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.

GABAA-mediated miniature IPSCs (mIPSCs) were recorded from layer V pyramidal neurons of the visual cortex using whole-cell patch-clamp recording in rat brain slices. At room temperature, the benzodiazepine site agonist zolpidem enhanced both the amplitude (to 138 +/- 26% of control value at 10 microM) and the duration (163 +/- 14%) of mIPSCs. The enhancement of mIPSC amplitude was not caused by an increase of the single-channel conductance of the postsynaptic receptors, as determined by peak-scaled non-stationary fluctuation analysis of mIPSCs. The effect of zolpidem on fast, synaptic-like (1 msec duration) applications of GABA to outside-out patches was also investigated. The EC50 for fast GABA applications was 310 microM. In patches, zolpidem enhanced the amplitude of currents elicited by subsaturating GABA applications (100-300 microM) but not by saturating applications (10 mM). The increase of mIPSC amplitude by zolpidem provides evidence that the GABAA receptors are not saturated during miniature synaptic transmission in the recorded cells. By comparing the facilitation induced by 1 microM zolpidem on outside-out patches and mIPSCs, we estimated the concentration of GABA seen by the postsynaptic GABAA receptors to be approximately 300 microM after single vesicle release. We have estimated a similar degree of receptor occupancy at room and physiological temperature. However, at 35 degreesC, zolpidem did not enhance the amplitude of mIPSCs or of subsaturating GABA applications on patches, implying that, in these neurons, zolpidem cannot be used to probe the degree of receptor occupancy at physiological temperature.

Although Ca 2ϩ-dependent exocytosis is considered to be a pathway for gliotransmitter release from astrocytes, the structural and functional bases of this process remain controversial. We studied the relationship between near-membrane Ca 2ϩ elevations and the dynamics of single astroglial vesicles with styryl (FM) dyes. We show that cultured astrocytes, unlike neurons, spontaneously internalize FM dyes, resulting in the labeling of the entire acidic vesicle population within minutes. Interestingly, metabotropic glutamate receptor activation did not affect the FM labeling. Most FM-stained vesicles expressed sialin, CD63/LAMP3, and VAMP7, three markers for lysosomes and late endosomes. A subset of lysosomes underwent asynchronous exocytosis that required both Ca 2ϩ mobilization from intracellular stores and Ca 2ϩ influx across the plasma membrane. Lysosomal fusion occurred within seconds and was complete with no evidence for kiss and run. Our experiments suggest that astroglial Ca 2ϩ -regulated exocytosis is carried by lysosomes and operates on a timescale orders of magnitude slower than synaptic transmission.

Neurotransmitter glutamate has been thought to derive mainly from glutamine via the action of glutaminase type 1 (GLS1). To address the importance of this pathway in glutamatergic transmission, we knocked out GLS1 in mice. The insertion of a STOP cassette by homologous recombination produced a null allele that blocked transcription, encoded no immunoreactive protein, and abolished GLS1 enzymatic activity. Null mutants were slightly smaller, were deficient in goal-directed behavior, hypoventilated, and died in the first postnatal day. No gross or microscopic defects were detected in peripheral organs or in the CNS. In cultured neurons from the null mutants, miniature EPSC amplitude and duration were normal; however, the amplitude of evoked EPSCs decayed more rapidly with sustained 10 Hz stimulation, consistent with an observed reduction in depolarization-evoked glutamate release. Because of this activitydependent impairment in glutamatergic transmission, we surmised that respiratory networks, which require temporal summation of synaptic input, would be particularly affected. We found that the amplitude of inspirations was decreased in vivo, chemosensitivity to CO 2 was severely altered, and the frequency of pacemaker activity recorded in the respiratory generator in the pre-Bötzinger complex, a glutamatergic brainstem network that can be isolated in vitro, was increased. Our results show that although alternate pathways to GLS1 glutamate synthesis support baseline glutamatergic transmission, the GLS1 pathway is essential for maintaining the function of active synapses, and thus the mutation is associated with impaired respiratory function, abnormal goal-directed behavior, and neonatal demise.

SUMMARY1. The effect of serotonin on inhibitory synaptic transmission was examined in forty-one CAI pyramidal neurones using intracellular voltage recordings in vitro.2. Serotonin (20-50 /LM) increased the synaptic noise of most (85%) neurones loaded with chloride (n = 33). The duration of this effect was enhanced with increasing concentrations of serotonin and was fully reversible within 5 min. When serotonin was applied at short intervals (less than 10 min), fading of the response was observed.3. The effect of serotonin on synaptic noise persisted in the presence of the glutamate NMDA and non-NMDA antagonists, APV (100,UM) and CNQX (10 /JM), but it was blocked (n = 5) by a GABAA antagonist, bicuculline (10 /IM). 6. These results suggest that serotonin directly excites GABAergic interneurones acting on a 5-HT3 receptor and consequently increasing the frequency of inhibitory synaptic events recorded in CAl pyramidal cells.

