The physiological and molecular features of nonpyramidal cells were investigated in acute slices of sensory-motor cortex using whole-cell recordings combined with single-cell RT-PCR to detect simultaneously the mRNAs of three calcium binding proteins (calbindin D28k, parvalbumin, and calretinin) and four neuropeptides (neuropeptide Y, vasoactive intestinal polypeptide, somatostatin, and cholecystokinin). In the 97 neurons analyzed, all expressed mRNAs of at least one calcium binding protein, and the majority (n ϭ 73) contained mRNAs of at least one neuropeptide. Three groups of nonpyramidal cells were defined according to their firing pattern. (1) Fast spiking cells (n ϭ 34) displayed tonic discharges of fast action potentials with no accommodation. They expressed parvalbumin (n ϭ 30) and/or calbindin (n ϭ 19) mRNAs, and half of them also contained transcripts of at least one of the four neuropeptides. (2) Regular spiking nonpyramidal cells (n ϭ 48) displayed a firing behavior characterized by a marked accommodation and presented a large diversity of expression patterns of the seven biochemical markers. (3) Finally, a small population of vertically oriented bipolar cells, termed irregular spiking cells (n ϭ 15), fired bursts of action potentials at an irregular frequency. They consistently co-expressed calretinin and vasoactive intestinal polypeptide. Additional investigations of these cells showed that they also co-expressed glutamic acid decarboxylase and choline acetyl transferase. Our results indicate that neocortical nonpyramidal neurons display a large diversity in their firing properties and biochemical patterns of co-expression and that both characteristics could be correlated to define discrete subpopulations.

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Glial cells of the nervous system directly influence neuronal and synaptic activities by releasing transmitters. However, the physiological consequences of this glial transmitter release on brain information processing remain poorly understood. We demonstrate here in hippocampal slices of 2-to 5-week-old rats that glutamate released from glial cells generates slow transient currents (STCs) mediated by the activation of NMDA receptors in pyramidal cells. STCs persist in the absence of neuronal and synaptic activity, indicating a nonsynaptic origin of the source of glutamate. Indeed, STCs occur spontaneously but can also be induced by pharmacological tools known to activate astrocytes and by the selective mechanical stimulation of single nearby glial cells. Bath application of the inhibitor of the glutamate uptake DL-threo-␤-benzyloxyaspartate increases both the frequency of STCs and the amplitude of a tonic conductance mediated by NMDA receptors and probably also originated from glial glutamate release. By using dual recordings, we observed synchronized STCs in pyramidal cells having their soma distant by Ͻ100 m. The degree of precision (Ͻ100 msec) of this synchronization rules out the involvement of calcium waves spreading through the glial network. It also indicates that single glial cells release glutamate onto adjacent neuronal processes, thereby controlling simultaneously the excitability of several neighboring pyramidal cells. In conclusion, our results show that the glial glutamate release occurs spontaneously and synchronizes the neuronal activity in the hippocampus.

Accumulative evidence indicates that microglial cells influence the normal development of brain synapses. Yet, the mechanisms by which these immune cells target maturating synapses and influence their functional development at early postnatal stages remain poorly understood. Here, we analyzed the role of CX3CR1, a microglial receptor activated by the neuronal chemokine CX3CL1 (or fractalkine) which controls key functions of microglial cells. In the whisker-related barrel field of the mouse somatosensory cortex, we show that the recruitment of microglia to the sites where developing thalamocortical synapses are concentrated (i.e., the barrel centers) occurs only after postnatal day 5 and is controlled by the fractalkine/CX3CR1 signaling pathway. Indeed, at this developmental stage fractalkine is overexpressed within the barrels and CX3CR1 deficiency delays microglial cell recruitment into the barrel centers. Functional analysis of thalamocortical synapses shows that CX3CR1 deficiency also delays the functional maturation of postsynaptic glutamate receptors which normally occurs at these synapses between the first and second postnatal week. These results show that reciprocal interactions between neurons and microglial cells control the functional maturation of cortical synapses.

