ALPHA-BUNGAROTOXIN BINDING-SITES IN RAT HIPPOCAMPAL AND CORTICAL CULTURES - INITIAL CHARACTERIZATION, COLOCALIZATION WITH ALPHA-7 SUBUNITS AND UP-REGULATION BY CHRONIC NICOTINE TREATMENT
Ge. Barrantes et al., ALPHA-BUNGAROTOXIN BINDING-SITES IN RAT HIPPOCAMPAL AND CORTICAL CULTURES - INITIAL CHARACTERIZATION, COLOCALIZATION WITH ALPHA-7 SUBUNITS AND UP-REGULATION BY CHRONIC NICOTINE TREATMENT, Brain research, 672(1-2), 1995, pp. 228-236
High density neuronal cultures from rat E18 hippocampus and cortex hav
e been characterised with respect to cholinergic binding sites. No spe
cific 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-Bungarotoxi
n (alpha Bgt) bound to hippocampal cultures with a B-max of 128 fmol/m
g 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 lab
elled, compared with only 36% of cortical neurons. Average densities o
f 4 X 10(4) and 2 x 10(4) binding sites/cell were calculated for hippo
campal and cortical cultures, respectively. Double labelling experimen
ts with mAb307 (which recognises the rat alpha 7 nicotinic receptor su
bunit) and rhodamine-alpha-Bgt gave coincident labelling patterns, sup
porting 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-sensiti
ve receptors.