Coherent Scientific, South Australia, Australia). The apparent reversal potential was corrected for access resistance according to EGABA = Erev – Irev * Ra. Using Clampex 8.1 junction potential calculator, we estimated the liquid junction potential in bicarbonate-free medium to be +3.5 mV and that in bicarbonate-based medium to be +3.2 mV. Because these numbers are small and would not affect the difference of ECl between different developmental stages, the reported reversal potentials were not corrected. If not otherwise noted, all drugs were obtained from Sigma/Aldrich/RBI (St. Louis, MO).
|
|
RESULTS |
|---|
|
We first determined the time course of developing GABAergic neurons in the mouse retina (Fig. 2). At P2, immunostaining for GABA was positive throughout the retinal layers, but certain cell somas, located near the inner plexiform layer, were more strongly stained. The staining in the inner plexiform layer was evenly distributed and significantly stronger than staining resulting from the secondary antibody itself (3 retina). At P4, most of the somas in the neuroblast layer were light, but some, located at the position of newly migrated horizontal cells, were weakly stained, and the amacrine cells were strongly stained. The inner plexiform layer was now stained more strongly in two distinct strata. P8 resembled P4 except for more frequent amacrine cells and more prominent staining of displaced amacrine cells. At P21, the staining resembled the adult pattern: the inner plexiform layer was now prominently stained in two thin strata and a wider one near the ganglion cell layer. The number of stained amacrine cells in the inner nuclear and ganglion cell layers was fewer than at P8, and horizontal cells were undetectable. Counting cell profiles at P8 and P21, we estimated a reduction of about 30% in the number of cells in the inner nuclear layer and 40% in the ganglion cell layer [2 retinas for each age; total cells counted at P8, N(INL) = 274; N(GCL) = 99].
|
To determine the time course of the GABA switch, we used fura-2 and recorded calcium responses from neurons located at the ganglion cell layer at different developmental stages. At P0-5, GABA increased calcium in over 99% of the cells (799 cells, 19 retinas; Fig. 3, A and C). The GABAA receptor agonist muscimol also increased calcium, and the response was indistinguishable from that evoked by GABA (154 cells, 6 retinas). The GABA or muscimol-evoked calcium increase was robustly and reversibly blocked by the GABAA receptor antagonist picrotoxin (by 92.5 ± 1.2%; 57 cells, 2 retinas). This confirmed that in the first postnatal week of the mouse retina, GABA is excitatory, and the effect is mediated by ionotropic GABA receptors. The effect of GABA started to change at P6. Then GABA increased calcium in three retinas (54 cells) but failed to do so in two retinas (28 cells), and in one retina, it elicited both responses (33 cells). From P7 to P11, GABA or muscimol consistently failed to increase calcium in over 99% of the cells tested (125 cells, 6 retinas; Fig. 3, B and C). This indicated that at P6 GABA's excitatory action had started to diminish and that the switch to inhibition was complete by P7.
|
GABA-evoked calcium rise requires extracellular calcium
To determine whether GABA-evoked calcium rise is due to calcium influx through plasma-membrane channels or calcium release from intracellular stores, we initially tried to block calcium channels with cadmium, a general voltage-gated calcium channel blocker. However, cadmium introduced a large artifact: it elevated the ratio of fura-2 fluorescence at 340 and 380 nm, consistent with its high binding affinity to fura-2 (Hinkle et al. 1992). Instead we tested the effect of zero extracellular calcium. After observing GABA's response under normal Ames medium, the retina was switched to zero-calcium solution for 3–4 min; then GABA was applied in the presence of zero-calcium; finally, the retina was switched to normal Ames medium and tested again for response to GABA. On switching to zero-calcium buffer, the intracellular calcium concentration slightly decreased, probably due to a resting calcium conductance. Because of the tendency of GABA-evoked calcium rise to desensitize, only those cells with significant response to GABA in washout were used for analysis. In all such cells (68 cells, 6 retinas), zero-calcium solution greatly diminished GABA-evoked calcium rise (Fig. 4A). Thus an influx of calcium through the plasma membrane is the main contributor to GABA-evoked calcium rise in early retina.
|
To test whether the GABA-evoked calcium rise requires an efflux of HCO3–, we tested the effect of CO2/HCO3–-free (HEPES-based) medium. At P3, GABA consistently evoked calcium rise in over 99% of cells in the absence of HCO3–, comparable to that of control (Fig. 4B, 165 cells, 7 retinas). Thus the early excitatory action of GABA does not require HCO3–, suggesting that the depolarization by GABA is not due to a high permeability to this ion.
