Roustem Khazipov

Ca2+ Oscillations Mediated by the Synergistic Excitatory Actions of GABAA and NMDA Receptors in the Neonatal Hippocampus

We asked whether GABA(A) and NMDA receptors may act in synergy in neonatal hippocampal slices, at a time when GABA exerts a depolarizing action. The GABA(A) receptor agonist isoguvacine reduced the voltage-dependent Mg2+ block of single NMDA channels recorded in cell-attached configuration from P(2-5) CA3 pyramidal neurons and potentiated the Ca2+ influx through NMDA channels. The synaptic response evoked by electrical stimulation of stratum radiatum was mediated by a synergistic interaction between GABA(A) and NMDA receptors. Network-driven Giant Depolarizing Potentials, which are a typical feature of the neonatal hippocampal network, provided coactivation of GABA(A) and NMDA receptors and were associated with spontaneous and synchronous Ca2+ increases in CA3 pyramidal neurons. Thus, at the early stages of development, GABA is a major excitatory transmitter that acts in synergy with NMDA receptors. This provides in neonatal neurons a hebbian stimulation that may be involved in neuronal plasticity and network formation in the developing hippocampus.

γ-Aminobutyric acid (GABA): a fast excitatory transmitter which may regulate the development of hippocampal neurones in early postnatal life

The properties of neonatal GABAergic synapses were investigated in neurones of the hippocampal CA3 region. GABA, acting on GABAA receptors, provides most of the excitatory drive on immature CA3 pyramidal neurones at an early stage of development, whereas glutamatergic synapses (in particular, those mediated by AMPA receptors) are mostly quiescent. Thus, during the first postnatal week of life, bicuculline fully blocked spontaneous and evoked depolarising potentials, and GABAA receptor agonists depolarised CA3 pyramidal neurones. GABAA mediated currents also had a reduced sensitivity to benzodiazepines. In the presence of bicuculline, between P0 and P4, increasing the stimulus strength reveals an excitatory postsynaptic potential which is mostly mediated by NMDA receptors. During the same developmental period, pre- (but not post) synaptic GABAB inhibition is present. Intracellular injections of biocytin showed that the axonal network of the GABAergic interneurones is well developed at birth, whereas the pyramidal recurrent collaterals are only beginning to develop. Finally, chronic bicuculline treatment of hippocampal neurones in culture reduced the extent of neuritic arborisation, suggesting that GABA acts as a trophic factor in that period. In conclusion, it is suggested that during the first postnatal week of life, when excitatory inputs are still poorly developed, GABAA receptors provide the excitatory drive necessary for pyramidal cell outgrowth. Starting from the end of the first postnatal week of life, when excitatory inputs are well developed, GABA (acting on both GABAA and GABAB receptors) will hyperpolarise the CA3 pyramidal neurones and, as in the adult, will prevent excessive neuronal discharges. Our electrophysiological and morphological studies have shown that hippocampal GABAergic interneurones are in a unique position to modulate the development of CA3 pyramidal neurones. Developing neurones require a certain degree of membrane depolarisation, and a consequent rise in intracellular calcium, for stimulating neurite outgrowth; the GABAergic network, which develops prior to the glutamatergic one, appears to provide this depolarisation. Starting from the end of the first postnatal week of life, at a time when excitatory pathways are developing, GABA (acting on both GABAA and GABAB receptors) would reverse its action, and start to play its well-known role as an inhibitory neurotransmitter.

Synchronization of GABAergic interneuronal network in CA3 subfield of neonatal rat hippocampal slices.

