Tyrosine 3-hydroxylase (TH, EC 1.14.16.2) catalyzes the first and rate-limiting step of the catecholamine biosynthetic pathway in the nervous and endocrine systems. The TH locus was disrupted in mouse embryonic stem cells by homologous recombination. Mice heterozygous for the TH mutation were apparently normal. In these mice, TH activity in the embryos and adult tissues was less than 50% of the wild-type values, but the catecholamine level was decreased only moderately in the developing animals and was maintained normally at adulthood, suggesting the presence of a regulatory mechanism for ensuring the proper catecholamine level during animal development. In contrast, the homozygous mutant mice died at a late stage of embryonic development or shortly after birth. Both TH mRNA and enzyme activity were lacking in the homozygous mutants, which thus explained the severe depletion of catecholamines. These changes, however, did not affect gross morphological development of the cells that normally express high catecholamine levels. Analysis of electrocardiograms of surviving newborn mutants showed bradycardia, suggesting an alteration of cardiac functions in the homozygous mice that may lead to the lethality of this mutation. In addition, transfer of a human TH transgene into the homozygous mice corrected the mutant phenotype, showing recovery of TH activity by expression of the human enzyme. These results indicate that TH is essential for survival of the animals during the late gestational development and after birth.Catecholaminergic neurons, which include dopaminergic, noradrenergic, and adrenergic neurons, are located in discrete regions in the central nervous system and have an important role in a wide range of brain functions, such as locomotion, behavior, sleep, memory, and learning. In the peripheral tissues, sympathetic neurons are noradrenergic, and adrenomedullary chromaffin cells produce noradrenaline and adrenaline as hormones. During vertebrate development, catecholaminergic phenotypes are generated from a given neuronal lineage. The primordial catecholaminergic neurons appear in the intermediate zone of the neural tube at the early embryonic stage (reviewed in Ref.

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The coexistence of immunoreactivities for tyrosine hydroxylase (TH) and glutamic acid decarboxylase (GAD) and/or gamma-aminobutyric acid (GABA) was revealed in various brain regions in colchicine-injected and untreated rats, using the peroxidase-antiperoxidase method. Consecutive 40 micron thick Vibratome sections were incubated in different antisera and those cells which were bisected by the plane of sectioning so as to be included at the paired surfaces of two adjacent sections were identified. The coexistence of the immunoreactivities for TH and GAD or GABA in the same cell could thus be determined by observing the immunoreactivity of the two halves of the cell incubated in two different antisera. In the olfactory bulb, retina, diencephalon, mesencephalic central grey and cerebral cortex, many TH-like immunoreactive neurons also showed GAD-like or GABA-like immunoreactivity, whereas in the substantia nigra, ventral tegmental area and locus ceruleus none of TH-like immunoreactive neurons showed either GAD-like or GABA-like immunoreactivity. In the olfactory bulb, retina and cerebral cortex, the majority of the TH-like immunoreactive neurons were also GAD-like or GABA-like immunoreactive. In the diencephalon of colchicine-injected rats, at least one-third of the TH-like immunoreactive neurons were GAD-like immunoreactive. Using serial 0.5 micron thick plastic-embedded sections, it was shown that immunoreactivities for three antigens, GAD, GABA and TH could occur in the same neurons in the olfactory bulb. These observations indicate the possible coexistence of two classical transmitters. GABA and catecholamine, in various brain regions of the rat.