Granule cell hyperexcitability in the early post-traumatic rat dentate gyrus: the 'irritable mossy cell' hypothesis.

1. Cytochemical and in vitro whole-cell patch clamp techniques were used to investigate granule cell hyperexcitability in the dentate gyrus 1 week after fluid percussion head trauma. 2. The percentage decrease in the number of hilar interneurones labelled with either GAD67 or parvalbumin mRNA probes following trauma was not different from the decrease in the total population of hilar cells, indicating no preferential survival of interneurones with respect to the non-GABAergic hilar cells, i.e. the mossy cells. 3. Dentate granule cells following trauma showed enhanced action potential discharges, and longer-lasting depolarizations, in response to perforant path stimulation, in the presence of the GABAA receptor antagonist bicuculline. 4. There was no post-traumatic alteration in the perforant path-evoked monosynaptic excitatory postsynaptic currents (EPSCs), or in the intrinsic properties of granule cells. However, after trauma, the monosynaptic EPSC was followed by late, polysynaptic EPSCs, which were not present in controls. 5. The late EPSCs in granule cells from fluid percussion-injured rats were not blocked by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV), but were eliminated by both the non-NMDA glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the AMPA receptor antagonist GYKI 53655. 6. In addition, the late EPSCs were not present in low (0.5 mM) extracellular calcium, and they were also eliminated by the removal of the dentate hilus from the slice. 7. Mossy hilar cells in the traumatic dentate gyrus responded with significantly enhanced, prolonged trains of action potential discharges to perforant path stimulation. 8. These data indicate that surviving mossy cells play a crucial role in the hyperexcitable responses of the post-traumatic dentate gyrus.

Temporal patterns and depolarizing actions of spontaneous GABAA receptor activation in granule cells of the early postnatal dentate gyrus.

Whole cell patch-clamp recordings were used to investigate the properties of the gamma-aminobutyric acid type A (GABAA) receptor-mediated spontaneous synaptic events in immature granule cells of the developing, early postnatal day (P0-P6) rat dentate gyrus. With Cs-gluconate-filled whole cell patch pipettes at 0 mV in control medium, spontaneous inhibitory postsynaptic currents (sIPSCs) occurred in prominent bursts (peak amplitude of the bursts 406.9 +/- 58.4 pA; intraburst IPSC frequency 71.0 +/- 12.4 Hz) at 0.05 +/- 0.02 Hz in every immature granule cell younger than P7. Between the bursts of IPSCs, lower frequency (1.7 +/- 0.7 Hz), interburst IPSCs could be observed. Bicuculline and picrotoxin as well as the intracellularly applied chloride-channel blockers CsF- and 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS) abolished the intraburst as well as the interburst IPSCs, indicating that the IPSCs were mediated by GABAA receptor channels. The bursts of IPSCs, but not the interburst IPSCs, were blocked by the simultaneous application of the glutamate receptor antagonists 2-amino-5-phosphovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione, indicating the importance of the glutamatergic excitatory drive onto the interneurons in the early postnatal dentate gyrus. The spontaneously occurring excitatory postsynaptic currents in immature granule cells, observable after the intracellular blockade of GABAA receptor channels with CsF- and DIDS, appeared exclusively as single events at low frequencies, i.e., they did not occur in prominent bursts. Gramicidin-based perforated patch-clamp recordings determined that the reversal potential for the burst of IPSCs (-46.6 +/- 3.1 mV) was more depolarized than the resting membrane potential (-54.2 +/- 4.2 mV) but more hyperpolarized than the action potential threshold (-41. 8 +/- 1.7 mV). The depolarizing action of the bursts of synaptic events most often evoked only a single action potential per burst. Simultaneous whole cell patch recordings, with KCl-filled patch pipettes at -60 mV in current clamp from pairs of immature granule cells of the developing dentate gyrus, determined that the bursts of IPSPs took place in a similar temporal pattern but with imperfect synchrony in neighboring granule cells (average lag between the onsets of the bursts between granule cell pairs 77.7 +/- 8.6 ms). These results show that the spontaneous activation of GABAA receptors in immature dentate granule cells displays unique properties that are distinct from the temporal patterns and biophysical features of spontaneous GABAA receptor activation taking place in the developing Ammon's horn and in the adult dentate gyrus.