1. Cell‐attached and whole‐cell recordings from interneurons localized in the stratum radiatum of the CA3 subfield (SR‐CA3) of neonatal (postnatal days 2‐5) rat hippocampal slices were performed to study their activity during the generation of GABAergic giant depolarizing potentials (GDPs) in CA3 pyramidal cells. 2. Dual recordings revealed that during the generation of GDPs in CA3 pyramidal cells, the interneurons fire bursts of spikes, on average 4.5 +/‐ 1.4 spikes per burst (cell‐attached mode). There bursts were induced by periodical large inward currents (interneuronal GDPs) recorded in whole‐cell mode. 3. Interneuronal GDPs revealed typical features of polysynaptic neuronal network‐driven events: they were blocked by TTX and by high divalent cation medium and they could be evoked in an all‐or‐none manner by electrical stimulation in different regions of the hippocampus. The network elements required for the generation of GDPs are present in local CA3 circuits since spontaneous GDPs were present in the isolated CA3 subfield of the hippocampal slice. 4. Interneuronal GDPs were mediated by GABAA and glutamate receptors, since: (i) their reversal potential strongly depended on [Cl‐]i; (ii) at the reversal potential of GABAA postsynaptic currents an inward component of GDPs was composed of events with the same kinetics as alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptor‐mediated EPSCs; and (iii) once GABAA receptors were blocked intracellularly by dialysis with F(‐)‐MgATP‐free solution, the remaining component of interneuronal GDPs reversed near 0 mV and rectified at membrane potentials more negative than ‐20 mV, suggesting an important contribution of NMDA receptors in addition to AMPA receptors. 5. In cell‐attached recordings from interneurons, electrical stimulation in the stratum radiatum evoked a burst of spikes that corresponded to evoked GDPs. Pharmacological study of this response revealed that excitation of SR‐CA3 interneurons during GDPs is determined by the co‐operative depolarizing actions mediated by GABAA and glutamate (AMPA and NMDA) receptors. Interestingly, after blockade of AMPA receptors, GABAA receptor‐mediated depolarization enabled the activation of NMDA receptors presumably via attenuation of their voltage‐dependent magnesium block. 6. It is concluded that synchronous activation of SR‐CA3 interneurons during generation of GDPs is mediated synaptically and is determined by the co‐operation of (i) excitatory GABAergic connections between interneurons and (ii) glutamatergic connections to interneurons originating presumably from the pyramidal cells.

Early Gamma Oscillations Synchronize Developing Thalamus and Cortex

Thalamic gamma rhythms help develop highly spatially and laminar-specific ascending cortical projections. During development, formation of topographic maps in sensory cortex requires precise temporal binding in thalamocortical networks. However, the physiological substrate for such synchronization is unknown. We report that early gamma oscillations (EGOs) enable precise spatiotemporal thalamocortical synchronization in the neonatal rat whisker sensory system. Driven by a thalamic gamma oscillator and initially independent of cortical inhibition, EGOs synchronize neurons in a single thalamic barreloid and corresponding cortical barrel and support plasticity at developing thalamocortical synapses. We propose that the multiple replay of sensory input in thalamocortical circuits during EGOs allows thalamic and cortical neurons to be organized into vertical topographic functional units before the development of horizontal binding in adult 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.

Dual Role of GABA in the Neonatal Rat Hippocampus

The effects of modulators of GABA-A receptors on neuronal network activity were studied in the neonatal (postnatal days 0–5) rat hippocampus in vitro. Under control conditions, the physiological pattern of activity of the neonatal hippocampal network was characterized by spontaneous network-driven giant depolarizing potentials (GDPs). The GABA-A receptor agonist isoguvacine (1–2 μM) and the allosteric modulator diazepam (2 μM) induced biphasic responses: initially the frequency of GDPs increased 3 to 4 fold followed by blockade of GDPs and desynchronization of the network activity. The GABA-A receptor antagonists bicuculline (10 μM) and picrotoxin (100 μM) blocked GDPs and induced glutamate (AMPA and NMDA)-receptor-mediated interictal- and ictal-like activities in the hippocampal slices and the intact hippocampus. These data suggest that at early postnatal ages GABA can exert a dual – both excitatory and inhibitory – action on the network activity.

New concepts in neonatal seizures.

The immature brain is more prone to seizures than the older brain as a result of an imbalance between excitatory and inhibitory input. The depolarizing, rather than hyperpolarizing effect of GABA(A) during the first week of life in the rodent, and the delay in postsynaptic GABA(B) inhibition coupled with the over-expression of glutamatergic synapses contribute to this increased propensity toward seizures. It is now clear that seizures can be injurious to the immature brain, although the pattern of seizure-induced injury is age-related. While the immature brain is resistant to acute seizure-induced cell loss, there are functional abnormalities following seizures with impairment of visual-spatial memory and reduced seizure threshold. Neonatal seizures are also associated with a number of activity-dependent changes in brain development including altered synaptogenesis and reduction in neurogenesis. These results argue that neonatal seizures should no longer be considered as benign events.