GABA switches from decreasing to increasing intracellular chloride around P6
Because the ionotropic GABA receptor conducts mainly chloride current, we next examined the effect of GABA directly on the intracellular chloride level with the chloride indicator MEQ (Fig. 5). At P0–P5, GABA or muscimol increased MEQ fluorescence in over 90% of the cells (230 cells, 9 retinas), indicating a decrease in intracellular chloride. At P6–P12, GABA or muscimol either decreased or caused no significant change in MEQ fluorescence (175 cells, 7 retinas).
|
ECl measured with gramicidin-perforated-patch recording
To test whether ECl indeed changes during development, we used the gramicidin-perforated-patch mode of recording on neurons in the ganglion cell layer. A cell was voltage clamped, its membrane potential was systematically varied using either a step or a ramp protocol, GABA was uncaged focally near the soma, and the reversal potential of the GABA response, EGABA, was calculated (Fig. 6, A and B). To test whether HCO3– contributes to EGABA, in some experiments, HCO3– was removed and replaced with HEPES. EGABA measured in either buffer was indistinguishable, suggesting HCO3– does not contribute significantly to EGABA in agreement with the calcium imaging results. Pooling data from all experiments, the average EGABA at P1–P3 was –45 ± 3 mV (7 cells), whereas at P7–P11, it was significantly lower with a mean value of –60 ± 2 mV (10 cells; P < 0.001, t-test; Fig. 6, C and D). After a perforated-patch recording, we disrupted the membrane using negative pressure, instituting the whole cell recording mode. This brought EGABA close to the chloride Nernst potential (5 mV) calculated using the composition of the intrapipette and extracellular media. This ensured that the perforated-patch recording mode was maintained throughout the recording period, and no occult breaking occurred. From our perforated-patch data, assuming that the predominant permeant ion for GABA responses is chloride, we calculate (using the Nernst equation) that the intracellular chloride concentration was 22 ± 2 mM for P1–P3 and 12 ± 1 mM for P7–P11.
|
To directly measure the intracellular chloride, we compared intracellular chloride to a set of specific reference chloride solutions. Reference chloride concentrations in these experiments were chosen according to intracellular chloride levels that would set ECl to values critically affecting chloride flux and its downstream effect on calcium flux or spiking (Table 2). There were three possible responses: if the initial intracellular chloride concentration was lower than the reference, fluorescence would be quenched by chloride influx; if intracellular chloride was higher than reference, fluorescence would increase with chloride efflux; if intracellular chloride was equal to reference, there would be no significant change (Fig. 7A). For each reference concentration, we counted the number of cells giving a certain response (Fig. 7B, Table 3). In general, all neighboring cells responded in a similar manner, suggesting amacrine and ganglion cells have similar chloride concentrations.
|
|
|
Fref (Eq. 1 in METHODS; see APPENDIX). We first estimated the average K50 value for retina from in vivo MEQ calibration experiments. For two retinas (P3 and P9), we obtained an average K50 of 75 mM (17 cells; see Fig. 1). We then measured the median Fref/F from each chloride reference experiment (Fig. 7A). Experiments with different reference solutions gave similar results, and were pooled. At P0–P5, the average intracellular chloride concentration was 29 mM with a range of 20–40 mM, at P6, it started to decline, averaging at 18 mM with a range of 10–30 mM, and after P6, it further declined to 14 mM with a range of 5–20 mM (Fig. 8B). |
|
DISCUSSION |
|---|
|
15 mV (from about –45 to –60 mV), and intracellular chloride level declines from 29 to 14 mM. Pros and cons of intracellular chloride measurement methods
Measurements of chloride are prone to various types of error. To address this, we used several methods and found reasonable agreement. Gramicidin-perforated patch measures GABA reversal potentials under conditions of unperturbed intracellular chloride. From these data, intracellular chloride can be calculated based on the assumption that EGABA = ECl. This is true only if the GABA receptor is relatively impermeant to bicarbonate ions. The accuracy of the measurement also relies on a good space clamp, which might be difficult to obtain, especially early in development when cells are highly coupled (Becker et al. 2002; Penn et al. 1994). In our experiment, we tested EGABA both in the presence and absence of HCO3– and alleviated the space-clamp problem by uncaging GABA focally onto the soma, so that much of the GABA-evoked current was close to the electrode.
