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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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...

Glial cells express a variety of neurotransmitter receptors. Notably, Bergmann glial cells in the cerebellum have Ca2+-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) assembled without the GluR2 subunit. To elucidate the role of these Ca2+-permeable AMPARs, we converted them into Ca2+-impermeable receptors by adenoviral-mediated delivery of the GluR2 gene. This conversion retracted the glial processes ensheathing synapses on Purkinje cell dendritic spines and retarded the removal of synaptically released glutamate. Furthermore, it caused multiple innervation of Purkinje cells by the climbing fibers. Thus, the glial Ca2+-permeable AMPARs are indispensable for proper structural and functional relations between Bergmann glia and glutamatergic synapses.

Glioblastoma multiforme is the most undifferentiated type of brain tumor, and its prognosis is extremely poor. Glioblastoma cells exhibit highly migratory and invasive behavior, which makes surgical intervention unsuccessful. Here, we showed that glioblastoma cells express Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors assembled from the GluR1 and/or GluR4 subunits, and that their conversion to Ca(2+)-impermeable receptors by adenovirus-mediated transfer of the GluR2 cDNA inhibited cell locomotion and induced apoptosis. In contrast, overexpression of Ca(2+)-permeable AMPA receptors facilitated migration and proliferation of the tumor cells. These findings indicate that Ca(2+)-permeable AMPA receptors have crucial roles in growth of glioblastoma. Blockage of these Ca(2+)-permeable receptors may be a useful therapeutic strategy for the prevention of glioblastoma invasion.

SUMMARY1. N-methyl-D-aspartate (NMDA)-, quisqualate-and kainate-induced currents were recorded in cultured rat hippocampal neurones using the whole-cell voltageclamp technique. To isolate the inward currents carried by Ca2+ and other divalent cations (Sr2+, Ba2+, Mn2+ and Mg2+), both Na+ and K+ in the control external solution were replaced with the impermeant cation N-methylglucamine (NMG).2. Replacement of Na+, K+ and Ca2+ with NMG abolished NMDA-, quisqualateand kinate-induced inward currents. In Na+-, K+-free (abbreviated simply as Na+-free) solution containing 10 mm-Caa2+ NMDA caused prominent inward currents at -60 mV. In this solution with the internal solution containing 165 mM-Cs+, the reversal potential of the NMDA-induced current was -5 0 + 0 7 mV (n = 36), indicating a value of Pca/Pcs = 6-2 for the ratio of the permeability coefficients of Ca2+ and Cs+ according to the constant-field equation.3. NMDA elicited inward current responses at -60 mV in Na+-, Ca2+-free solution containing 10 mm-Sr2+, Ba2+, or Mn2+, but not in Na+-free, 10 mM-Mg2+ solution.On the basis of reversal potential measurements, the permeability sequence of NMDA receptor channels among the divalent cations was determined to be Ba2+(1-2) > Ca2+(10) > Sr2+(0 8) > Mn2+(0 3) > Mg2+(< 0-02).4. The reversal potential of the quisqualate-induced current was more negative than -80 mV in Na+-free, 10 mM-Ca2+ solution, indicating a value of Pca/Pcs < 0-18.5. Kainate-induced current responses were classified into two types. In the type I response the reversal potential of the kainate-induced current was more negative than -80 mV in Na+-free, 10 mM-Ca2+ solution, indicating that the Ca2+ permeability of this type of kainate channel is as low as that of the quisqualate channel. In the neurones which showed a type I response, there was a tendency of outward rectification in the current-voltage plots of the kainate response in control solution.6. In the type II response kainate caused prominent inward currents at -60 mV in Na+-free, 10 mM-Ca2+ solution. The reversal potential was -23-3 + 5 6 mV (n = 17), indicating a permeability ratio Pca/Ics = 2-3. In the neurones which showed a type II response, a remarkable inward rectification was observed in the current-voltage plots of the kainate response in control solution. 7. Type II kainate channels showed relatively poor selectivity among divalent MS 7757 M. IINO, S. OZA WA AND K. TSUZUKI cations. The permeability sequence was Ba21(13) > Ca21(10) > Sr2+(0.9) > Mg2+(0-8) > Mn2+(0 7).

The cellular mechanisms by which neuronal nicotinic cholinergic receptors influence many aspects of physiology and pathology in the neocortex remain primarily unknown. Whole-cell recordings and single-cell reverse transcription (RT)-PCR were combined to analyze the effect of nicotinic receptor agonists on different types of neurons in acute slices of rat neocortex. Nicotinic receptor agonists had no effect on pyramidal neurons and on most types of interneurons, including parvalbuminexpressing fast spiking interneurons and somatostatinexpressing interneurons, but selectively excited a subpopulation of interneurons coexpressing the neuropeptides vasoactive intestinal peptide (VIP) and cholecystokinin. This excitation persisted in the presence of glutamate, GABA, and muscarinic receptor antagonists and in the presence of tetrodotoxin and low extracellular calcium, suggesting that the depolarization was mediated through the direct activation of postsynaptic nicotinic receptors. The responses were blocked by the nicotinic receptor antagonists dihydro-␤-erythroidine and mecamylamine and persisted in the presence of the ␣7 selective nicotinic receptor antagonist methyllycaconitine, suggesting that the involved nicotinic receptors lacked the ␣7 subunit. Single-cell RT-PCR analysis indicated that the majority of the interneurons that responded to nicotinic stimulation coexpressed the ␣4, ␣5, and ␤2 nicotinic receptor subunits. Therefore, these results provide a role for non-␣7 nicotinic receptors in the selective excitation of a subpopulation of neocortical interneurons. Because the neocortical interneurons expressing VIP have been proposed previously to regulate regional cortical blood flow and metabolism, these results also provide a cellular basis for the neuronal regulation of cortical blood flow mediated by acetylcholine. Key words: single-cell PCR; neuropeptides; calcium-binding proteins; methyllycaconitine; dihydro-␤-erythroidine; acetylcholine; mecamylamineNicotinic receptors are implicated in many important f unctions of the mammalian neocortex, including memory formation (Granon et al., 1995) and neuronal regulation of regional cerebral blood flow (Gitelman and Prohovnik, 1992;Uchida et al., 1997), as well as cortical pathologies such as Alzheimer's disease (Whitehouse et al., 1988;Newhouse et al., 1997), epilepsy (Steinlein et al., 1995, and Tourette's syndrome (Sanberg et al., 1997(Sanberg et al., , 1998. The cellular basis for the effects of nicotinic receptor stimulation is currently unknown but lies within its effects on the complex interactions between the excitatory glutamatergic pyramidal neurons and the inhibitory interneurons.A variety of different neuronal nicotinic receptor subunits have been cloned and named ␣2-␣9 and ␤2-␤4 (Elgoyhen et al., 1994;Le Novere and Changeux, 1995). C lassically, two broad subfamilies of nicotinic receptors have been characterized in neurons according to their sensitivity to ␣-bungarotoxin. Subunits ␣7 and ␣8 form a family of ␣-bungarotoxin-sensitive channels (Couturier...

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