 |
INTRODUCTION |
The GABAergic control of dentate granule cell discharges is an important regulator of the entorhinohippocampal interplay (Buzsáki et al. 1983
). In the adult dentate gyrus, distinct GABAergic interneuron classes supply the inhibitory innervation to specific, spatially segregated parts of granule cells, including the axon initial segment, soma, and proximal and distal dendrites (Halasy and Somogyi 1993
; Han et al. 1993
; Mott et al. 1997
), and these distinct interneuronal microcircuits regulating granule cell excitability are likely to serve separate functional roles (Buckmaster and Schwartzkroin 1995
; Cobb et al. 1995
; Miles et al. 1996
; Soltesz et al. 1995
). In contrast to the recent advancements in our knowledge about the nature of the synaptic organization of the dentate inhibitory system in the adult (Freund and Buzsáki 1996
), the temporal and spatial patterns of the functional interneuronal microcircuits in the developing dentate gyrus are not well known. Similarly, although GABAergic responses have been shown to occur in developing hippocampal and cortical neurons (Agmon et al. 1996
; Ben-Ari et al. 1989
; Blanton and Kriegstein 1991
; Fleidervish and Gutnick 1995
; Gaiarsa et al. 1990
; Hosokawa et al. 1994
; Janigro and Schwartzkroin 1988
; Kriegstein et al. 1987
; Luhman and Prince 1991; Mueller et al. 1984
; Owens et al. 1996
; Zhang et al. 1991
; Zhou and Hablitz 1997
) as well as in immature dentate granule cells (Draguhn and Heinemann 1996
; Hollrigel and Soltesz 1997
; Liu et al. 1996
) the functional properties of the early
-aminobutyric acid type A (GABAA) receptor-mediated synaptic transmission in the late-developing dentate granule cells are not well understood.
The question of early postnatal development of the GABAergic innervation of granule cells in the dentate gyrus is particularly interesting in light of the fact that in immature neurons GABA was shown to have several important developmental roles, including the modulation of neuronal phenotype (Marty et al. 1996
), regulation of neuronal process outgrowth (Behar et al. 1996
), and modulation of DNA synthesis (LoTurco et al. 1995
). In several neuronal systems, GABA is depolarizing early in life, leading to the opening of voltage-gated Ca2+ channels and increases in [Ca2+]i (Ben-Ari et al. 1997
; Cherubini et al. 1991
; Gaiarsa et al. 1995
; Garaschuk et al. 1998
; Obrietan and van den Pol 1995
; Owens et al. 1996
; Reichling et al. 1994
; Rohrbough and Spitzer 1996
; Yuste and Katz 1991
). The GABAA receptor-induced depolarizations in these early postnatal neurons may also increase [Ca2+]i through a reduction in the Mg2+ block of N-methyl-D-aspartate (NMDA) channels (Ben-Ari et al. 1997
; Leinekugel et al. 1997
). These spontaneous GABAA receptor activation-induced increases in [Ca2+]i in immature neurons are likely to be associated with the various developmental roles of GABA.
In hippocampal CA1 and CA3 pyramidal cells within the first postnatal week, the temporal pattern of the spontaneous activation of GABAA receptors occurs in characteristic bursts, named giant depolarizing potentials (Ben-Ari et al. 1989
). These giant depolarizing potentials were characterized in the Ammon's horn (Ben-Ari et al. 1989
; Gaiarsa et al. 1990
; McLean et al. 1995
; Strata et al. 1997
); however, no comparable data are available for the principal cells of the dentate gyrus, the granule cells. Developing dentate granule cells differ from immature hippocampal pyramidal cells in several aspects (Cowan et al. 1980
). One of the unique properties of the developing dentate is that 80% of dentate granule cells are generated postnatally (Bayer 1980
; Schlessinger et al. 1975
, 1978
; Soriano et al. 1989
); by contrast, the majority of the dentate GABAergic cells are born prenatally (Amaral and Kurz 1985; Dupuy and Houser 1996
, 1997
; Lubbers et al. 1985
; Lubbers and Frotscher 1988
; Schlessinger et al. 1978
). Second, unlike in adult pyramidal cells, GABAA receptor activation leads to depolarizing responses in adult dentate granule cells (Soltesz and Mody 1994
; Smith et al. 1995
; Staley 1992
, 1994
; Staley and Mody 1992
; Staley et al. 1992
). Third, GABAA receptor-mediated synaptic signals in immature granule cells from early postnatal rats are slower rising and slower decaying than the inhibitory postsynaptic currents (IPSCs) in granule cells in adult rats (Hollrigel and Soltesz 1997
), a kinetic difference that is likely to be associated with the distinct subunit composition of the GABAA receptor channels in immature versus adult granule cells (Fritschy et al. 1994
; Hollrigel and Soltesz 1997
; Killisch et al. 1991
; Laurie et al. 1992
; Poulter et al. 1992
).
