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The rate of DNA synthesis by human lymphocytes was studied in vitro by measuring unstimulated thymidine-2-14C incorporation (spontaneous lymphocyte blastogenesis; SLB). Freezing lymphocytes and extracting DNA after thawing did not alter the radioactive label count rate and was as efficient as extracting DNA immediately after culture. Omission of fetal calf serum also did not alter the rate of DNA synthesis. Standards established as optimal for studies of SLB were: cell concentration, 1.0 times 10(6)/ml/tube; 14C-TdR concentration, 0.4 mjCi/tube; duration of incubation, 8 hr. In sets of identical samples obtained by specimen division, the variation in counts was 6%. To achieve reproducibility of results; it was essential to count the lymphocytes, and then to ensure that each tube contained almost precisely known numbers of cells. Diurnal variations in the rate of DNA synthesis by circulating lymphocytes of healthy men were measured in vitro by SLB at 2-hr intervals for 24 hr. Leukocyte counts, hematocrit, hemoglobin, plasma cortisol, and body temperature were monitored concurrently. The DNA synthesis rate varied in a 24-hr cycle with peaks at 10 A.M. and 11:00 P.M.., depressions at 4 A.M. and 4 P.M. The rate was correlated with body temperature and hematocrit level, and inversely related to the absolute eosinophil count.

The locus ceruleus (LC) of the rhesus monkey (Macaca mulatta) was investigated using the histochemical fluorescence method and Nissl and ammoniacal silver stains. Caudally, a few fluorescent cells were observed in the lateral wall of the ventriculus quartus near the velum medullaris superior. Rostrally, the fluorescent cells were compactly clustered and reached their greatest density medial and, to a lesser extent, lateral to the tractus mesencephalicus n. trigemini at the level of the decussation of the nervus trochlearis. In the most rostral plane, fluorescent cells were more diffusely scattered ventral and medial to the pedunculus cerebellaris superior with a few cells situated near the nervus trochlearis. Nissl staining of tissue previously used for histochemical fluorescence showed that fluorescent cells were largely found in a region labelled LC in two rhesus monkey brain atlases and erroneously labelled nucleus tractus mesencephalicus n. trigemini in one brain atlas. Ammoniacal silver staining resulted in a dense accumulation of silver granules in the cells found to display a positive reaction for monoamines. The silver stains therefore offer an alternative to the histochemical fluorescence method for identifying the monoamine-containing LC neurons.

Self-stimulation performance of rats was tested with conditioning pulses to the anterior preoptic area of the medial forebrain bundle followed at various intervals by test pulses to the contralateral posterior hypothalamic area of this bundle. Alternatively, conditioning pulses were delivered through the posterior electrode and test pulses were sent through the anterior electrode. The animals' performance in these two test sequences was indicative of (i) synaptic facilitation and (ii) a posterior convergence site of "self-stimulation impulses" in the medial forebrain bundle.