The chloride-sensitive fluorescent dyes provide direct measurements of intracellular chloride and permit assessment of its spatial distribution. MEQ and its relatives MQAE and SPQ have been used to report relative chloride change and absolute chloride concentration in several brain regions (Fukuda et al. 1998; Ikeda et al. 2003; Inglefield and Schwartz-Bloom 1999; Kaneko et al. 2004; Marandi et al. 2002; Thoreson and Bryson 2004; Thoreson et al. 2000). MEQ is especially appealing among its relatives due to its improved cell retention and relatively high sensitivity, as reflected by a K50 of 50–100 mM found by this study and in other neurons (between 32 and 87 mM) (Chistina et al. 1999; Fukuda et al. 1998; Ikeda et al. 2003; Inglefield and Schwartz-Bloom 1998). However, chloride concentration measurement based on MEQ calibration carries a large error (30%) due to change in fluorescence baseline decay after introducing ionophores and high K+ (Nakamura et al. 1997). In addition, many cells do not survive the toxicity of long incubation in ionophores and high K+ (see also Kaneko et al. 2001).
To mitigate some of the problems associated with MEQ calibration, we developed the chloride reference method. It substantially shortens the experimental procedure and greatly enhances cell survival. It directly answers the question whether the intracellular chloride level is higher or lower than a certain level, which in our experiment was selected according to predictions made about GABA's function. Finally, if the reference concentration is chosen to be near the intracellular chloride level, the method can improve the accuracy of chloride measurement.
Intracellular chloride in the developing retina
By combining results from the different methods, we find that intracellular chloride can be divided into three stages. During the first postnatal week, intracellular chloride averaged 29 mM corresponding to an ECl of –39 mV, which is 21 mV more depolarized than average Erest (–60 mV). This is sufficiently depolarized to trigger most voltage-activated calcium and sodium channels (Table 2). Across cells, there was some variation in chloride concentration (20–40 mM). Thus some cells had a predicted ECl as hyperpolarized as –48 mV, which is below threshold for voltage-activated calcium channels. This might explain why a small percentage of cells failed to show a calcium rise at P1–P2.
During the transition period (P6), intracellular chloride in most cells exceeded 22 mM, which predicts an ECl above –46 mV, the threshold of low-voltage-activated calcium channels. This suggests that most cells would respond to GABA with a calcium increase, which is indeed what we found by fura-2 calcium imaging. At P7, although the average intracellular chloride was about 14 mM, corresponding to an ECl of –58 mV, there were still 50% of cells >22 mM; yet none responded with calcium increase. In rat, expression of major voltage-gated calcium channels shifts from low-voltage-activated calcium channels to high-voltage-activated calcium channels at around P6–P9 (Guenther et al. 1994; Schmid and Guenther 1996). Similar depolarizing shift in threshold for calcium activation have been observed for cultured hippocampal neuron (Ganguly et al. 2001). Thus the shift in calcium activation toward more depolarized potentials might account for the failure of GABA to evoke calcium increase at P7.
Finally, during the second postnatal week, intracellular chloride ranged from 5 to 20 mM, which predicts an ECl of –85 to –48 mV. This entire range of voltages would allow GABA to hyperpolarize or shunt the cell but would not trigger voltage-activated channels as was indeed demonstrated by calcium imaging during this developmental period. The average chloride concentration (14 mM) predicts an ECl of –58 mV and the perforated-patch recordings gave an average of –60 mV. These values are between the ECl measured in adult mudpuppy ganglion cells using a chloride-sensitive electrode (–49 mV) (Miller and Dacheux 1983) and in adult goldfish amacrine cells using gramicidin-perforated patch (–76 mV) (Watanabe et al. 2000). This might suggest that ECl approaches adult values by the end of the first postnatal week.
