Constancy of adult hypothalamic tyrosine hydroxylase after gonadal steroid treatment during development

Tyrosine hydroxylase (TH) was studied in the entire hypothalamus of 60-day-old male and female rats, and its activity was similar in both sexes. Administration of estrogen or androgen to male or female pups soon after birth and during development did not affect the kinetic parameters of TH. It is, thus, suggested that sexual dimorphism in the levels and turnover of catecholamines (CA) and their sensitivity to fluctuations in gonadal steroids during development do not depend on corresponding sex-related or steroid-sensitive differences in the synthesis of hypothalamic CA.     

Time-dependent effects of ovarian steroids on tyrosine hydroxylase activity in the limbic forebrain of female rats

Summary In this work, we have studied the time-course of the effects of pharmacological administration of ovarian steroids on tyrosine hydroxylase (TH) activity in the limbic forebrain of ovariectomized rats. Administration of estradiol produced a late decrease in TH activity. This effect was found 24 hours after the last steroid injection, disappearing at 32 hours. It was antagonized by progesterone, since a single injection of this steroid to estradiol-pretreated rats reversed to control values the estradiol-induced decrease. Nevertheless, the administration of progesterone after estradiol treatment caused a short-time decrease in the limbic activity of TH, which was observed 4 hours after the last steroid injection, disappearing subsequently. On the other hand, the administration of progesterone alone produced a biphasic effect, with a reduction at 24 hours, followed by an increase at 32 hours. These effects were only observed in the animals non-treated with estradiol, disappearing with a previous treatment with estrogens. Hence, it can be concluded that both ovarian steroids may affect the limbic TH activity. Thus, estradiol produced a late inhibitory effect on the activity of this enzyme, which was antagonized by progesterone. Administration of the last one to estradiol-treated rats produced a short-time inhibitory effect, whereas its administration to non-treated rats produced a late biphasic effect (inhibition followed by stimulation), which was not observed in estradiol-treated rats.     

Reserpine induction of tyrosine hydroxylase in paraplegia

Reserpine induction of tyrosine hydroxylase (TH) activity reflects the biochemical adaptability of sympathetic neurons. Midthoracic spinal cord transection in adult animals precludes TH induction in the sixth lumbar (L6) ganglion, a ganglion innervated by spinal segments caudal to the lesion. However, in animals receiving lesions as neonates, an elevation in L6 ganglion TH activity occurred after reserpine. This retained biochemical adaptability indicates an increased recuperative capacity of developing neurons.     

Adrenal influence on tyrosine hydroxylase activity in superior cervical ganglion

The involvement of adrenal hormones as regulatory factors in maintaining physiological levels of tyrosine hydroxylase (TH) was examined in mouse superior cervical ganglion. Following bilateral adrenalectomy, TH activity in the ganglion fell at a slow but steady rate, reaching 60-65% of the control levels after 2 weeks. Decentralization is known also to reduce TH activity in the ganglion. The effects of adrenalectomy and decentralization were therefore compared, and they were found to be additive, indicating different mechanisms in the two cases. The reduction of TH activity following adrenalectomy was not prevented by replacement with corticosterone (0.5 mg/kg, daily). However, replacement with epinephrine (4 mg/kg, daily) completely prevented the fall of TH activity in adrenalectomized animals. Isoproterenol, a beta-adrenergic receptor agonist, was as effective as epinephrine in preventing the reduction of TH activity following adrenalectomy. Furthermore, in intact animals, chronic administration of SKF 64139, an inhibitor of adrenal PNMT which depletes circulating epinephrine levels, also reduced ganglionic TH activity to the same level as that after adrenalectomy. These results indicate that epinephrine, but not corticosterone, is the adrenal factor required for physiological maintenance of normal levels of TH in the superior cervical ganglion.     

Monoclonal antibodies to tyrosine hydroxylase: production and characterization

Monoclonal antibodies were produced against tyrosine hydroxylase (TH), the enzyme catalyzing the rate limiting step in catecholamine neurotransmitter synthesis. An enzyme linked immunosorbant assay was used to detect the presence of specific antibody in culture. Specificity of the antibodies is characterized. These monospecific antibodies were used for immunoprecipitation and immunohistochemical staining of TH in brain.     

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.     

