AF-DX-384 BINDING IN RABBIT CINGULATE CORTEX, 2 SITE KINETICS AND SECTION AUTORADIOGRAPHY

Autoradiographic studies of muscarinic receptors are limited by the la ck of selective ligands. Inasmuch as AF-DX 384 has a higher affinity f or m2 than m4 receptors and pirenzepine (PZ) has a reverse affinity pr ofile, competition between these ligands was used to label m2 receptor s in homogenized and sectioned tissue. Rabbit cingulate cortex was use d because m2 receptors are expressed by anterior thalamic axons in pos terior cingulate cortex (PCC) and this region is easily deafferented w ith undercut lesions to demonstrate presynaptic binding, Saturation is otherms and Scatchard analysis of [H-3]AF-DX 384 binding showed one bi nding site with a K-D of 9 +/- 2.3 nM (mean +/- SEM) and a B-max of 14 05 +/- 146 fmol/mg protein. Competition studies with [H-3]AF-DX 384 (2 nM) and 10(-10)-10(-4) M PZ were performed in anterior cingulate cort ex (ACC) and PCC. In both regions, the best fit was a two site model f or low (B-L) and high (B-H) affinity binding in which B-max values wer e similar (ACC: B-L = 535 +/- 62 fmol/mg, B-H = 676 +/- 85; PCC: B-L = 552 +/- 41; B-H = 675 +/- 85). Although affinities for K-H were simil ar 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 [H-3]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 s ite. Autoradiography showed that [H-3]AF-DX 384 binding was similar in each layer of PCC and a PZ block reduced binding by 10 to 27%. Underc ut lesions reduced binding in layers I-II by 27 to 48%. Layer V had th e smallest PZ block and lesion effect indicating a high density of int rinsic m2 receptors. Binding in the anterior thalamic nuclei showed hi gh levels of [H-3]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 ax ons. 2) Binding of [H-3]AF-DX 384 (2 nM) with PZ (150 nM) is to a sing le site. 3) Undercut lesions and a PZ block uncover a high proportion of m4 receptors in layers Ib-II, extrinsic, presynaptic m2 receptors i n layers I-III and a high density of intrinsic m2 receptors in layer V .