Timing and functional implication of GABA's actions
GABA switches from excitatory to inhibitory at P6, the same time as bipolar cells start to invade the inner plexiform layer (Fig. 9). Such coincidence between the time of GABA's switch and the time of bipolar cell axonal arborizations also occurs in ferret, chicken, and rabbit (Catsicas and Mobbs 2001; Fischer et al. 1998; Zhou 2001). It is thus reasonable to suggest that, as in other brain regions (Ben Ari 2002), GABA provides the major excitatory role before glutamate released from bipolar cells takes over.
|
; Katz and Shatz 1996; Lichtman and Colman 2000; Wiesel 1982). In retina, we suggest that before the switch, GABA released from amacrine cells could contribute to activity-dependent refinement of synaptic connections from amacrine to ganglion cells. This hypothesis stems from several observations. First, GABAergic amacrine cells stratify before ganglion cells, suggesting they could provide laminar cues (Karne et al. 1997). Furthermore, when ganglion cells start to stratify, GABA is still excitatory (Fig. 9), suggesting the cues could result from local excitation by GABA. Second, excitation is indeed important for normal stratification as shown in later development, when glutamate provides the major excitation. At this stage of development, preventing glutamate release from ON bipolar terminals disrupts the segregation of ganglion cell dendrites into ON and OFF sublamina (Bodnarenko and Chalupa 1993; Bodnarenko et al. 1995). Third, early in development, the major excitation is provided by acetylcholine and GABA. Cholinergic amacrine cells may provide laminar cues for one type of ganglion cells but not for the others (Stacy and Wong 2003), leaving GABA as the main candidate. Fourth, similar role for GABA has been demonstrated in the outer retina, where GABA is required for the normal development of cone to horizontal cells synapses (Huang and Redburn 1996). Our findings suggest that disrupting the accumulation of intracellular chloride could be used to test the contribution of GABA's excitation to the development of the inner retina.
Later, when GABA ceases to evoke calcium rise, blocking ionotropic GABA receptor elicits synchronized calcium transients in all cells. Similar results were found in ferret (Fischer et al. 1998), turtle (Sernagor et al. 2003), and rabbit (Syed et al. 2004), suggesting a common role of GABA in restricting and terminating retinal waves later in development.
|
|
APPENDIX |
|---|
|
Chloride quenches MEQ fluorescence in a concentration-dependent manner according to the Stern-Volmer equation (Verkman 1990)
 | (A1) |
Where K50 is the reciprocal of the Stern-Volmer quenching constant and is equal to the concentration that quenches 50% of the maximum quenchable signal (Marandi et al. 2002); F0 and FCl are the baseline-corrected quenchable signals at zero chloride and at a given chloride concentration, respectively.
Rearranging the equation gives
 | (A2) |
Because the left side of the equation is constant for a given MEQ concentration, the right side can be applied to the steady states in initial [Cl–]i and [Cl–]ref; this gives
 | (A3) |
Where FCell and Fref are the steady-state fluorescence values in [Cl–]i and [Cl–]ref, respectively.
Rearranging Eq. A3 gives
 | (A:4) |
Subtracting 1 from both sides, and introducing Fref/F = (Fref – Fcell)/Fcell into Eq. A4 yields
 | (A5) |
Rearranging gives intracellular chloride concentration as a function of Fref/F
|
| (A6) |
Note that when Fref/F = 0, [Cl–]i = [Cl–]ref; when Fref/F is small, an error in K50 estimation has small consequence on [Cl–]i measurement; when [Cl–]ref = 0, Eq. A6 becomes the Stern-Volmer Eq. A1.
