Introduction

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-Aminobutyric acid (GABA) plays several trophic roles during development, including stimulation of outgrowth of neuronal processes (Behar et al., 1996), modulation of DNA synthesis (LoTurco et al., 1995), and regulation of neuronal phenotype and depolarization of immature neurons (Ben-Ari et al., 1994). The sequence of changes that result in transformation of GABA from a trophic (Behar et al., 1996), excitatory neurotransmitter (Ben-Ari et al., 1994; Ben-Ari et al., 1997) in the immature brain to the major inhibitory neurotransmitter in the forebrain involves changes in chloride ion reversal potential, changes in expression and distribution of glutamic acid decarboxylase 67 and 65 (Dupuy and Houser, 1996), and late coupling of GABA_B receptors to potassium channels (Ben-Ari et al., 1997). There is growing evidence that this transformation also may involve developmental changes in the subunit subtype composition and properties of GABA_A receptors.

The GABA_A receptor is a pentameric subunit complex that contains specific binding sites for GABA and multiple allosteric regulators, including picrotoxin, barbiturates, benzodiazepines (BZs), zinc, and the anesthetic steroids, and that forms a chloride ion channel (Macdonald and Olsen, 1994). Based on sequence similarity, six different GABA_A receptor subunit families have been identified in mammals (, , , , , and ) (Macdonald and Olsen, 1994; Davies et al., 1997; Whiting et al., 1997). Several of the subunit families have multiple subtypes (1-6, 1-3, and 1-3). Marked changes occur in the expression of GABA_A receptor subunit subtype mRNAs and receptor polypeptides and functional receptor properties during development (Laurie et al., 1992; Fritschy et al., 1994; Mathews et al., 1994; Thompson et al., 1996). Because the subunit composition of GABA_A receptors determines their pharmacological properties, it is likely that the pharmacological properties of GABA_A receptors change during development.

Granule cells of the hippocampal dentate gyrus provide a useful system in which to study GABA_A receptor development because many granule cells are born, proliferate, migrate, and mature in the postnatal period (Altman and Das, 1965; Altman and Das, 1966; Gould and Cameron, 1996). A recent study in dentate granule cells (Hollrigel and Soltesz, 1997) demonstrated that until the end of the second postnatal week, synaptic GABA_A receptor-mediated miniature inhibitory postsynaptic currents (mIPSC) displayed slower rise and decay kinetics than those of adult granule cells. It was proposed that these developmental changes in mIPSC kinetics reflected postnatal development of the properties of the GABA_A receptors.

We report the postnatal development of the properties of GABA_A receptors on dentate granule cells from 7 to 52 days of age. Pharmacological properties of GABA_A receptors on dentate granule cells acutely isolated from hippocampi of young rats (7-14 days) and adult rats (45-52 days) were characterized. In the past, we have characterized the pharmacological properties of GABA_A receptors present on dentate granule cells acutely isolated from 28- to 35-day-old rats (Kapur and Macdonald, 1996); the pharmacological properties of GABA_A receptors in these three age groups were compared.

	Materials and Methods

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Cell Isolation. Dentate granule cells were isolated from rats aged between 7 and 52 days according to the method described originally by Kay and Wong (1986) and later modified (Oh et al., 1995). The brain was dissected free, and the region containing the hippocampus was blocked and chilled in an oxygenated piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) -buffered medium (4°C) for 1 min. The PIPES buffer solution contained 120 mM NaCl, 2.5 mM KCl, 1.5 mM CaCl₂, 1 mM MgCl₂, 25 mM D-glucose, and 20 mM PIPES, pH 7.0. After blot-drying, the brain was mounted on a vibratome stage, and 500-µm coronal sections containing the hippocampus were cut. The sections were allowed to recover in oxygenated (95% O₂/5% CO₂) PIPES buffer for 30 to 60 min. Hippocampal sections were then incubated in oxygenated SIGMA type XXIII protease enzyme (Sigma Chemical Company, St. Louis, MO) in the buffer at 32°C for 30 to 45 min. The dentate gyrus was dissected out and cut into 0.5-mm cubes that were triturated in a cold (4°C) PIPES-buffered medium in fire-polished glass pipettes to isolate neurons. The isolated neurons were plated on poly-L-lysine-coated, 35-mm, polystyrene Petri dishes (Corning Glass Works, Corning, NY), and the recordings were made within 1 h of isolation.

