ALPHA-BUNGAROTOXIN BINDING-SITES IN RAT HIPPOCAMPAL AND CORTICAL CULTURES - INITIAL CHARACTERIZATION, COLOCALIZATION WITH ALPHA-7 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 [H-3]nicotine or [H-3]cytisine 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 [H-3]QNB were present at a density of 0.75 pmol/mg protein. [I-125]alpha-Bungarotoxin (alpha Bgt) 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 [I-125]alpha-Bgt binding sites. Fluorescence cytochemistry with rhodamine-alpha-Bgt indicated that 95% of hippocampal neurons were labelled, compared with only 36% of cortical neurons. Average densities of 4 X 10(4) and 2 x 10(4) binding sites/cell were calculated for hippocampal and cortical cultures, respectively. Double labelling experiments with mAb307 (which recognises the rat alpha 7 nicotinic receptor subunit) and rhodamine-alpha-Bgt gave coincident labelling patterns, supporting the correlation between the alpha 7 subunit and Bgt-sensitive neuronal nicotinic receptor. Treatment of hippocampal cultures with 10 mu M nicotine for 14 days elicited a 40% increase in the numbers of [I-125]alpha-Bgt binding sites, mimicking the up-regulation observed in in vivo studies. Primary cultures offer a useful in vitro system for investigating the expression and regulation of brain alpha-Bgt-sensitive receptors.