|
|
GRANTS |
|---|
|
|
|
ACKNOWLEDGMENTS |
|---|
|
|
|
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: N. Vardi, 123 Anatomy and Chemistry Bldg., University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6058 (E-mail: noga{at}retina.anatomy.upenn.edu)
|
|
REFERENCES |
|---|
|
Becker DL, Bonness V, Catsicas M, and Mobbs P. Changing patterns of ganglion cell coupling and connexin expression during chick retinal development. J Neurobiol 52: 280–293, 2002.[CrossRef][Web of Science][Medline]
Ben Ari Y. Developing networks play a similar melody. Trends Neurosci 24: 353–360, 2001.[CrossRef][Web of Science][Medline]
Ben Ari Y. Excitatory actions of GABA during development: the nature of the nurture. Nat Rev Neurosci 3: 728–739, 2002.[CrossRef][Web of Science][Medline]
Bodnarenko SR and Chalupa LM. Stratification of ON and OFF ganglion cell dendrites depends on glutamate-mediated afferent activity in the developing retina. Nature 364: 144–146, 1993.[CrossRef][Medline]
Bodnarenko SR, Jeyarasasingam G, and Chalupa LM. Development and regulation of dendritic stratification in retinal ganglion cells by glutamate-mediated afferent activity. J Neurosci 15: 7037–7045, 1995.[Abstract]
Bormann J, Hamill OP, and Sakmann B. Mechanism of anion permeation through channels gated by glycine and gamma-aminobutryic acid in mouse cultured spinal neurons. J Physiol 385: 243–286, 1987.
Catsicas M and Mobbs P. GABAB receptors regulate chick retinal calcium waves. J Neurosci 21: 897–910, 2001.
Chistina GA, Inglefield JR, Schwartz-Bloom RD, Devaud LL, and Morrow AL. Fluorescence imaging of GABAA receptor-mediated intracellular [Cl–] in P19-N cells reveals unique pharmacological properties. Brain Res 827: 1–11, 1999.[CrossRef][Web of Science][Medline]
Diao L, Sun W, Deng Q, and He S. Development of the mouse retina: emerging morphological diversity of the ganglion cells. J Neurobiol 61: 236–249, 2004.[CrossRef][Web of Science][Medline]
Ehrlich I, Lohrke S, and Friauf E. Shift from depolarizing to hyperpolarizing glycine action in rat auditory neurons is due to age-dependent Cl- regulation. J Physiol 520 Pt 1: 121–137, 1999.
Eilers J, Plant TD, Marandi N, and Konnerth A. GABA-mediated Ca2+ signaling in developing rat cerebellar Purkinje neurones. J Physiol 536: 429–437, 2001.
Firth SI, Wang CT, and Feller MB. Retinal waves: mechanisms and function in visual system development. Cell Calcium 37: 425–432, 2005.[CrossRef][Web of Science][Medline]
Fischer KF, Lukasiewicz PD, and Wong RO. Age-dependent and cell class-specific modulation of retinal ganglion cell bursting activity by GABA. J Neurosci 18: 3767–3778, 1998.
Fisher LJ. Development of synaptic arrays in the inner plexiform layer of neonatal mouse retina. J Comp Neurol 187: 359–372, 1979.[CrossRef][Web of Science][Medline]
Fukuda A, Tanaka M, Yamada Y, Muramatsu K, Shimano Y, and Nishino H. Simultaneous optical imaging of intracellular Cl– in neurons in different layers of rat neocortical slices: advantages and limitations. Neurosci Res 32: 363–371, 1998.[CrossRef][Web of Science][Medline]
Ganguly K, Schinder AF, Wong ST, and Poo M. GABA itself promotes the developmental switch of neuronal GABAergic responses from excitation to inhibition. Cell 105: 521–532, 2001.[CrossRef][Web of Science][Medline]
Guenther E, Rothe T, Taschenberger H, and Grantyn R. Separation of calcium currents in retinal ganglion cells from postnatal rat. Brain Res 633: 223–235, 1994.[CrossRef][Web of Science][Medline]
Guenther E, Schmid S, Reiff D, and Zrenner E. Maturation of intrinsic membrane properties in rat retinal ganglion cells. Vision Res 39: 2477–2484, 1999.[CrossRef][Web of Science][Medline]
Harms KJ, Tovar KR, and Craig AM. Synapse-specific regulation of AMPA receptor subunit composition by activity. J Neurosci 25: 6379–6388, 2005.
