Detection Of Tyrosine Hydroxylase Research Articles

Neurotensin terminals form synapses primarily with neurons lacking detectable tyrosine hydroxylase immunoreactivity in the rat substantia nigra and ventral tegmental area.

A light and electron microscopic double antigen localization technique was employed to examine the fine structural relationship between neurotensin-containing axon terminals and dopaminergic neurons in the substantia nigra and ventral tegmental area of the rat. At the light microscopic level, neurotensin-immunoreactive terminals were densely distributed throughout the substantia nigra pars compacta and ventral tegmental area in close proximity to tyrosine hydroxylase-immunoreactive somata and dendrites. On electron microscopic examination, direct synaptic connections were identified between neurotensin-immunoreactive axon terminals and tyrosine hydroxylase-immunopositive perikarya and dendrites. However, only 8.2% and 8.8% of the neurotensin-immunoreactive axonal profiles detected in the substantia nigra and ventral tegmental area, respectively, were found in direct apposition with tyrosine hydroxylase-immunostained elements. In turn, only 9.3% and 10.0% of tyrosine hydroxylase immunoreactive dendrites sampled from the substantia nigra and ventral tegmental area, respectively, were seen in contact with neurotensin immunopositive axon terminals. However, neurotensin-immunoreactive and tyrosine hydroxylase-immunolabelled elements were frequently identified in close anatomical proximity (less than 5 microns) to one another. These results are interpreted in light of the selective association of neurotensin receptors with dopaminergic neurons in the substantia nigra and ventral tegmental area to suggest a predominantly parasynaptic mechanism of action for neurotensin in the ventral midbrain.

Detection of tyrosine hydroxylase and phenylethanolamine-N-methyltransferase messenger RNAs in the mouse adrenal gland and the brain by in situ hybridization.

To study the expression of tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) genes in the mouse adrenal gland and the brain, we performed in situ hybridization studies by using several types of complementary DNA probes recognizing coding regions of human TH (THc), the 3'-end of the human region of TH (TH3'), and the coding region of the human PNMT (PNMTc). THc mRNA was detected in the chromaffin cells of the mouse adrenal medulla and brain catecholaminergic neurons including the substantia nigra and ventral tegmental area. TH-immunopositive neurons were located in a similar pattern in adjacent sections. However no positive signals were detected by the TH3' probe. Using the PNMTc probe, the majority of cells in the adrenal medulla demonstrated positive labelling. Although the mouse TH and PNMT genes have not been fully isolated and sequenced, the present study strongly suggests that the sequence of the coding regions of TH and PNMT are similar in human and mouse. The THc and PNMTc probes are particularly useful in investigating the loci of gene transcription in mouse tissues.

Quantitative Autoradiography of Tyrosine Hydroxylase Immunoreactivity in the Rat Brain

Levels of tyrosine hydroxylase (TH) were quantified in discrete areas of unfixed rat brain tissue sections using a rapid and sensitive radioimmunohistochemical method. The immunological reaction with the TH monoclonal antibody was revealed by a 35S-labelled secondary antibody and thus permitted autoradiographic detection of the enzyme. Autoradiograms were generated by apposition of tissue sections to high-sensitivity films or by dipping into autoradiographic emulsion. A detailed analysis of antibody concentration, incubation time, tissue section thickness, and exposure time of the film was undertaken to determine optimal conditions to produce a linear radiolabelling intensity with respect to the amount of antigen. Quantification of the antigen at regional levels was assessed by computer-assisted image analysis. Autoradiographic optical density of radiolabelling in brain areas was converted to enzyme concentrations by interpolation with a constructed TH calibration curve processed in parallel with tissue sections. The specificity of the labelling and the validity and reproducibility of the quantification were investigated. The distribution of TH radiolabelling was comparable to that described using immunofluorescence histochemistry or measuring TH enzymatic activity on homogenates. Using a 35S-labelled antibody, the detection of TH could be performed at the cellular level.