Whole Cell Recording. Whole cell GABA_A receptor currents were recorded from hippocampal dentate granule cells acutely isolated from 7- to 52-day-old rats using the technique described by Hamill et al., (1981). The extracellular recording solution consisted of 142 mM NaCl, 1.0 mM CaCl₂, 8 mM KCl, 6 mM MgCl₂, 10 mM glucose, and 10 mM HEPES, pH adjusted to 7.4 and osmolarity of 310 to 320 mOsM (all reagents from Sigma). Glass recording patch pipettes were filled with a solution consisting of 115 mM dibasic Trizma phosphate, 30 mM Trizma base, 11 mM EGTA, 2 mM MgCl₂, and 0.5 mM CaCl₂, pH 7.35. Recording pipettes also contained ATP (2 mM) unless otherwise specified. All recordings were obtained at room temperature (24°C). With a bathing solution containing a chloride concentration of 164 mM and whole cell recording pipettes containing a 5 mM chloride ion solution, the chloride ion concentration gradient produced a chloride ion equilibrium potential (E_Cl) of 76 mV. Granule cells were voltage-clamped to 0 mV; thus, application of GABA produced outward currents. Patch pipettes (resistance of 6-10 M) were pulled on P-87 Flaming Brown puller by a four-stage pull. Currents were recorded with an Axopatch 200 A amplifier (Axon Instruments, Foster City, CA) and low-pass filtered at 2 kHz with an eight-pole Bessel filter (Frequency Devices, Haverhill, MA) before digitization, storage, and display. Currents were displayed on a Gould 2400S chart recorder, and peak whole cell currents were measured manually from the chart paper. Currents were also recorded on a hard disk using the Axotape or Axoscope program (digitized at 208 Hz) and on a video cassette tape recorder (Sony SL-HF360) via a digital audio processor (Sony PCM-501 ES, 14-bit, 44 kHz).

Drug Application. GABA, zolpidem, and ZnCl₂ dissolved in extracellular solution were applied to neurons using a modified U-tube "multipuffer" rapid application system (Greenfield and Macdonald, 1996), with the tip of application pipette placed 100 to 200 µm from the cell. Diazepam and furosemide were dissolved first in dimethyl sulfoxide and then diluted in extracellular buffer; the final dimethyl sulfoxide dilution was at least 1:50,000. GABA, diazepam, and ZnCl₂ were obtained from Sigma. Zolpidem was obtained from Research Biochemicals, Inc. (Natick, MA).

Data Analysis. The magnitude of the enhancement or inhibition of GABA_A receptor current by a drug was determined by dividing the peak amplitude of GABA_A receptor current elicited in the presence of a given concentration of the drug and GABA by the peak amplitude of control current elicited by GABA alone and multiplying the fraction by 100 to express it as percentage control. The control response was 100%. Peak GABA_A receptor currents at various drug concentrations were fitted to a sigmoidal function using a four-parameter logistic equation (sigmoidal concentration-response) with a variable slope. The equation used to fit the concentration-response relationship was
where I is the GABA_A receptor current at a given GABA concentration, and I_max is the maximal GABA_A receptor current. Maximal current and concentration-response curves were obtained after pooling data from all neurons tested for GABA and for all drugs. The curve-fitting algorithm minimized the sum of the squares of the actual distance of points from the curve. Convergence was reached when two consecutive iterations changed the sum of squares by less than 0.01%. The curve fit was performed on an IBM PC-compatible personal computer using the program Prism (Graph Pad Software, Inc., San Diego, CA). All data is presented as mean ± S.E.M..

