α-Bungarotoxin binding sites in rat hippocampal and cortical cultures: initial characterisation, colocalisation with α7 subunits and up-regulation by chronic nicotine treatment

High density neuronal cultures from rat E18 hippocampus and cortex have been characterised with respect to cholinergic binding sites. No specific binding of [³H]nicotine or [³H]cyttine to live cells in situ was detected, although the limit for detection was estimated to be 30 fmol/mg protein. Muscarinic binding sites labelled with [³H]QNB were present at a density of 0.75 pmol/mg protein. [¹²⁵I]α-Bungarotoxin (αBgt) bound to hippocampal cultures with a B_max of 128 fmol/mg protein and a K_d of 0.6 nM; cortical cultures expressed five times fewer [¹²⁵I]α-Bgt binding sites. Fluorescence cytochemistry with rhodamine-α-Bgt indicated that 95% of hippocampal neurons were labelled, compared with only 36% of cortical neurons. Average densities of 4 × 10⁴ and 2 × 10⁴ binding sites/cell were calculated for hippocampal and cortical cultures, respectively. Double labelling experiments with mAb307 (which recognises the rat α7 nicotinic receptor subunit) and rhodamine-α-Bgt gave coincident labelling patterns, supporting the correlation between the α7 subunit and Bgt-sensitive neuronal nicotinic receptor. Treatment of hippocampal cultures with 10 μM nicotine for 14 days elicited a 40% increase in the numbers of [¹²⁵I]α-Bgt binding sites, mimicking the up-regulation observed in vivo studies. Primary cultures offer a useful in in vitro system for investigating the expression and regulation of brain α-Bgt-sensitive receptors.