Cellular localization of tyrosine hydroxylase mRNA and its regulation in the rat adrenal medulla and brain by in situ hybridization with an oligodeoxyribonucleotide probe

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines in neural tissues and adrenal medulla. To study the expression of the TH gene and its regulation in adult and developing neural tissues, we have synthesized an oligodeoxyribonucleotide probe (oligomer) that is specific for TH mRNA. Using Northern blot hybridization of polyadenylated RNAs from adrenal gland, brain stem, liver, and cerebral cortex with the 32P-labeled oligomer, a single TH mRNA of 1.9 kb was detected in adrenal gland and brain stem but not in liver and cerebral cortex. Using this TH-specific oligomer, TH mRNAs were localized to the chromaffin cells in the adrenal medulla and to catecholaminergic neurons in locus coeruleus and substantia nigra by in situ hybridization histochemistry. After reserpine administration, the intensity of hybridization signal was increased to threefold that of normal in sections of adrenal medulla and twofold that of normal in locus ceruleus. No difference in hybridization signal intensity was observed in the substantia nigra of normal and reserpine-treated animals. Use of this specific TH probe in in situ hybridization procedures represents a powerful approach to the study of regulation of TH gene expression at the cellular level.     

Effect of chronic cold exposure on tyrosine hydroxylase mRNA in rat adrenal gland

The effect of chronic stress on the levels of tyrosine hydroxylase (TH) RNA in rat adrenal gland was investigated by RNA-DNA hybridization using a cloned TH cDNA probe. Results of dot-blot hybridization experiments and northern analysis demonstrate that exposure of animals to cold for 1 week results in a 4-5-fold increase in the relative abundance of TH mRNA. This increase in TH mRNA level may underlie the increase in adrenal TH activity that is known to occur when rats are exposed to such cold stress.     

Different effects of insulin and 2-deoxy- -glucose administration on tyrosine hydroxylase gene expression in the locus coeruleus and the adrenal medulla in rats

The major brain norepinephrinergic nucleus, locus coeruleus, is an important integrating element of extero- and interoceptive stimuli in organisms facing different physiological challenges. We investigated the effects of single and repeated (seven times) exposure to immobilization stress (120 min daily), insulin (5 IU/kg, i.p. daily) or 2-deoxy- -glucose (500 mg/kg, i.p. daily) administration on tyrosine hydroxylase (TH) mRNA levels, the rate-limiting enzyme in catecholamine biosynthesis, by in situ hybridization in locus coeruleus and by Northern blot analysis in the adrenal medulla of rats. Both the single and repeated immobilization caused a significant increase in TH mRNA levels in the locus coeruleus (1.5–2-fold; p < 0.05) and in the adrenal medulla (about 4-fold; p < 0.05) when compared with unstressed controls. Hypoglycemia induced by a single or repeated insulin administration led to about fourfold (p < 0.01) elevation in adrenal medullary TH mRNA levels, whereas TH mRNA in locus coeruleus remained unchanged when compared with saline-treated controls. In contrast to the effect of insulin-induced hypoglycemia, cellular glucoprivation caused by a single or repeated 2-deoxy- -glucose administration significantly elevated TH mRNA levels in both the adrenal medulla (fourfold; p < 0.01) and the locus coeruleus (twofold; p < 0.01). Our data suggest that in contrast to immobilization or cellular glucoprivation caused by 2-deoxy- -glucose administration, insulin-induced hypoglycemia is not a specific or quantitatively sufficient stimulus for induction of TH gene expression in the locus coeruleus, although all these stressors strongly activate the process in the adrenal medulla.     

Chronic arthritis increases tyrosine hydroxylase mRNA levels in the pontine noradrenergic cell groups

In situ hybridization was used to examine the change of tyrosine hydroxylase(TH) mRNA levels in the pontine noradrenergic cell groups of chronic monoarthritic rats induced by adjuvant inoculation. The number of TH mRNA-expressing neurons and grains per labeled neuron in the A5, A6 and A7 cell groups on the ipsilateral and contralateral sides significantly increased 2 weeks after adjuvant inoculation into the left tibio-tarsal joint, compared to controls. These results suggest that noradrenalin in the pontine region may play a role in modulating chronic nociceptive stimuli.     

