Naturally expressed nicotinic acetylcholine receptors composed of ␣4 and ␤2 subunits (␣4␤2-nAChR) are the predominant form of high affinity nicotine binding site in the brain implicated in nicotine reward, mediation of nicotinic cholinergic transmission, modulation of signaling through other chemical messages, and a number of neuropsychiatric disorders. To develop a model system for studies of human ␣4␤2-nAChR allowing protein chemical, functional, pharmacological, and regulation of expression studies, human ␣4 and ␤2 subunits were stably introduced into the native nAChR-null human epithelial cell line SH-EP1. Heterologously expressed ␣4␤2-nAChR engage in high-affinity, specific binding of Rbϩ efflux assays indicate full efficacy of epibatidine, nicotine, and acetylcholine; partial efficacy for 1,1-dimethyl-4-phenyl-piperazinium, cytisine, and suberyldicholine; competitive antagonism by dihydro-␤-erythroidine, decamethonium, and methyllycaconitine; noncompetitive antagonism by mecamylamine and eserine; and mixed antagonism by pancuronium, hexamethonium, and d-tubocurarine. These results demonstrate utility of transfected SH-EP1 cells as models for studies of human ␣4␤2-nAChR, and they also reveal complex relationships between apparent affinities of drugs for radioligand binding and functional sites on human ␣4␤2-nAChR.

Temporal filtering is a fundamental operation of nervous systems. In peripheral sensory systems, the temporal pattern of spiking activity can encode various stimulus qualities, and temporal filtering allows postsynaptic neurons to detect behaviorally-relevant stimulus features from these spike trains. Intrinsic excitability, short-term synaptic plasticity, and voltage-dependent dendritic conductances have all been identified as mechanisms that can establish temporal filtering behavior in single neurons. Here we show that synaptic integration of temporally-summating excitation and inhibition can establish diverse temporal filters of presynaptic input. Mormyrid electric fish communicate by varying the intervals between electric organ discharges. The timing of each discharge is coded by peripheral receptors into precisely-timed spikes. Within the midbrain posterior exterolateral nucleus, temporal filtering by individual neurons results in selective responses to a particular range of presynaptic interspike intervals. These neurons are diverse in their temporal filtering properties, reflecting the wide range of intervals that must be detected during natural communication behavior. By manipulating presynaptic spike timing with high temporal resolution, we demonstrate that tuning to behaviorally-relevant patterns of presynaptic input is similar in vivo and in vitro. We reveal that GABAergic inhibition plays a critical role in establishing different temporal filtering properties. Further, our results demonstrate that temporal summation of excitation and inhibition establishes selective responses to high and low rates of synaptic input, respectively. Simple models of synaptic integration reveal that variation in these two competing influences provides a basic mechanism for generating diverse temporal filters of synaptic input.