In light of these unique features of the inhibitory circuits impinging on the dentate granule cells, this study was undertaken to answer the following questions. 1) Are the spontaneously occurring, large-amplitude bursts of GABAA receptor-mediated IPSCs, described in the developing Ammon's horn, present in early postnatal granule cells of the dentate gyrus? If yes, 2) are the IPSCs within a burst different in amplitude from the spontaneously occurring IPSCs outside the bursts? 3) Do the excitatory postsynaptic currents (EPSCs) in developing granule cells also occur in bursts, similar to the intraburst IPSCs? 4) Is the generation of the spontaneous bursts of IPSCs critically dependent on the glutamatergic excitatory drive onto interneurons? 5) What is the relationship among the resting membrane potential, the reversal potential of the IPSCs, and the action potential threshold in immature granule cells? 6) Do the spontaneous bursts of synaptic events evoke action potentials in immature granule cells? 7) Do the bursts of IPSPs occur synchronously in all granule cells? The answers to these questions are important because they will help us understand how the GABAA receptor-mediated system develops in the most important gating station of the entorhinohippocampal circuit.
 |
METHODS |
Slice preparation
Brain slices were prepared similar to what was previously described (Otis and Mody 1992
; Hollrigel and Soltesz 1997
; Staley et al. 1992
). Neonatal Wistar rats (P0-P6) were anesthetized by hypothermia and decapitated; the brains were removed and cooled in 4°C oxygenated (95% O2-5% CO2) artificial cerebral spinal fluid (ACSF) composed of (in mM) 126 NaCl, 2.5 KCl, 26 NaHCO3, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, and 10 glucose. Horizontal brain slices (450 µm for blind recording, 300 µm for visualized) (Hollrigel et al. 1996
, 1998
; Staley et al. 1992
; Toth et al. 1997
) were prepared with a vibratome tissue sectioner (Lancer Series 1000). The brain slices were sagittally bisected into two hemispheric components and incubated submerged in 32°C ACSF for 1 h.
Electrophysiology
Individual slices were transferred to a submersion-type recording chamber (Hollrigel et al. 1996
; Soltesz et al. 1995
) perfused with ACSF, in some experiments containing 10 µM 2-amino-5-phosphovaleric acid (APV) and/or 5 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or bicuculline (20 µM) or picrotoxin (100 µM). The brain slices rested on filter paper and were stabilized with platinum wire weights. The tissue was continuously superfused with humidified 95%O2-5%CO2, and the temperature of the perfusion solution was maintained at 36°C. All salts were obtained from Fluka. APV and CNQX were purchased from Tocris, bicuculline was obtained from Research Biochemicals International, and picrotoxin was obtained from Sigma. Drugs were applied via the perfusion system.
Patch pipettes were pulled from borosilicate (KG-33) glass capillary tubing (1.5 mm OD; Garner Glass) with a Narishige PP-83 two-stage electrode puller. Pipette solutions consisted of (in mM) 140 CsCl (KCl) or Cs-gluconate (K-gluconate), 2 MgCl2, and 10 N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid (HEPES), and in some cases 3 lidocaine N-ethyl bromide (QX-314). To record spontaneous EPSCs by blocking GABA-mediated IPSCs intracellularly, a pipette solution containing (in mM) 140 CsF, 2 MgCl2, 10 HEPES, and 1 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) was used. "Blind" whole cell recordings were obtained as previously described (Blanton et al. 1989
; Staley et al. 1992
). In some experiments, infrared video microscopy-aided visualized patch-clamp recordings were used (Axioscope FS, Zeiss) (Stuart et al. 1993
). Cell-attached recordings were obtained as above with K-gluconate filled pipettes, but negative pressure was not applied once a successful seal was obtained. Recordings were obtained with an Axopatch-200A amplifier (Axon Instruments) or with a NeuroData two-channel intracellular amplifier and digitized at 88 kHz (Neurocorder, NeuroData) before being stored on videotape. The series resistance (9.4 ± 0.5 M
) was monitored throughout the recordings, and the data were rejected if it increased significantly (all cells were <15 M
).
Perforated patch-clamp recordings were carried out as described by Kyrozis and Reichling (1995)
. The pipettes contained 140 KCl, 2 MgCl2, and 10 HEPES. Pipette tips were filled with this solution and then backfilled with one containing 10-20 µg/ml gramicidin (prepared from a 5 mg/ml stock solution in dimethyl sulfoxide). Series resistance was continuously monitored in voltage-clamp mode. At the end of the recording, negative pressure was applied to the pipette to establish the whole cell recording configuration, which was verified by the shift in the reversal potential of GABA-mediated IPSCs to 0 mV based on [Cl
]i = [Cl
]o. The membrane potentials were corrected off-line for the voltage drop across the series resistance (Ulrich and Huguenard 1997
) (e.g., average series resistance with gramicidin 77.4 ± 8.1 M
)
where Vcom is the command potential, Iclamp is the clamp current, and Rs is the series resistance.
Analysis
Recordings were filtered at 1-3 kHz before digitization at 2.5-10 kHz by a personal computer for analysis with Strathclyde Electrophysiology, and Synapse software. Detection of the IPSCs was performed with a software trigger similar to that previously described (Otis and Mody 1992
; Soltesz et al. 1995
). The sensitivity of the detector was carefully set so it could detect both the smaller, lower frequency interburst events and the higher frequency, larger intraburst IPSCs (see RESULTS). A "burst" was arbitrarily defined as
10 events with a frequency of the first 5 events at least five times greater than the previous 5 events. This definition of bursts corresponded well with what subjectively appeared as IPSC clusters on the basis of visual inspection of the records (e.g., Fig. 1). All of the detected events were analyzed, and any noise that spuriously met trigger specifications was rejected. The automatic detection had an upper limit of sensitivity of ~400 Hz (i.e., in the case of 2 events appearing closer than 2.5 ms, only the 1st was detected as an event); consequently, the measured frequencies of the intraburst IPSCs are underestimating the "real" frequency of occurrence for these events. A least-squares Simplex-based algorithm was used to fit the ensemble average of individual IPSCs (or EPSCs) with the sum of two (1 rising and 1 decaying) or three (1 rising and 2 decaying) exponentials
|
|
where I(t) is the miniature IPSC (mIPSC) as a function of time (t); A1 + A2 = A are constants; and
r,
D1, and
D2 are the rise, fast decay, and slow decay time constants, respectively. For single exponential decays, A1 was equal to 0. Statistical analyses were performed with SPSS for Windows or SigmaPlot, with a level of significance of P
0.05. Data are presented as means ± SE (n is number of cells).

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| FIG. 1.
