A long-standing question in neurobiology is whether astrocytes respond to the neuronal release of neurotransmitters in vivo. To address this question, acutely isolated hippocampal slices were loaded with the calcium-sensitive dye Calcium Green-1 and the responses of the astrocytes to electrical stimulation of the Schaffer collaterals were monitored by confocal microscopy. To confirm that the responsive cells were astrocytes, the slices were immunostained for the astrocytic marker glial fibrillary acidic protein. Stimulation of the Schaffer collaterals (50 Hz, 2 sec) resulted in increases in the concentration of intracellular calcium ([Ca 2ϩ ] i ) in the astrocytes located in the stratum radiatum of CA1. The astrocytic responses were blocked by the sodium channel blocker tetrodotoxin, the voltage-dependent calcium channel blocker -conotoxin-MVIIC, and the selective metabotropic glutamate receptor antagonist ␣-methyl-4-carboxyphenylglycine (MCPG). These results suggest that the astrocytic responses were induced by stimulation of metabotropic glutamate receptors on the astrocytes by neuronally released glutamate. The astrocytic responses to neuronal stimulation were enhanced in the presence of the K ϩ channel antagonist 4-aminopyridine (4-AP). Inhibition of the astrocytic responses in the presence of 4-AP required the presence of both MCPG and the ionotropic glutamate receptor antagonist kynurenic acid. These results suggest that higher levels of neuronal activity result in stimulation of both metabotropic and ionotropic glutamate receptors on the astrocytes. Overall, the results indicate that hippocampal astrocytes in situ are able to respond to the neuronal release of the neurotransmitter glutamate with increases in [Ca 2ϩ ] i .

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...

Sensory information, relayed through the thalamus, arrives in the neocortex as excitatory input, but rapidly induces strong disynaptic inhibition that constrains the cortical flow of excitation both spatially and temporally. This feedforward inhibition is generated by intracortical interneurons whose precise identity and properties were not known. To characterize interneurons generating feedforward inhibition, neurons in layers IV and V of mouse somatosensory ("barrel") cortex in vitro were tested in the cell-attached configuration for thalamocortically induced firing and in the whole-cell mode for synaptic responses. Identification as inhibitory or excitatory neurons was based on intrinsic firing patterns and on morphology revealed by intracellular staining. Thalamocortical stimulation evoked action potentials in ϳ60% of inhibitory interneurons but in Ͻ5% of excitatory neurons. The inhibitory interneurons that fired received fivefold larger thalamocortical inputs compared with nonfiring inhibitory or excitatory neurons. Thalamocortically evoked spikes in inhibitory interneurons followed at short latency the onset of excitatory monosynaptic responses in the same cells and slightly preceded the onset of inhibitory responses in nearby neurons, indicating their involvement in disynaptic inhibition. Both nonadapting (fast-spiking) and adapting (regular-spiking) inhibitory interneurons fired on thalamocortical stimulation, as did interneurons expressing parvalbumin, calbindin, or neither calcium-binding protein.Morphological analysis revealed that some interneurons might generate feedforward inhibition within their own layer IV barrel, whereas others may convey inhibition to upper layers, within their own or in adjacent columns. We conclude that feedforward inhibition is generated by diverse classes of interneurons, possibly serving different roles in the processing of incoming sensory information.

Extinction of conditioned fear is an active learning process involving inhibition of fear expression. It has been proposed that fear extinction potentiates neurons in the infralimbic (IL) prefrontal cortex, but the cellular mechanisms underlying this potentiation remain unknown. It is also not known whether this potentiation occurs locally in IL neurons as opposed to IL afferents. To determine whether extinction enhances the intrinsic excitability of IL pyramidal neurons in layers II/III and V, we performed whole-cell patch-clamp recordings in slices from naive, conditioned, or conditioned-extinguished rats. We observed that conditioning depressed IL excitability compared with slices from naive animals, as evidenced by a decreased number of spikes evoked by injected current and an increase in the slow afterhyperpolarizing potential (sAHP). Extinction reversed these conditioning-induced effects. Furthermore, IL neurons from extinguished rats showed increased burst spiking compared with naive rats, which was correlated with extinction recall. These changes were specific to IL prefrontal cortex and were not observed in prelimbic prefrontal cortex. Together, these findings suggest that IL intrinsic excitability is reduced to allow for expression of conditioning memory and enhanced for expression of extinction memory through the modulation of Ca 2ϩ -gated K ϩ channels underlying the sAHP. Inappropriate modulation of these intrinsic mechanisms may underlie anxiety disorders, characterized by exaggerated fear and deficient extinction.

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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...

Emotional arousal strengthens memory. This is most apparent in aversive conditioning, in which the stress-related neurotransmitter norepinephrine (NE) enhances associations between sensory stimuli and fear-inducing events. In contrast to conditioning, extinction decreases fear responses, and is thought to form a new memory. It is not known, however, whether NE is necessary for extinction learning. Previous work has shown that the infralimbic prefrontal cortex (IL) is a site of extinction consolidation. Here, we show that blocking noradrenergic ␤-receptors in IL before extinction training impaired retrieval of extinction the following day, consistent with a weakened extinction memory. We further found that the sequelae of ␤-receptor activation, including protein kinase A (PKA), gene transcription and translation in IL, are necessary for extinction. To determine whether activation of this cascade modulates IL excitability, we measured the response of IL pyramidal neurons to injected current. NE increased the excitability of IL neurons in a ␤-receptor-and PKA-dependent manner. We suggest that NE released in IL during fear extinction activates a PKA-mediated molecular cascade that strengthens extinction memory. Thus, emotional arousal evoked by conditioned fear paradoxically promotes the subsequent extinction of that fear, thereby ensuring behavioral flexibility.

In the rat neocortex, a subset of GABAergic interneurons express the neuropeptide vasoactive intestinal peptide (VIP). Previously, we demonstrated that a population of VIPergic interneurons could be accurately identified by their irregular spiking (IS) pattern and their bipolar morphology. IS interneurons were studied in neocortical slices from 16-22-day-old rats using whole-cell recordings, intracellular labelling and single-cell RT-PCR. In response to a depolarizing pulse, IS interneurons typically discharged a burst of action potentials followed by spikes emitted at an irregular frequency. Several seconds of depolarization, micromolar concentrations of 4-aminopyridine, and nanomolar concentrations of either dendrotoxin I or K converted this irregular pattern to a sustained discharge, suggesting the involvement of an ID-like K+ current. The main glutamate receptor subunits detected in IS cells were GluR1 flop and GluR2 flop, GluR5 and GluR6, and NR2B and NR2D for the alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartic acid (NMDA) subtypes, respectively. Paired whole-cell patch-clamp recordings indicated that pyramidal neurons provide intracortical glutamatergic inputs onto IS interneurons. Most connections had high probabilities of response and exhibited frequency-dependent paired pulse depression. Comparison of the amplitude distribution of paired responses suggested that most of these connections consisted of multiple functional release sites. Finally, two discrete subpopulations of IS cells could be identified based on the duration of the initial burst of action potentials and the differential expression of calretinin and choline acetyltransferase.