Amyloid-␤ (A␤) accumulation and aggregation are thought to contribute to the pathogenesis of Alzheimer's disease (AD). In AD, there is a selective decrease in the numbers of radioligand binding sites corresponding to the most abundant nicotinic acetylcholine receptor (nAChR) subtype, which contains human ␣4 and ␤2 subunits (h␣4␤2-nAChR). However, the relationships between these phenomena are uncertain, and effects of A␤ on h␣4␤2-nAChR function have not been investigated in detail. We first confirmed expression of h␣4 and h␤2 subunits as messenger RNA in transfected, human SH-EP1 cells by reverse transcription-polymerase chain reaction and mRNA fluorescence in situ hybridization analyses. Immunoprecipitation Western analyses confirmed ␣4 and ␤2 subunit protein expression and coassembly. Whole cell current recording demonstrated heterologous expression in SH-EP1-h␣4␤2 cells of functional h␣4␤2-nAChRs with characteristic responses to nicotinic agonists or antagonists. Nicotine-induced whole cell currents were suppressed by A␤ 1-42 in a dosedependent manner. Functional inhibition was selective for A␤ 1-42 compared with the functionally inactive, control peptide A␤ 40 -1 . A␤ 1-42 -mediated inhibition of h␣4␤2-nAChR function was non-competitive, voltage-independent, and use-independent. Pre-loading of cells with guanyl-5-yl thiophosphate failed to prevent A␤ 1-42 -induced inhibition, suggesting that down-regulation of h␣4␤2-nAChR function by A␤ 1-42 is not mediated by nAChR internalization. Sensitivity to A␤ 1-42 antagonism at 1 nM was evident for h␣4␤2-nAChRs, but not for heterologously expressed human ␣7-nAChRs, although both nAChR subtypes were functionally inhibited by 100 nM A␤ 1-42 , with the magnitude of functional block being higher for 100 nM A␤ 1-42 acting on h␣7-nAChRs. These findings suggest that h␣4␤2-nAChRs are sensitive and perhaps pathophysiologically relevant targets for A␤ neurotoxicity in AD.Alzheimer's disease (AD) 1 is a progressive, neurodegenerative disorder manifest as a severe impairment of learning and memory. Pathophysiological hallmarks of AD include extracellular deposits of ␤-amyloid peptide (A␤) in senile plaques, formation of intraneuronal neurofibrillary tangles, and cholinergic neuron death (1). Although the precise mechanisms of AD pathogenesis are only partially understood, it is now widely accepted that the accumulation and aggregation of A␤ 1-42 plays a key role in the disease (2). Evidence has indicated an interaction between A␤ and the cholinergic system (3). For example, very low concentrations (pico to nanomolar) of A␤ can directly induce cholinergic hypofunction (4 -6). It has been reported that solubilized A␤ inhibits several steps of acetylcholine synthesis and release (4, 7), inhibits cholinergic enzyme activity (6), impairs cholinergic metabolism and neurotransmission (8 -10), and depresses hippocampal synaptic function (11).Recent evidence suggests possible roles for nicotinic acetylcholine receptors (nAChRs) as central targets for A␤-induced neurotoxicity manifest as...

We examined a 7 b 2 -nicotinic acetylcholine receptor (a 7 b 2 -nAChR) expression in mammalian brain and compared pharmacological profiles of homomeric a 7 -nAChRs and a 7 b 2 -nAChRs. a-Bungarotoxin affinity purification or immunoprecipitation with anti-a 7 subunit antibodies (Abs) was used to isolate nAChRs containing a 7 subunits from mouse or human brain samples. a 7 b 2 -nAChRs were detected in forebrain, but not other tested regions, from both species, based on Western blot analysis of isolates using b 2 subunit-specific Abs. Ab specificity was confirmed in control studies using subunit-null mutant mice or cell lines heterologously expressing specific human nAChR subtypes and subunits. Functional expression in Xenopus oocytes of concatenated pentameric (a 7 ) 5 -, (a 7 ) 4 (b 2 ) 1 -, and (a 7 ) 3 (b 2 ) 2 -nAChRs was confirmed using two-electrode voltage clamp recording of responses to nicotinic ligands. Importantly, pharmacological profiles were indistinguishable for concatenated (a 7 ) 5 -nAChRs or for homomeric a 7 -nAChRs constituted from unlinked a 7 subunits. Pharmacological profiles were similar for (a 7 ) 5 -, (a 7 ) 4 (b 2 ) 1 -, and (a 7 ) 3 (b 2 ) 2 -nAChRs except for diminished efficacy of nicotine (normalized to acetylcholine efficacy) at a 7 b 2 -versus a 7 -nAChRs. This study represents the first direct confirmation of a 7 b 2 -nAChR expression in human and mouse forebrain, supporting previous mouse studies that suggested relevance of a 7 b 2 -nAChRs in Alzheimer disease etiopathogenesis. These data also indicate that a 7 b 2 -nAChR subunit isoforms with different a 7 /b 2 subunit ratios have similar pharmacological profiles to each other and to a 7 homopentameric nAChRs. This supports the hypothesis that a 7 b 2 -nAChR agonist activation predominantly or entirely reflects binding to a 7 /a 7 subunit interface sites.