Temporal clustering of inhibitory postsynaptic currents (IPSCs) in granule cells of the early postnatal dentate gyrus. A: Cs-gluconate recording at 0 mV shows the presence of prominent bursts of synaptic events, which occur in a recurrent manner but without obvious rhythmicity. Note the presence of individual spontaneous IPSCs occurring at lower frequencies between the bursts. B: high instantaneous frequency of the intraburst IPSCs is shown as determined by the automatic detection paradigm (note that the frequency is an underestimation of the "true" instantaneous frequency because of the limit of the resolution of the detector; see METHODS). A burst was defined as 10 or more events with a frequency of the 1st 5 events at least 5 times greater than the previous 5 events. This definition of bursts corresponded well with what subjectively appeared as IPSC clusters on the basis of visual inspection of the records in A. C: burst marked in A by an asterisk is shown at a faster timescale. D: corresponding instantaneous frequency plot of the same burst as in C (double asterisk in B) is shown at a similarly fast timescale as in C.
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|
 |
RESULTS |
Spontaneous "giant" IPSC bursts are present in granule cells of the early postnatal dentate gyrus
Spontaneous postsynaptic currents were recorded from dentate granule cells from P0-P6 rats (average age, 3 days after birth; n = 42 animals), voltage clamped at 0 mV, i.e., close to the reversal potential for the glutamatergic EPSCs) with Cs-gluconate-filled pipettes. [Throughout the experiments reported in this paper, to standardize our recording site, we restricted our recordings to the dorsal blade and the adjacent half of the crest of the granule cell layer (see also Hollrigel and Soltesz 1997
)]. Furthermore, the recordings, unless specifically stated otherwise, were restricted to granule cells situated in the middle and outer two-thirds of the layer to avoid recording from the youngest of the granule cells. The outward currents (IPSCs) appeared either in prominent clusters (i.e., bursts) or as single events (Fig. 1; for the definition of burst and for the upper limit of the automatic event detection routine, see METHODS). The bursts occurred at 0.05 ± 0.02 Hz, most often without any obvious rhythmicity (e.g., Fig. 1A), in all granule cells before P7 (n = 16). The bursts were large (peak amplitude 406.9 ± 58.4 pA), and lasted ~1 s (duration 1,230.5 ± 227.4 ms). By contrast, in the presence of the sodium channel blocker tetrodotoxin, the spontaneous mIPSCs typically occur as individual events (i.e., not in prominent, large bursts) at 0.8 ± 0.2 Hz (n = 13) with Cl
-filled pipettes in early postnatal dentate granule cells (Hollrigel and Soltesz 1997
), indicating that interneuronal firing plays an important part in the generation of these recurrent, spontaneous, large bursts of IPSCs.
In three cells that were selected on the basis of similarly low access resistance, the properties of the intraburst and interburst IPSCs were compared. The instantaneous frequency of the events within the bursts (the "intraburst IPSCs") was at least an order of magnitude higher (71.0 ± 12.4 Hz) than the frequency of the events between the bursts (the interburst IPSCs, 1.7 ± 0.7 Hz; note that the definition of the bursts per se only required that the intraburst events occur at 5 times higher frequencies than the interburst events, whereas these data indicate that the intraburst frequency is almost 40 times higher than the frequency of the interburst IPSCs). In addition to the higher frequency of occurrence of the intraburst IPSCs, the intraburst IPSCs were also larger than the interburst IPSCs (peak amplitudes; intraburst IPSC 74.9 ± 4.4 pA; interburst IPSCs 41.3 ± 1.9 pA; the distribution of the amplitudes of the intra- vs. interburst IPSCs was also significantly different, Kolmogorov-Smirnov test), consistent with the intraburst IPSCs resulting from GABA-release associated with the firing of presynaptic interneurons. (Because of the difficulty in selecting intraburst events with an uninterrupted decay phase caused by the high intraburst frequency, no further analysis of the kinetic properties of the intraburst events was carried out in this study; however, this is of no major consequence for the main points of the paper.)
In contrast to the situation during the first postnatal week, when all granule cells showed the giant bursts of IPSCs, the percentage of granule cells that displayed the large IPSC bursts decreased during the second week (e.g., in P8-P10 animals, only 6/13 cells, or 46.2%, showed the bursts of IPSCs). The progressive disappearance of the giant IPSC bursts from granule cells during the second week of life is similar to what was found in CA3 pyramidal neurons (Ben-Ari et al. 1989
).
