Biosynthesis of catecholamines in organotypic cultures of peripheral autonomic nervous system: modifications by biopterin and other agents.

Organotypic cultures of chick-embryo sympathetic ganglion chains maintained in vitro for 3-4 weeks rapidly synthesized catecholamines, as demonstrated by the conversion of L-[U-14C]tyrosine to catechol derivatives and by histofluorescence assay. The biosynthesis of catechols from radioactive L-tyrosine leveled off at 6 hr of incubation and dropped slightly at 10 hr. The addition of DL-alpha-methyl-p-tyrosine to the culture medium did not affect protein synthesis, but produced a complete block in the synthesis of catecholamines from L-tyrosine, with consequent loss of fluorescence in the bodies and proximal processes of adrenergic neurons in 2 hr, and essentially complete loss in 6 hr. Our observations suggest that a major portion of the catecholamines were synthesized in the perikarya and transported via neuronal processes to their terminals. The addition of monoamine oxidase inhibitors to the incubation medium produced a moderate to pronounced increase in fluorescence; reserpine caused a rapid and profound loss of catecholamines. When added to the culture medium, crude biopterin produced an increase in the synthesis of catechol derivatives from radioactive L-tyrosine and a marked increase in fluorescence, beginning in the neuronal perikarya. This effect was completely blocked by DL-alpha-methyl-p-tyrosine. The mechanism of biopterin's action in the synthesis of catecholamines in cultures of sympathetic ganglia is not completely elucidated from these studies, but may be related to the role it plays as cofactor for tyrosine hydrocylase.

Origin, development, and nature of intranuclear rodlets and associated bodies in chicken sympathetic neurons.

Correlative data are presented here on the developmental history, dynamics, histochemistry, and fine structure of intranuclear rodlets in chicken sympathetic neurons from in vivo material and long-term organized tissue cultures. The rodlets consist of bundles of approximately 70 +/- 10 A proteinaceous filaments closely associated with approximately 0.4-0.8 micro spheroidal, granulofibrillar (gf) bodies of a related nature. These bodies are already present in the developing embryo a week or more in advance of the rodlets. In early formative stages rodlets consist of small clusters of aligned filaments contiguous with the gf-bodies. As neuronal differentiation progresses these filaments increase in number and become organized into well-ordered polyhedral arrays. Time-lapse cinemicrography reveals transient changes in rodlet contour associated with intrinsic factors, changes in form and position of the nucleolus with respect to the rodlet, and activity of the gf-bodies. With the electron microscope filaments may be seen extending between the nucleolus, gf-bodies, and rodlets; nucleoli display circumscribed regions with fine structural features and staining reactions reminiscent of those of gf-bodies, We suggest that the latter may be derivatives of the nucleolus and that the two may act together in the assemblage and functional dynamics of the rodlet. The egress of rodlet filaments into the cytoplasm raises the possibility that these might represent a source of the cell's filamentous constituents.

Deuterium oxide: direct action on sympathetic ganglia isolated in culture.

Immature ganglia from chicks and rodents were maintained as organized, developing cultures for 2 months or more, during which time they were continuously exposed to deuterium oxide in their medium. Observations of the living cell communities with the light microscope indicated that deuteration within viable limits (up to 25 percent) accelerates and increases the growth of sympathetic neurons and favors their repeated subdivision as a very large size is attained, thus inducing them to recapitulate cyclically the early stages of neurogenesis. Living deuterated cells appear more opaque and heteromerous than control neurons; furthermore, electron micrographs reveal an unusual abundance of granular and fibrillar elements in the nuclei of both neurons and supporting cells. Sheaves of complexly organized fibrillar components appear in the neuronal perikaryon; and ribosomes, Golgi elements, and microtubules are conspicuously numerous. Both fine structure and function of these ganglia therefore appear to have been modified directly by action of the deuterium isotope.