Postnatal development of tyrosine hydroxylase mRNA-expressing neurons in mouse neostriatum

The striatum harbors a small number of tyrosine hydroxylase (TH) mRNA‐containing GABAergic neurons that express TH immunoreactivity after dopamine depletion, some of which reportedly resembled striatal medium spiny projection neurons (MSNs). To clarify whether the TH mRNA‐expressing neurons were a subset of MSNs, we characterized their postnatal development of electrophysiological and morphological properties using a transgenic mouse strain expressing enhanced green fluorescent protein (EGFP) under the control of the rat TH gene promoter. At postnatal day (P)1, EGFP‐TH⁺ neurons were present as clusters in the striatum and, thereafter, gradually scattered ventromedially by P18 without regard to the striatal compartments. They were immunonegative for calbindin, but immunopositive for enkephalin (54.5%) and dynorphin (80.0%). Whole‐cell patch‐clamp recordings revealed at least two distinct neuronal types, termed EGFP‐TH⁺ Type A and B. Whereas Type B neurons were aspiny and negative for the MSN marker dopamine‐ and cyclic AMP‐regulated phosphoprotein of 32 kDa (DARPP‐32), Type A neurons constituted 75% of the EGFP⁺ cells, had dendritic spines (24.6%), contained DARPP‐32 (73.6%) and a proportion acquired TH immunoreactivity after injections of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine and 3‐nitropropionic acid. The membrane properties and N‐methyl‐d ‐aspartate : non‐N‐methyl‐d ‐aspartate excitatory postsynaptic current ratio of Type A neurons were very similar to MSNs at P18. However, their resting membrane potentials and spike widths were statistically different from those of MSNs. In addition, the calbindin‐like, DARPP‐32‐like and dynorphin B‐like immunoreactivity of Type A neurons developed differently from that of MSNs in the matrix. Thus, Type A neurons closely resemble MSNs, but constitute a cell type distinct from classical MSNs.     

Δ9-tetrahydrocannabinol increases activity of tyrosine hydroxylase in cultured fetal mesencephalic neurons

The exposure of pregnant rats to Δ⁹-tetrahydrocannabinol (Δ⁹-THC), the main psychoactive constituent ofCannabis sativa, during gestation and lactation, affects the gene expression and the activity of tyrosine hydroxylase (TH) in the brain of their offspring, measured at fetal and early postnatal ages, when the expression of this enzyme plays an important role in neural development. In the present article, we have examined whether Δ⁹-THC is able to affect TH activity in cultured mesencephalic neurons obtained from fetuses at gestational d 14. Thus, TH activity increased approximately twofold in cells obtained from naive fetuses when exposed for 24 h to medium containing Δ⁹-THC. In addition, TH activity was also approx twofold higher in cells obtained from fetuses exposed daily to Δ⁹-THC from d 5 of gestation than in cells obtained from control fetuses, when both were exposed to basal media. This effect of Δ⁹-THC on TH activity seems to be produced via the activation of cannabinoid receptors, in particular the CB₁ subtype, which would presumably be located in these cells. This is because the exposure to medium containing both Δ⁹-THC and SR141716A, a specific antagonist for CB₁ receptors, abolished the effect observed with Δ⁹-THC alone. SR141716A alone was without effect on TH activity. Collectively, our results support the notion that Δ⁹-THC increased TH activity in cultured mesencephalic neurons, as previously observed in vivo, and that this effect was produced by activation of CB₁ receptors, which seem to be operative at these early ages. All this points to a role for the endogenous cannabimimetic system in brain development. Part of this work has been previously presented in abstract form at the 24th Meeting of the Federation of European Biochemical Societies, Barcelona, Spain, July, 1996.     

Reduced tyrosine hydroxylase activity in nigrostriatal system of sinoaortic-denervated rats

Tyrosine hydroxylase activity was measured in the caudate nucleus and substantia nigra (pars compacta) of sinoaortic-denervated (SAD) and sham-operated (SO) rats 3 days after operation. The SAD groups had approximately a 50%, statistically significant, reduction of enzyme activity in both regions and also a significant reduction of dopamine in caudate nucleus. These results suggest a probable link between cortico-somatomotor integration and neural input from arterial baroreceptor neural pathways.     

