INTRACELLULAR DISTRIBUTION OF C¹⁴-HEMICHOLINIUM-3 IN THE CANINE CAUDATE NUCLEUS AND HIPPOCAMPUS

¹ Neuropsychopharmacology Research Program, Departments of Pharmacology and Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan; Tulane University, New Orleans, Louisiana

C¹⁴-hemicholinium-3, methyl-labeled (C¹⁴-HC-3), was administered intraventricularly to acute dogs. At 30, 60 and 240 min postinjection, its intracellular distribution was determined in the caudate nucleus and hippocampus. Simultaneously the intracellular localization of succinate reductase, glutamine transferase, acetylcholinesterase (AChE) and aryl sulfatase was established and the validity of their use as subcellular markers for caudate and hippocampus was confirmed. Although C¹⁴-HC-3 accumulated in the heavy particulate fraction NEML (containing nerve endings, mitochondria and lysosomes) in both brain regions, the rate of its concentration was considerably higher in the caudate. Discontinuous density gradient centrifugation of fraction NEML yielded subfractions A, B, C, D and E. Although AChE showed enrichment in fractions B and C, up to 30% appeared in fraction D, which also exhibited (in the caudate) the highest relative specific concentration of C¹⁴-HC-3. Centrifugation of caudate fractions C and D in continuous density gradients confirmed the association of AChE and of C¹⁴-HC-3 with, and the real density difference between, the two granule populations comprising the fractions C and D. Experiments designed to assess the extent of the association of C¹⁴-HC-3 with nerveending membranes revealed that approximately 11% of the total NEML-associated C¹⁴-HC-3 resists attempts at extrusion and/or solubilization and, hence, denotes tightly bound C¹⁴-HC-3. The findings are interpreted to indicate that the majority of fraction C nerve endings contain few mitochondria, have a low affinity for HC-3 and exhibit high levels of AChE, while those of fraction D have most of the mitochondria-filled nerve endings, 30% of the AChE and a high affinity for HC-3. It is suggested that there are two populations of cholinergic nerve endings in the caudate nucleus.