	Results

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Maximal GABA_A Receptor Current Amplitude Increased During Development. GABA at concentrations ranging from 0.3 to 1000 µM was applied to granule cells isolated from 7- to 14-day-old rats with recovery intervals of at least 1 min (Fig. 1A). In these cells, the minimum concentration of GABA required for evoking currents was 1 µM, and the peak current elicited by 10 µM GABA was 40 ± 10 pA (n = 7). In contrast, GABA evoked larger currents from granule cells isolated from 45- to 52-day-old rats (Fig. 1B). In these cells, the minimum concentration of GABA required for evoking currents was 0.3 µM, and the peak current elicited by 10 µM GABA was 252 ± 64 pA (n = 8, p < .01, unpaired t test) (Fig. 1B). GABA concentration-response curves were obtained from individual granule cells isolated from 7- to 14- (n = 7) and 45- to 52-day-old rats (n = 8) (Fig. 2) for GABA concentrations ranging from 1 to 1000 µM. The maximal GABA_A receptor current increased with age. Maximal GABA_A receptor current elicited from granule cells isolated from younger rats was 476 ± 65 pA (n = 8), whereas maximal current elicited from granule cells isolated from older rats was 893 ± 160 pA (n = 7, p < .01, unpaired t test) (Figs. 1 and 2). GABA potency did not change significantly during this developmental period; the GABA EC₅₀ value for granule cells from younger rats was 40 ± 8 µM, whereas the GABA EC₅₀ value for granule cells from older rats was 31 ± 10 µM (p > .05) (Fig. 2).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Larger GABAA receptor peak currents were recorded from dentate granule cells isolated from 45- to 52-day-old rats than in granule cells isolated from 7- to 14-day-old rats. GABAA receptor currents were recorded from dentate granule cells isolated from a 10-day-old rat (A) and a 50-day-old rat (B). The duration and concentration of GABA applied are indicated by the bar below each bottom trace.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   There was higher efficacy, but no change in potency, of GABA during development. GABA concentration-GABAA receptor peak current relationships were plotted for groups of hippocampal dentate granule cells acutely isolated from 7- to 14-day-old rats (n = 7, ) and from 45- to 52-day-old rats (n = 8, ). Each point represents the mean of peak currents and error bars show the S.E.M. The line is the best fit of data to a sigmoid function. The maximal current (Imax) and EC50 values were derived from the equation for the sigmoid function that best fit the data.

BZ Sensitivity of Granule Cell GABA_A Receptors Increased During Development. Diazepam (100 nM) was coapplied with 10 µM GABA to granule cells isolated from 7- to 14-day-old rats and compared with peak currents elicited by GABA alone. There was minimal enhancement of peak currents by 100 nM diazepam [12 ± 8% larger than control currents (n = 3)] (Fig. 3A). In contrast, in granule cells isolated from 45- to 52-day-old rats, 100 nM diazepam enhanced GABA_A receptor currents elicited by 10 µM GABA by 53 ± 13%, (p < .01, n = 4, ANOVA with post-test Newman-Keuls multiple comparison test, compared with enhancement by 100 or 1000 nM diazepam in cells from 7- to 14-day-old rats) (Fig. 3B).

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Sensitivity of GABAA receptor currents to enhancement by diazepam increased during development. GABAA receptor currents were recorded from dentate granule cell isolated from a 7-day-old rat (A) and a 52-day-old rat (B). The duration of GABA- and diazepam-application are indicated by the bar below each trace. The drug concentration indicated beneath the bottom trace applies to the top trace also. C, diazepam concentration-GABAA receptor peak current relationships were plotted for groups of hippocampal dentate granule cells acutely isolated from 7- to 14-day-old (n = 4, ) and 45- to 52-day-old (n = 5, ) rats. Each point represents the mean enhancement of peak current elicited by 10 µM GABA and error bars show the S.E.M. The line is the best fit of data to a sigmoid function. The Imax and EC50 values were derived from the equation for sigmoid function that best fit the data In granule cells acutely isolated from 7- to 14-day-old rats the data were fit poorly to a sigmoidal function largely because application of high diazepam concentrations (>1 µM) inhibited GABAA receptor currents and a plateau of maximal enhancement was not obtained. The diazepam EC50 for 7- to 14-day-old rats is marked with an asterisk to indicate poor data fit.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Sensitivity of GABAA receptor currents to enhancement by zolpidem increased during development. GABAA receptor currents were recorded from dentate granule cell isolated from a 14-day-old rat (A) and a 48-day-old rat (B). The durations of GABA and zolpidem application are indicated by the bar below each trace. The drug concentration indicated beneath the bottom trace applies to the top trace also. C, zolpidem concentration-GABAA receptor peak current relationships were plotted for a group of hippocampal dentate granule cells acutely isolated from 45 to 52 day old rats (n = 3, ). Each point represents the mean enhancement of peak currents elicited by 10 µM GABA and error bars show the S.E.M.. The line is the best fit of data to a sigmoid function. The Imax and EC50 values were derived from the equation for sigmoid function that best fit the data.