In vitro effects of catecholamines and catecholestrogens on brain tyrosine hydroxylase activity and kinetics in the female catfish Heteropneustes fossilis

Effects of catecholamines and catecholestrogens on tyrosine hydroxylase (TH) activity and kinetics were investigated in the telencephalon and hypothalamus of female Heteropneustes fossilis in gonad quiescent (resting) and recrudescent (preparatory) phases. Dopamine, noradrenaline and adrenaline and the catecholestrogen, 2-hydroxyestradiol-17 beta inhibited TH activity in a concentration-dependent manner in both resting and preparatory phases, with a higher effect in the resting phase. Two- methoxyestradiol-17 beta did not alter TH activity in any season. The catecholamines inhibited TH in a competitive manner increasing apparent K(m) values significantly without altering the apparent V(max). Two-hydroxyestradiol-17 beta inhibited significantly the enzyme in a noncompetitive manner and decreased apparent V(max) without altering apparent K(m) values. The apparent K(i) is higher for dopamine than noradrenaline or adrenaline. The apparent K(i) for 2-hydroxyestradiol-17 beta is not significantly different from that of noradrenaline. The present results suggest an interaction between oestradiol-17beta (E2) and catecholamine metabolism at the level of tyrosine hydroxylation and E2 effects on catecholamines may be mediated through its 2-hydroxylation.     

Castration Reduces the Nocturnal Rise of Pineal Melatonin Levels in the Male Rat by Impairing its Noradrenergic Input

The effects of castration and testosterone treatment on pineal day-night rhythms were studied in male rats. Bilateral gonadectomy was performed at 21 days of age. Testosterone propionate was given subcutaneously to castrated animals in a dose of 10 μg/100 g body weight during two consecutive days before sacrifice. Animals were killed 40 days after gonadectomy at four different times of a 12:12 h light-dark cycle (1600, 2400, 0400 and 0800h). Tyrosine hydroxylase activity was measured in individual pineals by means of high-performance liquid chromatography determination of L-DOPA formed. Pineal levels of norepinephrine, dopamine, 5-hydroxytryptamine and 5-hydroxyindole acetic acid were determined by high-performance liquid chromatography with amperometric detection, while pineal melatonin content was measured by radioimmunoassay. Castration abolished the day-night rhythms of pineal tyrosine hydroxylase activity and norepinephrine content, both by elevating their daytime levels and by blocking their nocturnal rise. In addition, gonadectomy drastically modified pineal indoleamine metabolism by increasing daytime levels of both 5-hydroxytryptamine and 5-hydroxyindole acetic acid, and by reducing the nocturnal elevation of pineal melatonin content. Testosterone treatment was unable to prevent the effect of orchidectomy on pineal rhythms of tyrosine hydroxylase activity, 5-hydroxytryptamine or 5-hydroxyindole acetic acid content, however it partially restored the day-night pineal rhythms of both norepinephrine and melatonin content. These results are indicative of a possible participation of reproductive hormones in the control of pineal rhythmic activity in the male rat. Apparently, since gonadectomy abolished the nocturnal rise of both pineal tyrosine hydroxylase activity and norepinephrine content, the primary site of action of reproductive hormones could be at the level of the superior cervical ganglion.     

Effects of dexamethasone and other glucocorticoid steroids on tyrosine hydroxylase activity in the superior cervical ganglion

Dexamethasone is known to elicit an increase of tyrosine hydroxylase activity in the superior cervical ganglion. The details of such a glucocorticoid effect were investigated in the present study. Of 4 glucocorticoids (dexamethasone, corticosterone, hydrocortisone and triamcinolone) examined in rats, only the synthetic steroid dexamethasone was found to be effective in increasing ganglionic tyrosine hydroxylase activity (by 50% at 48 h after drug administration). Corticosterone even at doses as high as 50 mg/kg failed to show an effect. Since recent reports indicate that a cytoplasmic glucocorticoid receptor is not present in the sympathetic ganglion, it is unlikely that the dexamethasone effect involves a receptor-mediated mechanism. Moreover, the dexamethasone effect was totally blocked by chlorisondamine, a nicotinic cholinergic receptor antagonist. The possibility of an enhanced impulse flow from the CNS, however, was excluded by the finding that decentralization immediately prior to dexamethasone administration did not prevent the increase of ganglionic tyrosine hydroxylase activity, although earlier decentralization (24 h or longer) abolished the steroid effect. Significantly, in the freshly decentralized ganglia, the increase of tyrosine hydroxylase activity by dexamethasone was still blocked by chlorisondamine. Since synaptic activity in terminals is known to continue for a brief period following nerve transection, our data support the contention that the primary site of the dexamethasone effect may be the preganglionic terminals.     