Effects of stress and adrenalectomy on tyrosine hydroxylase mRNA levels in the locus ceruleus by in situ hybridization

The locus ceruleus-norepinephrine system is one of the principal effectors of the stress response. Acute stress induces norepinephrine synthesis and release, and noradrenergic cells compensate by increasing the activity of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. Here we use in situ hybridization histochemistry to show the effects of acute and chronic intermittent stress on the expression of tyrosine hydroxylase mRNA in the rat locus ceruleus. Restraint stress increased tyrosine hydroxylase mRNA in the locus ceruleus but not in dopaminergic nuclei such as the substantia nigra or ventral tegmental area. One hour of footshock or restraint caused a rapid increase in locus ceruleus tyrosine hydroxylase mRNA which returned to basal levels within 24 h. Chronic intermittent stress (1 hour of restraint or footshock per day for 14 days) produced no change in tyrosine hydroxylase mRNA. Neither adrenalectomy nor dexamethasone replacement significantly affected mRNA expression. These findings indicate that acute stress can increase the expression of tyrosine hydroxylase mRNA in the locus ceruleus but that adaptation occurs to repeated stress, and that the expression of tyrosine hydroxylase mRNA in the locus ceruleus is independent of direct glucocorticoid modulation.     

Different effects of insulin and 2-deoxy-D-glucose administration on tyrosine hydroxylase gene expression in the locus coeruleus and the adrenal medulla in rats

The major brain norepinephrinergic nucleus, locus coeruleus, is an important integrating element of extero- and interoceptive stimuli in organisms facing different physiological challenges. We investigated the effects of single and repeated (seven times) exposure to immobilization stress (120 min daily), insulin (5 IU/kg, i.p. daily) or 2-deoxy-d-glucose (500 mg/kg, i.p. daily) administration on tyrosine hydroxylase (TH) mRNA levels, the rate-limiting enzyme in catecholamine biosynthesis, by in situ hybridization in locus coeruleus and by Northern blot analysis in the adrenal medulla of rats. Both the single and repeated immobilization caused a significant increase in TH mRNA levels in the locus coeruleus (1.5–2-fold; p < 0.05) and in the adrenal medulla (about 4-fold; p < 0.05) when compared with unstressed controls. Hypoglycemia induced by a single or repeated insulin administration led to about fourfold (p < 0.01) elevation in adrenal medullary TH mRNA levels, whereas TH mRNA in locus coeruleus remained unchanged when compared with saline-treated controls. In contrast to the effect of insulin-induced hypoglycemia, cellular glucoprivation caused by a single or repeated 2-deoxy-d-glucose administration significantly elevated TH mRNA levels in both the adrenal medulla (fourfold; p < 0.01) and the locus coeruleus (twofold; p < 0.01). Our data suggest that in contrast to immobilization or cellular glucoprivation caused by 2-deoxy-d-glucose administration, insulin-induced hypoglycemia is not a specific or quantitatively sufficient stimulus for induction of TH gene expression in the locus coeruleus, although all these stressors strongly activate the process in the adrenal medulla.     

Reduced tyrosine hydroxylase and GTP cyclohydrolase mRNA expression, tyrosine hydroxylase activity, and associated neurochemical alterations in Nurr1-null heterozygous mice