Association of iron‐containing astrocytes with dopaminergic neurons of the arcuate nucleus

Specialized astrocytes, identified by cytoplasmic granules that are electron-dense and vividly stained by toluidine blue due to the presence in the granules of SH molecules and molecules of iron, have long been known to be present within the arcuate nucleus of the hypothalamus. Their function, however, is obscure. To determine whether or not these specialized astrocytes are in contact with dopaminergic neurons, rat brain sections were stained to detect tyrosine hydroxylase (TH) immunoreactive neurons by immunocytochemistry and were examined by both light and electron microscopy. Iron-rich astrocytes were located in the same general portion of the arcuate nucleus as were TH+ neurons, and most appeared closely associated with TH+ structures (somas, dendrites, and fibers) at the light-microscopic level. At the ultrastructural level, close contact between TH + neurons and processes of iron-rich glia was confirmed. This unique anatomical association suggests a functional relationship between the two cell types that may be related to unusual histochemical features of both cell types and/or to the location of these cells in an area with a highly permeable blood-brain barrier.

Neuropeptide Y-like immunoreactivity in rat cranial parasympathetic neurons: coexistence with vasoactive intestinal peptide and choline acetyltransferase.

Neuropeptide Y (NPY) is widely distributed in the sympathetic nervous system, where it is colocalized with norepinephrine. We report here that NPY-immunoreactive neurons are also abundant in three cranial parasympathetic ganglia, the otic, sphenopalatine, and ciliary, in the rat. High-performance liquid chromatographic analysis of the immunoreactive material present in the otic ganglion indicates that this material is very similar to porcine NPY and indistinguishable from the NPY-like immunoreactivity present in rat sympathetic neurons. These findings raise the possibility that NPY acts as a neuromodulator in the parasympathetic as well as the sympathetic nervous system. In contrast to what has been observed for sympathetic neurons, NPY-immunoreactive neurons in cranial parasympathetic ganglia do not contain detectable catecholamines or tyrosine hydroxylase (EC 1.14.16.2) immunoreactivity, and many do contain immunoreactivity for vasoactive intestinal peptide and/or choline acetyltransferase (EC 2.3.1.6). These findings suggest that there is no simple rule governing coexpression of NPY with norepinephrine, acetylcholine, or vasoactive intestinal peptide in autonomic neurons. Further, while functional studies have indicated that NPY exerts actions on the peripheral vasculature which are antagonistic to those of acetylcholine and vasoactive intestinal peptide, the present results raise the possibility that these three substances may have complementary effects on other target tissues.

Testosterone inhibition of tyrosine hydroxylase expression in the hypothalamic arcuate nucleus

Immunohistochemistry was used to detect tyrosine hydroxylase (TH)-containing neurons in the hypothalamic arcuate nucleus in male rats. Two weeks following castration, the number of TH-positive cells was significantly greater than in intact controls. The castration-responsive TH-positive cells were uniformly distributed throughout the mediolateral extent of the arcuate nucleus. Treatment with testosterone significantly suppressed the castration response, whereas neither estradiol nor 5α-dihydrotestosterone were effective. The number of TH-positive arcuate neurons in the female was similar to that in the male. Ovariectomy did not affect the number of TH-positive neurons. These findings indicate that TH expression is tonically inhibited in the tuberoinfundibular dopaminergic system of the male rat by testosterone.

Absence of tyrosine hydroxylase activity and dopamine β-hydroxylase immunoreactivity in intrinsic nerves of the guinea-pig ileum

The activity of tyrosine hydroxylase and the localization of dopamine β-hydroxylase were determined in the myenteric and submucous plexuses of the normal guinea-pig ileum and in these plexuses after extrinsic denervation. In the normal ileum, the distribution of axons showing immunoreactivity for dopamine β-hydroxylase was not distinguishable from the distribution of noradrenergic axons determined by the fluorescence histochemical localization of catecholamines. The distribution of tyrosine hydroxylase in the different layers of the intestine correlated well with the distribution of dopamine β-hydroxylase and noradrenaline, tyrosine hydroxylase activity being most concentrated in the myenteric and submucous plexuses. Extrinsic denervation resulted in the complete disappearance of both biochemically detectable tyrosine hydroxylase and immunohistochemically demonstrable dopamine β-hydroxylase. It is concluded that if the amine-handling neurons which are known to be intrinsic to the intestine synthesize an aromatic amine, it is almost certainly not a catecholamine, and is probably an indoleamine.