AF-DX 384 binding in rabbit cingulate cortex: Two site kinetics and section autoradiography

Autoradiographic studies of muscarinic receptors are limited by the lack of selective ligands. Inasmuch as AF-DX 384 has a higher affinity for m2 than m4 receptors and pirenzepine (PZ) has a reverse affinity profile, competition between these ligands was used to label m2 receptors in homogenized and sectioned tissue. Rabbit cingulate cortex was used because m2 receptors are expressed by anterior thalamic axons in posterior cingulate cortex (PCC) and this region is easily deafferented with undercut lesions to demonstrate presynaptic binding. Saturation isotherms and Scatchard analysis of [³H]AF- DX 384 binding showed one binding site with a K(D) of 9 ± 2.3 nM (mean ± SEM) and a B(max) of 1405 ± 146 fmol/mg protein. Competition studies with [³H]AF-DX 384 (2 nM) and 10^-10-10^-4 M PZ were performed in anterior cingulate cortex (ACC) and PCC. In both regions, the best fit was a two site model for low (B(L)) and high (B(H)) affinity binding in which B(max) values were similar (ACC: B(L) = 535 ± 62 fmol/mg, B(H) = 676 ± 85; PCC: BL = 552 ± 41; B(H) = 675 ± 85). Although affinities for K(H) were similar in each region (ACC: K(H) = 4.69 ± 1.36 nM; PCC: K(H) = 8.53 ± 3.69 nM), those for K(L) were significantly different (ACC: 181 ± 15.4 nM; PCC: 285 ± 42; P = .018). Binding of [³H]AF-DX 384 with PZ (150 nM) was best fit with a single site model (K(D) = 6 ± 0.01 nM; B(max) = 688 ± 31 fmol/mg), suggesting that PZ blocks the lower affinity site. Autoradiography showed that [³H]AF-DX 384 binding was similar in each layer of PCC and a PZ block reduced binding by 10 to 27%. Undercut lesions reduced binding in layers I-II by 27 to 48%. Layer V had the smallest PZ block and lesion effect indicating a high density of intrinsic m2 receptors. Binding in the anterior thalamic nuclei showed high levels of [³H]AF-DX 384/PZ binding. These conclusions were drawn: 1) The low affinity PZ site has a high K(L) in PCC compared to ACC and may represent m2 receptor binding on glutamatergic, thalamocortical axons. 2) Binding of [³H]AF-DX 384 (2 nM) with PZ (150 nM) is to a single site. 3) Undercut lesions and a PZ block uncover a high proportion of m4 receptors in layers Ib-II, extrinsic, presynaptic m2 receptors in layers I-III and a high density of intrinsic m2 receptors in layer V.