Yoko Fujiwara-Tsukamoto

Excitatory gaba input directly drives seizure-like rhythmic synchronization in mature hippocampal CA1 pyramidal cells

GABA, which generally mediates inhibitory synaptic transmissions, occasionally acts as an excitatory transmitter through intense GABA(A) receptor activation even in adult animals. The excitatory effect results from alterations in the gradients of chloride, bicarbonate, and potassium ions, but its functional role still remains a mystery. Here we show that such GABAergic excitation participates in the expression of seizure-like rhythmic synchronization (afterdischarge) in the mature hippocampal CA1 region. Seizure-like afterdischarge was induced by high-frequency synaptic stimulation in the rat hippocampal CA1-isolated slice preparations. The hippocampal afterdischarge was completely blocked by selective antagonists of ionotropic glutamate receptors or of GABA(A) receptor, and also by gap-junction inhibitors. In the CA1 pyramidal cells, oscillatory depolarizing responses during the afterdischarge were largely dependent on chloride conductance, and their reversal potentials (average -38 mV) were very close to those of exogenously applied GABAergic responses. Moreover, intracellular loading of the GABA(A) receptor blocker fluoride abolished the oscillatory responses in the pyramidal cells. Finally, the GABAergic excitation-driven afterdischarge has not been inducible until the second postnatal week. Thus, excitatory GABAergic transmission seems to play an active functional role in the generation of adult hippocampal afterdischarge, in cooperation with glutamatergic transmissions and possible gap junctional communications. Our findings may elucidate the cellular mechanism of neuronal synchronization during seizure activity in temporal lobe epilepsy.

Prototypic Seizure Activity Driven by Mature Hippocampal Fast-Spiking Interneurons

A variety of epileptic seizure models have shown that activation of glutamatergic pyramidal cells is usually required for rhythm generation and/or synchronization in hippocampal seizure-like oscillations in vitro. However, it still remains unclear whether GABAergic interneurons may be able to drive the seizure-like oscillations without glutamatergic transmission. Here, we found that electrical stimulation in rat hippocampal CA1 slices induced a putative prototype of seizure-like oscillations (“prototypic afterdischarge,” 1.8–3.8 Hz) in mature pyramidal cells and interneurons in the presence of ionotropic glutamate receptor antagonists. The prototypic afterdischarge was abolished by GABAA receptor antagonists or gap junction blockers, but not by a metabotropic glutamate receptor antagonist or a GABAB receptor antagonist. Gramicidin-perforated patch-clamp and voltage-clamp recordings revealed that pyramidal cells were depolarized and frequently excited directly through excitatory GABAergic transmissions in each cycle of the prototypic afterdischarge. Interneurons that were actively spiking during the prototypic afterdischarge were mostly fast-spiking (FS) interneurons located in the strata oriens and pyramidale. Morphologically, these interneurons that might be “potential seizure drivers” included basket, chandelier, and bistratified cells. Furthermore, they received direct excitatory GABAergic input during the prototypic afterdischarge. The O-LM cells and most of the interneurons in the strata radiatum and lacunosum moleculare were not essential for the generation of prototypic afterdischarge. The GABA-mediated prototypic afterdischarge was observed later than the third postnatal week in the rat hippocampus. Our results suggest that an FS interneuron network alone can drive the prototypic form of electrically induced seizure-like oscillations through their excitatory GABAergic transmissions and presumably through gap junction-mediated communications.

Synaptic interactions between pyramidal cells and interneurone subtypes during seizure-like activity in the rat hippocampus

We have recently reported that excitatory GABAergic and glutamatergic mechanisms may be involved in the generation of seizure‐like (ictal) rhythmic synchronization (afterdischarge), induced by a strong synaptic stimulation of the CA1 pyramidal cells in the mature rat hippocampus in vitro. To clarify the network mechanism of this neuronal synchronization, dual whole‐cell patch‐clamp recordings of the afterdischarge responses were performed simultaneously from a variety of interneurones and their neighbouring pyramidal cells in hippocampal CA1‐isolated slice preparations. According to morphological and electrophysiological criteria, the recorded interneurones were then classified into distinct subtypes. The non‐fast‐spiking interneurones located in the strata lacunosum‐moleculare and radiatum hardly discharged during the afterdischarge, whereas most of the fast‐spiking and non‐fast‐spiking interneurones in the strata oriens and pyramidale, including the basket, chandelier and bistratified cells, exhibited prominent firings that were precisely synchronous with oscillatory responses in the pyramidal cells. Field potential recordings showed that excitatory synaptic transmissions might take place primarily in the strata oriens and pyramidale during the afterdischarge. Restricted lesions in the strata oriens and pyramidale, but not in the other layers, resulted in the complete desynchronization of afterdischarge activity, and also, local application of glutamate receptor antagonists to these layers blocked the expression of afterdischarge. The present findings indicate that the neuronal synchronization of epileptic afterdischarge may be accomplished in a ‘positive feedback circuit’ formed by the excitatory GABAergic interneurones and the glutamatergic pyramidal cells within the strata oriens and/or pyramidale of the hippocampal CA1 region.