Hinkle PM, Shanshala ED, and Nelson EJ. Measurement of intracellular cadmium with fluorescent dyes. Further evidence for the role of calcium channels in cadmium uptake. J Biol Chem 267: 25553–25559, 1992.
Huang BO and Redburn DA. GABA-induced increases in [Ca2+]i in retinal neurons of postnatal rabbits. Vis Neurosci 13: 441–447, 1996.[Web of Science][Medline]
Ikeda M, Yoshioka T, and Allen CN. Developmental and circadian changes in Ca2+ mobilization mediated by GABAA and NMDA receptors in the suprachiasmatic nucleus. Eur J Neurosci 17: 58–70, 2003.[CrossRef][Web of Science][Medline]
Inglefield JR and Schwartz-Bloom RD. Activation of excitatory amino acid receptors in the rat hippocampal slice increases intracellular Cl– and cell volume. J Neurochem 71: 1396–1404, 1998.[Web of Science][Medline]
Inglefield JR and Schwartz-Bloom RD. Fluorescence imaging of changes in intracellular chloride in living brain slices. Methods 18: 197–203, 1999.[CrossRef][Web of Science][Medline]
Kaila K, Pasternack M, Saarikoski J, and Voipio J. Influence of GABA-gated bicarbonate conductance on potential, current and intracellular chloride in crayfish muscle fibers. J Physiol 416: 161–181, 1989.
Kaneko H, Nakamura T, and Lindemann B. Noninvasive measurement of chloride concentration in rat olfactory receptor cells with use of a fluorescent dye. Am J Physiol Cell Physiol 280: C1387–C1393, 2001.
Kaneko H, Putzier I, Frings S, Kaupp UB, and Gensch T. Chloride accumulation in mammalian olfactory sensory neurons. J Neurosci 24: 7931–7938, 2004.
Karne A, Oakley DM, Wong GK, and Wong RO. Immunocytochemical localization of GABA, GABAA receptors, and synapse-associated proteins in the developing and adult ferret retina. Vis Neurosci 14: 1097–1108, 1997.[Web of Science][Medline]
Katz LC and Shatz CJ. Synaptic activity and the construction of cortical circuits. Science 274: 1133–1138, 1996.
Koizumi A, Jakobs TC, and Masland RH. Inward rectifying currents stabilize the membrane potential in dendrites of mouse amacrine cells: patch-clamp recordings and single-cell RT-PCR. Mol Vis 10: 328–340, 2004.[Web of Science][Medline]
Krapf R, Berry CA, and Verkman AS. Estimation of intracellular chloride activity in isolated perfused rabbit proximal convoluted tubules using a fluorescent indicator. Biophys J 53: 955–962, 1988.[Web of Science][Medline]
Leinekugel X, Tseeb V, Ben Ari Y, and Bregestovski P. Synaptic GABAA activation induces Ca2+ rise in pyramidal cells and interneurons from rat neonatal hippocampal slices. J Physiol 487: 319–329, 1995.
Lichtman JW and Colman H. Synapse elimination and indelible memory. Neuron 25: 269–278, 2000.[CrossRef][Web of Science][Medline]
Liu J, Morrow AL, Devaud L, Grayson DR, and Lauder JM. GABAA receptors mediate trophic effects of GABA on embryonic brainstem monoamine neurons in vitro. J Neurosci 17: 2420–2428, 1997.
Marandi N, Konnerth A, and Garaschuk O. Two-photon chloride imaging in neurons of brain slices. Pfluegers 445: 357–365, 2002.
Marty S, Berninger B, Carroll P, and Thoenen H. GABAergic stimulation regulates the phenotype of hippocampal interneurons through the regulation of brain-derived neurotrophic factor. Neuron 16: 565–570, 1996.[CrossRef][Web of Science][Medline]
Miller RF and Dacheux RF. Intracellular chloride in retinal neurons: measurement and meaning. Vision Res 23: 399–411, 1983.[CrossRef][Web of Science][Medline]
Nakamura T, Kaneko H, and Nishida N. Direct measurement of the chloride concentration in newt olfactory receptors with the fluorescent probe. Neurosci Lett 237: 5–8, 1997.[CrossRef][Web of Science][Medline]
Olney JW. Centripetal sequence of appearance of receptor-bipolar synaptic structures in developing mouse retina. Nature 218: 281–282, 1968.[CrossRef][Medline]
Owens DF, Boyce LH, Davis MB, and Kriegstein AR. Excitatory GABA responses in embryonic and neonatal cortical slices demonstrated by gramicidin perforated-patch recordings and calcium imaging. J Neurosci 16: 6414–6423, 1996.
