Characterization and Regional Distribution of a Class of Synapses with Highly Concentrated cAMP Binding Sites in the Rat Brain

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Abstract
A class of putative synaptic terminals with concentrated cAMP binding sites are labelled in unfixed sections of rat brain by means of the ligand 8-thioacetamido fluorescein cAMP (SAF-cAMP), a fluorescent analogue of cAMP. The labelled terminals appear as sharply delimited bouton-like structures in close proximity but external to the cell body of neurons. The SAF-cAMP binding, measured at equilibrium in competition with other nucleotides, indicates that the binding site recognizes the cAMP moiety of SAF-cAMP. In the labelled terminals of the frontal cortex the concentration of SAF-cAMP binding sites is estimated to be in the millimolar range (at least 2.1 +/- 1.0 mM). In a brain homogenate, labelled terminals are visualized only in the membrane fraction enriched in synaptosomes. The cAMP binding activity of the synaptosomes is insoluble in high and in low ionic strength solution and is only partially solubilized by detergents, suggesting that the binding sites are intrinsic membrane proteins and/or proteins associated with the cytoskeleton. There is the possibility that SAF-cAMP labels new cAMP binding sites highly concentrated in a class of synaptic terminals. SAF-cAMP labelling is prominent in well defined regions of the rat brain: (i) the frontal and entorhinal areas of the cortex; (ii) the field CA1 of the hippocampus; (iii) the olfactory system; (iv) the medial nuclei of the thalamus; (v) the parabrachial nuclei and other less defined regions of the reticular substance; (vi) the substantia gelatinosa of Rolando in the spinal cord; and (vii) the neo- and paleocerebellum in the Purkinje cell layer, the archicerebellum in the granular cell layer. SAF-cAMP labelling is absent in specific motor and sensory structures, with the exception of the olfactory system. It is proposed that SAF-cAMP binding sites single out a new type of synaptic terminals involved in complex nervous functions.