The nuclear receptor Nurr1 is essential for the development of midbrain dopamine neurons and appears to be an important regulator of dopamine levels as adult Nurr1-null heterozygous (+/-) mice have reduced mesolimbic/mesocortical dopamine levels. The mechanism(s) through which reduced Nurr1 expression affects dopamine levels has not been determined. Quantitative real-time PCR revealed a significant reduction in tyrosine hydroxylase (TH) and GTP cyclohydrolase (GTPCH) mRNA in ventral midbrain of +/- mice as compared to wild-type mice (+/+). The effect on TH expression was only observed at birth, while reduced GTP cyclohydrolase was also observed in the adult ventral tegemental area. No differences in dopamine transporter, vesicular monoamine transporter, dopamine D2 receptor or aromatic amino acid decarboxylase were observed. Since TH and GTPCH are both involved in dopamine synthesis, regulation of in vivo TH activity was measured in these mice. In vivo TH activity was reduced in nucleus accumbens and striatum of the +/- mice (24.7% and 15.7% reduction, respectively). In the striatum, gamma-butyrolactone exacerbated differences on +/- striatal TH activity (29.8% reduction) while haloperidol equalized TH activity between the +/+ and +/-. TH activity in the nucleus accumbens was significantly reduced in all conditions measured. Furthermore, dopamine levels in the striatum of +/- mice were significantly reduced after inhibition of dopamine synthesis or after haloperidol treatment but not under basal conditions while dopamine levels in the nucleus accumbens were reduced under basal conditions. Based on these data the +/- genotype results in changes in gene expression and impairs dopamine synthesis which can affect the maintenance of dopamine levels, although with differential effects between mesolimbic/mesocortical and nigrostriatal dopamine neurons. Together, these data suggest that Nurr1 may function to modify TH and GTPCH expression and dopamine synthesis.     

Acute effects of reserpine on tryptophan hydroxylase activity and mRNA in rat brain

The present study examined changes in tryptophan hydroxylase (TPH) activity in the rat brainstem 1–4 days after i.p. reserpine administration. TPH enzyme activity was significantly decreased day 1 posttreatment and followed by a small but significant increase on days 2–4. Maximal TPH activity on day 4 was elevated only 42% above control level. This small increase in TPH activity was not preceded by any change of in situ hybridization signal of TPH message on day 1. In contrast, adrenal tyrosine hydroxylase (TH) activity in these same reserpinized animals was 2.5-fold higher than control on 4 day posttreatment. In addition, TH message was correspondingly increased day 1 posttreatment. Taken together, these results indicate a trend of small increase in TPH activity and no alteration in message after reserpine treatment unlike catecholaminergic systems.     

A cholinergic antagonist blocks cold stress-induced alterations in rat adrenal tyrosine hydroxylase mRNA

The role of nicotinic cholinergic transmission in cold stress-induced alterations in rat adrenomedullary tyrosine hydroxylase (TH) mRNA was investigated by RNA dot-blot hybridization, using a cloned TH cDNA probe. Chlorisondamine, a ganglionic blocking agent, greatly attenuated the induction of TH mRNA levels caused by cold exposure, whereas carbachol and nicotine, cholinergic agonists, increased TH mRNA in control animals. These results suggest that cholinergic nicotinic receptors play a key role in the transsynaptic induction of adrenal TH gene expression.     

Saline ingestion stimulates the in situ molar activity of tyrosine hydroxylase in the median eminence and superior cervical ganglion

The effects of drinking saline for 7 days on the mass and in situ activity of tyrosine hydroxylase (TH) in the median eminence (ME) and superior cervical ganglion (SCG) of rats were investigated. TH mass was quantified by immunoblot assay. In situ TH activity was calculated from the rate of intracellular accumulation of L-dihydroxyphenylalanine (DOPA). In rats that drank 10 mM, 30 mM, and 100 mM NaCl for 7 days, TH activity in the ME was 34 +/- 4, 36 +/- 5, and 45 +/- 3 (mean and S.E.M.) mol of DOPA.h-1.mol of TH-1, respectively, compared to 30 +/- 2 for rats that drank water. The activity of TH in the SCG of animals that drank 10 mM, 30 mM, and 100 mM NaCl was 143 +/- 24, 167 +/- 12, and 272 +/- 13 mol DOPA.h-1.mol TH-1, respectively, compared to 119 +/- 10 for animals that drank water. The mass of TH in the ME and SCG decreased as a function of the concentration of NaCl in the drinking water. In animals that drank water, 10 mM, 30 mM, and 100 mM NaCl, the amounts (pmol) of TH were, respectively, 0.28 +/- 0.03, 0.31 +/- 0.04, 0.23 +/- 0.02, and 0.21 +/- 0.01 per ME and 0.67 +/- 0.06, 0.72 +/- 0.11, 0.37 +/- 0.01, and 0.34 +/- 0.02 per SCG. TH activity in the ME or SCG was unaffected by treatment for 7 days with arginine vasopressin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Combination of alkaline phosphatase in situ hybridization with immunohistochemistry: colocalization of calretinin-mRNA with calbindin and tyrosine hydroxylase immunoreactivity in rat substantia nigra neurons