Distinct types of ionic modulation of GABA actions in pyramidal cells and interneurons during electrical induction of hippocampal seizure-like network activity

It has recently been shown that electrical stimulation in normal extracellular fluid induces seizure‐like afterdischarge activity that is always preceded by GABA‐dependent slow depolarization. These afterdischarge responses are synchronous among mature hippocampal neurons and driven by excitatory GABAergic input. However, the differences in the mechanisms whereby the GABAergic signals in pyramidal cells and interneurons are transiently converted from hyperpolarizing to depolarizing (and even excitatory) have remained unclear. To clarify the network mechanisms underlying this rapid GABA conversion that induces afterdischarges, we examined the temporal changes in GABAergic responses in pyramidal cells and/or interneurons of the rat hippocampal CA1 area in vitro. The extents of slow depolarization and GABA conversion were much larger in the pyramidal cell group than in any group of interneurons. Besides GABAA receptor activation, neuronal excitation by ionotropic glutamate receptors enhanced GABA conversion in the pyramidal cells and consequent induction of afterdischarge. The slow depolarization was confirmed to consist of two distinct phases; an early phase that depended primarily on GABAA‐mediated postsynaptic Cl– accumulation, and a late phase that depended on extracellular K+ accumulation, both of which were enhanced by glutamatergic neuron excitation. Moreover, extracellular K+ accumulation augmented each oscillatory response of the afterdischarge, probably by further Cl– accumulation through K+‐coupled Cl– transporters. Our findings suggest that the GABA reversal potential may be elevated above their spike threshold predominantly in the pyramidal cells by biphasic Cl– intrusion during the slow depolarization in GABA‐ and glutamate‐dependent fashion, leading to the initiation of seizure‐like epileptiform activity.

A network mechanism underlying hippocampal seizure-like synchronous oscillations.

The hippocampus is a remarkable neural structure that displays a variety of synchronous oscillations that may be physiological or pathophysiological, such as theta rhythms and epileptic seizures. Electrically induced seizure-like afterdischarges are an excellent system for elucidating the network mechanisms underlying neuronal synchronization and rhythm generation of epileptic synchronous oscillations in extremely hyperactive hippocampal networks. In this Update Article, we review key findings of studies on these electrically induced seizure-like afterdischarges in vitro. During these afterdischarges, GABAergic responses become transiently depolarizing and even excitatory as chloride rapidly accumulates postsynaptically in pyramidal cells. Glutamate and potassium enhance this transient GABAergic excitation. Neuronal synchronization of afterdischarge is achieved by GABAergic and glutamatergic excitation of pyramidal cells and interneurons localized in the stratum pyramidale and stratum oriens. Rhythm generation in seizure-like synchronous oscillations is not yet understood but is the subject of intensive study.

Reinforcing operandum: rapid and reliable learning of skilled forelimb movements by head-fixed rodents.

Stereotaxic head fixation plays a necessary role in current physiological techniques, such as in vivo whole cell recording and two-photon laser-scanning microscopy, that are designed to elucidate the cortical involvement in animal behaviors. In rodents, however, head fixation often inhibits learning and performance of behavioral tasks. In particular, it has been considered inappropriate for head-fixed rodents to be operantly conditioned to perform skilled movements with their forelimb (e.g., lever-press task), despite the potential applicability of the task. Here we have solved this problem conceptually by integrating a lever (operandum) and a rewarding spout (reinforcer) into one ″spout-lever″ device for efficient operant learning. With this device, head-fixed rats reliably learned to perform a pull manipulation of the spout-lever with their right forelimb in response to an auditory cue signal (external-trigger trial, namely, Go trial) within several days. We also demonstrated stable whole cell recordings from motor cortex neurons while the rats were performing forelimb movements in external-trigger trials. We observed a behavior-related increase in the number of action potentials in membrane potential. In the next session, the rats, which had already learned the external-trigger trial, effortlessly performed similar spout-lever manipulation with no cue presentation (internal-trigger trial) additionally. Likewise, some of the rats learned to keep holding the spout-lever in response to another cue signal (No-go trial) in the following session, so that they mastered the Go/No-go discrimination task in one extra day. Our results verified the usefulness of spout-lever manipulation for behavioral experiments employing cutting-edge physiological techniques.

Large‐scale analysis reveals populational contributions of cortical spike rate and synchrony to behavioural functions

There have been few systematic population‐wide analyses of relationships between spike synchrony within a period of several milliseconds and behavioural functions. In this study, we obtained a large amount of spike data from > 23,000 neuron pairs by multiple single‐unit recording from deep layer neurons in motor cortical areas in rats performing a forelimb movement task. The temporal changes of spike synchrony in the whole neuron pairs were statistically independent of behavioural changes during the task performance, although some neuron pairs exhibited correlated changes in spike synchrony. Mutual information analyses revealed that spike synchrony made a smaller contribution than spike rate to behavioural functions. The strength of spike synchrony between two neurons was statistically independent of the spike rate‐based preferences of the pair for behavioural functions.

Three-dimensional reconstruction of electron micrographs reveals intrabulbar circuit differences between accessory and main olfactory bulbs

Three-dimensional (3D) reconstruction of synaptic arrangement on a particular dendrite provides essential information regarding neuronal properties and neural microcircuits. Unconventional synapses are particularly good candidates for such steric attribution. In main and accessory olfactory bulbs (MOBs and AOBs), there are dendrodendritic reciprocal synapses (RSs) between excitatory projection neurons and inhibitory interneurons. Although the fine structure and configuration of these synapses have been investigated in MOB, their characteristics in AOB were unknown. In this study, we performed 3D AOB reconstruction using serial section transmission electron microscopy. We found numerous RSs on primary dendrites from glomeruli to mitral/tufted (MT) cell somas. These synapses formed between dendritic shafts of MT cells and large dendritic spines, or so-called gemmules, of granule (Gr) cells. This indicates that chemical signals received by a glomerulus are regulated in the primary dendrite of an MT cell before reaching its soma. In MOB, RSs are located on secondary dendrites and act as lateral and self-inhibiting following mitral cell depolarization. Our results indicate that AOB intrabulbar microcircuitry is quite different from that in the MOB.