A parturition-associated nonsynaptic coherent activity pattern in the developing hippocampus.

Correlated neuronal activity is instrumental in the formation of networks, but its emergence during maturation is poorly understood. We have used multibeam two-photon calcium microscopy combined with targeted electrophysiological recordings in order to determine the development of population coherence from embryonic to postnatal stages in the hippocampus. At embryonic stages (E16-E19), synchronized activity is absent, and neurons are intrinsically active and generate L-type channel-mediated calcium spikes. At birth, small cell assemblies coupled by gap junctions spontaneously generate synchronous nonsynaptic calcium plateaus associated to recurrent burst discharges. The emergence of coherent calcium plateaus at birth is controlled by oxytocin, a maternal hormone initiating labour, and progressively shut down a few days later by the synapse-driven giant depolarizing potentials (GDPs) that synchronize the entire network. Therefore, in the developing hippocampus, delivery is an important signal that triggers the first coherent activity pattern, which is silenced by the emergence of synaptic transmission.

Spontaneous epileptic manifestations in a DCX knockdown model of human double cortex.

Previous reports indicate that in utero knockdown of doublecortin (DCX) results in the genesis of a subcortical heterotopia reminiscent of the doublecortex observed in female patients with DCX mutations. It has also been shown that these rats display an increased susceptibility to convulsant agents and increased cortical neurons excitability; but it is presently unknown whether they display spontaneous seizures. Furthermore, the link between the size of heterotopia and the clinical manifestation remained to be elucidated. Using video-electrocorticogram recordings, we now report that DCX knockdown induces frequent spontaneous seizures commonly associated with myoclonic jerks in adult rats. Surprisingly, epilepsy occurred even in rats with very small subcortical heterotopias, as revealed by histological analysis of recorded animals. Moreover, the severity of the epileptic manifestations was positively correlated with both the size of the subcortical heterotopia and the age of recorded animals; thus, epileptic features were not detected in immature affected rats. In conclusion, our data demonstrate for the first time that subtle alterations can yield epilepsy and reveal a strong correlation between thicknesses of subcortical heterotopia, age of affected individuals and clinical impairment.

Inhibitory actions of the gamma-aminobutyric acid in pediatric Sturge-Weber syndrome.

OBJECTIVE: The mechanisms of epileptogenesis in Sturge-Weber syndrome (SWS) are unknown. We explored the properties of neurons from human pediatric SWS cortex in vitro and tested in particular whether gamma-aminobutyric acid (GABA) excites neurons in SWS cortex, as has been suggested for various types of epilepsies. METHODS: Patch-clamp and field potential recordings and dynamic biphoton imaging were used to analyze cortical tissue samples obtained from four 6- to 14-month-old pediatric SWS patients during surgery. RESULTS: Neurons in SWS cortex were characterized by a relatively depolarized resting membrane potential, as was estimated from cell-attached recordings of N-methyl-D-aspartate channels. Many cells spontaneously fired action potentials at a rate proportional to the level of neuronal depolarization. The reversal potential for GABA-activated currents, assessed by cell-attached single channel recordings, was close to the resting membrane potential. All spontaneously firing neurons recorded in cell-attached mode or imaged with biphoton microscopy were inhibited by GABA. Spontaneous epileptiform activity in the form of recurrent population bursts was suppressed by glutamate receptor antagonists, the GABA(A) receptor agonist isoguvacine, and the positive allosteric GABA(A) modulator diazepam. Blockade of GABA(A) receptors aggravated spontaneous epileptiform activity. The NKCC1 antagonist bumetanide had little effect on epileptiform activity. INTERPRETATION: SWS cortical neurons have a relatively depolarized resting membrane potential and spontaneously fire action potentials that may contribute to increased network excitability. In contrast to previous data depicting excitatory and proconvulsive actions of GABA in certain pediatric and adult epilepsies, GABA plays mainly an inhibitory and anticonvulsive role in SWS pediatric cortex.

Synaptic kainate receptors tune oriens-lacunosum moleculare interneurons to operate at theta frequency.