Bursts of IPSCs are depolarizing GABAA receptor-mediated events
The intraburst IPSCs as well as the interburst IPSCs were blocked by the GABAA receptor antagonist bicuculline in immature granule cells of the dentate gyrus in a reversible manner (not shown; bicuculline: 20 µM; n = 7 granule cells with switching of the control perfusate to ACSF containing bicuculline; similarly, no IPSCs could be observed in granule cells from slices that were bathed in bicuculline, n = 3; in addition, the intraburst and the interburst IPSCs were also abolished when the perfusate was switched to ACSF containing 100 µM picrotoxin, n = 2). These data indicate that the bursts of IPSCs were mediated by the activation of GABAA receptors.
To determine the relationship between the reversal potential for the bursts of IPSCs and the resting membrane potential (Vm) in developing granule cells without disturbing the intracellular Cl
concentration, gramicidin-based perforated patch-clamp recordings (Abe et al. 1994
; Brickley et al. 1996
; Ebihara et al. 1995
; Kyrozis and Reichling 1995
; Owens et al. 1996
; Spruston and Johnston 1992
; Ulrich and Huguenard 1997
) (see METHODS) were obtained from immature granule cells. With gramicidin-containing patch pipettes, the spontaneous bursts of IPSCs reversed at
46.6 ± 3.1 mV (n = 8; Fig. 2A). After the measurement of the reversal potential of the burst of IPSCs, the resting membrane potentials of the recorded cells were also determined, in current-clamp mode. The resting membrane potential was
54.3 ± 4.2 mV; therefore the bursts of GABAA receptor-mediated synaptic events will lead to depolarizations in immature granule cells at rest.

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| FIG. 2.
Bursts of IPSCs reverse at a potential that is more depolarized than the resting membrane potential in immature granule cells. A: examples of recordings at various membrane potentials obtained from an immature granule cell with gramicidin-filled patch pipettes. The bursts reversed at 46.6 ± 3.1 mV (n = 8 cells). The reversal potential for the bursts of IPSCs was more depolarized than the resting membrane potential ( 54.3 ± 4.2 mV) in the same cells. B: at the end of the perforated patch-clamp recordings, the perforated patch was ruptured, and the whole cell configuration was established. Note that, in the perforated patch configuration at 40 mV, the bursts appeared outward and that they switched polarity after the establishment of the whole cell configuration.
|
|
At the end of the recording, negative pressure was applied to the pipette to establish the whole cell recording configuration, which invariably resulted in a prominent shift of the reversal potential of the bursts of IPSCs to 0 mV (Owens et al. 1996
), as expected from GABAA receptor-mediated and Cl
-dependent events recorded with Cl
-filled pipettes ([Cl
]i = [Cl
]o, because the electrodes contained 140 KCl, in addition to gramicidin; see METHODS). Consequently, the bursts of IPSCs, which appeared as outward currents at
40 mV in the perforated patch mode, became prominent inward currents at
40 mV after the establishment of the whole cell configuration (Fig. 2B).
Role of AMPA and NMDA receptors in the generation of the IPSC bursts
Next, the role of the ionotropic glutamate receptors in the generation of the bursts of IPSCs in immature dentate granule cells was tested. As shown in Fig. 3A, switching the perfusing solution from control ACSF to one containing 10 µM APV and 5 µM CNQX resulted in the reversible abolishment of the bursts of IPSCs (n = 6; similarly, the IPSC bursts could not be observed in cells obtained from slices bathed in APV and CNQX, n = 4). However, the glutamate receptor antagonists did not block the interburst IPSCs (Fig. 3A, middle panel). These results indicate that the glutamatergic excitatory drive onto interneurons plays a crucial role in the generation of the bursts of IPSCs. Interestingly, when either APV or CNQX were applied alone, the bursts of IPSCs could still be recorded, albeit at a much reduced frequency. In fact, occasional bursts of IPSCs were observed even at relatively high concentrations of the antagonist and after prolonged (>45 min) incubation of the slices in the drug (Figs. 3, B and C; APV: 20 µM, n = 2 cells; 100 µM, n = 3 cells from slices bathed in the drug; frequency of the bursts in 100 µM APV: 0.006 ± 0.0002 Hz; in 10 µM CNQX: 0.013 ± 0.007 Hz). The dramatic reduction in frequency by APV or CNQX alone did not appear to be paralleled by a similar decrease in the amplitude or duration of the bursts (peak amplitude of the bursts in control: 406.9 ± 58.4 pA; in 100 µM APV: 449.2 ± 14.2 pA; in 10 µM CNQX: 640.5 ± 361.0 pA; duration of the bursts in control: 1,230.5 ± 227.4 ms; in 100 µM APV: 1,853.8 ± 574.8 ms; in 10 µM CNQX: 1,064.3 ± 159.1 ms; however, it should be emphasized that, because of the extremely low frequency of the bursts in individual granule cells in the presence of APV or CNQX, these data cannot be expected to detect the presence or absence of small changes, e.g., reduction, in burst amplitude or duration by these drugs). Taken together, these results indicate that the
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and NMDA-receptor-mediated excitatory glutamatergic drive onto interneurons plays a crucial role in the generation of the large bursts of IPSCs in immature granule cells during early postnatal development; however, sufficient excitatory drive can be provided to the interneurons through either of these two glutamate receptor types to result in the generation, albeit at severely reduced frequencies, of the bursts of IPSCs in the granule cells.

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| FIG. 3.
