Neuron, Vol. 9, 247-258, August, 1992, Copyright 0 1992 by Cell Press AMPA Receptor Subunits Expressed by Single Ptirkinje Cells Bertrand Lambolez,* Etienne Audinat,+ Pascal Bochet,* Francis CrepeI,+ and Jean Rossier* *Institut Alfred Fessard Centre National de la Recherche Scientifique 91198 Gif-sur-Yvette France +Laboratoire de Neurobiologie et de Neuropharmacologie du Dhveloppement Centre National de la Recherche Scientifique 91405 Orsay France Summary Several subunits of the glutamate receptor of the AMPA subtype have been cloned recently. These subunits, named GIuRl, GIuR2, CluR3, and CIuR4, 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 reac- tion, 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 GluRl and CluR2 as well as GluR3flip, CluR2 being the most abundant. In addition, GluR3flop and GluR4flip were scarcely expressed in half of these neurons, and GluR4flop was never detected. Introduction Molecular cloning and expression studies have dem- onstrated that the subtype diversity of neurotrans- mitter receptors is much larger than expected from pharmacological studies. In particular, for neuro- transmitter-gated channels, the number of subunits already cloned is always larger than the number of subunits required to construct functional receptors in expression systems. A crucial question is therefore to determine the actual subunit composition of native receptor-channels expressed by a given neuron un- der functional investigation. The regional distribution in thecentral nervous sys- tem of receptor-channel subunits can be mapped by in situ hybridization. However, this technique does not allow one to determine the subunit composition of the receptor-channels at the single-cell level, since it is impossible to hybridize 1 given cell with several probes. In addition, in situ hybridization does not pro- vide the molecular analysis with functional correlates. Recently, several subunits of a glutamate receptor- ‘channel have been cloned. These subunits, named ~GluRI, 2,3, and 4 (GIuRl-4), exist in two versions (flip and flop) generated by alternative splicing. Functional expression of homomeric or heteromeric combina- tions of these subunits generates receptors respon- sive to glutamate, quisqualate (QA), kainate (KA), and a-amino-3-hydroxy-5-methyl-4-isoxazolepropio- nate (AMPA) and on which quinoxalinediones act as competitive antagonists (Hollmann et al., 1989; Boul- ter et al., 1990; Dawson et al., ‘1990; Keininen et al., 1990; Lambolez et al., 1991; Nakanishi et al., 1990; Saki- mura et al., 1990; Sommer et al., 1990). This pharma- cological profile is characteristic of the glutamate receptor of the AMPA subtype as opposed to the N-methyl-o-aspartate and high affinity KA subtypes (Barnard and Henley, 1990; Huettner, 1990; Miller, 1991; Patneau and Mayer, 1991). A biochemical analysis of the AMPA receptor-chan- nel purified from rat brain has shown that this recep- tor is a pentameric structure composed of combi- nations of the GIuRl-4 subunits; the presence of additional subunits is very unlikely (Wenthold et al., 1992). However, the subunit composition of native AMPA receptors involved in single-neuron responses is still unknown. To address this issue, we have combined whole-cell patch-clamp recordings (Hamill et al., 1981) and the amplification, by means of the polymerase chain reac- tion (PCR), of the mRNAs harvested from the single neuron under investigation. This method was applied here to determine, after electrophysiological investi- gation, the AMPA receptor subunits expressed by in- dividual Purkinjecells in both cerebellar and olivocer- ebellar slice cultures. Purkinje cells were chosen because they can be easily identified in this prepara- tion, and the pharmacology of their responses to ex- citatory amino acids is well characterized (Audinat et al., 1990; Crkpel et al., 1982; Kniipfel et al., 1990; Perkel et al., 1990; Farrant and Cull-Candy, 1991; Llano et al., 1991). Results Electrophysiological Properties of the AMPA Receptors of Purkinje Cells Cerebellar cells, grown in organotypic slice cultures, were recorded in the whole-cell configuration of the patch-clamp technique and subsequently used to de- termine, for each of them, the content of mRNA en- coding the GluRl-4 subunits of the AMPA receptor (see Figure 1). Purkinje cells were identified according to their lo- calization in the slice culture and their morphology as seen in phase-contrast microscopy (Audinat et al., 1990). In cerebellar monocuItures,when Purkinjecells were voltage clamped at a holding potential of -60 mV, 50 PM QA induced an initial peak current, which rapidly decreased to a steady-state desensitized level (Figure2A, lower row; seealso Geoffroyet al., 1991). In