Etienne Audinat

Molecular and Physiological Diversity of Cortical Nonpyramidal Cells

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 = 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 = 34) displayed tonic discharges of fast action potentials with no accommodation. They expressed parvalbumin (n = 30) and/or calbindin (n = 19) mRNAs, and half of them also contained transcripts of at least one of the four neuropeptides. (2) Regular spiking nonpyramidal cells (n = 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 = 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.

AMPA receptor subunits expressed by single purkinje cells

Several subunits of the glutamate receptor of the AMPA subtype have been cloned recently. These subunits, named GluR1, GluR2, GluR3, and GluR4, exist as two splicing variants (flip and flop). We have determined the subset of AMPA receptor subunits expressed by single cerebellar Purkinje cells in culture. This was achieved by combining whole-cell patch-clamp recordings and a molecular analysis, based on the polymerase chain reaction, of the messenger RNAs harvested into the patch pipette at the end of each recording. We found that each single cell expresses the messenger RNAs encoding the following five subunits: the flip and flop versions of GluR1 and GluR2 as well as GluR3flip, GluR2 being the most abundant. In addition, GluR3flop and GluR4flip were scarcely expressed in half of these neurons, and GluR4flop was never detected.

Subunit composition at the single-cell level explains functional properties of a glutamate-gated channel

The diversity of known glutamate-gated channels has been markedly increased by the discovery of multiple subunits and their spliced and edited variants. These subunits can potentially form different oligomeric complexes with diverging properties. A crucial question is therefore to determine the actual subunit composition of naturally occurring glutamate receptors. We have coupled patch-clamp recordings and reverse transcription followed by PCR amplification to correlate the presence of mRNAs for each subunit and the functional properties of native glutamate receptors at the single-cell level. In a homogeneous population of functionally identified hippocampal neurons (type II) in culture bearing a glutamate receptor of the AMPA subtype with a high calcium permeability, we found that, among the multiple subunits, only two, the flop forms of GluR1 and GluR4, were expressed. In particular, GluR2 was never detected. This composition explains the uncommon properties of AMPA receptors in type II neurons.

Selective Excitation of Subtypes of Neocortical Interneurons by Nicotinic Receptors

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 parvalbumin-expressing fast spiking interneurons and somatostatin-expressing 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-β-erythroidine and mecamylamine and persisted in the presence of the α7 selective nicotinic receptor antagonist methyllycaconitine, suggesting that the involved nicotinic receptors lacked the α7 subunit. Single-cell RT-PCR analysis indicated that the majority of the interneurons that responded to nicotinic stimulation coexpressed the α4, α5, and β2 nicotinic receptor subunits. Therefore, these results provide a role for non-α7 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.

Properties of bipolar VIPergic interneurons and their excitation by pyramidal neurons in the rat neocortex

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 α‐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.

Activity-dependent regulation of N-methyl-D-aspartate receptor subunit expression in rat cerebellar granule cells.

The glutamate receptor channels of the N‐methyl‐d‐aspartate (NMDA) subtype are composed of different subunits named NR1 and NR2A‐D. These subunits can combine in different oligomers with diverging properties and their expression is developmentally regulated. We have used rat cerebellar slice cultures to test the involvement of bioelectrical activity and synaptic transmission in the changes in NR2A‐C expression observed in developing granule cells. A correlation between the functional properties of the NMDA receptors and expression of the NR2A‐C mRNAs was obtained in single granule cells by coupling patch‐clamp recording and reverse transcription followed by polymerase chain reaction. Granule cells grown under standard culture conditions expressed mainly NR2A mRNA when examined after 15–40 days in vitro. Consistent with this observation, their responses to NMDA were only weakly reduced by 3 μM ifenprodil, a non‐competitive antagonist which discriminates between NR2A and NR2B subunits in expression systems. In cerebellar cultures chronically exposed to tetrodotoxin to eliminate spontaneous electrical activity, granule cells maintained a predominant expression of NR2B subunits and their responses to NMDA were largely inhibited by 3 μM ifenprodil. These results provide evidence that the expression of the NR2A and B subunits is regulated through an activity‐dependent mechanism leading to the formation of NMDA receptors with different pharmacological properties. Finally, the NR2C subunit, abundantly expressed in vivo by adult granule cells, was only rarely detected in slice cultures, even when excitatory synapses were formed between granule cells and fibres originating from co‐cultured brainstem explants. These data suggest that the induction of NR2C expression observed in vivo requires an additional factor(s) that remains to be identified.

Single cell RT-PCR proceeds without the risk of genomic DNA amplification

We have previously described a method for detection of mRNAs expressed in single cells after patch-clamp recordings. The method, termed single cell RT-PCR, involves aspiration of the cell content, a reverse transcription (RT) step, and a polymerase chain reaction (PCR) using specific primers. Since the nucleus is frequently harvested together with the cytosol, genomic DNA may generate false positive results. Thus, we demonstrated that dilutions containing a few copies of plasmid could be detected by PCR in a range which, according to the Poisson law, suggests that the PCR method can amplify from the two genomic alleles. We performed single cell RT-PCR of intronless GluR2 or GluR5 fragments by comparing cerebellar cell types where these mRNAs are known to be present or absent. For each cell the nucleus was harvested together with the cytosol. Following RT-PCR with GluR5 primers, all Purkinje cells (n = 6) yielded the expected PCR product, whereas it was not generated from any of the granule cells (n = 5). In corresponding experiments with GluR2 primers, we obtained the GluR2 product from all Purkinje cells (n = 5), but not from any of the glial cells (n = 5). These results are in agreement with the known cellular expression of GluR2 and GluR5 mRNAs. We conclude that the single cell RT-PCR method does not amplify the genomic DNA when the nucleus is aspirated together with the cytosol. We suggest that genomic DNA amplification is avoided, because the genomic alleles are not exposed during the procedure.

Patch-Clamp Recording and RT-PCR on Single Cells

The technique described in this chapter provides electrophysiologists using patch-clamp with a convenient method to link electrophysiological data to a molecular analysis of the mRNAs expressed in a single cell. This molecular analysis can be used either to correlate cell responses with their molecular basis or to identify cell types according to the expression of specific markers. The core of the molecular analysis is polymerase chain reaction (PCR), which makes it fast, sensitive, and simple. Figure 1 outlines the general procedure followed in the experiments. Briefly, after recording of a cell with a patchclamp electrode, the cell content is aspirated through the tip of the electrode and expelled into a test tube with the whole content of the patch electrode. Reagents are then added to perform first strand cDNA synthesis from the mRNA present in the cell. After completion of the reverse transcription (RT) reaction, further reagents are added to the tube to enable a PCR reaction to amplify the cDNA(s) under investigation. Basically, one tube corresponds to one cell, since no change of tubes and very few biochemical manipulations are required. After the first PCR the amplified DNA is analyzed on agarose gel electrophoresis. In some instances, the DNA product from the first PCR is reamplified through a second

Diversity of glutamate receptors in neocortical neurons: Implications for synaptic plasticity

The biochemical and functional characteristics of the AMPA subtype of the glutamate receptors expressed by pyramidal and non-pyramidal neurons of the neocortex have been studied in acute slices by means of single-cell RT-PCR and fast applications of glutamate on outside-out patches. Our results suggest that the predominant expression of the flop splice variants of the GluR1-4 AMPA subunits contributes to the faster desensitization of these receptors in non-pyramidal neurons compared to pyramidal cells where flip variants of GluR1-4 are dominant. Alternative splicing of AMPA receptors may therefore play an important role in regulating synaptic function in a cell-type specific manner.