GABAergic interneurons of the hippocampus play an important role in the generation of behaviorally relevant network oscillations. Among this heterogeneous neuronal population, somatostatin (SOM)-positive oriens-lacunosum moleculare (O-LM) interneurons are remarkable because they are tuned to operate at theta frequencies (6-10 Hz) in vitro and in vivo. Recent studies show that a high proportion of glutamatergic synapses that impinge on O-LM interneurons are mediated by kainate receptors (KA-Rs). In the present study, we thus tested the hypothesis that KA-Rs transmit afferent inputs in O-LM neurons during synaptic stimulation at theta frequency. We combined multibeam two-photon calcium imaging in hippocampal slices from SOM-enhanced green fluorescent protein (EGFP) mice, to record the activity of SOM cells as well as hundreds of neurons simultaneously, and targeted electrophysiological recordings and morphological analysis to describe the morphofunctional features of particular cells. We report that EGFP-positive O-LM neurons are the only subtype of interneuron that reliably follows synaptic stimulation of the alveus in the theta frequency range. Electrophysiological recordings revealed the crucial contribution of KA-Rs to the firing activity and to the glutamatergic response to theta stimuli in O-LM cells compared with other cell types. The reliable activation of O-LM cells in the theta frequency range did not simply result from the longer kinetics of KA-R-mediated postsynaptic events (EPSP(KA)) but presumably from a specific interaction between EPSP(KA) and their intrinsic active membrane properties. Such preferential processing of excitatory inputs via KA-Rs by distally projecting GABAergic microcircuits could provide a key role in theta band frequency oscillations.

Antiepileptic drugs and brain maturation: fetal exposure to lamotrigine generates cortical malformations in rats.

Intake of antiepileptic drugs (AEDs) during pregnancy can provoke severe and subtle fetal malformations associated with deleterious sequelae, reflecting the need for experimental investigations on the comparative teratogenic potential of these agents. We recently reported that prenatal exposure to vigabatrin and valproate, two AEDs which act through GABAergic mechanisms, induces hippocampal and cortical dysplasias in rodents. We have now investigated the effects of phenobarbital (PB, 30 mg/kg day) i.p.), a drug also endowed with GABAergic effects, and the new generation AEDs lamotrigine (LTG, 5-20mg/kg/day i.p.), topiramate (TPM, 10mg/kg/day i.p.), and levetiracetam (LEV, 50mg/kg/day i.p.) on brain development. Prenatal exposure to LTG induced hippocampal and cortical malformations in a dose-dependent manner, at maternal plasma concentrations within the clinically occurring range. These abnormalities were not observed after exposure to PB, TP and LEV. These observations raise concerns about potential clinical correlates and call for detailed comparative investigations on the consequences of AED use during pregnancy.

Glutamate acting on AMPA but not NMDA receptors modulates the migration of hippocampal interneurons.

Paracrine GABA and glutamate acting, respectively, on GABAA and NMDA receptors modulate the migration of hippocampal pyramidal cells. Using corticohippocampal organotypic explants from glutamic acid decarboxylase (GAD) 67-enhanced green fluorescent protein (EGFP) knock-in embryos, we now report that, in contrast to pyramidal neurons, the blockade of AMPA but not NMDA receptors exerts important actions on the migration of GABAergic interneurons. In addition, the blockade of GABAA receptors fails to modify the migration rates of GABAergic interneurons. Immunohistochemical analyses of GAD67-EGFP embryos (from embryonic day 14 to birth) reveal that interneurons colonize the hippocampal primordium by embryonic day 15. At that stage, the hippocampal primordium is already composed of pioneer glutamatergic neurons, including (1) Cajal-Retzius cells, immunopositive to calretinin and reelin, and (2) other presumptive pioneer pyramidal cells that are immunopositive to betaIII-tubulin and vesicular glutamate transporter 3 and immunonegative to GABA or GAD67. Therefore, the migrations of pyramidal neurons and GABAergic interneurons are cross-modulated: glutamate released from pioneer glutamatergic neurons facilitates the migration of GABAergic interneurons, which in turn would release GABA, facilitating the migration of glutamatergic neuroblasts. This general sequence may provide a retroactive positive loop needed to construct the hippocampal network. It might constitute a primitive homeostatic mechanism in the developing brain that acts to balance GABA-glutamate contributions to network construction and activity.

Ongoing epileptiform activity in the post-ischemic hippocampus is associated with a permanent shift of the excitatory-inhibitory synaptic balance in CA3 pyramidal neurons.

