Synaptic GABA(A) activation inhibits AMPA-kainate receptor-mediated bursting in the newborn (P0-P2) rat hippocampus.

The mechanisms of synaptic transmission in the rat hippocampus at birth are assumed to be fundamentally different from those found in the adult. It has been reported that in the CA3-CA1 pyramidal cells a conversion of "silent" glutamatergic synapses to conductive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) synapses starts gradually after P2. Further, GABA via its depolarizing action seems to give rise to grossly synchronous yet slow calcium oscillations. Therefore, GABA is generally thought to have a purely excitatory rather than an inhibitory role during the first postnatal week. In the present study field potential recordings and gramicidin perforated and whole cell clamp techniques as well as K(+)-selective microelectrodes were used to examine the relative contributions of AMPA and GABA(A) receptors to network activity of CA3-CA1 pyramidal cells in the newborn rat hippocampus. As early as postnatal day (P0-P2), highly coherent spontaneous firing of CA3 pyramidal cells was seen in vitro. Negative-going extracellular spikes confined to periodic bursts (interval 16 +/- 3 s) consisting of 2.9 +/- 0.1 spikes were observed in stratum pyramidale. The spikes were accompanied by AMPA-R-mediated postsynaptic currents (PSCs) in simultaneously recorded pyramidal neurons (7.6 +/- 3.0 unitary currents per burst). In CA1 pyramidal cells synchronous discharging of CA3 circuitry produced a barrage of AMPA currents at >20 Hz frequencies, thus demonstrating a transfer of the fast CA3 network activity to CA1 area. Despite its depolarizing action, GABA(A)-R-mediated transmission appeared to exert inhibition in the CA3 pyramidal cell population. The GABA(A)-R antagonist bicuculline hypersynchronized the output of glutamatergic CA3 circuitry and increased the network-driven excitatory input to the pyramidal neurons, whereas the GABA(A)-R agonist muscimol (100 nM) did the opposite. However, the occurrence of unitary GABA(A)-R currents was increased after muscimol application from 0.66 +/- 0.16 s(-1) to 1.43 +/- 0.29 s(-1). It was concluded that AMPA synapses are critical in the generation of spontaneous high-frequency bursts in CA3 as well as in CA3-CA1 transmission as early as P0-P2 in rat hippocampus. Concurrently, although GABA(A)-R-mediated depolarization may excite hippocampal interneurons, in CA3 pyramidal neurons it can restrain excitatory inputs and limit the size of the activated neuronal population.

Fast network oscillations in the newborn rat hippocampus in vitro.

Spontaneous neural activity is crucial for the formation of the intricate patterns of cortical connectivity during development. In particular, temporal correlations in presynaptic and postsynaptic activity have been hypothesized to be a critical determinant in the selection of neurons that are to become wired together. To date, however, temporally correlated activity in the neonatal brain has been believed to take place with a precision of tens of milliseconds to seconds. Here we describe a novel type of a fast network oscillation associated with millisecond synchronization of pyramidal cell firing in newborn rat hippocampus in vitro. Individual pyramidal neurons fired mainly at lower gamma frequencies (20-40 Hz) but were synchronized into a high-frequency (100-400 Hz) population oscillation that was reflected in field potential spikes and intracellular AMPA-kainate receptor-mediated currents. The high-frequency population oscillation was patterned by a gamma-frequency modulatory oscillation. The gamma modulation was imposed by GABAergic currents, which exerted an inhibitory action on pyramidal neurons. Patterned activity based on GABAergic inhibition and glutamatergic excitation thus occurs already in newborn hippocampus. The network oscillations described here may be a mechanism for selective coincidence detection with a millisecond range temporal precision to shape the patterns of connectivity within the emerging hippocampal synaptic circuitry.

Maturation of kainate-induced epileptiform activities in interconnected intact neonatal limbic structures in vitro.

In vivo studies suggest that ontogenesis of limbic seizures is determined by the development of the limbic circuit. We have now used the newly-developed in vitro intact interconnected neonatal rat limbic structures preparation to determine the developmental profile of kainate-induced epileptiform activity in the hippocampus and its propagation to other limbic structures. We report gradual alterations in the effects of kainate during the first postnatal week on an almost daily basis; from no epileptiform activity at birth, through interictal seizures around postnatal day (P) 2 and ictal seizures by the end of the first week. The developmental profile of kainate-induced hippocampal seizures is paralleled by the expression of postsynaptic kainate receptor-mediated currents in CA3 pyramidal cells. Intralimbic propagation of the hippocampal seizures is also age-dependent: whereas seizures readily propagate to the septum and to the contralateral hippocampus via the commissures on P2, propagation to the entorhinal cortex only takes place from P4 onwards. Finally, repeated brief applications of kainate to the hippocampus induce recurrent spontaneous glutamatergic ictal and interictal discharges which persist for several hours after the kainate is washed away and which replace the physiological pattern of network activity. Paroxysmal activities are thus generated by kainate in the hippocampus at an early developmental stage and are initially restricted to this structure. Before the end of the first week of postnatal life, kainate generates the epileptiform activities that may perturb activity-dependent mechanisms that modulate neuronal development. Although at this stage neurons are relatively resistant to the pathological effects of kainate, the epileptiform activities that it generates will perturb activity-dependent mechanisms that modulate neuronal development.

