GABAA Currents in Immature Dentate Gyrus Granule Cells

Ying Bing Liu, Gui-Lan Ye, Xue-Song Liu, Joseph F. Pasternak, and Barbara L. Trommer

From the Division of Pediatric Neurology, Evanston Hospital, Evanston, 60201; Departments of Pediatrics and Neurology, Northwestern University Medical School, Chicago, Illinois 60611

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Liu, Ying Bing, Gui-Lan Ye, Xue-Song Liu, Joseph F. Pasternak, and Barbara L. Trommer. GABAA Currents in immature dentate gyrus granule cells. J. Neurophysiol. 80: 2255-2267, 1998. We used whole cell patch clamp and gramicidin perforated patch recordings in hippocampal slices to study gamma -aminobutyric acid (GABA) currents in granule cells (GCs) from juvenile rat dentate gyrus (DG). GCs are generated postnatally and asynchronously such that they can be detected at different stages of their maturation in DG within the first month. In contrast, inhibitory interneurons are generated embryonically, and their circuitry is well developed even as their target GCs and GC excitatory connections are still being formed. In this study, two GABA currents evoked in GCs by medial perforant path stimulation are compared. The first, pharmacologically isolated by glutamate receptor blockade, is the product of direct activation of GABA interneurons with monosynaptic input to the recorded GC (monosynaptic GABAA). Monosynaptic GABAA displays slight outward rectification of its current-voltage relation, is 97% eliminated by 10 µM bicuculline and coincides temporally with the excitatory components of GC postsynaptic currents as has been described for GABAA currents in other brain regions. The second is a novel GABA response that is detectable in 10 µM bicuculline and is present on GCs only at the earliest stages of their maturation. Unlike monosynaptic GABAA, this transient GABA is eliminated by glutamate receptor blockade and hence is likely to be generated by interneurons activated via an intervening glutamatergic synapse (polysynaptically). It is predominantly chloride mediated, has a relative bicarbonate/chloride permeability ratio of 26%, and is unchanged by bath-applied saclofen and strychnine or by intracellular calcium chelation. It is 97% antagonized by 100 µM picrotoxin and 99% antagonized by 100 µM bicuculline. This current is thus a relatively bicuculline (BMI)-resistant GABAA current (BMIR-GABAA). Compared with monosynaptic GABAA, BMIR-GABAA has a later peak, slower time course of decay, and marked outward rectification. Its reversal potential is 7-8 mV depolarized to that of monosynaptic GABAA whether recorded in whole cell or with gramicidin perforated patch to preserve native internal chloride concentration. Together these data may suggest that BMIR-GABAA is evoked by an anatomically segregated population of interneurons activating a unique, developmentally regulated GABAA receptor. Further, the transient nature of this current coupled with its temporal characteristics that preclude overlap with the excitatory components of the synaptic response are consistent with a role that is trophic or signaling rather than primarily inhibitory.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The dentate gyrus (DG) undergoes postnatal development that is unique within the cerebral cortex. Neuroblasts destined to generate DG granule cells (GCs) migrate from the periventricular region to a secondary proliferative zone in what will become the DG hilus, and postmitotic immature neurons then further migrate to stratum granulosum. The vast majority of GCs are generated between postnatal days 5 and 7 (Schlessinger et al. 1975). When they first arrive in stratum granulosum, these immature neurons have a small soma and only short, simple, often bipolar processes (Cowan et al. 1980). During the next few days, the apical dendrites elongate into the molecular layer to make contact with the axons arriving from entorhinal cortex via the perforant path (Cowan et al. 1980; Rihn and Claiborne 1990). In addition, some of the postmigrational cells located along the hilar border of stratum granulosum retain proliferative potential and continue to generate new GCs locally (Rickmann et al. 1987; Schlessinger et al. 1978).

Because of this prominent and asynchronous postnatal proliferation, it is possible to record from GCs of markedly differing postgenerational ages within an anatomically restricted region of DG at a given animal age. We have demonstrated previously that in GCs recorded from the middle portion of the suprapyramidal limb of DG during the first postnatal month (Liu et al. 1996a), a reasonable estimate of cell maturity can be obtained from measurement of input resistance (IR). In particular, GCs with high IR (>= 1,000 MOmega ), tended to have depolarized resting membrane potentials and low-amplitude, long-duration action potentials, similar to immature neurons in other brain regions (Huguenard et al. 1988; Spigelman et al. 1992). Examination of a subset of high IR neurons filled with biocytin revealed short apical dendrites that just penetrated into the proximal molecular layer as previously described for immature GCs (Cowan et al. 1980; Lubbers and Frotscher 1988). GCs with low IR (<= 350 MOmega ) had more hyperpolarized resting potentials, higher amplitude, briefer duration action potentials, and extensive apical dendritic arborization extending throughout the entire molecular layer to the hippocampal fissure that is characteristic of mature GCs (Cowan et al. 1980; Rihn and Claiborne 1990; Seay-Lowe and Claiborne 1992). Consistent with this classification scheme, the proportion of high IR GCs decreased and low IR GCs increased with advancing animal age (Liu et al. 1996a).

DG inhibitory interneurons are generated prenatally, mainly between embryonic days 14 and 18 (Amaral and Kurz 1985; Lubbers et al. 1985) and are morphologically mature by postnatal day 7 (Seay-Lowe and Claiborne 1992). In consequence, an extensive plexus of GABAergic axons is already well developed when their target GCs and GC excitatory connections are still being formed (Lubbers and Frotscher 1988).

We previously have examined postsynaptic currents (PSCs) evoked in GCs of different maturities via medial perforant path stimulation (Liu et al. 1996a). gamma -Aminobutyric acid-A (GABAA)-, alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and N-methyl-D-aspartate (NMDA) receptor-mediated responses contributed to the PSCs in all GCs. The GABAA component was dominant at all cell maturities but proportionately greatest in immature GCs as might be predicted from the known discrepancy in GC and interneuron maturation.

