Characterization of muscarinic acetylcholine receptors in rat cerebral cortex slices with concomitant morphological and physiological assessment of tissue viability.
We have begun studies on regulatory mechanisms of muscarinic acetylcholine receptors (mAChRs) in slices of rat cerebral cortex. This paper, the first of two, deals with the viability of the cells in the slices (a prerequisite for studying receptor regulation) and provides a characterization of binding sites for [3H]N-methyl scopolamine ([3H]NMS) and [3H]quinuclidinyl benzylate ([3H]QNB) in this preparation. Trypan blue exclusion tests in 400-microns-thick cortical slices showed a number of dead cells in a 100 microns zone from each cut edge, for a total of about 15-30% of all cells in the slice. In agreement with previous reports, electron microscopy revealed healthy tissue in the middle of the slice, but after incubation for several hours, swollen cells and dendrites were seen without cytoplasmic organelles. Axon terminals, however, were still seen to synapse upon these processes. Electrophysiological single unit recordings showed spontaneous action potentials in the slices. For receptor binding experiments, slices were incubated with either [3H]NMS, a hydrophilic mAChR ligand which does not penetrate the cell membrane, or the lipophilic ligand [3H]QNB which readily enters cells. For both ligands, equilibrium binding was reached after 8 h at 4 degrees C, and after 3 h at 30 degrees C. Binding of both ligands could be displaced by unlabelled atropine sulphate, NMS or QNB. Saturation binding curves yielded a Bmax of 2187 fmol/mg protein for [3H]QNB (reflecting all mAChRs) and 1335 fmol/mg protein for [3H]NMS (only mAChRs on the cell surface) at 30 degrees C. Kd values were 8.2 and 5.2 nM for [3H]QNB and [3H]NMS, respectively. These values are high compared with values obtained from homogenates, frozen sections or dissociated cells, and presumably reflect the use of intact, living tissue. These data are probably a better reflection of the actual, in vivo mAChR number and affinity than those obtained from dead tissue. This slice preparation suggests itself as a simple but effective method with which to study the regulation of mAChRs in living brain tissue.
van Huizen
F
F
Department of Ophthalmology, University of British Columbia, Vancouver, Canada.
Shaw
C
C
Wilkinson
M
M
Cynader
M S
MS
D000818:Animals; D002470:Cell Survival; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D003712:Dendrites / Q000378:metabolism / Q000648:ultrastructure; D004594:Electrophysiology; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011976:Receptors, Muscarinic / Q000378:metabolism*
D011976:Receptors, Muscarinic