The onset of puberty defines a developmental stage when some learning processes are diminished, but the mechanism for this deficit remains unknown. We found that, at puberty, expression of inhibitory α4βδ γ-aminobutyric acid type A (GABA A ) receptors (GABAR) increases perisynaptic to excitatory synapses in CA1 hippocampus. Shunting inhibition via these receptors reduced Nmethyl-D-aspartate receptor activation, impairing induction of long-term potentiation (LTP). Pubertal mice also failed to learn a hippocampal, LTP-dependent spatial task that was easily acquired by δ−/− mice. However, the stress steroid THP (3αOH-5α[β]-pregnan-20-one), which reduces tonic inhibition at puberty, facilitated learning. Thus, the emergence of α4βδ GABARs at puberty impairs learning, an effect that can be reversed by a stress steroid.Certain learning and cognitive processes decline at the onset of puberty (1-3). The pubertal process that shapes this developmental decline is unknown but is likely to involve the hippocampus, which is widely regarded as the site for learning (4-6). In addition to excitatory input, the inhibitory GABAergic (GABA, γ-aminobutyric acid) system plays a pivotal role in shaping developmental plasticity, as in the visual cortex (7), where drugs that target the γ-aminobutyric acid type A (GABA A ) receptor (GABAR) alter the timing of the critical period. The GABAR mediates most central nervous system inhibition and consists of diverse subtypes with distinct properties. Of these, α4βδ GABARs increase at pubertal onset in the mouse hippocampus (8), suggesting that they may shape plasticity here.We employed immunocytochemical, electron microscopic techniques (9) to localize and quantify α4 and δ GABAR subunits on CA1 hippocampal pyramidal cells across the pubertal state of female mice, because females exhibit greater deficits in learning at puberty than males † To whom correspondence should be addressed. sheryl.smith@downstate.edu. * These authors contributed equally to this work. (10,11). We detected immunostaining of both subunits perisynaptic to asymmetric synapses on the plasma membrane of spines of the apical dendrite, which increased up to 700% at puberty ( Fig. 1, A to C, and fig. S1; α4, P = 0.0048; δ, P = 0.00091) (9). In contrast, α4 and δ immunoreactivity on the dendritic shaft increased by less than 100% at puberty ( fig. S2). Functional expression of δ-containing GABAR at puberty was demonstrated by robust responses of pyramidal cells at puberty to 100 nM gaboxadol, which, at this concentration, is selective for this receptor (Fig. 1, D and E) (12). Gaboxadol had no effect before puberty and only a modest effect in the adult hippocampus (Fig. 1, D and E), where α4 and δ expression is lower than at puberty ( fig. S3).Extrasynaptic α4β2δ GABARs on spines could impair voltage-triggered Mg++ unblock of Nmethyl-D-aspartate (NMDA) receptors. Thus, we used whole-cell voltage clamp techniques with blockade of synaptic GABARs (13) to record evoked NMDA excitatory postsynaptic currents (EPSCs) from CA1 ...

SUMMARY1. Current mediated by GABAA receptors was examined in pyramidal cells acutely dissociated from the hippocampus of mature guinea-pigs. Current responses were measured using whole-cell voltage-clamp recordings. An internal perfusion technique was used to change the intracellular contents during recording.2. Application of GABA (100-300 /M) by short duration pressure pulses produced outward current responses at a holding potential of -10 mV. When recordings were made with intracellular solutions which did not contain Mg-ATP, GABA responses progressively decreased to less than 10% of their initial values after 10 min. This 'run-down' of the GABA response could not be accounted for by desensitization since the rate of run-down was not dependent upon agonist application.3. The run-down of the GABAA response was reversed when Mg2+ (4 mM) and ATP (2 mM) were introduced into the intracellular perfusate. In addition to the presence of Mg-

Gamma aminobutyric acid (GABA) mediates fast synaptic inhibition in the central nervous system by activating the chloride-permeable GABAA channel. The GABAA conductance progressively diminishes with time when the intracellular contents of hippocampal neurons are perfused with a minimal intracellular medium. This "run down" of the GABA-activated conductance can be prevented by the inclusion of magnesium adenosine triphosphate and calcium buffer in the intracellular medium. The amount of chloride conductance that can be activated by GABA is determined by competition between a calcium-dependent process that reduces the conductance and a phosphorylation process that maintains the conductance.