The GABAA response was isolated pharmacologically using glutamate receptor blockade and therefore was produced by direct activation of GABA interneurons with monosynaptic input onto the recorded GC (monosynaptic GABA); it was almost entirely eliminated by addition of 10 µM bicuculline (BMI). We also recorded the pharmacologically isolated AMPA current (in 10 µM BMI, 50 µM 2-D-aminophosphonopentanoic acid, AP5) and found the expected early and fast PSCs that reversed near 0 mV. However, in some experiments we also found a later, unexpected, slower PSC component that was large when recording from immature neurons and small or absent when recording from mature neurons. In the experiments described in this report, we sought to characterize this late current. We found that it is also a GABAA current, distinct from the monosynaptic GABAA. Portions of this data have been presented in abstract form (Liu et al. 1996b).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Hippocampal slices were prepared from Sprague Dawley rats of both genders (8-32 days). Animals were anesthetized with isoflurane (>= 15 days only) and decapitated. The brains were removed quickly and placed in chilled (0-5°C) artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 124 NaCl, 3 KCl, 2.4 CaCl2, 1.3 MgSO4, 1.25 NaH2PO4, 26 NaHCO3, and 10 D-glucose (gassed with 95%O2-5%CO2, pH 7.4). After removal of the lateral cerebral convexities by parasaggital cuts, brains were bisected along the interhemispheric fissure, trimmed, and glued at the lateral surface to the specimen tray of a vibrating tissue slicer (Vibratome) with cyanoacrylate. Slices were cut at a thickness of 300-400 µm and the hippocampus subsequently was dissected free. Slices were stored in room-temperature gassed ACSF until ready for use.

Recording pipettes were pulled from borosilicate glass capillaries (OD 1.2, ID 0.68, World Precision Instruments) using a Flaming Brown model P-97 horizontal puller and had tip impedances 3-5 MOmega . Standard internal solutions contained (in mM) 117.5 potassium or cesium gluconate, 17.5 potassium methylsulfate or cesium methanesulfonate, 8 NaCl, 3 Mg2ATP, 0.2 GTP, 0.2 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 10 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) (pH 7.2, osmolarity adjusted to 290 mOsmol). In experiments requiring buffering of intracellular calcium, EGTA was eliminated and 10 mM 5,5'-dimethyl-bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) was added to the cesium standard internal solution. For experiments in which internal and external chloride concentrations were symmetrical, 123.8 mM CsCl was substituted for the cesium gluconate, and cesium methanesulfonate was decreased to 11.2 mM. The high bicarbonate/pH 8.28 internal solution contained (in mM) 90 cesium bicarbonate, 27.5 cesium gluconate, 17.5 cesium methanesulfonate, 8 NaCl, 0.2 EGTA, and 3 Mg2ATP 3 and the HEPES buffer was replaced by a bicarbonate/CO2 buffer. Gramicidin-containing internal solution (5 µg/ml) was prepared by adding 10 µl of gramicidin stock [0.05% in dimethyl sulfoxide (DMSO)] to 1 ml of the equimolar chloride internal solution just before use. Pipette tips were prefilled with gramicidin-free solution. Liquid junction potentials were determined to be 10 mV for standard solutions (Neher 1992), and 0 mV for the equimolar chloride and high-bicarbonate internal solutions; all reported membrane potentials were corrected appropriately.

Recordings were made in the suprapyramidal limb of the dentate gyrus granule cell layer approximately midway between the crest and the distal end of the limb. Slices were superfused with ACSF (3-4 ml/min) maintained at 30°C. Bipolar platinum stimulating electrodes (Rhodes Medical Instruments) were placed in the middle third of the molecular layer to activate the medial perforant path. PSCs were evoked with interstimulus intervals of 0.1, 0.05, or 0.033 Hz depending on the data being collected but were not varied within a given experimental protocol. Whole cell blind-patch recordings (Blanton et al. 1989) were obtained using an Axoclamp 2-A amplifier and pClamp6 software (Axon Instruments). Cell access was obtained in current clamp ("bridge") mode, and resting membrane potential was measured at the time of break-in. Input resistance was measured in response to hyperpolarizing pulses of 0.05-0.1 nA before switching to continuous single-electrode voltage-clamp mode. In gramicidin patch experiments, a giga seal was obtained in bridge mode, and recordings were monitored continuously until a stable resting membrane potential was evident (~10 min after gigaseal formation) after which recording was switched to voltage clamp at holding potential -70 mV. Current measurements were amplified 50 times before digitization using a custom-built amplifier. Whole cell recordings were rejected if the initial series resistance measured in current clamp was >30 MOmega , if the series resistance measured at the end of the experiment had changed, or if DC offset exceeded 5 mV after withdrawal from the cell. Gramicidin patch experiments were rejected if the series resistance at the end of the experiment exceeded 50 MOmega or if DC offset exceeded 5 mV after withdrawal. In some experiments, whole cell patch recordings were made visually using a Leitz Laborlux S microscope with water-immersion lens, an Axopatch 1-D amplifier, and PClamp6 software. In these experiments, cells were accessed in voltage-clamp mode, and input resistance was calculated from the steady-state current in response to a 5-mV, 35-ms duration hyperpolarizing pulse delivered 60 ms before each stimulus. Series resistance was monitored throughout using the extrapolated peak of the transient produced in response to the same pulse and was uncompensated. In GABA agonist experiments, GABA was added to the bath solution for 30 s at the indicated concentration and holding potential, and the peak current was measured.

Drugs were applied by bath perfusion. Drugs used were AP5 (40-50 µM, Tocris Cookson), BMI (5-10 µM, Sigma), picrotoxin (PTX, 100 µM, Sigma), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM, Tocris Cookson), strychnine (5 µM, Sigma), saclofen (500 µM, Tocris Cookson), tetrodotoxin (TTX, 1 µM; Sigma), and GABA (RBI). AP5, BMI, and GABA and TTX were prepared as stock solutions (50, 10, 50, and 1 mM, respectively) in distilled water; PTX was prepared as a stock solution in EtOH at 50 mM; CNQX was prepared as a stock solution in DMSO at 20 mM; strychnine was prepared as a stock solution in HCl at 25 mM; and saclofen was prepared as a stock solution in NaOH at 250 mM.

