Measuring nicotinic receptors with characteristics of alpha 4 beta 2, alpha 3 beta 2 and alpha 3 beta 4 subtypes in rat tissues by autoradiography

Comparison of [ super(125) I]epibatidine and 5-[ super(125) I]iodo-3-(2-azetidinylmethoxy)pyridine ([ super(125) I]A-85380) autoradiography showed evidence for nicotinic receptor heterogeneity. To identify the receptor subtypes, we performed [ super(125) I]epibatidine autoradiography in the presence of cytisine or A-85380. By comparing these results with binding data from human embryonic kidney (HEK) 293 cells stably transfected with different combinations of rat nicotinic receptor subunits, we were able to quantify three distinct populations of [ super(125) I]epibatidine binding sites with characteristics of alpha 4 beta 2, alpha 3 beta 2 and alpha 3 beta 4 receptors. Although the predominant subtype in rat brain was alpha 4 beta 2, non- alpha 4 beta 2 binding sites were prominent in many regions. In the habenulo-peduncular system, cerebellum, substantia gelatinosa, and many medullary nuclei, alpha 3 beta 4-like binding accounted for more than 40% of [ super(125) I]epibatidine binding, and nearly all binding in superior cervical ganglion and pineal gland. Other regions enriched in alpha 3 beta 4-like binding included locus ceruleus, dorsal tegmentum, subiculum and anteroventral thalamic nucleus. Regions enriched in alpha 3 beta 2-like binding included the habenulo-peduncular system, many visual system structures, certain geniculate nuclei, and dopaminergic regions. The combination of autoradiography using a broad spectrum radioligand in the presence of selective competitors, and data from binding to defined receptor subtypes in expression systems, allowed us to quantify the relative populations of these three subtypes.