Gender-specific regulation of tyrosine hydroxylase in thymocyte differentiation antigen-1 knockout mice

Thymocyte differentiation antigen-1 (Thy-1) is a cell surface glycoprotein found on T cells and neurons and is involved in cell-to-cell interactions. In addition, Thy-1 knockouts (KO) are a potential mouse model of restless legs syndrome (RLS) based on clinical observations and the role of dopamine in the disease. In this study, we analyzed the activity and quantity of tyrosine hydroxylase (TH; the rate-limiting enzyme in dopamine production) and determined phosphorylation levels for the enzyme phosphoserine-40 (pSer-40). There was no significant difference in the total TH activity and pSer-40 TH levels between Thy-1 KO and control groups; however, TH specific activity was significantly lower (by 26%) in Thy-1 KO mice. This difference is due in part to increased TH protein levels in this group (increased by 29%). When analyzed by gender, Thy-1 KO female mouse striata contained less TH specific activity compared with control females (decreased by 41%) and male control or Thy-1 KO animals (decreased by 30%). TH specific activity and pSer-40 TH levels in male Thy-1 KO and control displayed no differences. However, pSer-40 TH was significantly higher in control females (38%) compared with control or Thy-1 KO males. The Thy-1 KO females exhibited significantly lower (28%) pSer-40 TH (normalized to GAPDH or TH) than control females. Indeed, the Thy-1 KO females had 50% of the pSer-40 TH found in controls. Our results suggest a gender effect on TH specific activity, TH protein levels, and serine-40 phosphorylation of TH in Thy-1 KO female mice.     

Long-term regulation of tyrosine hydroxylase activity in the superior cervical ganglion in organ culture: Effects of nerve stimulation and dexamethasone

The rat superior cervical ganglion was stimulated in vitro via its preganglionic trunk and then maintained in organ culture for 2-3 days. Following nerve stimulation, the specific activity of tyrosine hydroxylase (TH) increased with a lag period of at least 12 h, and this increased enzyme activity was maintained throughout the culture period. The magnitude of the increase in TH activity depended on the frequency and duration of preganglionic nerve stimulation. This transsynaptic increase in enzyme activity could be completely blocked by the nicotinic antagonist, hexamethonium. The synthetic glucocorticoid dexamethasone, at concentrations above 1 nM, increased TH activity in unstimulated ganglia with a lag period of at least 24 h. The increase in enzyme activity produced by dexamethasone was not affected by nicotinic antagonists or by prior decentralization of the superior cervical ganglion, in contrast to the findings of previous workers. These data indicate that dexamethasone can stimulate ganglionic TH activity in the absence of cholinergic stimulation. In addition, experiments combining dexamethasone with nerve stimulation demonstrate that the steroid can potentiate the effects on TH activity of brief periods of nerve stimulation.     

Ultrastructural immunocytochemical localization of tyrosine hydroxylase in the neostriatum

The morphology and synaptic associations of dopaminergic axons in the n. caudate-putamen (neostriatum) of the adult rat brain are examined. Identification of dopaminergic axons is based upon the electron microscopic immunocytochemical localization of the catecholamine synthesizing enzyme, tyrosine hydroxylase. Immunoreactivity for the enzyme is detected in unmyelinated axons and axon terminals in serial sections collected throughout the neostriatum. The labeled terminals range from 0.1 to 1.5 micron in diameter and have peroxidase reaction product located around closely packed, round vesicles with a diameter of 40-60 nm. The tyrosine hydroxylase containing axon terminals constitute approximately 21% of the total number of terminals in the n. caudatus-putamen and include 3 types which differ in size and synaptic specializations. The most prevalent (82% of total), type I, is small (0.15-0.39 micron in diameter) and forms symmetric junctions with dendrites and dendritic spines. The other two terminal types (II and III) have a medium to large diameter (0.4-1.5 micron) and show either no membrane specializations or asymmetric junctions with dendrites. The axon terminals without observable membrane densities are occasionally oriented so as to suggest an association with dopaminergic and non-dopaminergic axon terminals. These findings indicate that while the dopaminergic terminals may form axoaxonic connections, the primary synaptic contacts are with dendrites of intrinsic neurons in all regions of n. caudatus-putamen.     

Homospecific activity (activity per enzyme protein) of tyrosine hydroxylase increases in parkinsonian brain

Summary Tyrosine hydroxylase (TH) contents in the caudate nucleus, putamen, and substantia nigra from control and parkinsonism brains were measured for the first time by a sandwich enzyme immunoassay. Both the TH protein content and TH activity (V_max) were decreased in parallel in the parkinsonian brains as compared with those of the control brains. In contrast, TH homospecific activity (activity per enzyme protein) was significantly increased in the parkinsonian brains. The results indicate that the decrease of TH activity in parkinsonian brains is due to the decrease of TH protein content as a result of cell death. The increase in the homospecific activity of residual TH in parkinsonian brain suggests such molecular changes in TH molecules as result in a compensatory increase in TH activity.     