Stimulus-evoked PSCs were collected on the hard drive of an IBM-compatible 486 microcomputer, stored on external disk drive cartridges (SyQuest), and analyzed using the Clampfit subroutines of pClamp software. GABA agonist experiments were recorded using a Gould Windowgraf 900 chart recorder, and current amplitude was measured manually from the traces. Data were imported into Systat software (SPSS, Chicago) for statistical computations and graphing. The peak synaptically evoked current was measured as maximum amplitude of the current, and total charge was measured as the area under the curve described by the current trace. Cells were classified as mature if IR was <= 350 MOmega , immature if IR was >= 1,000 MOmega , and intermediate if 350 MOmega  < IR < 1,000 MOmega . We previously have demonstrated that this classification by IR can provide an estimate of cell maturity as determined morphologically (Liu et al. 1996a). In particular, cells with high IR appear immature, tending to have short dendritic arbors that penetrate only minimally into the molecular layer. In contrast, cells with low IR appear mature, with a more elaborated dendritic arbor that extends to the hippocampal fissure. Approximately a third of the cells in the present experiments were filled with biocytin (0.5%) to permit confirmation of cell maturity assignment by morphological criteria. Representative examples of each IR/maturity classification are illustrated in Fig. 1.


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FIG. 1. Representative biocytin-filled granule cells (GCs) of different maturities. A: immature GC [input resistance (IR) 2,300 MOmega ] has soma near hilar border of stratum granulosum and short dendritic branches that just penetrate stratum moleculare. B: intermediate GC (IR 800 MOmega ) has longer dendrites with deeper penetration into stratum moleculare; the wide angle subtended by the dendrites departing the soma is characteristic of immature/intermediate cells. C: mature GC (IR 280 MOmega ). Dendritic arbor subtends a narrower angle than in less mature cells, but the branches are elongated and most extend through the entire extent of stratum moleculare to the hippocampal fissure. Calibration bars are 40 µM.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Unique PSC in immature GCs

We evoked pharmacologically isolated GABAA PSCs in DG GCs by stimulating the medial perforant path in bath applied 10 µM CNQX and 50 µM AP5 (Fig. 2A). These GABAA PSCs likely were evoked by direct electrical activation of a subpopulation of GABA interneurons because the CNQX and AP5 would be expected to eliminate polysynaptically evoked GABA inhibition with an intervening glutamate synapse (such as feed-forward and feedback inhibition). For simplicity, we will refer to this pharmacologically isolated GABAA as the monosynaptic GABAA (mGABAA). mGABAA PSCs were evoked in GCs of all maturities. Overall, 10 µM BMI antagonized the mGABAA PSC by 94.2 + 1.5% (n = 12) when determined by measuring the peak currents and by 96.8 + 0.9% (n = 12) when determined by measuring total charge, and no differences in this level of antagonism were detected among GC of different maturities (P > 0.5)


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FIG. 2. Comparison of monosynaptic gamma -aminobutyric acid-A (GABAA) with the relatively bicuculline (BMI)-resistant GABA (BMIR-GABA) in immature and mature neurons. A: monosynaptic GABAA 1: families of synaptically evoked currents recorded in 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 50 µM 2-D-aminophosphonopentanoic acid (AP5) to isolate the monosynaptic GABAA current (mGABAA) demonstrate its similarity in immature and mature GCs. Holding potentials (in millivolts) are represented on or to left of each trace. 2: mGABAA is sensitive to 10 µM BMI. In this immature GC recorded at Vholding = +10 mV, the top trace is the compound postsynaptic current (PSC) [in artificial cerebrospinal fluid (ACSF)]; superimposed is the monosynaptic GABAA (in 10 µM CNQX and 50 µM AP5), which is almost completely eliminated by 10 µM BMI. B: families of synaptically evoked currents recorded in 50 µM AP5 and 10 µM BMI. Relatively bicuculline-resistant GABA is the late, outwardly rectifying current that follows the alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) current in the immature GC shown at the left. Inset: I-V relation for the GABA (bullet ) and AMPA (black-square) currents in this cell. In the mature GC (right) recorded under the same conditions, only the AMPA current is seen. Note difference in scale bars. Each trace represents the average of 4-6 sweeps.

When recording PSCs in bath-applied 10 µM BMI and 50 µM AP5, we found the expected early, fast AMPA PSC. In addition, we found that a late-occurring, unidentified PSC followed the AMPA in many GCs. This late PSC was seen more consistently and was of larger magnitude in immature than intermediate or mature GCs (Fig. 2B). It was blocked by 100 µM picrotoxin (96.8 ± 4.1% antagonism, mean ± SD; n = 6), 100 µM BMI (98.7 ± 1.7% antagonism, n = 4), and CNQX (98.4%, n = 3) but not by 500 µM saclofen (n = 4), 5 µM strychnine (n = 6), or addition of 10 µM BAPTA to the patch pipette (n = 4) (Fig. 3). The antagonism by 100 µM BMI was reversible. (Fig. 4). These pharmacological data suggested that the late PSC was likely a GABAA current and that it was relatively resistant to antagonism by BMI because we had evoked it in bath applied 10 µM BMI. For convenience, we will refer to this component current as the BMI-resistant (BMIR)-GABAA.


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FIG. 3. Pharmacological sensitivity of the BMIR-GABAA. A: BMIR-GABA is not eliminated by bath applied strychnine or saclofen or by including BAPTA in the recording electrode. It is thus not mediated by glycine or GABAB receptors nor by a calcium-activated chloride channel. These examples are recorded in immature GCs at Vholding = 0 mV. Total GABA is recorded with 50 µM AP5 in the bath. B: BMIR-GABAA (recorded in these immature neurons at Vholding = 0 mV in 10 µM BMI and 50 µM AP5) is eliminated by the subsequent addition of 100 µM picrotoxin (PTX) (left) and 100 µM BMI (middle). Unlike the mGABAA, which is evoked by direct activation of interneurons, BMIR-GABAA is eliminated by the addition of 10 µM CNQX (right), suggesting that it requires an intervening glutamatergic synapse for activation.


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FIG. 4. Reversibility of blockade of the BMIR-GABAA by 100 µM BMI. In this immature GC, the top trace represents the BMIR-GABAA recorded at Vholding = 0 mV in 10 µM BMI. PSC is eliminated by 100 µM BMI (bottom trace) and partially recovered after wash to 10 µM BMI. All recordings were made in ACSF containing 50 µM AP5.