Zinc Sensitivity of Granule Cell GABA_A Receptors Declined during Development. The BZ modulation of GABA_A receptor currents in dentate granule cells isolated from 7- to 14-day-old rats suggested the presence of BZ 3 receptors on these cells. Recombinant receptors containing an 4 or 6 subtype, with  and 2 subtypes, have BZ 3 pharmacology (Wieland et al., 1992) with moderate zinc sensitivity [zinc 50% inhibitory concentration (IC₅₀) value of 30-70 µM for inhibition of GABA_A receptor currents] (Knoflach et al., 1996; Saxena and Macdonald, 1996). In contrast, BZ modulation of GABA_A receptor currents in dentate granule cells isolated from 45- to 52-day-old rats suggested the presence of BZ 1 receptors. Recombinant 1x2 receptors were minimally zinc sensitive (zinc IC₅₀ value of > 100 µM for inhibition of GABA_A receptor currents) (Saxena and Macdonald, 1996).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Sensitivity of GABAA receptor currents on dentate granule cells to zinc inhibition declined during development. GABAA receptor currents were recorded from a dentate granule cell isolated from an 8-day-old rat (A) and a 52-day-old rat (B). The durations of GABA and zinc application are indicated by the bar below each trace. The drug concentration indicated beneath the bottom trace applies to the top trace also. C, zinc concentration-GABAA receptor peak current inhibition relationships were plotted for groups of hippocampal dentate granule cells acutely isolated from 7- to 14-day-old (n = 10, ) and 45- to 52-day-old (n = 7, ) rats. Each point represents the mean inhibition of peak currents elicited by 30 µM GABA and error bars show the S.E.M. The line was the best fit of data to a sigmoid function. The Imax and EC50 values were derived from the equation for sigmoid function that best fit the data.

Furosemide Sensitivity of Granule Cell GABA_A Receptor Currents Declined during Development. Dentate granule cells from 7- to 14-day-old rats expressed GABA_A receptors that were relatively insensitive to diazepam and zolpidem but were sensitive to Ro-15-4513 (data not shown), which suggests BZ 3 pharmacology. In contrast, GABA receptors from 45- to 52-day-old rats expressed GABA_A receptors with high diazepam and zolpidem sensitivity, consistent with BZ 1 pharmacology. Recombinant GABA_A receptors that contained the 1 subtype and had BZ 1 pharmacology also had low sensitivity to furosemide inhibition of GABA_A receptor currents, whereas 4 or 6 subtype-containing diazepam-insensitive receptors with BZ 3 pharmacology had high sensitivity to furosemide (Wafford et al., 1996).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Sensitivity of GABAA receptor currents on dentate granule cells to furosemide inhibition declined during development. GABAA receptor currents were recorded from a dentate granule cell isolated from a 14-day-old rat (A) and a 52-day-old rat (B). The durations of GABA and furosemide application are indicated by the bar below each trace; the drug concentration indicated beneath the bottom trace applies to the top trace also. C, furosemide concentration-GABAA receptor peak current inhibition relationships were plotted for groups of hippocampal dentate granule cells acutely isolated from 7- to 14-day-old (n = 3, ), 18- to 30-day-old (n = 6, ), and 45- to 52-day-old (n = 4, ) rats. Each point represents the mean inhibition of peak currents elicited by 30 µM GABA and error bars show the S.E.M.. The line was the best fit of data to a sigmoid function. The maximal inhibition Imax and EC50 values were derived from the equation for sigmoid function that best fit the data. The data from neurons isolated from 18- to 30-day-old rats were not fit to a sigmoidal function.