Penn AA, Riquelme PA, Feller MB, and Shatz CJ. Competition in retinogeniculate patterning driven by spontaneous activity. Science 279: 2108–2112, 1998.
Penn AA, Wong RO, and Shatz CJ. Neuronal coupling in the developing mammalian retina. J Neurosci 14: 3805–3815, 1994.[Abstract]
Redburn DA. Development of GABAergic neurons in the mammalian retina. Prog Brain Res 90: 133–147, 1992.[Web of Science][Medline]
Rothe T, Juttner R, Bahring R, and Grantyn R. Ion conductances related to development of repetitive firing in mouse retinal ganglion neurons in situ. J Neurobiol 38: 191–206, 1999.[CrossRef][Web of Science][Medline]
Sassoè-Pognetto M and Wässle H. Synaptogenesis in the rat retina: subcellular localization of glycine receptors, GABAA receptors, and the anchoring protein gephyrin. J Comp Neurol 381: 158–174, 1997.[CrossRef][Web of Science][Medline]
Schmid S and Guenther E. Developmental regulation of voltage-activated Na+ and Ca2+ currents in rat retinal ganglion cells. Neuroreport 7: 677–681, 1996.[Web of Science][Medline]
Sernagor E, Young C, and Eglen SJ. Developmental modulation of retinal wave dynamics: shedding light on the GABA saga. J Neurosci 23: 7621–7629, 2003.
Shatz CJ and Stryker MP. Prenatal tetrodotoxin infusion blocks segregation of retinogeniculate afferents. Science 242: 87–89, 1988.
Sherry DM, Wang MM, Bates J, and Frishman LJ. Expression of vesicular glutamate transporter 1 in the mouse retina reveals temporal ordering in development of rod vs. cone and ON vs. OFF circuits. J Comp Neurol 465: 480–498, 2003.[CrossRef][Web of Science][Medline]
Stacy RC and Wong RO. Developmental relationship between cholinergic amacrine cell processes and ganglion cell dendrites of the mouse retina. J Comp Neurol 456: 154–166, 2003.[CrossRef][Web of Science][Medline]
Syed MM, Lee S, Zheng J, and Zhou ZJ. Stage-dependent dynamics and modulation of spontaneous waves in the developing rabbit retina. J Physiol 560: 533–549, 2004.
Thoreson WB and Bryson EJ. Chloride equilibrium potential in salamander cones. BMC Neurosci 5:53, 2004.[CrossRef][Medline]
Thoreson WB, Nitzan R, and Miller RF. Chloride efflux inhibits single calcium channel open probability in vertebrate photoreceptors: chloride imaging and cell-attached patch-clamp recordings. Vis Neurosci 17: 197–206, 2000.[CrossRef][Web of Science][Medline]
Ueda Y, Iwakabe H, Masu M, Suzuki M, and Nakanishi S. The mGluR6 5' upstream transgene sequence directs a cell-specific and developmentally regulated expression in retinal rod and ON-type cone bipolar cells. J Neurosci 17: 3014–3023, 1997.
Verkman AS. Development and biological applications of chloride-sensitive fluorescent indicators. Am J Physiol Cell Physiol 259: C375–C388, 1990.
Vu TQ, Payne JA, and Copenhagen DR. Localization and developmental expression patterns of the neuronal K-Cl cotransporter (KCC2) in the rat retina. J Neurosci 20: 1414–1423, 2000.
Wang GY, Olshausen BA, and Chalupa LM. Differential effects of apamin- and charybdotoxin-sensitive K+ conductances on spontaneous discharge patterns of developing retinal ganglion cells. J Neurosci 19: 2609–2618, 1999.