Role of the excitatory drive onto interneurons in the generation of the bursts of IPSCs in immature granule cells of the dentate gyrus. A, left panel: Cs-gluconate recordings at 0 mV show the bursts of IPSCs, interspersed with the lower frequency interburst IPSCs. Middle panel: application of the glutamate receptor antagonists 2-amino-5-phosphovaleric acid (APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) abolished the prominent bursts of IPSCs, whereas the individual "interburst" IPSCs could still be observed. Right panel: after a switch back to the control perfusate, recovery of the bursts of IPSCs could be obtained. B and C: although the individual (i.e., separate) application of APV or CNQX severely reduced the frequency of the bursts, none of these drugs completely abolished them. B: example of a rare burst in APV. C: example of a rare burst recorded in the presence of CNQX. Taken together, these data indicate the importance of the excitatory drive onto the interneurons in generating the IPSC bursts and that sufficient excitatory drive onto the interneurons can be provided by either the NMDA or the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors alone to sustain the bursts of IPSCs in immature granule cells.
|
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Because of the difficulty in detecting a possible small reduction in the amplitude of the bursts in either APV or CNQX, from these data alone it was not possible to determine whether EPSCs contribute significantly to the bursts of IPSCs in these neurons. Therefore an alternative strategy was applied, namely, instead of blocking the EPSCs in the whole network and trying to detect a small change in the amplitude of the bursts, in the next series of experiments, GABAA receptors were blocked in the recorded immature granule cells by the Cl
-channel blockers CsF
and DIDS (Leinekugel et al. 1997
; Nelson et al. 1994
; Somers et al. 1995
) (see METHODS). Application of CsF
and DIDS resulted in the fast (<3 min) abolishment of the inter- as well as the intraburst IPSCs, and the remaining EPSCs appeared as fast inward currents at
60 mV (Fig. 4; n = 6; EPSC amplitude: 18.3 ± 1.7pA;
R= 0.33 ± 0.05 ms;
D1= 2.20 ± 0.26 ms;
D2= 18.43 ± 6.29 ms). Importantly, the EPSCs did not occur in prominent bursts (Fig. 4A; frequency: 2.0 ± 0.7 Hz; similarly, no high-frequency bursts of spontaneous EPSCs could be observed when the cells were held at + 30 mV; note that the y-scale in Fig. 4 is set to equal the y-scale used in Fig. 1A for the IPSCs to emphasize the difference between the two populations of synaptic events). These results indicate that prominent bursts of fast EPSCs do not contribute to a significant degree to the large, prolonged bursts of IPSCs recorded from immature granule cells. Naturally, however, these results do not preclude that individual EPSCs may contribute to the depolarizations during the bursts; in fact, because the EPSCs occur at ~2 Hz, and the bursts last ~1 s, it is likely that about two individual EPSCs would take place during any given burst just by chance. Furthermore, as indicated by recent results obtained from hypothalamic neurons (Gao et al. 1998
), EPSCs may serve an important, time-dependent role in eliciting action potentials during depolarizing GABAA receptor mediated bursts during development.

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| FIG. 4.
E x c i t a t o r y p o s t s y n a p t i c c u r r e n t s (EPSCs) do not occur in prominent bursts in immature granule cells. A: instantaneous frequency plot for the EPSCs detected after the intracellular blockade of -aminobutyric acid type A (GABAA) receptor channels with the Cl channel blockers CsF and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). The y-scale was purposely set to be the same as in Fig. 1B to emphasize the difference between the frequencies of the IPSCs and the EPSCs. B: example of a recording obtained after the intracellular blockade of the GABAA receptor channels with CsF and DIDS. The inward currents appeared to have fast kinetics; the first decay time constant of the double exponential decay is indicated in C (note that the decay kinetics of these EPSCs is different from the slower decay kinetics of IPSCs in these immature cells) (Hollrigel and Soltesz 1997 ).
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Relationship of the giant depolarizing bursts and action potential discharges in immature granule cells
Next, the question of whether the depolarizing bursts lead to action potential discharges was tested. This question is important, because it relates to the possible functions of the giant depolarizing bursts. As mentioned above, the reversal potential of the bursts of IPSCs in gramicidin recordings was determined to be
46.6 ± 3.1 mV, ~8-10 mV higher than the resting membrane potential (
54.3 ± 4.2 mV) in these early postnatal dentate granule cells. Further experiments with gramicidin-based perforated patch-clamp pipettes determined that the giant depolarizing bursts in dentate granule cells lead to no or only one or two action potential(s) per burst (on average, there was 0.8 ± 0.1 spikes/burst; n = 55 bursts examined from 10 granule cells; the recorded granule cells were not firing spontaneously between the depolarizing bursts; Fig. 5A). Interestingly, the immature granule cells were intrinsically capable of firing at higher frequencies, as determined by the ability of depolarizing current pulses delivered during the gramicidin recordings to cause the firing of action potentials at >10 Hz (not shown). In addition, these experiments also determined that the action potential threshold (
41.8 ± 1.7 mV, determined with depolarizing current pulses in n = 10 cells) in the immature dentate granule cells appeared to be slightly more depolarized than the reversal potential of the bursts (
46.6 ± 3.1 mV). Thus the GABAergic events alone (i.e., without the contribution of intrinsic conductances or EPSCs) would not be expected to cause high-frequency action potential discharges in developing granule cells of the dentate gyrus.

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| FIG. 5.
Effect of the bursts of IPSPs on action potential discharge in immature granule cells. A: gramicidin-based perforated patch-clamp recording shows the depolarizing bursts, which evoke most often only a single action potential per burst. B: similar results were obtained when granule cells were recorded in the cell-attached mode, as illustrated by the instantaneous frequency plot and by the trace below the plot. C: by contrast, hilar cells in cell-attached recordings displayed intense bursts of action potentials.