In order to compare the frontal cortex of rat and macaque monkey, cortical and subcortical afferents to subdivisions of the medial frontal cortex (MFC) in the rat were analyzed with fluorescent retrograde tracers. In addition to afferent inputs common to the whole MFC, each subdivision of the MFC has a specific pattern of afferent connections. The dorsally situated precentral medial area (PrCm) was the only area to receive inputs from the somatosensory cortex. The specific pattern of afferents common to the ventrally situated prelimbic (PL) and infralimbic (IL) areas included projections from the agranular insular cortex, the entorhinal and piriform cortices, the CA1-CA2 fields of the hippocampus, the subiculum, the endopiriform nucleus, the amygdalopiriform transition, the amygdalohippocampal area, the lateral tegmentum, and the parabrachial nucleus. In all these structures, the number of retrogradely labeled cells was larger when the injection site was located in area IL. The dorsal part of the anterior cingulate area (ACd) seemed to be connectionally intermediate between the adjacent areas PrCm and PL; it receives neither the somatosensory inputs characteristic of area PrCm nor the afferents characteristic of areas PL and IL, with the exception of the afferents from the caudal part of the retrosplenial cortex. A comparison of the pattern of afferent and efferent connections of the rat MFC with the pattern of macaque prefrontal cortex suggests that PrCm and ACd areas share some properties with the macaque premotor cortex, whereas PL and IL areas may have characteristics in common with the cingulate or with medial areas 24, 25, and 32 and with orbital areas 12, 13, and 14 of macaques.

The neurons responsible for the onset of sleep are thought to be located in the preoptic area and more specifically, in the ventrolateral preoptic nucleus (VLPO). Here we identify sleep-promoting neurons in vitro and show that they represent an homogeneous population of cells that must be inhibited by systems of arousal during the waking state. We find that two-thirds of the VLPO neurons are multipolar triangular cells that show a low-threshold spike. This proportion matches that of cells active during sleep in the same region. We then show, using single-cell reverse transcriptase followed by polymerase chain reaction, that these neurons probably contain gamma-aminobutyric acid (GABA). We also show that these neurons are inhibited by noradrenaline and acetylcholine, both of which are transmitters of wakefulness. As most of these cells are also inhibited by serotonin but unaffected by histamine, their overall inhibition by transmitters of wakefulness is in agreement with their relative inactivity during waking with respect to sleep. We propose that the reciprocal inhibitory interaction of such VLPO neurons with the noradrenergic, serotoninergic and cholinergic waking systems to which they project is a key factor for promoting sleep.

A classification of fusiform neocortical interneurons (n ‫؍‬ 60) was performed with an unsupervised cluster analysis based on the comparison of multiple electrophysiological and molecular parameters studied by patch-clamp and single-cell multiplex reverse transcription-PCR in rat neocortical acute slices. The multiplex reverse transcription-PCR protocol was designed to detect simultaneously the expression of GAD65, GAD67, calbindin, parvalbumin, calretinin, neuropeptide Y, vasoactive intestinal peptide (VIP), somatostatin (SS), cholecystokinin, ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, N-methyl-D-aspartate, and metabotropic glutamate receptor subtypes. Three groups of fusiform interneurons with distinctive features were disclosed by the cluster analysis. The first type of fusiform neuron (n ‫؍‬ 12), termed regular spiking nonpyramidal (RSNP)-SS cluster, was characterized by a firing pattern of RSNP cells and by a high occurrence of SS. The second type of fusiform neuron (n ‫؍‬ 32), termed RSNP-VIP cluster, predominantly expressed VIP and also showed firing properties of RSNP neurons with accommodation profiles different from those of RSNP-SS cells. Finally, the last type of fusiform neuron (n ‫؍‬ 16) contained a majority of irregular spiking-VIPergic neurons. In addition, the analysis of glutamate receptors revealed cell-type-specific expression profiles. This study shows that combinations of multiple independent criteria define distinct neocortical populations of interneurons potentially involved in specific functions. B ecause, in part, of their diversity, the function of neuron subtypes in the physiology of the neocortex is still poorly understood. A better knowledge of the different neuronal populations that compose this heterogeneous brain structure may therefore contribute to elucidating their specific role.Attempts to classify neurons rely on several independent criteria (morphological, physiological, and molecular). In the neocortex, neurons are classified as pyramidal cells or nonpyramidal cells according to their morphology. Pyramidal cells accumulate glutamate and constitute the main class of excitatory projecting neurons (1). In contrast, nonpyramidal cells, also termed interneurons, are mainly inhibitory ␥-aminobutyric acid-ergic neurons with a short axon involved in local circuits (2) and have a large diversity of morphology (3). The expression of biochemical markers has been used to define different classes of nonpyramidal cells. The distribution of three calcium binding proteins, calbindin (CB), parvalbumin (PV), and calretinin (CR), and four neuropeptides, neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), somatostatin (SS), and cholecystokinin (CCK), defines partially overlapping groups of interneurons (4 -12). In addition to this morphological and molecular diversity, nonpyramidal cells also have a large repertoire of firing behaviors (13-18), such as fast spiking (FS), regular spiking nonpyramidal (RSNP), or irregular spiking (IS).Some types of interneurons have b...

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