Watanabe S, Koizumi A, Matsunaga S, Stocker JW, and Kaneko A. GABA-mediated inhibition between amacrine cells in the goldfish retina. J Neurophysiol 84: 1826–1834, 2000.
Wiesel TN. Postnatal development of the visual cortex and the influence of environment. Nature 299: 583–591, 1982.[CrossRef][Medline]
Wong WT and Wong RO. Changing specificity of neurotransmitter regulation of rapid dendritic remodeling during synaptogenesis. Nat Neurosci 4: 351–352, 2001.[CrossRef][Web of Science][Medline]
Yamada J, Okabe A, Toyoda H, Kilb W, Luhmann HJ, and Fukuda A. Cl– uptake promoting depolarizing GABA actions in immature rat neocortical neurons is mediated by NKCC1. J Physiol 557: 829–841, 2004.
Young TL and Cepko CL. A role for ligand-gated ion channels in rod photoreceptor development. Neuron 41: 867–879, 2004.[CrossRef][Web of Science][Medline]
Yuste R and Katz LC. Control of postsynaptic Ca2+ influx in developing neocortex by excitatory and inhibitory neurotransmitters. Neuron 6: 333–344, 1991.[CrossRef][Web of Science][Medline]
Zheng JJ, Lee S, and Zhou ZJ. A developmental switch in the excitability and function of the starburst network in the mammalian retina. Neuron 44: 851–864, 2004.[CrossRef][Web of Science][Medline]
Zhou ZJ. Direct participation of starburst amacrine cells in spontaneous rhythmic activities in the developing mammalian retina. J Neurosci 18: 4155–4165, 1998.
Zhou ZJ. A critical role of the strychnine-sensitive glycinergic system in spontaneous retinal waves of the developing rabbit. J Neurosci 21: 5158–5168, 2001.
This article has been cited by other articles:
|
|
 |
|
  S. Di Marco, V. A. Nguyen, S. Bisti, and D. A. Protti Permanent Functional Reorganization of Retinal Circuits Induced by Early Long-Term Visual Deprivation J. Neurosci., October 28, 2009; 29(43): 13691 - 13701. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. Cook and D. L. Becker Gap-Junction Proteins in Retinal Development: New Roles for the "Nexus" Physiology, August 1, 2009; 24(4): 219 - 230. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X.-p. Tong, X.-y. Li, B. Zhou, W. Shen, Z.-j. Zhang, T.-l. Xu, and S. Duan Ca2+ signaling evoked by activation of Na+ channels and Na+/Ca2+ exchangers is required for GABA-induced NG2 cell migration J. Cell Biol., July 13, 2009; 186(1): 113 - 128. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-T. Wang, A. G. Blankenship, A. Anishchenko, J. Elstrott, M. Fikhman, S. Nakanishi, and M. B. Feller GABAA Receptor-Mediated Signaling Alters the Structure of Spontaneous Activity in the Developing Retina J. Neurosci., August 22, 2007; 27(34): 9130 - 9140. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Achilles, A. Okabe, M. Ikeda, C. Shimizu-Okabe, J. Yamada, A. Fukuda, H. J. Luhmann, and W. Kilb Kinetic Properties of Cl Uptake Mediated by Na+-Dependent K+-2Cl Cotransport in Immature Rat Neocortical Neurons J. Neurosci., August 8, 2007; 27(32): 8616 - 8627. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L.-L. Zhang, E. Delpire, and N. Vardi NKCC1 Does Not Accumulate Chloride in Developing Retinal Neurons J Neurophysiol, July 1, 2007; 98(1): 266 - 277. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
TOP
ABSTRACT
INTRODUCTION
, the median 
, putative amacrine cells (AM) or displaced amacrine cells (DA); 
, putative horizontal cells (HC). Note staining in IPL is diffuse at P2 and stratified at P4. NBL, neuroblast layer; IPL, inner plexiform layer; GCL, ganglion cell layer; OPL, outer plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; DA, displaced amacrine cells.
22 mM (
) and 30 mM (
) vs. age. Certain data are binned into a 2-day window (P2–P3, P7–P8. and P10–P11). Dotted line, concomitant change of calcium responses to GABA (from 
) are from 