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|
In a further effort to determine the spontaneous firing frequency of the immature granule cells, granule cells were recorded in cell-attached mode as described elsewhere (Leinekugel et al. 1997
). As shown in Fig. 5B, and as expected from the gramicidin-data presented previously, granule cells most often fired only at low frequency (Fig. 5B; n = 6). By contrast, hilar neurons frequently fired in intense bursts (Fig. 5C; n = 5) (Strata et al. 1997
). In addition, because in all the experiments described previously the recording sites were restricted to the mid- and outer thirds of granule cell layer, further experiments were conducted to exclude the possibility that the youngest granule cells may exhibit burst firing. The newly generated granule cells are situated in the inner third of the granule cell layer, closest to the hilus, and based on the infrared video image, the position of the recorded cells could be easily determined. The data, obtained with whole cell pipettes in current-clamp mode with K-gluconate electrodes, showed that these youngest granule cells did not exhibit burst firing either, similar to their more mature counterparts (n = 4 granule cells in the inner third of the granule cell layer; consistent with these cells being younger than those in the mid- and outer thirds of the layer, their resting membrane potential was more depolarized, Vm =
43.3 ± 2.3 mV, compared with the granule cells in the mid- and outer thirds of the layer recorded under the same conditions, Vm =
55.0 ± 2.1, n = 3; in contrast to the lack of burst firing in both the younger and older developing granule cells, 3 out of 5 hilar cells of the early postnatal dentate gyrus, recorded under the same conditions, did fire at rest) (Strata et al. 1997
). In conclusion, these perforated, whole cell and cell-attached patch-clamp data show that the intense depolarizing bursts of IPSPs do not cause high-frequency firing in immature dentate granule cells.
GABAergic bursts occur in a similar temporal pattern in neighboring granule cells, but with imperfect synchrony
Simultaneous infrared-video microscopy-aided visualized whole cell patch-clamp recordings were obtained in current-clamp mode with Cl
-filled pipettes (QX-314 was also included to avoid action potential discharges) from pairs of immature granule cells of the dentate gyrus to determine whether the bursts of IPSPs occur synchronously. These experiments determined that invariably each time a burst occurred in one granule cell, the other simultaneously recorded granule cell also showed a burst (Fig. 6). However, the bursts did not occur in perfect synchrony (Fig. 6; average difference between the onsets of the bursts was 77.7 ± 8.6 ms; n = 3 pairs, n = 49 bursts measured; the pairs of cells were either immediate neighbors or within 2-4 cell bodies away from each other). The delay (lag) between the bursts within any one pair of granule cells did not appear to be constant from cycle to cycle [latency range between the onset of the bursts, with the onset of the burst in the "leading" cell being time 0: pair 1: 36.5-272.7 ms; pair 2:
4.6 ms (the negative sign indicates that the predominantly "follower"cell exhibited a burst before the burst occurred in the leader cell) to 259.1 ms; pair 3:
92-145.4 ms)]. Interestingly, large delays in the order of 100 ms were observed even between granule cells that were right next to each other in the layer (as determined by the infrared image). These data indicate that the IPSP bursts in immature dentate granule cells occur in a similar temporal pattern but with imperfect synchrony.

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| FIG. 6.
Temporal properties of the bursts of IPSPs in pairs of granule cells. A: simultaneous whole cell patch-clamp recordings of the bursts of IPSPs from a pair of granule cells show that the bursts occurred in a similar temporal pattern (i.e., when a burst occurred in cell 1, a burst could be invariably observed in cell 2). However, the bursts were not completely synchronous, as shown by the example of a pair of burst in B (indicated by the single or double asterisks in the recordings above; the delay between these 2 bursts was~70 ms). In this case, cell 1 was the typical "leader" cell, whereas cell 2 was a "follower" cell. As described in the text, large delays (lags) of several hundred milliseconds between the onsets of the bursts and the typically leading cell could occasionally become a follower cell.
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DISCUSSION |
The main findings of this study are that 1) large, GABAA receptor-mediated bursts of IPSCs are present in granule cells of the early postnatal dentate gyrus; 2) the intraburst IPSCs occur at higher frequencies and are larger in amplitude compared with the interburst IPSCs; 3) the EPSCs do not occur in bursts in these developing cells; 4) the generation of the bursts of IPSCs are critically dependent on glutamatergic excitatory drive onto interneurons; 5) the reversal potential for the bursts of IPSCs is more depolarized than the resting membrane potential in the immature granule cells, and the bursts are depolarizing at rest; 6) the reversal potential for the bursts of IPSCs is more hyperpolarized than the threshold for action potential generation, and the depolarizing bursts evoke only low-frequency action potential discharge in granule cells; and 7) the GABAergic bursts occur in a similar temporal pattern but not in perfect synchrony in neighboring granule cells.
