Functional and molecular characterization of neuronal nicotinic ACh receptors in rat CA1 hippocampal neurons.

Neuronal nicotinic acetylcholine receptors (nAChRs) are members of the ligand-gated ion channel superfamily, along with the GABAA, glycine and 5-HT3 receptors. Currently there are at least 10 nAChR subunits known to be expressed in the rat CNS (see McGehee & Role, 1995; Boyd, 1997; Jones et al. 1999). The nAChRs are widely expressed throughout the CNS and have been suggested to play roles in such diverse phenomena as Alzheimer's disease (Perry et al. 1995), epilepsy (Steinlein et al. 1995), schizophrenia (Freedman et al. 1997), cognition (Levin, 1992; Changeux et al. 1998), tobacco addiction (Pontieri et al. 1996) and even cell proliferation and differentiation in the developing CNS (McFarland et al. 1991). However, many of the cellular and molecular mechanisms involved in these diverse actions are currently unknown. Recent reports have clearly demonstrated a postsynaptic role for nAChRs in the hippocampus (Frazier et al. 1998a; Alkondon et al. 1998). These nAChRs are involved in fast synaptic transmission in CA1 stratum radiatum and stratum oriens interneurons. There is cholinergic innervation to the hippocampus from the medial septum-diagonal band complex (MSDB) of the basal forebrain (Frotscher & Leranth, 1985), and there is also evidence for intrinsic cholinergic synapses between hippocampal interneurons as well (Matthews et al. 1987; Freund & Buzsaki, 1996). Hippocampal interneurons can have GABAergic projections onto the more numerous CA1 pyramidal neurons, causing inhibition of pyramidal cell excitation. The neuronal nAChRs on hippocampal interneurons are in a key position to be involved in conditions previously linked to both the hippocampus and nicotine, such as cognition, schizophrenia and epilepsy (see Jones et al. 1999 for review). Like other ligand-gated ion channels, the subunit composition of a nAChR determines its functional properties (e.g. ligand sensitivity, activation and desensitization rates, ion permeability and sensitivity to pharmacological agents; see Boyd, 1997 and McGehee, 1999, for reviews). The subunit composition of nAChRs in hippocampal neurons in vivo has yet to be determined. Previous studies have used pharmacological agents that suggest the involvement of particular nAChR subunits in the nAChR-mediated responses of these neurons (Jones & Yakel, 1997; Alkondon et al. 1998; Frazier et al. 1998b; McQuiston & Madison, 1999). These reports clearly suggest that α7-containing nAChRs are functionally expressed in most hippocampal interneurons, but the incomplete block by α7-specific antagonists also suggests that there is a class of non-α7-containing receptors for which the precise subunits involved are currently unknown. The composition of these non-α7-containing receptors has been difficult to establish using conventional pharmacological techniques, since there are not many agents which block nAChR subtypes that show subunit-specific selectivity for individual subunits other than α7. Therefore, we have used single-cell reverse-transcription polymerase chain reaction (single-cell RT-PCR; Eberwine et al. 1992; Lambolez, 1992; Jonas et al. 1994) to examine the subunit mRNA expression pattern in CA1 neurons (e.g. interneurons from the stratum radiatum and stratum oriens, as well as pyramidal neurons) in rat hippocampal slices to identify nAChR subunits that are involved in channel formation. Additionally, we performed whole-cell patch-clamp recordings of nAChR-mediated responses in these neurons as an assay of functional receptor properties. Using statistical analysis, we looked for functional and molecular correlations between nAChR subunit mRNA expression and functional nAChR channel properties using these combined techniques. We have found distinct patterns of expression for the various nAChR subunits in the three different neuronal populations examined, and have found statistically significant correlations between the kinetics of nAChR-mediated responses in neurons from brain slices and the mRNA expression of specific nAChR subunits.