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Opioid receptors can be labeled with [3H]ligands in lightly fixed tissue sections mounted on microscope slides. The preparation of these sections does not seem to alter any of the known characteristics of opioid receptors. The light microscopic autoradiographic distribution of these binding sites can be observed if one attaches emulsion-coated coverslips to these slides to obtain autoradiograms. The distribution of [3H]diprenorphine binding sites determined by this in vitro method is identical to the distribution found in earlier studies utilizing in vivo labeling of opioid receptors. In addition, [3H]opioid peptide binding sites and [3H]dihydromorphine binding sites may be similar, perhaps identical, to those for [3H]diprenorphine, an opiate antagonist. This method has several important advantages over earlier methods for determining the autoradiographic localization of receptors. It is possible, by washing, to reduce nonspecific binding to low levels. Since receptor labeling is performed with single tissue sections mounted on slides, one can examine different receptors in different but adjacent sections. One can use ligands that are not entirely suitable for in vivo labeling (For example, one can use [3H]peptides which do not normally cross the blood-brain barrier). One can perform autoradiographic studies after labeling tissues under a wide variety of conditions (For example, one can examine the effects of various ions and nucleotides on ligand binding distributions). This technique seems to be free of various artifacts, such as edge artifacts, which were common by techniques used in our laboratory earlier. Since one does not have to load an entire animal with radioactive ligand as one must with in vivo labeling, this approach is economically advantageous. One can use postmortem tissues, including that from humans to study receptor distribution with a high anatomical resolution.

Opiate receptor distribution, determined by the autoradiographic localization of stereospecific [3H]diprenorphine binding sites, was examined in the telencephalon. Areas showing very dense or dense localization of receptors included parts of the presubiculum and amygdala, patchy areas in the caudate-putamen and accumbens, the subfornical organ, the interstriatal nucleus of the striae terminalis and the anterior olfactory nucleus, pars externa. Lower densities were found in other parts of the hippocampal formation, the deeper part of the cerebral cortex, the entopeduncular nucleus, globus pallidus, nucleus triangularis septi and nucleus paratenialis. The significance of these findings is discussed in terms of the biochemical and physiological actions of opiates.

Subcellular fractionation of rat cerebral cortical slices on sucrose density gradients provides evidence for the existence of a unique synaptosomal fraction (enriched in pinched-off nerve endings) that selectively accumulates glutamic and aspartic acids. The particles in this fraction sediment to a less dense portion of sucrose gradients than do particles that accumulate aromatic, basic, and neutral (large and small) amino acids. Particles that store gamma-aminobutyric acid are even less dense than those that contain exogenous glutamic and aspartic acids. The distribution of endogenous glutamic acid encompasses both that of exogenous glutamic acid and that of the neutral and basic amino acids. These findings provide neurochemical support for the suggestion that glutamic and/or aspartic acid has a specialized synaptic function, perhaps as a neurotransmitter, in the mammalian brain.

This communication describes ultrastructural variations among synaptosomal fractions isolated from the corpus striatum of the rat by incomplete equilibrium sedimentation in sucrose density gradients, and attempts to relate the variations to neurotransmitter-specific synaptosomes. The concentration of synaptosomes in each fraction of the density gradient was found to be correlated with the concentration of potassium, a marker for cytoplasm occluded within synaptosomes. Monoamine oxidase activity was found to be correlated with the incidence of free mitochondria in the gradients. Synaptosomes from denser gradient fractions showed a markedly increased frequency of adherent postsynaptic elements and intraterminal mitochondria. These denser gradient fractions were rich in synaptosomes containing norepinephrine, dopamine, serotonin, and acetylcholine, while synaptosomes in lighter portions of the gradients were rich in gamma-aminobutyric acid and other amino acids. These data suggest that significant morphological variations may exist among different neurotransmitter-specific nerve terminals in the brain.