Depolarizing nature of GABAA receptor-mediated IPSCs in immature and adult granule cells
In adult granule cells of the dentate gyrus, the relationship between the reversal potential for GABAA receptor activation and the resting membrane potential was measured by sharp electrode- and whole cell recordings as well as by noninvasive methods with cell-attached recordings of K+-channels (Soltesz and Mody 1994
; Staley and Mody 1992
; Staley et al. 1992
). In adult granule cells, the reversal potential for GABAA receptor activation is ~16 mV more depolarized than the resting membrane potential (Vm =
82.5 ± 2.7 mV) (Soltesz and Mody 1994
). The data presented in this paper show that in developing granule cells of the early postnatal dentate gyrus the resting membrane potential is also more hyperpolarized, by ~8 mV, than the reversal potential for the GABAA receptor-mediated events. Therefore these results indicate that during development the resting membrane potential and the reversal potential for GABAA receptor-mediated events both undergo prominent shifts in the hyperpolarizing direction in dentate granule cells (Liu et al. 1996
; Soltesz and Mody 1994
; Spruston and Johnston 1992
; Staley and Mody 1992
; Staley et al. 1992
). Specifically, early postnatal granule cells rest at around
54 mV and the resting membrane potential shifts to about
82 mV (~30 mV hyperpolarization) during development. The reversal potential for GABAA events hyperpolarizes from about
46 to
64 mV (a change of approximately
20 mV). Consequently, GABAA receptor activation causes larger depolarizations in membrane potential in granule cells from adult animals than in immature granule cells; however, the depolarization after GABAA receptor activation in immature granule cells can still bring the membrane potential close to firing threshold (
5 mV, because, as described previously, the reversal potential for the IPSCs is
46 mV, which is close to the action potential firing threshold of about
42 mV).
GABAA receptor-mediated large depolarizations and action potential discharge in immature granule cells
It is a remarkable feature of the developing interneuronal-granule cell system that the relationship between the resting membrane potential and the reversal potential for GABAA receptor-mediated events is so finely tuned that the depolarization during the intense bursts reaches its peak just below firing threshold. It is interesting to note that adult granule cells reach firing threshold also only infrequently. For example, during
oscillations in vivo, adult granule cells receive prominent, rhythmic barrages of depolarizing IPSPs in bouts of 40 Hz (Buzsáki et al. 1983
; Soltesz and Deschênes 1993
); however, they reach firing threshold only rarely during
oscillations, as determined with either extracellular (Jung and McNaughton 1993
; Mizumori et al. 1989
) or intracellular (Ylinen et al. 1995
) in vivo electrophysiological recordings. Therefore, irrespective of the age of the granule cell, depolarizing GABAA-receptor mediated bursts of IPSPs seem to be bringing the membrane potential closer to firing threshold, but when and which granule cells actually discharge is likely to be determined by additional depolarizations, e.g., those provided by EPSPs.
Recent results from developing hypothalamic neurons (Gao et al. 1998
) determined that the temporal relationship between the onset of the GABAA receptor-mediated depolarization and the glutamatergic input is a crucial determinant of whether the EPSP can result in action potential discharge during the GABAergic depolarization. Specifically, at the peak of the fast GABAergic depolarization, the glutamatergic input is severely shunted (see also Staley and Mody 1992
); however, after the peak and during the decay phase of the GABAA receptor-mediated depolarization, the same glutamatergic input can discharge the neuron. GABAA receptor-mediated IPSCs in immature granule cells are slower rising and slower decaying than their counterparts in adults (Hollrigel and Soltesz 1997
), which may help to maintain a relatively large window for the EPSPs to potentially discharge the neuron during the decay phase of the GABAergic burst. In addition, as pointed out before (Hollrigel and Soltesz 1997
), the slower kinetics of the IPSCs during development in dentate granule cells may also increase the probability of summation of the individual GABAA receptor-mediated events into large depolarizing bursts described in this paper. By contrast, presynaptic GABAB receptors (but not the postsynaptic GABAB receptors) appear functional early in postnatal development, resulting in the effective inhibitory control of the amplitude of the IPSCs during high-frequency firing of the presynaptic interneuron (Fukuda et al. 1993
; Gaiarsa et al. 1995
), which, in turn, is likely to shorten the decay phase of the depolarizing bursts (McLean et al. 1996
). Furthermore, GABAA receptors in granule cells of the developing dentate gyrus (but not in the adult) (Buhl et al. 1996
) are sensitive to zinc (Hollrigel and Soltesz 1997
), which is present in and is released from the mossy fiber terminals, at least in the adult (Assaf and Chung 1984
; Frederickson and Moncrieff 1994
; Howell et al. 1984
); although the developmental profile of the zinc content in and the zinc release from the mossy fiber terminals is not yet clear, the zinc-based Timm stain reveals mossy fibers as early as P1-P3 (Zimmer and Haug 1978
). Consequently, it is possible that the amplitude and the decay time constant of the IPSPs during the depolarizing bursts in early postnatal dentate granule cells may be regulated by the activity levels of the granule cells themselves. Therefore several pre- and postsynaptic factors play various, sometimes opposing, roles in shaping the morphology of the GABAergic bursts, which, in addition to supplying the bulk of the depolarization needed for the action potential discharge during development, may also contribute to the spatial and temporal gating of the intracellular Ca2+ signals in immature neurons (Ben-Ari et al. 1997
; Cherubini et al. 1991
; Gaiarsa et al. 1995
; Garaschuk et al. 1998
; Obrietan and van den Pol 1995
; Owens et al. 1996
; Reichling et al. 1994
; Rohrbough and Spitzer 1996
; Yuste and Katz 1991
).