Developmental regulation of sensitivity to BMI

PSCs evoked in GCs by medial perforant path stimulation in the presence of AP5 (50 µM) at Vholding = 0 mV (to make the AMPA component negligible) include both the mGABAA and BMIR-GABAA PSCs (compound PSC). To assess maturational changes in the proportion of the BMIR-GABAA component, we measured BMI sensitivity in a series of GCs of different maturities. To normalize the data across different GCs, we compared the magnitude of the PSC evoked before and after the addition of 10 µM BMI. We found that in immature GCs, 31 ± 14% of the compound PSC (total charge) remained after addition of 10 µM BMI compared with only 8 ± 5% in mature GCs (Table 1).

 
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TABLE 1. Relatively BMI-resistant GABA

To demonstrate the differences in BMI sensitivity between the exclusively monosynaptically evoked GABAA PSC and the compound PSCs, we obtained dose response curves. Immature neurons were used exclusively in these experiments to maximize the BMIR-GABAA. We demonstrated that the dose response curve for the mGABAA recorded at Vholding = 0 in 50 µM AP5 and 10 µM CNQX differed from the corresponding curve for the compound PSC recorded in 50 µM AP5 at Vholding = 0 (Fig. 5).


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FIG. 5. Dose-response curves for the mGABAA PSC (black-square) and the compound GABAA PSC (bullet ). For mGABAA each point represents the mean ± SE of 2-4 cells. For total GABAA, each point represents the mean ± SE of 4-6 cells. Whereas the mGABAA was 94% antagonized by 5 µM BMI and 97% antagonized by 10 µM BMI, the total GABAA was only 69-72% antagonized at these concentrations. The total GABAA PSC was 97% antagonized by 50 µM BMI. For uniformity, only immature GCs were used in both dose response curves. - - -, calculated dose response curve for the polysynaptically mediated GABAA, which was derived from the other 2 curves (see text) and which differs from the mGABAA because of the contribution of the BMIR-GABAA.

To make the comparison more specific, we also plotted a calculated dose-response curve for the polysynaptically evoked PSC. We have shown previously that the compound GABA PSC in immature GCs is 37% monosynaptically and 63% polysynaptically mediated (Liu et al. 1996a). Therefore the fraction of polysynaptic PSC remaining after addition of BMI was derived from the mGABAA and compound PSC dose-response curves by subtracting 37% of the fraction of remaining mGABAA from the fraction of remaining compound PSC and dividing the difference by 0.63 for each concentration. By definition, the BMIR PSC is included entirely in the polysynaptic component because it was detected in a concentration of BMI (10 µM) that eliminated virtually all of the mGABAA. The precise BMI sensitivity of the BMIR-GABAA cannot be determined from this protocol because the makeup of the polysynaptic component remains unknown. For example, it may consist of two currents, one completely resistant to 10 µM BMI and the other sensitive or, alternatively, a single current with intermediate sensitivity to 10 µM BMI. Nevertheless the difference in BMI sensitivity between mGABAA and the polysynaptic PSC results from the contribution of the BMIR-GABAA.

Reversal potentials, ionic conductance, and rectification of the BMIR-GABAA

We compared the I-V relations of the mGABAA and BMIR-GABAA PSCs. For these and subsequent I-V relations, the mGABAA amplitude was measured at its peak at each holding potential; the BMIR-GABAA amplitude at each holding potential was measured at the latency corresponding to the peak current where it was most readily identified (i.e., at the most depolarized holding potential). The mGABAA I-V relation showed slight outward rectification (Fig. 6A), whereas the BMIR-GABAA I-V relation showed more marked outward rectification with an inflection near its reversal (Fig. 6B). Both reversed near the calculated Cl- reversal potential (-71 mV for our preparation): -69 ± 5 mV (n = 18) for the mGABAA PSC versus -61 ± 5 mV (n = 8) for the BMIR-GABAA (Table 2). The differences in mGABAA and BMIR-GABAA reversal potentials were statistically significant, (P < 0.0001), raising the possibility that they are mediated by different relative ionic conductances. For example, if we assume that the GABAA PSCs are mediated by conductances only for Cl- and HCO-3, the -69 mV reversal implies a relative HCO-3 to Cl- conductance of 4%, whereas a -61 mV reversal implies a relative HCO-3 to Cl- conductance of 29%.


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FIG. 6. I-V relations for mGABAA and BMIR-GABAA recorded using standard gluconate-containing internal solution in whole cell mode. A: I-V relation for the monosynaptic GABAA current shows slight outward rectification and a reversal potential of -69 ± 5 mV (each point = mean ± SE for n = 18 cells; current normalized to peak current at Vholding = -50 mV). B: I-V relation for the BMIR-GABAA showing prominent outward rectification and reversal potential of -61 ± 5 mV (each point = mean ± SE for n = 8 cells; current normalized to peak current at Vholding = +30 mV).

 
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TABLE 2. Effects of internal solution on reversal potentials of BMIR-GABAA and mGABAA

To pursue this possibility, we performed a number of experiments to confirm or refute a difference in the relative Cl- to HCO-3 conductance of the mGABAA and BMIR PSCs. To assess their Cl- dependence, we used a patch-pipette solution that contained Cl- equimolar to the extracellular solution (i.e., 131.8 meq) to evoke mGABAA PSCs (n = 5) or BMIR PSCs (n = 6). Both PSCs reversed near 0 mV as would be expected if they are mediated by Cl- (Table 2, and Fig. 7A). Further, the I-V relation of the BMIR PSC in these experiments (i.e., when CsCl was substituted for the cesium gluconate in the internal solution to render the chloride concentrations symmetrical) showed only minimal outward rectification in contrast to the marked rectification that was apparent using the standard electrode solution (Fig. 7A).