The application of tetanic electrical stimuli to the stratum radiatum fibre pathway in the hippocampus in vitro produces an NMDA (N-methyl-D-aspartate) receptor-dependent enhancement of synaptic efficacy. Repeated application of such stimuli produces a progressive enhancement of synaptic efficacy leading to the genesis of spontaneous and stimulation-evoked epileptiform discharges. We have used this in vitro approach to explore the cellular mechanisms which underlie the kindling model of epilepsy. Kindling of the stratum radiatum fibre pathway in vitro induced a progressive, long-lasting reduction of both spontaneous and stimulation-evoked GABAergic (gamma-aminobutyric acid-mediated) inhibitory postsynaptic potentials (i.p.s.ps). The reduction of i.p.s.ps by kindling was associated with a profound decrease in the sensitivity of CA1 pyramidal neurons to ionophoretically applied GABA and an increase in sensitivity to NMDA. The reduction of i.p.s.ps and GABA sensitivity was prevented by kindling in the presence of the NMDA receptor antagonist D-2-amino-5-phosphonovalerate (D-APV). These results demonstrate that kindling-like stimulus patterns produce a reduction of GABAergic inhibition in the hippocampus resulting from a stimulus-induced postsynaptic activation of NMDA receptors. The modulation of GABAergic inhibition by NMDA receptors may cause the synaptic plasticity which underlies the kindling model of epilepsy.

Long-term potentiation (LTP) in the CA1 region of the hippocampus is widely believed to occur through In spite of the attention excitatory transmission processes have received in connection with LTP, potentiation of excitatory responses after tetanization could theoretically occur through impairment of synaptic inhibition. Type A y-aminobutyric acid (GABAA) receptor-mediated synaptic inhibition plays a critical role in the control of excitation in the mammalian central nervous system (6). Several studies have reported short-term changes of synaptic inhibition in connection with high-frequency stimulation (7-9). During the maintenance phase of LTP, however, orthodromically evoked early inhibitory postsynaptic potentials (IPSPs) are generally not reduced (10-12). Furthermore, postsynaptic GABA sensitivity was found to be unchanged during LTP in an extracellular study (13), and in several studies the excitability of GABAergic interneurons was found to be unchanged or even increased during LTP (14)(15)(16) Fig. 2).Stimulation. Evoked responses were elicited by stimulation of stratum radiatum Schaffer collateral/commissural fibers through a pair of insulated tungsten bipolar electrodes (stimulation range, 15-400 1uA). Tetanic stimulation (50 or 100 Hz; 1 s; single-pulse duration, 80 As) was applied via the same stimulation electrode.Drugs. Bicuculline, picrotoxin (PTX), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 2-amino-5-phosphonopentanoic acid (AP5), and saclofen were applied by bath perfusion. CNQX, D-AP5, and saclofen were purchased from Tocris Neuramin (Bristol, U.K.); all other drugs were from Sigma. Iontophoresis. lontophoretic GABAA responses were elicited by application of GABAA-receptor agonists muscimol (50 mM, pH 3.5, in extracellular solution) or GABA (1 M, pH 3.5, in the presence of bath-applied GABAB-receptor antagonist saclofen, 100 pM) in CA1 pyramidal cells through a double-or triple-barreled extracellular iontophoretic electrode with one channel containing extracellular solution for current balancing (ejecting currents, +5 to +140 nA; retaining currents, -3 to -20 nA). The iontophoretic electrode was positioned by an independent micromanipulator as close as possible to the respective recording site.Data Acquisition. Data (voltage responses from the recording electrode) were digitized and stored on disk (Nicolet 410 oscilloscope) for subsequent off-line analysis.LY Staining and Histology. LY staining (26-28) was performed in 17 (of 57) apical dendritic recordings (29,30) and 26 (of 55) interneuron recordings (31) for morphological confirmation of cell type. The tip of the electrode was back-filled with LY (1.5% in 1 M Li2SO4 or LiCl) whereas the shaft was filled with 1 M Li2SO4 or LiCl alone. Dye injection was implemented by repetitive 400-ms hyperpolarizing current pulses (between -0.5 and -1 nA DC; 5-10 min; 0.5 Hz)