Giant Depolarizing Potentials: the Septal Pole of the Hippocampus Paces the Activity of the Developing Intact Septohippocampal Complex In Vitro

In neonatal hippocampal slices, recurrent spontaneous giant depolarizing potentials (GDPs) provide neuronal synchronized firing and Ca2+ oscillations. To investigate the possible role of GDPs in the synchronization of neuronal activity in intact neonatal limbic structures, we used multiple simultaneous electrophysiological recordings in the recently described preparation of intact neonatal septohippocampal complex in vitro. Combined whole-cell (in single or pairs of cells) and extracellular field recordings (one to five simultaneous recording sites) from the CA3 hippocampal region and various parts of the septum indicated that spontaneous GDPs, which can be initiated anywhere along the longitudinal hippocampal axis, are most often initiated in the septal poles of hippocampus and propagate to medial septum and temporal poles of both hippocampi simultaneously. GDPs were abolished in the medial septum but not in the hippocampus after surgical separation of both structures, suggesting hippocampal origin of GDPs. The preferential septotemporal orientation of GDP propagation observed in the intact hippocampus was associated with a corresponding gradient of GDP frequency in isolated portions of hippocampus. Accordingly, most GDPs propagated in the septotemporal direction in both septal and temporal hippocampal isolated halves, and whereas GDP frequency remained similar in the septal part of hippocampus after its surgical isolation, it progressively decreased in more temporally isolated portions of the hippocampus. Because GDPs provide most of the synaptic drive of neonatal neurons, they may modulate the development of neuronal connections in the immature limbic system.

Seizures accelerate anoxia‐induced neuronal death in the neonatal rat hippocampus

Seizures occurring in infants with hypoxia are frequently associated with an ominous prognosis. There is, however, no direct evidence that seizures are involved in the pathogenesis of hypoxia‐induced neuronal damage. Here, we report that seizures significantly aggravate the hypoxic state by accelerating rapid anoxic depolarization (AD) and associated neuronal death in preparations of the intact hippocampus of neonatal rats in vitro. Under control conditions, prolonged episodes of anoxia/aglycemia induced rapid suppression of synaptic activity followed sequentially by brief bursts of epileptiform activity and then by rapid AD. AD was associated with irreversible neuronal damage manifested by irreversible loss of the membrane potential, synaptic responses, and neuronal degeneration. Aggravation of electrographic seizure activity during anoxic episodes by the adenosine A1 receptor antagonists DPCPX and caffeine or the γ‐aminobutyric acid‐A receptor antagonist bicuculline or pretreatment with 4‐aminopyridine accelerated AD and associated neuronal death by up to twofold, whereas blockade of seizure activity by the glutamate receptor antagonists or tetrodotoxin significantly delayed the onset of AD. This report provides direct evidence for the need to prevent seizures during neonatal brain hypoxia. Ann Neurol 2000;48:632–640

In vivo blockade of neural activity alters dendritic development of neonatal CA1 pyramidal cells

During development, neural activity has been proposed to promote neuronal growth. During the first postnatal week, the hippocampus is characterized by an oscillating neural network activity and a rapid neuronal growth. In the present study we tested in vivo, by injecting tetanus toxin into the hippocampus of P1 rats, whether this neural activity indeed promotes growth of pyramidal cells. We have previously shown that tetanus toxin injection leads to a strong reduction in the frequency of spontaneous GABA and glutamatergic synaptic currents, and to a complete blockade of the early neural network activity during the first postnatal week. Morphology of neurobiotin‐filled CA1 pyramidal cells was analyzed at the end of the first postnatal week (P6–10). In activity‐reduced neurons, the total length of basal dendritic tree was three times less than control. The number, but not the length, of basal dendritic branches was affected. The growth impairment was restricted to the basal dendrites. The apical dendrite, the axons, or the soma grew normally during activity deprivation. Thus, the in vivo neural activity in the neonate hippocampus seems to promote neuronal growth by initiating novel branches.