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FIG. 7. Both mGABAA and BMIR-GABAA are carried by chloride and bicarbonate ions in similar proportions. A: I-V relations recorded with internal solution containing chloride concentration 131.8 meq, equimolar to the extracellular bath solution. 1: mGABAA PSC reversed at -0.2 ± 2.2 mV (n = 5). 2: BMIR-GABAA reversed at 1.4 ± 1.7 mV (n = 6). Currents are normalized to the peak current recorded at Vholding = +50 mV. Insets: representative families for each GABAA PSC. B: I-V relations recorded with internal solution containing high (82 mM, pH 8.0) bicarbonate. 1: BMIR-GABAA reversal potential was -45 ± 3 mV (n = 6). 2: mGABAA PSC reversal potential was -45 ± 5 mV (n = 6). Currents are normalized to the peak current recorded at Vholding = -10 mV (mGABAA) or + 20 mV (BMIR-GABAA). Insets: representative families for each PSC.

We next investigated the HCO-3 dependence of both PSCs by using a patch solution with high (82 mM, pH 8.0) HCO-3 (Fig. 7B). With this high HCO-3 patch solution, the reversal potential of the mGABAA PSC was -45 ± 5 mV (n = 6), and the reversal potential of the BMIR-GABAA was -45 ± 3 mV (n = 6). Because with this preparation the calculated HCO-3 reversal is +31 mV, a reversal of -45 mV corresponds to a relative HCO-3 conductance of 26% for both currents. Outward rectification of the I-V relation of the BMIR-GABAA was again apparent (Fig. 7B).

We measured the I-V relationships of the mGABAA and BMIR-GABAA using gramicidin perforated-patch recording configuration to preserve physiological intraneuronal Cl- (Ebihara et al. 1995; Kyrozis et al. 1995). In immature neurons, the BMIR-GABAA reversal potential (-42 ± 4 mV, n = 8, Fig. 8A) was positive to the mGABAA PSC reversal potential (-49 ± 6 mV, n = 8, Fig. 8B1). We used the PSCs evoked at Vholding = -10 mV in these gramicidin experiments to further quantitate the differences between the two currents in immature GCs. These differences are likely to reflect several factors including synaptic circuitry and receptor location as well as receptor kinetics. We found (Table 3) that both the decay time (from peak to half-maximal current) and the latency (from stimulus artifact to peak) were longer in the BMIR-GABAA than the corresponding values for the mGABAA and that the peak amplitude of the BMIR-GABAA was smaller.


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FIG. 8. I-V relations recorded using perforated-patch technique (gramicidin) to determine reversal potentials with native intracellular chloride. A: BMIR-GABAA reversal potential (recorded in immature neurons only, n = 8) was -42 ± 4 mV. B: mGABAA reversal potential was maturity dependent. 1: in immature neurons (n = 8), the mGABAA PSC reversal potential was -49 ± 6 mV. 2: in mature neurons (n = 5), the mGABAA PSC reversal potential was -65 ± 6 mV. Currents were normalized to the peak current recorded at Vholding = -10 mV in the immature neurons and -30 mV in mature neurons.

 
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TABLE 3. Properties of BMIR-GABAA and mGABAA PSCs using gramicidin perforated patch

Finally, we demonstrated that with native chloride/gramicidin perforated patch recordings the mGABAA reversal potential in immature GCs (-49 ± 6 mV, n = 8) differed from that of mature GCs (-65 ± 6 mV, n = 8, Fig. 8B2) measured in the same manner (P < 0.001). Assuming an intracellular pH of 7.2 and a bicarbonate-to-chloride permeability ratio of 0.26, these results correspond to an internal chloride concentration of 16 mM in immature cells versus 7 mM in mature cells.

Agonist experiments

As indicated in the preceding text, we have used the term BMIR-GABAA because both the pharmacological profile and the ionic dependence of this late PSC in immature GCs suggest that it is a GABAA-receptor-mediated current. In this context, however, its sensitivity to CNQX bears further investigation. In particular, it is possible that CNQX could be directly antagonizing the PSC at the receptor and that the late PSC is a glutamate-receptor-mediated Cl- current unique to immature GCs. We therefore investigated whether a comparable current could be evoked directly by bath application of GABA and whether this current was sensitive to CNQX. To simulate the physiological conditions in which the synaptically evoked BMIRGABAA was detected, we held cells at 0 mV, included AP5 (50 µM) in the perfusate, and sought a GABA concentration that would evoke a robust current in 10 µM BMI but no current or minimal current in 100 µM BMI. A dose-response curve (100 µM to 1 mM GABA) was constructed in 100 µM BMI using immature GCs (n = 8) and normalizing the evoked current at each concentration to the maximum current evoked by 1 mM GABA (Fig. 9; mean maximum current = 241 ± 126 pA). Under these conditions, no current was evoked by 100-150 µM GABA in 100 µM BMI, and only 4.7 ± 2.6% of the maximum current was evoked by 200 µM GABA. We therefore used bath application of 200 µM GABA in 10 µM BMI to construct I-V curves in immature GCs (n = 9). I-V curves obtained in the presence (n = 5) or absence (n = 4) of TTX (1 µM) did not differ and were pooled. As is illustrated in Fig. 10, this GABA-evoked current showed marked outward rectification and had a reversal potential of approximately -70 mV, the calculated chloride reversal potential using the cesium gluconate-containing electrode solution. In three additional cells, we measured the current evoked by 200 µM GABA in 10 µM BMI before and after application of CNQX (10 µM) and found that the peak amplitude of the current was unchanged (Fig. 11). Thus in immature GCs, GABA evokes a chloride current with rectification properties similar to those of the BMIRGABAA under comparable conditions of BMI antagonism. This current is insensitive to CNQX. These data suggest that the synaptically evoked BMIR-GABA is produced by activation of a GABAA receptor and that CNQX antagonizes this current at a point different from the receptor itself.


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FIG. 9. BMI (100 µM) antagonizes 200 µM GABA nearly completely. To construct this dose-response curve, immature GCs (n = 8) were held at Vholding = 0 mV and perfused with ACSF containing 100 µM BMI and 50 µM AP5. Six 30-s GABA applications were delivered to each cell, and the peak current at each concentration was normalized to the maximum peak current evoked by application of 1 mM GABA for each cell. No current was evoked by 100-150 µM GABA; 200 µM GABA evoked a current that was 4.7 ± 2.6% of the maximum.