Fraction of Granule Cells Sensitive to Loreclezole Increased during Development. The antiepileptic drug loreclezole has been shown to enhance recombinant GABA_A receptor currents via a specific modulatory site on GABA_A receptor  subunits. The action of loreclezole depended on the  subtype expressed. Isoforms containing 2 or 3 subtypes had a 300-fold lower EC₅₀ value for loreclezole enhancement of GABA_A receptor current than isoforms containing the 1 subtype (Wafford et al., 1994). Additionally, at concentrations above 6 µM, loreclezole enhanced the degree and rate of apparent desensitization in a concentration-dependent manner (Donnelly and Macdonald, 1996). This inhibitory effect of loreclezole occurred regardless of subunit composition of the receptor.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Loreclezole effects on granule-cell GABAA receptor currents changed during development. GABAA receptor currents were recorded from dentate granule cells isolated from a 14-day-old rat (A) and a 52-day-old rat (B). The durations of GABA and loreclezole application are indicated by the bar below each trace. The drug concentration indicated beneath the bottom trace applies to the top trace also. Note inhibition of peak current by loreclezole in A and enhancement of the apparent current desensitization in trace B (see text for details).

	Discussion

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Multiple changes in the pharmacological properties of dentate granule-cell GABA_A receptors occurred during the first 52 days of postnatal development: maximal GABA-evoked current increased with postnatal age; GABA_A receptors changed from type BZ 3 in young rats to type BZ 1 in adult rats; furosemide and zinc inhibited GABA_A receptor currents in young rats but not in adult rats; and the fraction of cells expressing loreclezole sensitive GABA_A receptors increased with postnatal age. This transformation was gradual, and granule cells isolated from 28- to 35-day-old rats expressed receptors with pharmacological properties that were intermediate between younger and older rats.

Prolonged Postnatal Development of Dentate Granule Cell GABA_A Receptors. The findings of this study and our previously published study (Kapur and Macdonald, 1996) suggest that the pharmacological properties of dentate granule-cell GABA_A receptors undergo prolonged development lasting up to at least postnatal day 52. There was prolonged postnatal development of GABA_A receptor-mediated, paired-pulse inhibition in the dentate gyrus (Bronzino et al., 1996). The prolonged postnatal maturation of GABA_A receptors was quite similar to the morphological maturation of dentate granule cells (Bayer and Altman, 1974). The granule cell population of the dentate gyrus has the distinctive characteristic of neuronal birth, migration, and death occurring over an extended period of time beginning in gestation and extending to adulthood (Altman and Das, 1965; Gould and Cameron, 1996). The proliferation and migration of granule cells reached its peak in rats in first 2 postnatal weeks and then began to decline (Bayer and Altman, 1974; Schlessinger et al., 1975). The maximum number of immature postnatal granule cells was present at 2 weeks of age and declined to a low level at 2 months of age. Mature granule cells accumulated from the first week; their number reached an asymptotic level at 30 to 70 days.

Postnatal Development of GABA_A Receptor Currents. The maximal current elicited by GABA increased significantly from day 14 to day 28 and then remained stable at age 52 days (Table 1). The increase in the magnitude of whole cell GABA_A receptor currents observed during postnatal development could have been caused by an increased receptor density present at each synapse, an increase in the density of extrasynaptic receptors, an increase in the number of GABAergic synapses on each dentate granule cell, or a combination of these factors. An increase in the density of synaptic GABA_A receptors seemed unlikely, because the peak amplitude of miniature IPSCs did not change during development (Hollrigel and Soltesz, 1997) and the miniature IPSC peak current was determined by the number of postsynaptic GABA_A receptors (Edwards et al., 1989; Mody et al., 1994). An increase in the size of granule cells with an associated proportional increase in synaptic and extrasynaptic GABA_A receptors could explain the larger whole cell GABA_A receptor currents in cells from older rats. Histological studies demonstrated that immature granule cells were small, whereas mature cells could be small or large, and the number of mature granule cells continued to increase up to postnatal day 70 (Bayer and Altman, 1974). This could result in increased GABAergic synapses on mature granule cells and increased extrasynaptic GABA_A receptors. However, the current study did not systematically evaluate cell dentate granule cell volume at various ages by capacitance measurement. Thus the increase in GABA_A receptor currents could have resulted from a combination of these factors.

