A CNS catecholaminergic cell line, Cath.a, was established by targeted oncogenesis in transgenic mice. Cath.a cells express neuronal properties but lack neuronal morphology. Here, we describe a variant of Cath.a, called CAD (Cath.a-differentiated), in which reversible morphological differentiation can be initiated by removal of serum or exogenously added protein from the medium. In serum- or protein-free media, CAD cells stop proliferating and extend long processes. Differentiated CAD cells can be maintained without serum or protein for at least 6 weeks. CAD cells are distinct from Cath.a cells; most significant, the original immortalizing oncogene, SV40 T antigen, was spontaneously lost. By immunostaining or immunoblotting, we show that CAD cells express neuron-specific proteins, such as class III beta-tubulin, GAP-43, SNAP-25, and synaptotagmin, but not GFAP. Ultrastructurally, processes from differentiated CAD cells have abundant parallel microtubules and intermediate filaments, and bear varicosities that contain both large dense-core vesicles/granules (120-160 nm) and smaller clear vesicles (60-80 nm). Additionally, CAD cells express enzymatically active tyrosine hydroxylase and accumulate L-DOPA. CAD cells exhibit biochemical and morphological characteristics of primary neurons and provide an unique tool for studying neuronal differentiation.

Development of a culture system for mammalian olfactory epithelium has permitted the process of neuro genesis to be examined in vitro. Antibody markers allowing the unambiguous identification of putative neuroepitheiiil stem cells (keratin+ basal cells) and differentiated neurons (N-CAM+ olfactory receptor neurons) are described. In combination with t3H]thymidine uptake analysis, these antibodies have been used to characterize the existence, proliferation, and differentiation of the immediate neuronal precursor in this system. This cell is distinct from basal cells and rapidly sorts out from them, dividing as it migrates. Data are presented which suggest that the precursor follows a simple lineage program, dividing to give rise to two N-CAM+ daughter neurons. Although this precursor efficiently generates neurons in defined medium, neurogenesis subsequently ceases because new precursors are not produced, suggesting that epigenetic factors may regulate continual nemogenesis in this system.

Glucocorticoid and cyclic AMP increase tyrosine hydroxylase (TH) activity and mRNA levels in pheochromocytoma cultures. The transcriptional activity of the TH gene, as measured by nuclear run-on assay, is also increased when cultures are treated with the synthetic glucocorticoid dexamethasone or agents that increase intracellular cyclic AMP, such as forskolin and 8-BrcAMP. Both inducers effect transcriptional changes within 10 min after treatment and are maximal after 30 min for forskolin and after 60 min for dexamethasone. The 5' flanking sequences of the TH gene were fused to the bacterial gene chloramphenicol acetyltransferase (CAT), and the hybrid gene was transfected into pheochromocytoma cultures and GH4 pituitary cells. In both cell lines, a region of the TH gene containing bases -272 to +27 conferred induction of CAT by cyclic AMP, but not by glucocorticoid. The same results were found when a region of the TH gene containing -773 to +27 was used. Thus, the sequences required for induction of TH by cyclic AMP are contained within 272 bases of 5' flanking sequence, but sequences sufficient for glucocorticoid regulation are not contained within 773 bases.The rate of biosynthesis of specific neurotransmitters can be modulated by a variety of environmental, neuronal, and hormonal stimuli. In catecholamine biosynthesis, the rate of flow through the pathway is largely dependent on the activity of the initial enzyme, tyrosine hydroxylase (TH), which is expressed in the adrenal medulla, sympathetic ganglia, and certain defined nuclei of the brain. Activity of tyrosine hydroxylase can be modulated both by changes in the synthetic rate of new tyrosine hydroxylase polypeptide and by posttranslational modification of preexisting enzyme molecules.The synthesis of TH is influenced by a variety of factors; in vivo, TH is induced in response to environmental stimuli, such as stress (1), and this induction can be mimicked pharmacologically by treatment of animals with agents that deplete cellular catecholamine stores, such as reserpine (2). In cultures of adrenal chromaffin or pheochromocytoma cells, tyrosine hydroxylase activity is induced by a number of effectors, including glucocorticoid (3, 4), cyclic AMP (5, 6), epidermal growth factor (7), and nerve growth factor (6,8,9).Whether these effectors act through similar mechanisms and whether changes are modulated transcriptionally or posttranscriptionally is not known. Using a cloned cDNA probe for TH, we have previously shown increases in the mRNA for tyrosine hydroxylase following treatment of clonal rat pheochromocytoma cells from an adrenal medullary tumor with analogs of cyclic AMP and glucocorticoid (10). The TH RNA is also increased in adrenal glands and superior cervical ganglia in vivo when animals are subjected to cold stress or reserpine treatment (11-14). In this communication, we have extended those studies by demonstrating that both glucocorticoid and cyclic AMP stimulate the transcriptional activity of the TH gene. In addition, we report that the 5...