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FIG. 10. GABA evokes a current similar to the BMIR-GABAA in immature GCs. A: I-V relation for the current evoked by 30-s bath applications of 200 µM GABA was recorded in immature GCs using the standard cesium gluconate internal solution. Order of Vholding (depolarized 1st vs. hyperpolarized 1st) was alternated to control for possible rundown of the current over time. I-V relation shows the marked outward rectification characteristic of the BMIR-GABAA evoked synaptically in immature GCs. (Each point represents mean ± SE for n = 9 cells; current normalized to peak current at Vholding = 0 mV;). Bath solution contained 10 µM BMI and 50 µM AP5 in all cells. In n = 5 cells, 1 µM tetrodotoxin (TTX) also was included. Presence or absence of TTX did not affect the results, which therefore were pooled. B: representative example of current traces from a cell used in A in which TTX was included in the perfusate. Numbers above each trace indicate Vholding .


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FIG. 11. GABA-evoked current is not altered by CNQX. Top: recorded in 10 µM BMI and 50 µM AP5. Middle: recorded after addition of CNQX (10 µM). Bottom: recorded after the addition of 100 µM BMI to the perfusate. GABA application bar applies to all traces.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The major result of this study is that GABAA currents in DG granule cells at their earliest stages of maturation demonstrate a novel response to medial perforant path stimulation. In addition to the previously described monosynaptically activated GABAA current (mGABAA) (Liu et al. 1996a), these cells exhibit a GABAA current (BMIRGABAA) that is relatively resistant to BMI, has more prominent rectification properties, has a slow, late time course, and is not seen when AMPA receptors are blocked by CNQX.

Although BMIR-GABAA is dependent on intact AMPA receptors, this dependence is not likely to implicate a glutamate-receptor-mediated chloride current: BMIR-GABAA could be simulated by direct application of GABA in BMI even in the presence of CNQX. However, the AMPA receptor-dependence of the synaptically evoked BMIR does imply that the responsible interneurons must be activated by intervening glutamatergic synapses (i.e., polysynaptically), particularly because the concentration of BMI (10 µM) in which it is seen eliminates virtually all (97%) of the mGABAA PSC. This contrasts with the mGABAA PSC, which is evoked by interneurons directly activated by the stimulating electrode, because this current is detected in drugs (CNQX and AP5) that would be expected to eliminate intervening glutamatergic synapses.

Several observations confirm that the BMIR-GABA, like the monosynaptic GABA, is GABAA mediated. In both currents, the reversal potential shifted to near 0 mV in symmetric chloride solutions, indicating that chloride is the major permeant anion. Further, BMIR-GABAA was sensitive to 100 µM PTX, and could be eliminated by BMI in high enough (100 µM) concentrations. BMIR-GABAA was not eliminated by bath-applied strychnine or saclofen or BAPTA in the internal solution, suggesting that it is not mediated by either glycine or GABAB receptors and is not a calcium-activated chloride conductance. It is also unlikely to be activated by the GABAC receptor because the latter is insensitive to 100 µM BMI (Qian and Dowling 1995). The reversal potentials for both currents showed a comparable shift in the depolarizing direction when internal bicarbonate was increased. The relative bicarbonate/chloride permeability determined from this shifted reversal potential was similar for both currents (26%) and was consistent with that previously reported (20-30%) for postsynaptic GABAA receptor channels in rat neocortical neurons (Kaila et al. 1993).

Despite these similarities, the marked differences in kinetics and sensitivity to BMI and CNQX between BMIRGABAA and mGABAA raise important questions about the anatomic localization and subunit composition of their respective receptors. In particular, it is possible that BMIR-GABAA is mediated by a novel synaptic receptor unique to developing granule cells. Alternatively, the two currents may be mediated by the same receptor, and the characteristic features of BMIR-GABAA may reflect its anatomic segregation (e.g., a distal dendritic entry point) and/or functional segregation (activation by a restricted population of afferent interneurons). Finally, it is possible that BMIR-GABAA is mediated by a receptor that is both unique in subunit composition and functionally and anatomically separate from the mGABAA receptor.

There is considerable anatomic evidence for segregation of GABAA subpopulations. For example, in mature DG there are at least five types of GABAergic neurons (Freund and Buzsaki 1996; Nusser et al. 1995; Soltesz et al. 1995) that terminate on largely mutually exclusive GC domains. In particular, axo-axonic cells exclusively innervate the axon initial segment, whereas basket cells make contact with the somata and proximal dendrites of granule cells. Evidence for corresponding functional segregation in DG is more limited, although Soltesz et al. (1995) used a combination of whole cell patch-clamp and computational techniques to demonstrate that proximal but not distal synapses are responsible for tonic action-potential independent inhibition. In CA1 pyramidal cells Pearce (1993) demonstrated two spatially segregated physiologically and pharmacologically distinct BMI sensitive GABAA PSCs: a furosemide-sensitive response that enters near the soma, rapidly curtails the excitatory response, and decays in 3-8 ms and a furosemide-insensitive dendritic response that decays over 30-70 ms and underlies the conventional early inhibitory postsynaptic potential. These two currents were attributed to two different GABAA receptor subtypes thought to be contacted by different populations of interneurons (Banks et al. 1998; Pearce 1993).

We hypothesize that the glutamate dependence of BMIR-GABAA results from its activation by an anatomically and/or functionally distinct set of interneurons that require an intervening glutamatergic synapse. Further, because BMIR-GABAA regresses with GC maturation, we speculate that these interneurons activate a novel GABAA receptor that is characteristic of immature granule cells. Several studies have demonstrated that GABAA subunit composition is regulated developmentally, that the switchover from immature to mature subunit composition requires GABAA receptor activity, and that the presynaptic interneuron provides the cues for synaptic clustering of receptor subtypes (Brooks-Kayal and Pritchett 1993; Fritschy et al. 1994; Hollrigel and Soltesz 1997; Mohler et al. 1996; Nusser et al. 1996; Poulter et al. 1997).

