Modulation of tyrosine hydroxylase gene expression in the central nervous system visualized by in situ hybridization.

Abstract

A rat tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] cDNA probe was used for in situ hybridization studies on histological sections through the locus coeruleus, substantia nigra, and the ventral tegmental area of the rat brain. Experimental conditions were established that yielded no background and no signal when pBR322 was used as a control probe. Using the tyrosine hydroxylase probe, we ascertained the specificity of the labeling over catecholaminergic cells by denervation experiments and comparison of the hybridization pattern with that of immunoreactivity.The use of35S-labeled probe enabled the hybridization signal to be resolved at the cellular level. A single injection of reserpine into the rat led to an increase of the intensity of the autoradiographic signal over the locus coeruleus area, confirming an RNA gel blot analysis. The potential of in situ hybridization to analyze patterns of modulation of gene activity as a result of nervous activity is discussed.Catecholaminergic cells of the central nervous system are found in small nuclei, mostly in the brain stem and mesencephalon (1, 2). In spite of their paucity (probably <5 x 104 cells) these neurons send projections to nearly every region of the brain and play a crucial role in its function. Tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2], the rate-limiting enzyme in catecholamine synthesis, has been intensively investigated, and its expression has been found to be controlled by a variety of external factors, both in the developing and adult organism (3, 4). For instance, prolonged stimulation of TyrOHase activity has been observed following increased neuronal activity (5, 6). These long-term changes result from a rise in TyrOHase mRNA levels, most probably due to an increase in TyrOHase gene transcription (7,8).In the last decade, the use of TyrOHase antibodies has been instrumental in revealing the organization ofvarious adrenergic, noradrenergic, and dopaminergic cell clusters and pathways in the brain. In the near future, it can confidently be predicted that the histochemical approach will greatly benefit from the availability of cloned cDNA probes. We report here the direct visualization of gene activity and its modulation on rat brain sections at the level ofthe locus coeruleus, the ventral tegmental area, and the substantia nigra. The complementarity of this approach with immunohistochemistry to analyze brain function and plasticity is discussed.MATERIALS AND METHODS Preparation of Probes. The construction and characterization of the recombinant pTH51 clone in plasmid pBR322 have been described (9, 10). The 1758-base-pair insert containing the complete coding and 3'-untranslated sequences was purified by electroelution after agarose gel electrophoresis and labeled by nick-translation (11) with 35S-substituted adenosine and cytidine 5'-[a-thio]-tr...

Differential Role of Galanin Receptors in the Regulation of Depression-Like Behavior and Monoamine/Stress-Related Genes at the Cell Body Level

“…The stressful event (forced swimming) combined with repeated i.p. injections resulted in increased activity of the LC NA system, evidenced by an increase of TH and galanin mRNA levels in the LC, in agreement with previous findings (Biguet et al, 1986;Berod et al, 1987;Richard et al, 1988;Austin et al, 1990;Holmes et al, 1995). Stress-induced hyperactivity of the LC neurons and increased release of NA have been proposed to contribute to development of human depression (Nestler et al, 1990; see Mongeau et al, 1997;Grant and Weiss, 2001;Morilak and Frazer, 2004).…”

Growth Hormone-Releasing Hormone-Synthesizing Neurons Are a Subpopulation of Somatostatin Receptor-Labelled Cells in the Rat Arcuate Nucleus: A Combined in situ Hybridization and Receptor Light-Microscopic Radioautographic Study

“…GHRH in situ hybridization was carried out as previously de scribed [3]. Hybridization was performed on the frozen sections ad jacent to that used for l25I-SR!H binding.…”

Coexistence and gene expression of phenylethanolamine N-methyltransferase, tyrosine hydroxylase, and neuropeptide tyrosine in the rat and bovine adrenal gland: effects of reserpine.

“…6) have been cloned and, by using in situ hybridization, the localization of PNMTase mRNA has been studied in the adrenal gland and the brain (6). Also TyrOHase mRNA has been studied with in situ hybridization (7)(8)(9). Neuropeptide tyrosine (NPY) (10) coexists with epinephrine and norepinephrine in the rat adrenal medulla, and its expression as well as that of TyrOHase is known to be increased in stress and after injection of the catecholamine-depleting drug reserpine (for references, see ref.…”