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We describe here a protocol developed to detect specific mRNAs by in situ hybridization using tissue sections that were not treated to inactivate RNase and were stored in cryoprotectant solution for several years. Brains from rats, monkeys and humans were sectioned at 50 microns and stored free floating in an ethylene glycol based cryoprotective solution at -20 degrees C. Rat brain sections were kept in cryoprotective solution for 3 days, 1 month and 2 months. Control sections were cut and mounted immediately on gelatin-coated slides and stored at -80 degrees C. Monkey brain sections were stored in cryoprotective solution for up to 5 years. Human sections were tested after storage for one year. Oligonucleotide probes that were complementary to human preproenkephalin mRNA (amino acid sequences 130-145), rat preproenkephalin mRNA (sequences 388-435) and rat tyrosine hydroxylase mRNA (sequences 1441-1488) were labeled with 35S-dATP and terminal deoxynucleotidyl transferase. To prevent possible RNase contamination from mounting the tissue sections onto gelatin coated slides, the in situ hybridization was performed in sterile culture dishes. Following each step, solutions were aspirated out of the dish. The amount of probe necessary for each section was 45 microliters (rat), 450 microliters (monkey), and 350 microliters (human). Using this protocol, the detection of specific mRNAs in rat brain sections was more specific with less non-specific background as compared to control sections that were processed after they were mounted onto gelatin-coated sides. Excellent resolution was also obtained from monkey brain sections that were stored in cryoprotectant for up to 31 months and in human brain sections stored for 12 months.(ABSTRACT TRUNCATED AT 250 WORDS)

Small injections of the lectin Phaseolus vulgaris-leucoagglutinin (PHA-L) in the external segment of the pallidum (GPe) in the squirrel monkey (Saimiri sciureus) and in the rhesus monkey (Macaca mulatta) led to anterograde labeling of fibers in the internal segment of the pallidum (GPi). These fibers formed numerous large varicosities reminiscent of terminal boutons, which closely surrounded GPi cell bodies and primary dendrites. Conversely, PHA-L injections in the GPi of squirrel monkeys produced anterograde fiber labeling in the GPe. However, in contrast to fibers in GPi, those in GPe did not make prominent pericellular contacts. Instead, they displayed a rather linear course, had long intervaricose segments, and appeared to contact several GPe neurons along their course by close appositions on cell bodies and primary dendrites. These results suggest the existence of a reciprocal connection between the two pallidal segments, which may play a crucial role in the functional organization of the basal ganglia in primates.

Twelve weeks after focal ventrolateral cerebrocortical suction lesions (ca. 12 X 4 mm) were made in rats, concentrations of the monoamines norepinephrine (NE), dopamine (DA), and serotonin (5-HT) and their metabolites were measured in several cortical and subcortical brain regions using high performance liquid chromatography with electrochemical detection. Widespread changes in the concentrations of monoamines, their metabolites, and metabolite:monoamine ratios were found in the hemisphere ipsilateral to unilateral (right) lesions, and bilaterally in animals with bilateral lesions. NE was decreased in undamaged dorsolateral cortex and hippocampus, and tended to be increased in striatum and midbrain ipsilateral to lesions. DA was increased in the hypothalamus of bilaterally lesioned animals, and also tended to be increased in striatum and midbrain. The changes of greatest magnitude and anatomical extent were found in the serotonin system: 5-HT was generally increased, and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) and the 5-HIAA:5-HT ratio were decreased throughout the cerebral hemispheres ipsilateral to lesions. These widespread changes in cerebral 5-HT metabolism were qualitatively different and smaller than those previously found at 6 days after cortical lesions, and suggest a biphasic response of the ipsilateral 5-HT system to ventrolateral cortical injury.