The pro-convulsant actions of corticotropin-releasing hormone in the hippocampus of infant rats.

Whole-cell patch-clamp and extracellular field recordings were obtained from 450-microns-thick brain slices of infant rats (10-13 days postnatal) to determine the actions of corticotropin-releasing hormone on glutamate- and GABA-mediated synaptic transmission in the hippocampus. Synthetic corticotropin-releasing hormone (0.15 microM) reversibly increased the excitability of hippocampal pyramidal cells, as determined by the increase in the amplitude of the CA1 population spikes evoked by stimulation of the Schaffer collateral pathway. This increase in population spike amplitude could be prevented by the corticotropin-releasing hormone receptor antagonist alpha-helical (9-41)-corticotropin-releasing hormone (10 microM). Whole-cell patch-clamp recordings revealed that, in the presence of blockers of fast excitatory and inhibitory synaptic transmission, corticotropin-releasing hormone caused only a small (1-2 mV) depolarization of the resting membrane potential in CA3 pyramidal cells, and it did not significantly alter the input resistance. However, corticotropin-releasing hormone, in addition to decreasing the slow afterhyperpolarization, caused an increase in the number of action potentials per burst evoked by depolarizing current pulses. Corticotropin-releasing hormone did not significantly change the frequency, amplitude or kinetics of miniature excitatory postsynaptic currents. However, it increased the frequency of the spontaneous excitatory postsynaptic currents in CA3 pyramidal cells, without altering their amplitude and single exponential rise and decay time constants. Corticotropin-releasing hormone did not change the amplitude of the pharmacologically isolated (i.e. recorded in the presence of GABAA receptor antagonist bicuculline) excitatory postsynaptic currents in CA3 and CA1 pyramidal cells evoked by stimulation of the mossy fibers and the Schaffer collaterals, respectively. Current-clamp recordings in bicuculline-containing medium showed that, in the presence of corticotropin-releasing hormone, mossy fiber stimulation leads to large, synchronized, polysynaptically-evoked bursts of action potentials in CA3 pyramidal cells. In addition, the peptide caused a small, reversible decrease in the amplitude of the pharmacologically isolated (i.e. recorded in the presence of glutamate receptor antagonists) evoked inhibitory postsynaptic currents in CA3 pyramidal cells, but it did not significantly alter the frequency, amplitude, rise and decay time constants of spontaneous or miniature inhibitory postsynaptic currents. These data demonstrate that corticotropin-releasing hormone, an endogenous neuropeptide whose intracerebroventricular infusion results in seizure activity in immature rats, has diverse effects in the hippocampus which may contribute to epileptogenesis. It is proposed that the net effect of corticotropin-releasing hormone is a preferential amplification of those incoming excitatory signals which are strong enough to reach firing threshold in at least a subpopulation of CA3 cells. These findings suggest that the actions of corticotropin-releasing hormone on neuronal excitability in the immature hippocampus may play a role in human developmental epilepsies.

Enhanced bursts of IPSCs in dentate granule cells in mice with regionally inhibited long-term potentiation.