Postnatal Development of BZ, Zinc, and Furosemide Modulation of Dentate Granule Cell GABA_A Receptor Currents. Dentate granule cells from 7- to 14-day-old rats expressed GABA_A receptor currents that were poorly enhanced by diazepam and zolpidem, whereas GABA_A receptor currents from 45- to 52-day-old rats were enhanced with high affinity by diazepam and zolpidem (Table 1). These data suggested that BZ 3 receptors were expressed early in postnatal development and were transformed to BZ 1 receptors by 45 to 52 days of age. GABA_A receptors present on cells from 28- to 35-day-old rats (Table 1) had intermediate BZ sensitivity and could not be classified as BZ 1, BZ 2, or BZ 3 receptors (for details, see Kapur and Macdonald, 1996). Data from the previous study combined with those reported here suggest that transformation of granule-cell GABA_A receptors from BZ 3 to BZ 1 receptors is a gradual process with a clear intermediate state.

Possible Molecular Bases for Developmental Changes in BZ, Zinc, and Furosemide Modulation of Dentate Granule Cell GABA_A Receptor Currents. The most likely explanation for changes in the pharmacological properties of dentate granule-cell GABA_A receptors during development was that different receptor isoforms were expressed at 7 to 14, 28 to 35, and 45 to 52 days of age. The pharmacological properties of GABA_A receptors in granule cells from 7- to 14-day-old rats were similar to those of recombinant receptors containing 4, 2, and 1 or 3 subtypes. The presence of an 4 or 6 subtype along with a 2 subtype would explain the low affinity for diazepam, moderate affinity for zinc, and relatively high sensitivity to furosemide. GABA_A receptors in 45- to 52-day-old rats had high diazepam and zolpidem sensitivities but were relatively insensitive to zinc and furosemide. Recombinant receptors containing 1, 2, and x subtypes also have these properties.

Development of Loreclezole Sensitivity. The action of loreclezole on recombinant GABA_A receptors depends on the  subtype expressed. Isoforms containing 2 or 3 subtypes had a 300-fold lower EC₅₀ value for loreclezole enhancement of GABA_A receptor current than isoforms containing the 1 subtype (Wafford et al., 1994). High loreclezole sensitivity depends on the presence of a 2 or 3 subtype without a 1 subtype (Fisher and Macdonald, 1997). This suggests that 30% of granule cells in 7- to 14-day-old rats, 50% of granule cells in 28- to 35-day-old rats, and 70% of granule cells in 45- to 52-day-old rats expressed GABA_A receptor with only 2 and/or 3 subtypes. The continued presence of granule cells expressing GABA_A receptors not enhanced by loreclezole at 45 to 52 days of age may reflect continued presence of immature granule cells to this age (Bayer and Altman, 1974). In addition to enhancement of peak GABA_A receptor currents, loreclezole was demonstrated to enhance the degree and rate of apparent desensitization in a concentration-dependent manner (Donnelly and Macdonald, 1996). This inhibitory effect of loreclezole occurred regardless of subunit composition of the receptor and was apparent in cells isolated from all three age groups.

GABA_A Receptor Development in Other Regions of the Brain. During development, changes in the properties of GABA_A receptors present in many different regions of the brain have been described. The properties and subunit composition of GABA_A receptors on cultured cerebellar granule cells changed during development (Mathews et al., 1994). Cerebellar granule cells expressed BZ-sensitive 1-subtype-containing GABA_A receptors after 5 to 7 days in culture, but mature granule cells, 21 to 25 days in culture, expressed 6-containing BZ-insensitive GABA_A receptors. In hippocampal CA3 neurons in slices from young animals before 15 days of age, zolpidem enhanced IPSPs poorly and BZ action was mediated through BZ 2 receptors, whereas in slices from adult animals, BZ 1 receptors were present (Rovira and Ben-Ari, 1993). GABA_A receptors present on rat thalamic reticular neurons (Gibbs, III et al., 1996) and principal neurons (Oh et al., 1995) also undergo developmental regulation with changes in maximal current and BZ regulation.