Synaptic activation of GABAA receptors induces neuronal uptake of Ca2+ in adult rat hippocampal slices.

Synaptically evoked transmembrane movements of Ca2+ in the adult CNS have almost exclusively been attributed to activation of glutamate receptor channels and the consequent triggering of voltage-gated calcium channels (VGCCs). Using microelectrodes for measuring free extracellular Ca2+ ([Ca2+]o) and extracellular space (ECS) volume, we show here for the first time that synaptic stimulation of gamma-aminobutyric acid-A (GABAA) receptors can result in a decrease in [Ca2+]o in adult rat hippocampal slices. High-frequency stimulation (100-200 Hz, 0.4-0.5 s) applied in stratum radiatum close (

The K+/Cl- co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation.

GABA (gamma-aminobutyric acid) is the main inhibitory transmitter in the adult brain, and it exerts its fast hyperpolarizing effect through activation of anion (predominantly Cl-)-permeant GABA(A) receptors. However, during early neuronal development, GABA(A)-receptor-mediated responses are often depolarizing, which may be a key factor in the control of several Ca2+-dependent developmental phenomena, including neuronal proliferation, migration and targeting. To date, however, the molecular mechanism underlying this shift in neuronal electrophysiological phenotype is unknown. Here we show that, in pyramidal neurons of the rat hippocampus, the ontogenetic change in GABA(A)-mediated responses from depolarizing to hyperpolarizing is coupled to a developmental induction of the expression of the neuronal (Cl-)-extruding K+/Cl- co-transporter, KCC2. Antisense oligonucleotide inhibition of KCC2 expression produces a marked positive shift in the reversal potential of GABAA responses in functionally mature hippocampal pyramidal neurons. These data support the conclusion that KCC2 is the main Cl- extruder to promote fast hyperpolarizing postsynaptic inhibition in the brain.

A novel in vitro preparation: the intact hippocampal formation.

The intact hippocampal formation (IHF) of neonatal or young rats can be kept alive for an extended period in a fully submerged chamber with excellent morphological preservation. Field or patch-clamp recordings, intracellular Ca2+ measurements, and 3-D reconstruction of biocytin-filled neurons can be performed routinely. The generation and propagation of network-driven activities can be studied within the IHF or between connected intact structures such as the septum and the hippocampus or two hippocampi, and the use of a dual chamber enables the application of drugs separately to each structure. This preparation will be useful to study intact neuronal networks in the developing hippocampus in vitro.

Ionic mechanisms of spontaneous GABAergic events in rat hippocampal slices exposed to 4-aminopyridine.

Ionic mechanisms of spontaneous GABAergic events in rat hippocampal slices exposed to 4-aminopyridine. J. Neurophysiol. 78: 2582-2591, 1997. Ion-selective (H+ and K+) microelectrode techniques as well as conventional extra- and intracellular recordings were used to study the ionic mechanisms of propagating spontaneous GABAergic events (SGEs) in rat hippocampal slices exposed to 4-aminopyridine (4-AP, 50-100 mu M). All experiments were made in the presence of antagonists of ionotropic glutamate receptors [10 mu M 6-nitro-7-sulphamoylbenzoquinoxaline-2,3-dione (NBQX) and 40 mu M -2-amino-5-phosphonopentanoic acid (AP5)]. The SGEs were composed of a negative-going change in field potential with a temporally coincident increase (0.7 +/- 0.3 mM; mean +/- SE) in extracellular K+ ([K+]o) and an alkaline transient (0.01-0.08 units) in extracellular pH (pHo) in stratum radiatum of the area CA1. Simultaneous intracellular recordings showed a triphasic hyperpolarization-depolarization-late hyperpolarization response in pyramidal cells. Application of pentobarbital sodium (PB, 100 mu M) decreased the interval between SGEs from a mean value of 35 to approximately 20 s and shortened the period of refractoriness of stimulus-evoked propagating events. This was accompanied by an increase in the amplitude of the field potential response of the [K+]o and the pHo shifts and of the depolarizing phase of the pyramidal-cell response. The SGEs were completely blocked by the gamma-aminobutyric acid-A (GABAA) receptor antagonist, picrotoxin (PiTX; 100 mu M). The amplitudes of the negative-going field potential and of the depolarizing phase of the pyramidal-cell response as well as the ionic shifts associated with SGEs were strongly suppressed in the nominal absence of CO2/HCO-3. There was a five-fold increase in the interevent interval, and propagating SGEs could not be evoked by stimuli given at intervals shorter than approximately 2-3 min. Exposure to inhibitors of carbonic anhydrase, benzolamide (BA; 10 micro M) or ethoxyzolamide (EZA; 50 mu M) fully blocked the alkaline pHo transients and turned them into acid shifts. The poorly membrane-permeant BA had no discernible effect on the other components of the SGEs, but application of EZA had effects reminiscent to those of CO2/HCO-3-free medium. Addition of the GABAA receptor-permeant weak-acid anion, formate (20 mM) reestablished the SGEs that were first suppressed by exposure to the CO2/HCO-3-free medium. No SGEs were seen in the presence of a similar concentration of the GABAA receptor-impermeant anion propionate. Unlike the alkaline transients associated with HCO-3-driven SGEs, those supported by formate were not blocked by BA. The present data suggest that an inward current carried by bicarbonate is necessary for the generation of SGEs and that the GABAA receptor-mediated excitatory coupling among GABAergic interneurons is essentially dependent on the availability of intracellular bicarbonate.