Until recently, most studies on the synaptic-cellular basis of learning and memory concentrated on the activity-dependent changes occurring in principal cells such as hippocampal pyramidal cells and dentate granule cells. However, the ability of the inhibitory interneurons to regulate synaptic plasticity remains less understood. This study tested the hypothesis that the gamma-aminobutyric-acid (GABA)-mediated inhibitory neurotransmission is enhanced in mice that show no detectable long-term potentiation in the dentate gyrus in the absence of the GABAA receptor antagonist bicuculline. Patch clamp recordings were made from dentate granule cells in brain slices from wild-type and Thy-1 knockout (KO) mice. The frequency, amplitude and kinetics of miniature inhibitory postsynaptic currents (mIPSCs, generated by the action potential-independent release of GABA) was not different between animals. However, bursts of spontaneous IPSCs (sIPSCs, generated by both action potential-independent and -dependent GABA release) in KO mice were associated with larger synaptic charge transfers and increased durations. When pairs of IPSCs were evoked at varying intervals, the amplitude of the second response with respect to the first was significantly larger in KO animals. These results further support the concept that enhancement of interneuronal functions in cortical structures can have profound effects on the activity-dependent synaptic plasticity observed in principal cells.

Instantaneous perturbation of dentate interneuronal networks by a pressure wave-transient delivered to the neocortex.

Whole-cell patch-clamp recordings and immunocytochemical experiments were performed to determine the short- and long-term effects of lateral fluid percussion head injury on the perisomatic inhibitory control of dentate granule cells in the adult rat, with special reference to the development of trauma-induced hyperexcitability. One week after the delivery of a single, moderate (2.0-2.2 atm) mechanical pressure wave to the neocortex, the feed-forward inhibitory control of dentate granule cell discharges was compromised, and the frequency of miniature IPSCs was decreased. Consistent with the electrophysiological data, the number of hilar parvalbumin (PV)- and cholecystokinin (CCK)-positive dentate interneurons supplying the inhibitory innervation of the perisomatic region of granule cells was decreased weeks and months after head injury. The initial injury to the hilar neurons took place instantaneously after the impact and did not require the recruitment of active physiological processes. Furthermore, the decrease in the number of PV- and CCK-positive hilar interneurons was similar to the decrease in the number of the AMPA-type glutamate receptor subunit 2/3-immunoreactive mossy cells, indicating that the pressure wave-transient causes injurious physical stretching and bending of most cells that are large and not tightly packed in a cell layer. These results reveal for the first time that moderate pressure wave-transients, triggered by traumatic head injury episodes, impact the dentate neuronal network in a unique temporal and spatial pattern, resulting in a net decrease in the perisomatic control of granule cell discharges.

Slow kinetics of miniature IPSCs during early postnatal development in granule cells of the dentate gyrus.

Whole-cell patch-clamp recordings were used to investigate the properties of GABAA receptor-mediated postsynaptic currents during development in dentate gyrus granule cells from neonatal [postnatal day 0 (P0)] to adult rats in brain slices. The frequency of miniature IPSCs (mIPSCs) was low at birth and increased progressively with age. The mIPSCs of all ages could be satisfactorily fitted with the sum of a single exponential rise and single exponential decay. From P0 to P14, both the rise time and the decay time constants were significantly longer than in the adult. The mIPSC rise and decay kinetics did not change during the first 2 postnatal weeks, but during the third week the kinetics sped up and by P21 attained adult values. In contrast, the amplitude of the mIPSCs did not change during development. The synaptic GABAA receptors in immature and adult cells showed differential sensitivity to modulators. The subunit-specific benzodiazepine agonist zolpidem increased the decay time constant of the IPSCs of immature granule cells with a reduced potency compared with the adult. Furthermore, zinc decreased the amplitude and decay time constant of mIPSCs from developing granule cells, whereas it had no effect on mIPSCs in adult neurons. The results reveal for the first time that until the end of the second postnatal week the synaptic GABAA receptor-mediated currents in dentate granule cells display slower rise and decay kinetics but similar amplitudes compared with adult, resulting in a net decrease in synaptic charge transfer during development.

Neuroprotection by propofol in acute mechanical injury: role of GABAergic inhibition.