We describe a method to combine non-radioactive in situ hybridization using alkaline phosphatase (AP) labelled oligonucleotide-probes with immunohistochemistry on the same thin paraffin section. The simultaneous detection of calretinin-mRNA and calbindin- or tyrosine hydroxylase-like immunoreactivity in neurons of rat substantia nigra, pars compacta, was used as a test system to develop the method. Brains were fixed by perfusion with 4% paraformaldehyde and embedded in paraffin. Five-μm-thick sections were processed for non-radioactive in situ hybridization with a 33-base alkaline phosphatase conjugated synthetic oligonucleotide complementary to calretinin mRNA. After hybridization and colour reaction to visualize calretinin mRNA, sections were incubated with antibodies against calbindin D_28K or tyrosine hydroxylase. Immunoreaction was visualized using the avidin-biotin-complex-technique and diaminobenzidine. As the colour of both reaction products differ markedly, the distribution of calretinin mRNA-containing neurons (purple-blue, alkaline phosphatase product) and calbindin/tyrosine hydroxylase immunopositive cells (brown peroxidase product) could be differentiated easily on the same section. Calbindin- and tyrosine hydroxylase-like immunoreactivity was found in the majority of calretinin mRNA-containing cells within the substantia nigra, pars compacta, indicating that in this nucleus a proportion of the dopaminergic neurons contain both calcium binding proteins calbindin and calretinin. In conclusion, non-radioactive in situ hybridization using alkaline phosphatase labelled oligonucleotide probes can be readily combined with immunohistochemistry.     

Coexistence of oxytocin and tyrosine hydroxylase in the rat hypothalamus,an immunocytochemical study

Summary Immunocytochemical double labelling was used to determine the structural relationship of oxytocin (OT) and tyrosine hydroxylase (TH) containing perikarya and processes in the rat hypothalamus. Extrahypothalamic TH fibers, as well as parvocellular TH neurons were found to form contacts with OT cells. A fraction of the OT neurons contained TH immunoreactivity. It is likely that in addition to the classical mesencephalic afferences also hypothalamic interneurons and magnocellular dopaminergic neurons control the hypothalamo neurohypophysial system.     

Bradykinin elevates tyrosine hydroxylase and dopamine β-hydroxylase mRNA levels in PC12 cells

Bradykinin is known to rapidly elevate intracellular calcium leading to secretion of neurotransmitters and short term activation of tyrosine hydroxylase (TH). In this study we examined the effect of bradykinin on mRNA levels of two catecholamine biosynthetic enzymes. Treatment of PC12 cells with 1 μM bradykinin for 3 h markedly elevated both TH and dopamine β-hydroxylase (DBH) mRNA levels.     

Immunocytochemical localization of tyrosine hydroxylase in rat adrenal medulla by the peroxidase labeled antibody method: effects of enzyme activation on ultrastructural distribution of the enzyme

A monospecific antibody against tyrosine hydroxylase (TH), purified from a transplantable rat pheochromocytoma, was produced in rabbits. Immunohistochemical techniques were employed in order to determine if a relationship exists between the subcellular distribution of TH and the level of activation of the enzyme in the rat adrenal medulla. Tyrosine hydroxylase activity in adrenals removed from non-stressed rats following pentobarbital anesthesia was found to be 12.9 +/- 1.0 nmol DOPA formed x mg protein-1. The use of ether anesthesia (17.9 +/- 2.0 nmol DOPA formed x mg protein-1), and the administration of electroconvulsive shock (ECS) followed by decapitation (35.9 +/- 2.0 nmol DOPA formed x mg protein-1) was associated with an acute activation of adrenal TH. The subcellular distribution of TH within the cytosol of chromaffin cells from animals subjected to anesthesia or ECS, as determined by immunocytochemical techniques, was similar. In all treatment groups chromaffin cells were found which had TH associated with some chromaffin granules. The percentage of chromaffin granules which appeared to contain TH was lower in animals subjected to ECS plus decapitation as compared with anesthetized animals. These observations suggest that the activation of adrenal medullary TH is not associated with a shift in the subcellular distribution of the enzyme from the cytosol to membranous structures.     