Interneuronal network synchrony and the depolarizing bursts in developing granule cells
The generation of the depolarizing bursts in developing neurons presumably requires the regulation of the timing, amplitude, and kinetics of the IPSCs as well as the timing and synchronization of the firing of the presynaptic interneurons (Traub et al. 1998). Recent data suggest that interneurons in the dentate hilus may be the ultimate drivers of the large depolarizing bursts of GABAergic events throughout the CA1 and CA3 regions of the hippocampus (Strata et al. 1997
). Many hilar interneurons synapse with dentate granule cells, and, in turn, these GABAergic cells also receive synaptic inputs from the granule cells (Freund and Buzsáki 1996
; Halasy and Somogyi 1993
; Han et al. 1993
). Indeed, our results show that the generation of the bursts of IPSCs in the immature granule cells of the developing dentate gyrus is determined by the glutamatergic drive onto the interneurons. The glutamatergic drive may arise from the granule cells as well as hilar non-GABAergic cells and CA3c pyramidal cells and from perforant path fibers (Kneisler and Dingledine 1995a
,b
).
Importantly, the data presented in this paper also indicate that the glutamatergic drive onto interneurons in the developing dentate gyrus is more important in maintaining the sIPSC frequency in granule cells compared with adult. Specifically, in the adult dentate gyrus, APV and CNQX has negligible effect on the frequency of the sIPSCs recorded from granule cells (Buhl et al. 1996
; Otis and Mody 1992
; Thompson 1994
), indicating the low level of the spontaneous excitatory drive on the interneuronal networks of the dentate gyrus in the adult animal. By contrast, APV and CNQX caused a dramatic reduction in the frequency of the sIPSCs in immature granule cells (by blocking the sIPSC bursts, and hence the intraburst IPSCs). Therefore these data reveal a developmental decrease in the excitatory drive onto the interneurons of the dentate gyrus. It is interesting that this developmental decrease in the excitatory drive on interneurons can be reversed in the adult dentate gyrus, for example, by seizures (Buhl et al. 1996
).
The paired recording experiments showed that the bursts of IPSPs occur in a similar temporal pattern in neighboring granule cells, in the sense that when a burst occurred in one granule cell the other granule cell of the pair also showed a burst. However, the onset of the bursts was not perfectly synchronous; large (
272 ms) delays could be observed. This phase delay (lag) between the bursts suggests the intriguing possibility, to be followed up in subsequent studies, that distinct subsets of presynaptic interneurons may provide the depolarizing GABAergic inputs to particular subsets of granule cells. On the other hand, the similar temporal pattern of the bursts indicates that these hypothetical subsets of interneurons are coupled to each other or to a common "conductor" cell type (e.g., such as the calretinin- or vasoactive intestinal polypeptide-positive interneurons in the adult, which primarily contact other interneurons) (Freund and Buzsáki 1996
), resulting in a complex temporal and spatial distribution of the spontaneous release of GABA onto the postsynaptic membranes of the developing granule cells.
Possible differences between the maturation of inhibitory systems in pyramidal cells and granule cells
Comparison of the results presented in this paper with published data from hippocampal pyramidal cells indicate that prominent differences may exist between the spontaneous giant depolarizing bursts in the developing dentate gyrus and Ammon's horn. First, the giant depolarizing bursts in CA3 and CA1 pyramidal cells were reported to lead to the burst-firing of action potentials (Ben-Ari et al. 1989
; Garaschuk et al. 1998
; Leinekugel et al. 1997
; Strata et al. 1997
), in contrast with the low-frequency spontaneous action potential discharge observed in immature granule cells of the developing dentate gyrus. It is interesting to note that, similarly to dentate granule cells, spontaneous depolarizing bursts of IPSPs also lead to only low-frequency action potential discharge in immature granule cells of the developing cerebellar cortex, as determined with gramicidin-containing pipettes (Brickley et al. 1996
). Second, the depolarizing bursts were reported to take place more synchronously between pairs of pyramidal cells (Leinekugel et al. 1997
; Strata et al. 1997
) than in dentate granule cells. For example, Strata et al. (1997)
determined that the delay between the onset of the depolarizing events between CA3 pyramidal cells depended on the distance between the cells, ranging from a minimum of 5 ms when the distance was <0.5 mm to a maximum of 24 ms when the distance was 2 mm (average time delay = 14.4 ± 8.3 ms). By contrast, in the dentate granule cell layer even neighboring granule cells can exhibit prominent delays in the order of several hundred milliseconds, and there is also large (
300 ms) cycle-to-cycle variability in the delays of the onsets of the bursts in the case of simultaneously recorded granule cells. Third, the kinetics of the GABAA receptor-mediated currents were not reported to show developmental alterations in pyramidal cells, in contrast with dentate granule cells (Hollrigel and Soltesz 1997
). Finally, the early depolarizing GABAA responses become hyperpolarizing in adult pyramidal cells, whereas they remain depolarizing in granule cells of the adult dentate gyrus (Liu et al. 1996
; Soltesz and Mody 1994
; Staley and Mody 1992
). These data indicate that the developing GABAergic system may exhibit a staggering variety of phenotypes (Gibbs et al. 1996
; Kraszewski and Grantyn 1992
; Oh et al. 1995
; Tia et al. 1996
), even in closely interlinked brain regions such as the dentate gyrus and the Ammon's horn, indicating that GABAA receptor-mediated synaptic transmission may play developmental roles (Behar et al. 1996
; Hansen et al. 1984
1988; LoTurco et al. 1995
; Marty et al. 1996
) that are specific to various brain areas. Defining these time- and location-specific roles of the developing inhibitory system will be of great interest for future efforts aimed at determining the construction of intricate neuronal microciruits during mammalian development.