alpha-Bungarotoxin binding sites in rat hippocampal and cortical cultures: initial characterisation, colocalisation 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 [3H]nicotine or [3H]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 [3H]QNB were present at a density of 0.75 pmol/mg protein. [125I]alpha-Bungarotoxin (alpha Bgt) bound to hippocampal cultures with a Bmax of 128 fmol/mg protein and a Kd of 0.6 nM; cortical cultures expressed five times fewer [125I]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 microM nicotine for 14 days elicited a 40% increase in the numbers of [125I]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.
Barrantes
G E
GE
School of Biology and Biochemistry, University of Bath, UK.
Rogers
A T
AT
Lindstrom
J
J
Wonnacott
S
S
D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D007150:Immunohistochemistry; D009538:Nicotine / Q000494:pharmacology*; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D051381:Rats / Q000196:embryology; D017207:Rats, Sprague-Dawley; D011978:Receptors, Nicotinic / Q000378:metabolism; D013997:Time Factors; D014018:Tissue Distribution; D015854:Up-Regulation
D002038:Bungarotoxins; D011978:Receptors, Nicotinic; D011813:Quinuclidinyl Benzilate; D009538:Nicotine