1. Whole cell patch-clamp and extracellular field recordings were obtained from granule cells of the dentate gyrus in 400-microns-thick brain slices of the adult rat to determine the actions of the intravenous general anesthetic 2,6-diisopropylphenol (propofol) on acute neuronal survival and preservation of synaptic integrity after amputation of dendrites (dendrotomy), and to determine the role of gamma-aminobutyric acid-A (GABAA)-receptor-mediated inhibition in the neuroprotective effects of propofol. The actions of propofol were compared with those exerted by another widely used intravenous general anesthetic, 5-ethyl-5-[1-methylbutyl]-2-thiobarbituric acid (thiopental). 2. Propofol (10 microM) increased the frequency (control: 5.9 +/- 0.9 Hz, mean +/- SE; propofol: 10.5 +/- 1.3 Hz) and the single-exponential decay time constant (tau D) (control: 4.5 +/- 0.2 ms; propofol: 15.3 +/- 1.5 ms) of miniature inhibitory postsynaptic currents (mIPSCs) recorded in control neurons. Thiopental (25 microM) also increased the tau D (14.3 +/- 0.9 ms) of mISPCs, but had no effect on mIPSC frequency. Both anesthetics potentiated mIPSCs at low concentrations (propofol: 5 microM; thiopental: 1 microM). Propofol and thiopental did not change the peak amplitude and rise times of mIPSCs. 3. Propofol (10 microM) was able to depress the excitability of control granule cells, as determined by the reduction in the amplitude of the orthodromic population spikes. This depression could be prevented by the GABAA receptor antagonist bicuculline (50 microM), indicating that propofol reduces excitability via GABAA receptor functions. 4. Propofol and thiopental were neuroprotectant (assessed by antidromic population responses 2-5 h after injury) if present before and during the amputation of the granule cell dendrites. The protective actions were dose dependent, and at high doses (propofol: 200 microM; thiopental: 400 microM) the anesthetics were as neuroprotective against dendrotomy-induced cell death as 2-amino 5-phosphovaleric acid (APV) and 6-cyano-7-nitroquinoxaline-2,3 dione (CNQX). The protective effects of the anesthetics were completely blocked with the GABAA receptor antagonists picrotoxin or bicuculline, and were mimicked by the GABAA receptor agonist muscimol (100 microM). 5. Propofol, in contrast to APV and CNQX, could not prevent the dendrotomy-induced Ca(2+)-dependent and long-lasting changes in mIPSC decay kinetics (appearance of a double-exponential, prolonged decay). 6. The protective effects of the anesthetics and those of APV and CNQX on neuronal survival were not significant when the drugs were applied after dendrotomy, indicating that dendrotomy carried out 150-200 microns from the soma without neuroprotective agents rapidly induces irreversible acute degeneration in most injured neurons. The failure to rescue cells from dendrotomy-induced injury did not result from a decreased sensitivity of the GABAA receptors to the anesthetics, because the potentiating effects of the anesthetics on mIPSCs from control and dendrotomized neurons were not different. 7. These data indicate that propofol potentiates synaptic inhibition pre- and postsynaptically, and, when present during dendrotomy, it can protect neurons from acute mechanical-injury induced cell death via potentiation of GABAA receptor functions. However, propofol fails to provide neuroprotection against dendrotomy-induced changes in synaptic physiology.

Response properties of units in the posterior auditory field deprived of input from the ipsilateral primary auditory cortex.

The influence of the ipsilateral primary auditory field (AI) on the response properties of neurons in the posterior auditory field (Field P) was examined in three cats anesthetized with sodium pentobarbital. Rate/level functions were obtained, by extracellular recording, from single units in Field P before (n = 38) and after (n = 50) subpial aspiration of AI. The ablations were primarily confined to the medial ectosylvian gyrus, although in one case extended into the high-frequency portion of the anterior auditory field. Comparisons between the behavior of units isolated before and after AI ablation failed to demonstrate any changes in the response properties of neurons in Field P attributable to the ablation. Nonmonotonic response profiles, first spike latency, variability in latency, threshold and maximal discharge rates of the units to acoustic stimuli were not significantly altered by the AI ablation. These results indicate that the basic response properties of neurons in Field P do not depend on input from the ipsilateral AI. This suggests that these properties are most likely determined by thalamic input or by circuitry within Field P.