In the cortex fast excitatory synaptic currents onto excitatory pyramidal neurons and inhibitory nonpyramidal neurons are mediated by a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors exhibiting cell-type-specific differences in their kinetic properties. AMPA receptors consist of four subunits (GluR1-4), each existing as two splice variants, flip and flop, which critically affect the desensitization properties of receptors expressed in heterologous systems. Using single cell reverse transcription PCR to analyze the mRNA of AMPA receptor subunits expressed in layers I-HI neocortical neurons, we find that 90%Y of the GluR1-4 in nonpyramidal neurons are flop variants, whereas 92% ofthe GluRl-4 in pyramidal neurons are flip variants. We also find that nonpyramidal neurons predominantly express GluRl mRNA (GluR1/GluRl-4 = 59%o), whereas pyramidal neurons contain mainly GluR2 mRNA (GluR2/GluR1-4 = 59%o). However, the neuron-type-specific splicing is exhibited by all four AMPA receptor subunits. We suggest that the predominance ofthe flop variants contributes to the faster and more extensive desensitization in nonpyramidal neurons, compared to pyramidal cells where flip variants are dominant. Alternative splicing of AMPA receptors may play an important role in regulating synaptic function in a cell-typespecific manner, without changing permeation properties.Cortical neurons in superficial layers can be classified as regular spiking (RS) pyramidal cells and fast spiking (FS) nonpyramidal cells (1, 2). FS nonpyramidal neurons contain y-aminobutyric acid and glutamic acid decarboxylase and are thought to be inhibitory interneurons (1, 3). Pyramidal neurons accumulate glutamate and are presumably the main excitatory neurons (4, 5). The fast excitatory synaptic transmission onto both cell types is mediated by a-amino-3-hydroxy-5-methylisoxasole-4-propionic acid (AMPA) receptors (6, 7). AMPA receptors desensitize faster and to a greater extent in FS nonpyramidal cells than in RS pyramidal cells (8,9). Furthermore, AMPA receptors in layer IV FS nonpyramidal neurons are more permeant to calcium than in layer V pyramidal neurons (9) and layers 11/111 nonpyramidal neurons exhibit larger single channel conductance than layers II/III pyramidal neurons (8). These findings suggest that structural differences may underlie the functional differences among AMPA receptors in cortical pyramidal and nonpyramidal neurons.AMPA receptors are multimeric assemblies of four subunits GluR1-4 (reviewed in refs. 10 and 11). The expression of the GluR2 subunit in heterologous systems endows AMPA receptors with low Ca2+ permeability (12, 13). Further diversity is generated by alternative splicing, giving either flip or flop variants of all subunits, and RNA editing (14-16). The kinetic properties of AMPA receptors depend on subunit composiThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely...

The disruptive effect of excessive serotonin (5-HT) levels on the development of cortical sensory maps is mediated by 5-HT1B receptors, as shown in barrelless monoamine oxidase A knockout mice, in which the additional inactivation of 5-HT1B receptors restores the barrels. However, it is unclear whether 5-HT1B receptors mediate their effect on barrel formation by a trophic action or an activity-dependent effect.To test for a possible effect of 5-HT1B receptors on activity, we studied the influence of 5-HT on the thalamocortical (TC) synaptic transmission in layer IV cortical neurons. In TC slices of postnatal day 5 (P5)-P9 neonate mice, we show that 5-HT reduces monosynaptic TC EPSCs evoked by low-frequency internal capsule stimulation and relieves the short-term depression of the EPSC evoked by high-frequency stimulation. We provide evidence that 5-HT decreases the presynaptic release of glutamate: 5-HT reduces similarly the AMPA-kainate and NMDA components and the paired pulse depression of TC EPSCs. We show also that 5-HT1B receptors mediate exclusively the effect of 5-HT: first, the effect of 5-HT on the TC EPSC is correlated with the transient expression of 5-HT1B receptor mRNAs in the ventrobasal thalamic nucleus during postnatal development; second, it is mimicked by a 5-HT1B agonist; third, 5-HT has no effect in 5-HT1B receptor knock-out mice. Our results show that in the developing barrel field of the neonatal mice, 5-HT1B receptors mediate an activitydependent regulation of the TC EPSC that could favor the propagation of high-frequency TC activity.

SUMMARY1. Recordings were made in vitro from chloride-loaded CAI rat hippocampal pyramidal neurones in the presence of tetrodotoxin (TTX) to examine miniature inhibitory postsynaptic currents (IPSCs).2. Most spontaneous synaptic events recorded before TTX was applied, and all events that were resolved in the presence of TTX, were blocked by the GABAA receptor antagonist bicuculline.3. At 25°C, averaged miniature IPSCs had a time to peak of about 3 ms and in most cases decayed with a single time constant close to 25 ms.4. With a driving force for chloride ions between 70 and 80 mV, the mean miniature IPSC amplitude was 19-6-27-9 pA, yielding a conductance of 258-326 pS. The mean amplitude of unitary IPSCs recorded before TTX was applied was in the range of 31-73 pA.5. When intervals between miniature IPSCs were compared with an exponential distribution, there was an excess of events at intervals shorter than 5 ms. Some individual events appeared to represent the nearly simultaneous release of two inhibitory quanta.6. Miniature IPSC amplitude distributions were better fitted with the sum of two Gaussians than with one Gaussian. The variance in amplitude of a single quantal event exceeded that of the baseline noise.7. Comparison of the conductance changes corresponding to the first Gaussian distribution with single GABA channel data suggests that one inhibitory quantum opens twelve to twenty chloride channels and that GABA molecules bind once to a postsynaptic receptor.