Contrasting molecular composition and channel properties of AMPA receptors on chick auditory and brainstem motor neurons

Neurons in the brainstem auditory pathway exhibit a number of specializations for transmitting signals reliably at high rates, notably synaptic AMPA receptors with very rapid kinetics. Previous work has not revealed a common structural pattern shared by the AMPA receptors of auditory neurons that could account for their distinct functional properties. We have used whole-cell patch-clamp recordings, mRNA analysis, immunofluorescence, Western blots and agonist-evoked cobalt uptake to compare AMPA receptors on the first-, second- and third-order neurons in the chick ascending auditory pathway with those on brainstem motor neurons of the glossopharyngeal/vagal nucleus, which have been shown to have very slow desensitization kinetics. The results indicate that the AMPA receptors of the cochlear ganglion, nucleus magnocellularis and nucleus laminaris share a number of structural and functional properties that distinguish them from the AMPA receptors of brainstem motor neurons, namely a lower relative abundance of glutamate receptor (GluR)2 transcript and much lower levels of GluR2 immunoreactivity, higher relative levels of GluR3 flop and GluR4 flop, lower relative abundance of the C-terminal splice variants GluR4c and 4d, less R/G editing of GluR2 and 3, greater permeability to calcium, predominantly inwardly rectifying I–V relationships, and greater susceptibility to block by Joro spider toxin. We conclude that the AMPA receptors of auditory neurons acquire rapid kinetics from their high content of GluR3 flop and GluR4 flop subunits and their high permeability to Ca2+ from selective post-transcriptional suppression of GluR2 expression. Ionotropic glutamate receptors (GluRs) of the AMPA subtype are assembled from four protein subunits, termed GluR1-4 or A-D. Native AMPA receptors are assembled from a variety of subunit, splice variant and mRNA editing combinations that result in diverse functional properties (Borges & Dingledine, 1998). Although some types of neuron are reported to show great cell-to-cell variation in AMPA receptor function (Angulo et al. 1997; Washburn et al. 1997), other cell classes are reported to display a predominant functional type of AMPA receptor. Rat CA3 pyramidal neurons, for example, express slowly desensitizing receptors with low permeability to calcium, and the AMPA receptors of Bergmann glial cells desensitize rapidly and show relatively high calcium permeability (Geiger et al. 1995). Since AMPA receptors mediate most rapid synaptic transmission in the CNS (Collingridge & Lester, 1989), understanding how different classes of neuron develop and maintain characteristic information-processing functions will require detailed analysis of the properties of their AMPA receptors. Auditory neurons show a number of striking morphological and functional specializations that can be related to their roles in hearing (Trussell, 1999). The AMPA receptors of auditory neurons in birds and mammals also exhibit specializations. Several types of auditory neuron have AMPA receptors with unusually high permeability to divalent cations (Otis et al. 1995; Zhou et al. 1995; Caicedo et al. 1998) and very rapid desensitization rates – almost fivefold faster than the AMPA receptors of brainstem motor neurons, for example (Raman et al. 1994). The molecular bases for these AMPA receptor specializations are poorly understood. A few studies have analysed AMPA receptor subunit expression within certain auditory centres using in situ hybridization (Hunter et al. 1993; Sato et al. 1993; Niedzelski & Wenthold, 1995), mRNA analysis (Niedzelski & Wenthold, 1995) or immunohistochemistry (Petralia & Wenthold, 1992; Petralia et al. 1996, 1997; Levin et al. 1997; Wang et al. 1998; Caicedo & Eybalin, 1999). Geiger et al. (1995) included one type of auditory neuron in their study of the correlation of AMPA receptor functional properties with GluR mRNA profiles. Each of these studies has provided a partial characterization of AMPA receptor structure and there are discrepancies in their results which make it unclear whether there is a common structural pattern shared by the AMPA receptors of auditory neurons. To address this issue, we used mRNA analysis, whole-cell patch-clamp recordings, immunofluorescence, Western blots and agonist-evoked cobalt uptake to compare the molecular and functional properties of AMPA receptors in the first three neural centres of the chick auditory pathway – the cochlear ganglion (CG), nucleus magnocellularis (NM) and nucleus laminaris (NL) – with those of motor neurons in the glossopharyngeal/vagal nucleus (NIX/X), which Raman et al. (1994) have shown to have very slow desensitization kinetics.