Long-lasting GABA-mediated depolarization evoked by high-frequency stimulation in pyramidal neurons of rat hippocampal slice is attributable to a network-driven, bicarbonate-dependent K+ transient.

Biphasic GABAA-mediated postsynaptic responses can be readily evoked in CA1 pyramidal neurons of rat hippocampal slices by high-frequency stimulus (HFS) trains in the presence of ionotropic glutamate receptor antagonists. In the present experiments with sharp microelectrodes, whole-cell techniques, and K+-selective microelectrodes, an HFS train (40 pulses at 100 Hz) applied in stratum radiatum close to the recording site evoked a brief hyperpolarizing IPSP (hIPSP), which turned into a prolonged (2-3 sec) depolarization (GABA-mediated depolarizing postsynaptic potential; GDPSP). The I-V relationships of the postsynaptic currents (hIPSC and GDPSC) had distinct characteristics: the hIPSC and the early GDPSC showed outward rectification, whereas the late GDPSC was reduced with positive voltage steps to zero or beyond (inward rectification), but often no clear reversal was seen. That two distinct currents contribute to the generation of the GDPSP was also evident from the finding that a second HFS train at peak or late GDPSP induced a prompt GABAA-mediated hyperpolarization. The GDPSP/C was dependent on the availability of bicarbonate, but not on interstitial or intrapyramidal carbonic anhydrase activity. The HFS train evoked a rapid GABAA-mediated bicarbonate-dependent increase in the extracellular K+ concentration ([K+]o), and the GDPSP followed the K+ transient in a sub-Nernstian manner. The spatial and pharmacological characteristics of the [K+]o shift indicated that it is generated by a local network of GABAergic interneurons. The brief ascending phase of the GDPSP is linked to a K+-dependent accumulation of intracellular Cl-. Thereafter, a nonsynaptic mechanism, a direct depolarizing effect of the [K+]o shift, is responsible for the most conspicuous characteristics of the GDPSP: its large amplitude and prolonged duration.

Posttetanic excitation mediated by GABA(A) receptors in rat CA1 pyramidal neurons.

The contributions of gamma-aminobutyric acid (GABA) receptors to posttetanic excitation of CA1 pyramidal neurons in rat hippocampal slices were studied using extracellular and intracellular recording techniques. Synaptic responses were evoked on tetanic stimulation (100-200 Hz, 40-100 pulses) applied in stratum radiatum close (300-600 microm) to the recording site. Under control conditions, tetanic stimulation resulted in a triphasic depolarization/hyperpolarization/sustained depolarization sequence in area CA1 pyramidal cells. The late depolarization usually gave rise to a prolonged (< or = 3 s) spike firing. The late depolarization and the associated spike firing were blocked both specifically and completely (within a time window of 3-6 min starting from picrotoxin application) by the GABA(A) receptor antagonist picrotoxin (PiTX, 100 microM). Paradoxically, at this early stage of PiTX application, overall neuronal firing was attenuated to a higher degree than what was achieved by ionotropic glutamate antagonists. Complete block of ionotropic glutamate receptors by the antagonists D-2-amino-5-phosphonopentoate (AP5, 80 microM), 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX, 10 microM), and ketamine (50 microM) blocked the initial fast depolarization and suppressed the late one. Exposure to a permeable inhibitor of carbonic anhydrase, ethoxyzolamide (EZA, 50 microM) inhibited the late, apparently GABA-mediated depolarization. It is concluded that GABA can provide the main posttetanic excitatory drive in the adult hippocampus. The present results suggest that intense activation of GABAergic interneurons may accentuate the excitation of principal neurons and, hence, play an important facilitatory role in the induction of long-term potentiation (LTP) and epileptogenesis.