Ischemic strokes are often associated with late-onset epilepsy, but the underlying mechanisms are poorly understood. In the hippocampus, which is one of the regions most sensitive to ischemic challenge, global ischemia induces a complete loss of CA1 pyramidal neurons, whereas the resistant CA3 pyramidal neurons display a long-term hyperexcitability several months after the insult. The mechanisms of this long-term hyperexcitability remain unknown despite its clinical implication. Using chronic in vivo EEG recordings and in vitro field recordings in slices, we now report spontaneous interictal epileptiform discharges in the CA3 area of the hippocampus from post-ischemic rats several months after the insult. Whole-cell recordings from CA3 pyramidal neurons, revealed a permanent reduction in the frequency of spontaneous and miniature GABAergic IPSCs and a parallel increase in the frequency of spontaneous and miniature glutamatergic postsynaptic currents. Global ischemia also induced a dramatic loss of GABAergic interneurons and terminals together with an increase in glutamatergic terminals in the CA3 area of the hippocampus. Altogether, our results show a morpho-functional reorganization in the CA3 network several months after global ischemia, resulting in a net shift in the excitatory-inhibitory balance toward excitation that may constitute a substrate for the generation of epileptiform discharges in the post-ischemic hippocampus.

Recurrent mossy fibers establish aberrant kainate receptor-operated synapses on granule cells from epileptic rats.

Glutamatergic mossy fibers of the hippocampus sprout in temporal lobe epilepsy and establish aberrant synapses on granule cells from which they originate. There is currently no evidence for the activation of kainate receptors (KARs) at recurrent mossy fiber synapses in epileptic animals, despite their important role at control mossy fiber synapses. We report that KARs are involved in ongoing glutamatergic transmission in granule cells from chronic epileptic but not control animals. KARs provide a substantial component of glutamatergic activity, because they support half of the non-NMDA receptor-mediated excitatory drive in these cells. KAR-mediated EPSC(KA)s are selectively generated by recurrent mossy fiber inputs and have a slower kinetics than EPSC(AMPA). Therefore, in addition to axonal rewiring, sprouting of mossy fibers induces a shift in the nature of glutamatergic transmission in granule cells that may contribute to the physiopathology of the dentate gyrus in epileptic animals.

A noncanonical release of GABA and glutamate modulates neuronal migration.

Immature neurons express GABA and glutamate receptors before synapse formation, and both transmitters are released at an early developmental stage. We have now tested the hypothesis that the ongoing release of GABA and glutamate modulates neuronal migration. Using 5-bromo-2'-deoxyuridine labeling and cocultures of hippocampal slices obtained from naive and green fluorescent protein-transgenic mice, we report that migration is severely affected by GABA(A) or NMDA receptor antagonist treatments. These effects were also present in munc18-1 knock-out slices in which soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent vesicular secretion of transmitters has been deleted. GABA(A) antagonists were more efficient than NMDA antagonists to reduce cell migration, in keeping with the earlier maturation of GABAergic mechanisms. We conclude that GABA and, to a lesser degree, glutamate released in a SNARE-independent mechanism exert a paracrine action on neuronal migration.

Fetal exposure to GABA-acting antiepileptic drugs generates hippocampal and cortical dysplasias.

PURPOSE: The management of epilepsy during pregnancy entails a number of concerns. While seizures may affect adversely maternal and fetal outcome, antiepileptic drugs (AEDs) may increase the incidence of congenital abnormalities and possibly affect postnatal cognitive development in the offspring. Experimental animal studies can aid in assessing teratogenic features associated with individual AEDs and/or with seizures, and to identify the mechanisms involved. The purpose of this study was to investigate the consequences of prenatal exposure to (a) different AEDs and (b) maternal seizures on brain maturational processes in rats. METHODS: Pregnant rats received from embryonic days 14 to 19 intraperitoneal injections of carbamazepine (20 mg/kg/day), vigabatrin (200 mgkg/day), and valproate (100 mg/kg/day) at doses not widely different from those used clinically. Pups exposed to AEDs in utero were analyzed postnatally. Animals born to "kindled" pregnant animals that had experienced one generalized convulsive seizure per day during the same gestational period were analyzed in parallel. RESULTS: Prenatal exposure to vigabatrin and valproate, which act on GABA signaling, induced hippocampal and cortical dysplasias, which were likely to result from a neuronal migration defect and neuronal death. By contrast, offspring of rats exposed to carbamazepine (which at the dose used produced low plasma concentrations) or to generalized convulsive seizures showed no clear-cut evidence of dysplasias. CONCLUSIONS: We suggest that AEDs that increase the extracellular concentration of GABA might induce severe neuronal migration disorders. Drugs acting through other molecular targets would also perturb cortical maturation. The potential clinical relevance of these results should be a subject of future research.