The prominent outward rectification of BMIR-GABAA compared with mGABAA is consistent with its dependence on a novel receptor. In our experiments, mGABAA showed modest outward rectification similar to that previously reported in response to applied GABA in whole cell or single-channel recordings from hippocampal pyramidal and granule cells, spinal neurons, and reconstituted GABAA channels in transfected human embryonic kidney cells as well as in synaptically evoked inhibitory PSCs in hippocampal neurons in culture (Ashwood et al. 1987; Barker and Harrison 1988; Birnir et al. 1994; Bormann et al. 1987; Curmi et al. 1993; Fatima-Shad and Barry 1993; Gray and Johnston 1985; Verdoorn et al. 1990). Rectification of BMIR-GABAA was much greater: in the standard (low chloride) internal solution, there was minimal current flow below its reversal potential. There is considerable evidence from molecular studies that rectification is influenced by subunit composition. For example, in recombinant rat GABAA receptors expressed in human embryonic kidney cells, outward rectification appears to be conferred largely by the beta 2 subunit (Verdoorn et al. 1990) and mutation-induced outward rectification has been used to deduce stochiometric relations among alpha , beta , and gamma  subunits (Backus et al. 1993). In contrast, normal outward rectification is abolished in recombinant receptors by a newly described human GABAA subunit (epsilon ), which, by virtue of its high density of basic residues, is thought to sequester chloride ions near the intracellular face of the ion channel (Davies et al. 1997). In the present study, I-V curves for both mGABAA and BMIR-GABAA were constructed in comparable populations of GCs using comparable internal solutions. Not only did BMIR-GABAA show much more dramatic rectification in the standard solution (Figs. 6B and 7B), but its I-V relation reverted to nearly linear in symmetrical chloride (Fig. 7A). Because preparation of symmetrical chloride solutions also entailed elimination of gluconate in the pipette, one possibility is that the gluconate itself caused a voltage-dependent block of the BMIR-GABAA receptor channel and did not alter the mGABAA-associated channel. An analogous role has been proposed for cytoplasmic polyamine cations (spermine, spermidine) in the intrinsic gating of cationic currents through inward rectifier potassium channels (Ficker et al. 1994; Lopatin et al. 1994) and calcium permeable AMPA channels (Williams 1997).

Despite the molecular evidence presented above, rectification is clearly not determined exclusively by subunit composition and may be influenced by constant field considerations (Hammill et al. 1983), voltage-dependent alterations in channel kinetics or conductance (Ashwood et al. 1987; Gray and Johnston 1985) and/or voltage-dependent coupling of GABA-activated cochannels (Curmi et al. 1993). Thus the hypothesis that mGABAA and BMIR-GABAA are mediated by the same receptor despite their differential rectification properties is tenable with a considerable number of constraints. For example, it is conceivable that the two currents have spatially segregated entry points and that intracellular chloride gradients lead to a difference in rectification that is exaggerated in low internal chloride and diminished in equimolar chloride because of a constant field effect. It is also possible that the rectification of BMIR-GABAA is not a property of the receptor/ionophore complex per se, but that the entry point for this current is selectively located near an active voltage-sensitive inwardly rectifying chloride conductance that expels much of the intracellular chloride at holding potentials below ECl. Such a conductance has been proposed to account for apparent outward rectification of GABA responses in mature CA1 cells but was not seen in dentate GCs (Staley 1994).

We also considered that the relatively depolarized reversal potential of BMIR-GABAA might suggest a novel receptor. Although small, the difference in reversal potential was statistically significant and was consistent whether recorded with standard internal solution (-61 vs. -69 mV) or using gramicidin perforated patch (-42 vs. -49 mV). Hypothetically, such a difference could result if the novel receptor were more permeable to bicarbonate than the mGABAA receptor (Bonnet and Bingmann 1995), but our data were not consistent with this explanation. The possibility that the depolarized reversal of BMIR-GABAA represents contamination by AMPA current seems unlikely. The duration of the AMPA response (from stimulus artifact to recovery) isolated at hyperpolarized potentials in these cells, as well its duration in other immature cells recorded in PTX (unpublished), was ~20 ms. This is very much shorter than the latency at which the peak BMIR-GABAA current was measured (45 ± 22 ms) in the present study. The most likely explanation may be that BMIR-GABAA has a more distal dendritic entry point than mGABAA. In this scenario, an intracellular chloride gradient would have to exist such that the chloride equilibrium potential was maintained more positive in distal dendrites than in proximal dendrites or soma. The existence of such a gradient maintained by spatially segregated inward and outward chloride pumps has been postulated in adult CA1 and CA3 pyramidal cells. Both manifest a biphasic hyperpolarizing (reversal potential -70 mV)/depolarizing (reversal potential -50 mV) GABAA-mediated response to GABA application with the depolarizing response predominating when the GABA application is dendritic (Alger and Nicoll 1979; Misgeld et al. 1986; but see Lambert et al. 1991; Perkins and Wong 1996). In adult GCs, both GABA-evoked and synaptic mGABAA PSCs are depolarizing (Liu et al. 1996a; Misgeld et al. 1986; Staley and Mody 1992), and thus although both inward and outward chloride pumps have been postulated, their spatial distribution is thought to be homogeneous (Misgeld et al. 1986). No comparable data are available for immature GCs the fine distal dendritic branches of which make it more difficult to eliminate the possibility of a reversed chloride gradient (Lambert et al. 1991) and which may differ from mature GCs with respect to the presence/distribution of factors such as voltage-gated chloride channels (Staley et al. 1996) that regulate the transmembrane anionic concentration gradient. If BMIR-GABAA has a distal dendritic entry point, its apparent reversal potential measured at the soma could be shifted further in the depolarizing direction by a contribution from nonisopotential synapses secondary to inadequate voltage clamp. Such a contribution is likely to be small, however, because of the short simple dendritic arbor in immature GCs and because the reversal potential of BMIR-GABAA was measured near zero (1.4 mV) in symmetrical chloride.

Neither the relative BMI insensitivity of BMIR-GABAA nor its time course is strongly suggestive of differential subunit composition. BMI sensitivity to concentrations of 5-100 µM did not distinguish among receptors containing the isoforms alpha 1, beta 1, gamma 2S, and gamma 2L (Krishek et al. 1996). Because BMI is a competitive GABAA inhibitor, it is possible that the relative BMI insensitivity reflects differences in the amount of presynaptic GABA release or factors that differentially restrict access to the postsynaptic membrane. The late peak and smaller peak amplitude of BMIR-GABAA are most likely the result of the asynchronous GABA release implied by polysynaptic activation.