The response to afferent fibre stimulation ('orthodromic response') is a commonly used measure of excitability of neuronal populations. An excitatory (EPSP) and inhibitory (IPSP) component was identified by studies of the orthodromic response in hippocampal pyramidal cells in vivo (Kandel et al. 1961;Andersen et al. 1963Andersen et al. , 1964. This basic sequence of orthodromic EPSP-IPSP has been confirmed since then: the response that is elicited by stimulation of the Schaffer collateral-commissural pathway consists of a fast EPSPÏEPSC mediated primarily by AMPA receptors, followed by an early IPSPÏIPSC mediated by GABAA receptors and a late IPSPÏIPSC mediated by GABAB receptors. Despite such clear separation of postsynaptic potentials (PSPs) or currents (PSCs), it is conceivable that inhibitory influences may be present long before the population IPSP is expressed. If so, how large is the temporal overlap with the orthodromic EPSPÏEPSC?This study has focused on three points. First, the kinetics of dendritic inhibition were studied. Several previous studies have reported that the kinetics of dendritic inhibitory responses were slower than those of somatic responses in pyramidal cells (Miles, 1990b;Pearce, 1993;Buhl et al. 1994a). Second, kinetics of different inhibitory responses, i.e. IPSPs, IPSCs and conductance changes, were investigated. It was suggested that inhibitory conductance changes are faster than IPSPs (Araki et al. 1960) or inhibitory currents (Koch, 1985). Third, time courses of various components of the circuitry were studied: a monosynaptic inhibitory response is elicited via direct electrical stimulation of interneuron processes (Davies et al. 1990). Activation of afferent fibres results in a feedforward inhibitory response in CA1 pyramidal cells via synaptic activation of CA1 interneurons (Knowles & Schwartzkroin, 1981;Alger & Nicoll, 1982;Buzsaki & Eidelberg, 1982; Ashwood et al. Journal of Physiology (1999) but > 3·0 ms after the peak of the pyramidal cell EPSP. 6. Orthodromically evoked interneuron spikes were observed at stimulation intensities that were below the threshold for eliciting EPSPs in concomitantly recorded pyramidal cells. The activation of feedforward inhibitory responses by weakest excitatory input, and the large temporal overlap between feedforward inhibition and evoked excitation, suggest that in situ any excitatory input in CA1 is effectively controlled by fast synaptic inhibition. Keywords:

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Spontaneous activity was monitored during pharmacological blockade of GABAA receptor function in the CA1 minislice (CA3 was cut off). Synaptic inhibition was blocked by competitive GABAA antagonists bicuculline‐methiodide (Bic) or GABAZINE (GBZ) and the chloride channel blocker picrotoxin (PTX). Extra‐ and intracellular recordings using sharp electrodes were carried out in stratum radiatum and pyramidale. At low antagonist concentrations (Bic, GBZ: 1‐10 μm; PTX: < 100 μm), synchronized bursts (< 500 ms in duration, interictal activity) were seen as described previously. However, in the presence of high concentrations (Bic, GBZ: 50‐100 μm; PTX: 100‐200 μm), seizure‐like, ictal events (duration 4‐17 s) were observed in 67 of 88 slices. No other experimental measures to increase excitability were applied: cation concentrations ([Ca2+]o= 2 mm, [Mg2+]o= 1.7 mm, [K+]o= 3 mm) and recording temperature (30‐32 °C) were standard and GABAB‐mediated inhibition was intact. In whole‐slice recordings prominent interictal activity, but fewer ictal events were observed. A reduced ictal activity was also observed when interictal‐like responses were evoked by afferent stimulation. Ictal activity was reversibly blocked by antagonists of excitatory transmission, CNQX (40 μm) or d‐AP5 (50 μm). Disinhibition‐induced ictal development did not rely on group I mGluR activation as it was not prevented in the presence of group I mGluR antagonists (AIDA or 4CPG). (RS)‐3,5‐DHPG prevented the induction and reversed the tertiary component of the ictal event through a group I mGluR‐independent mechanism.