Effects of paraformaldehyde fixation on nicotinic acetylcholine receptor binding in adult and developing rat brain sections

Abstract

In vitro receptor autoradiography requires unfixed tissue sections, but incubation and washing procedures often result in substantial tissue damage in sections from developing brain, hindering quantitative and qualitative analysis. Formaldehyde fixation greatly preserves morphology. However, fixation can interfere with pharmacological properties of receptors, increase in non-specific background labeling, or even destroy ligand binding sites. Two mild fixation protocols, 0.2% paraformaldehyde (pFA) and pFA vapor fixation, were compared for their ability to improve tissue morphology in postnatal day 7 (P7) brain slices and maintain binding of [ 125I]-epibatidine and [ 125I]-α-bungarotoxin to heteromeric and homomeric nicotinic acetylcholine receptors, respectively. Fixation greatly improved the ability of P7 brain slices to withstand incubation and washing procedures during binding, resulting in minimal or no loss of tissue after prior 0.2% pFA or vapor fixation, respectively. In adults, distribution pattern of [ 125I]-epibatidine was identical in fixed and unfixed slices, with no difference in total and non-specific labeling. Distribution of [ 125I]-αBTX labeling was similarly unaffected by 0.2% pFA fixation, but vapor fixation increased total and non-specific binding signal. Thus, mild fixations greatly improve tissue quality during receptor binding procedures and can preserve pharmacological properties of nicotinic acetylcholine receptors. However, different receptors or ligands might exhibit differential sensitivity to fixation protocols.