Sensitive non-radioisotopic in situ hybridization histochemistry: demonstration of tyrosine hydroxylase gene expression in rat brain and adrenal

An alkaline phosphatase-labelled anti-sense oligonucleotide probe specific for tyrosine hydroxylase (TOH) mRNA has been used for visualisation of TOH mRNA in the rat brain and adrenal gland. Both ribonuclease pre-treatment and the use of excess non-labelled probe abolished the specific hybridization signal. Furthermore the TOH mRNA-positive signal was only found in cells known from earlier studies to react with anti-TOH antibodies. To determine if the alkaline phosphatase-labelled probe could be used in a semiquantitative manner for measurement of the density of TOH mRNA signal, we used reserpine pre-treatment which induces TOH mRNA expression. The results revealed a significant increase in TOH mRNA signal in locus coeruleus and substantia nigra neurons, and in adrenal medulla chromaffin cells. The increased signal in these areas agreed with the increase in TOH mRNA signal previously observed by Northern analysis and suggests that this type of alkaline phosphatase-labelled probe allows sensitive detection of changes in TOH gene expression.     

So-called interplexiform cells immunoreactive to tyrosine hydroxylase or somatostatin in rat retina

The morphology of so-called interplexiform (IP) cells immunoreactive to tyrosine hydroxylase (TH) or somatostatin (SOM) in the rat retina was described in comparison with those in the carp retina. In frozen cross-sections of the rat retina, many processes of TH-like immunoreactive cells were found to extend toward the outer plexiform layer (OPL), forming a thin layer of network fibers. A few of them further extended into the photoreceptor cell layer; such fibers were never found in the carp retina. Some processes of SOM-like immunoreactive cells in the rat retina were found to travel across the inner nuclear layer and appeared to poorly develop a network at the OPL. In the carp retina, on the other hand, only one exceptional cross-section contained such an ascending process.     

Prolactin inhibits the activity of tyrosine hydroxylase in cultured bovine adrenal chromaffin cells in a dose-dependent manner

Prolactin can modulate the adrenal medulla function, but it has not yet been established whether its action is directly exerted on the adrenal medulla cells. In this work, we have studied the effect of several concentrations of prolactin on the synthesis, storage and release of norepinephrine and epinephrine using cultured bovine adrenal chromaffin cells. In these cells, prolactin inhibited the activity of tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine synthesis, in a dose-dependent manner, from a concentration above 50 ng/ml of prolactin in the incubation medium. Surprisingly, this dose-dependent decrease was not accompanied by changes in the catecholamine release, since the secretion of both norepinephrine and epinephrine as well as the total catecholamine secretion were not significantly altered by the different prolactin concentrations. Moreover, the cellular content of both catecholamines was not altered by prolactin. In summary, these observations allow us to conclude that prolactin exerts a direct inhibitory effect on the tyrosine hydroxylase activity in cultured adrenal chromaffin cells without altering catecholamine release.     

Trans-synaptic increase in RNA coding for tyrosine hydroxylase in a rat sympathetic ganglion

To begin examining molecular mechanisms underlying trans-synaptic regulation, tyrosine hydroxylase (TH) and its messenger RNA (mRNA) were examined in the superior cervical sympathetic ganglion (SCG) of adult rats. Basal levels of TH mRNA were detectable in control ganglia by RNA dot hybridization, using the ³²P nick translated PstI-KpnI restriction fragment of pTH.4 as a probe. Reserpine induced a 3-fold rise in TH activity per μg protein, and a simultaneous 3-fold increase in ganglion TH mRNA. As expected, ganglion decentralization (denervation) prevented the trans-synaptic induction of TH. In addition, decentralization prevented the increase in TH mRNA, suggesting that the increase in message was dependent on trans-synaptic stimulation. Northern blot analysis indicated that the cDNA (complementary DNA) probe hybridized to a single band of approximately 1900 nucleotides, which was the same size in all ganglia. Our observations indicate that induction of TH is associated with a trans-synaptic increase in mRNA coding for the enzyme. Consequently, trans-synaptic increases in impulse activity may induce TH by increasing neuronal levels of TH mRNA in the SCG.