Thus far we have considered only one explanation for the glutamate dependence of BMIR-GABAA: that it results from activation by a segregated population of interneurons activated polysynaptically. An alternative hypothesis is that in the presence of 10 µM BMI and with AMPA receptors unblocked, dentate excitatory projections are activated so intensely that interneurons fire in bursts. Most of the immature GCs in this study were recorded in 8- to 15-day-old animals. At this age, GABA removal (rather than properties of the receptor/ionophore complex) is believed to be rate-limiting for decay of mGABAA PSCs in DG, but during this time period the mechanism of removal is in transition from diffusion (postnatal days 4-6) to reuptake (days 13-16) (Draguhn and Heinemann 1996). Thus in these young rats, the synchronized recruitment of large numbers of GABA synapses (interneuron bursting) may be coupled with an immature GABA reuptake system. In this situation the synaptic GABA concentration could be sufficiently high to generate a synaptic GABA response despite the 10 µM BMI and also to diffuse sufficiently far to activate neighboring synapses or extrasynaptic receptors (Roepstorff and Lambert 1994). The absence of BMIR-GABAA from mature GCs might reflect the greater efficiency of the uptake system because the mature cells were recorded from older (26-28 day) rats. Extrasynaptic GABAA receptors have been demonstrated anatomically in adult cat DG (Nusser et al. 1995). Immature GCs may have more such receptors or may have different extrasynaptic receptors with particularly high affinity for GABAA such as the alpha 6 subunit-containing receptor, which recently has been shown to promote spillover-mediated inhibitory synaptic transmission in cerebellar granule cells (Rossi and Hamann 1998).

Whatever its receptor or mechanism of activation, BMIR-GABAA is a late-occurring relatively depolarizing current. Together these factors suggest that its function may not be primarily inhibitory. The relationships among GABAA reversal potentials, resting membrane potentials, and action potential threshold were not systematically measured in individual neurons in the present study. However, in a prior study of 27 immature and 53 mature GCs (Liu et al. 1996a), group mean resting membrane potential was -54 ± 6 mV immature GCs and 82 ± 7 mV in mature GCs. Group mean action potential threshold was -28 ± 5 mV in immature GCs and -43 ± 4 mV in mature GCs. The mGABAA reversal potential in our mature GCs is similar to that previously reported for adult rat GCs in which a monosynaptic GABAA current activated by lateral perforant path stimulation reversed at approximately -66 mV (Staley and Mody 1992). This current, the reversal potential of which was both depolarized with respect to resting membrane potential (-85 mV) and hyperpolarized to mean action potential threshold (-49 mV), was shown to participate in shunting inhibition (Staley and Mody 1992). In the present study, both mGABAA and BMIR-GABAA in immature GCs have reversal potentials that are depolarized compared with rest. However, only the mGABAA occurs with the time course that would enable it to participate in shunting the excitatory component of a given compound evoked postsynaptic potential (Qian and Sejnowski 1990) and thus be inhibitory.

If BMIR-GABAA is depolarizing but not primarily inhibitory, it may be a trophic or regulatory signal as has been postulated previously for GABA currents early in development (Obrietan and van den Pol 1995). For example, GABA activity is associated with increases in dendritic length, number of branch points, and number of primary neurites of hippocampal neurons in culture (Barbin et al. 1993; Ben-Ari et al. 1994). In neocortex, depolarizing GABA currents regulate neuronogenesis by inhibiting DNA synthesis in cortical progenitor cells (LoTurco et al. 1995). GABA regulatory functions are believed to operate via elevations in intracellular calcium (Leinekugel et al. 1995; Reichling et al. 1994; Wang et al. 1994) which in turn appear to be triggered by activation of high-threshold voltage gated calcium channels (LoTurco et al. 1995; Obrietan and van den Pol 1995; Reichling et al. 1994). Interestingly, calcium channel activation occurs with rather modest depolarizations (e.g., 12 mV above rest to a mean of -50 mV in cultured rat dorsal horn neurons, Reichling et al. 1994) and does not require action potential generation (Obrietan and van den Pol 1995).

Previous demonstrations of maturational changes in GABA currents have shown two patterns. The first, as indicated above, is seen in neocortical, hypothalamic, and CA1 pyramidal neurons and is marked by the conversion of a BMI-sensitive GABAA current from depolarizing in immature animals to hyperpolarizing in mature animals (Chen et al. 1996; Luhmann and Prince 1991; Zhang et al. 1991). A second pattern is the downregulation or disappearance over time of a current that is present only in very young animals. This is seen, for example, in CA3 where a depolarizing response to GABA is expressed only during the first two postnatal weeks and has been attributed to a novel, transiently active BMI- and baclofen-insensitive receptor (Martina et al. 1995; Strata and Cherubini 1994). Further studies are warranted to determine whether BMIR-GABAA is comparable in immature GCs from animals of different postnatal ages. If so, this is a candidate third pattern in which a developmentally regulated current is expressed transiently only at the earliest stages of individual neuronal development.

    ACKNOWLEDGEMENTS

  We are grateful to P. Lio and A. Weible for help with data analysis in the early stages of these experiments, to S. Tilwalli for participation in histological processing, to T. Roberts for photography, and to Dr. Nelson Spruston for helpful discussion during the preparation of the manuscript.

  This work was supported in part by National Institute of Neurological Disorders and Stroke Grant KO8-NS-01498 to B. L. Trommer and generous gifts from The Ruggles Research Fund, The Emanuel Carton Neurology Fund, The Simms Family Foundation, Open Hearts for Retarded Children, and The Crown Family.

  Y. B. Liu and G.-L. Ye contributed equally to this journal article.

    FOOTNOTES

  Address for reprint requests: B. L. Trommer, Division of Neurology, Evanston Hospital, 2650 Ridge Ave., Evanston, IL 60201.

  Received 13 August 1997; accepted in final form 28 July 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society