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.     

Plasticity of tyrosine hydroxylase and serotonergic systems in the regenerating spinal cord of adult zebrafish

Monoaminergic innervation of the spinal cord has important modulatory functions for locomotion. Here we performed a quantitative study to determine the plastic changes of tyrosine hydroxylase-positive (TH1(+); mainly dopaminergic), and serotonergic (5-HT(+)) terminals and cells during successful spinal cord regeneration in adult zebrafish. TH1(+) innervation in the spinal cord is derived from the brain. After spinal cord transection, TH1(+) immunoreactivity is completely lost from the caudal spinal cord. Terminal varicosities increase in density rostral to the lesion site compared with unlesioned controls and are re-established in the caudal spinal cord at 6 weeks post lesion. Interestingly, axons mostly fail to re-innervate more caudal levels of the spinal cord even after prolonged survival times. However, densities of terminal varicosities correlate with recovery of swimming behavior, which is completely lost again after re-lesion of the spinal cord. Similar observations were made for terminals derived from descending 5-HT(+) axons from the brain. In addition, spinal 5-HT(+) neurons were newly generated after a lesion and transiently increased in number up to fivefold, which depended in part on hedgehog signaling. Overall, TH1(+) and 5-HT(+) innervation is massively altered in the successfully regenerated spinal cord of adult zebrafish. Despite these changes in TH and 5-HT systems, a remarkable recovery of swimming capability is achieved, suggesting significant plasticity of the adult spinal network during regeneration.     

Central administration of serotonin decreases tyrosine hydroxylase catalytic activity and messenger ribonucleic Acid signal levels in the hypothalamus of female rats

We investigated the effect of central serotonin (5-hydroxytryptamine, 5-HT) administration on hypothalamic tuberoinfundibular dopamine neurons and related changes in neuronal activity to circulating prolactin (PRL) levels. Ovariectomized rats were treated with either vehicle or 5-HT through a lateral ventricular cannula in one of two dose paradigms: 1) a bolus of 20 μg, with tissues taken at 30 min, or 2) the same bolus immediately followed by 20 μg/30 min via a syringe pump for 120 min, and tissues taken at 120 min. Blood samples were taken throughout experiments and plasma PRL determined by radioimmunoassay. Under both paradigms, NSD 1015, a dihydroxyphenylalanine (DOPA) decarboxylase inhibitor (25 mg/kg intraarterially) was injected 10 min before decapitation and brain excision followed by stalk-median eminence dissection. The rate of DOPA accumulation, determined by measuring DOPA levels in the stalk-median eminence by high-performance liquid chromatography with electrochemical detection was used as a measure of tyrosine hydroxylase (TH) catalytic activity. Stalk-median eminence DOPA accumulation in control rats was 29.9 ± 4.2 and 28.8 ± 4,4 ng/mg protein (30 and 120 min experiments, respectively). DOPA accumulation in 5-HT-treated rats was significantly reduced (P<0.05) after 30 min to 17.8 ± 1.2 ng/mg protein, but it was similar (21.7 ± 3.9) to controls after 120 min of 5-HT infusion. 5-HT levels in the stalk-median eminence of rats treated with 5-HT were 13- to 17-fold greater than controls (16.9 to 18.5 ng/mg protein). Plasma PRL levels in both groups increased 10-fold after 5-HT treatment with a peak at 5 min, returning to baseline by 120 min. TH mRNA levels were determined by in situ hybridization in a second group of rats which were treated with the 20μg bolus and subsequent 120 min infusion of 5-HT. TH mRNA signal levels in the arcuate nucleus of control rats averaged 144 ± 21 grains/cell. After treatment with 5-HT, TH mRNA levels in the arcuate nucleus were significantly lower (P<0.0001) with 69±14 grains/cell. In a third group of rats, the effects of the 30 min 5-HT treatment on TH catalytic activity and circulating PRL levels was challenged with two 5-HT(2) receptor antagonists, LY53857 (5 mg/kg intraperitoneally) or ketanserin (10 mg/kg intraperitoneally). Neither the 5-HT-induced decrease in TH catalytic activity nor the increase in PRL was altered by pretreatment (120 min) with 5-HT(2) antagonists. These data suggest that central 5-HT is capable of decreasing TH activity and TH mRNA levels in the tuberoinfundibular dopamine neurons and that the decrease in dopaminergic neuronal activity may contribute to the 5-HT-induced PRL rise. The changes in TH catalytic activity and PRL after intracerebroventricular administration of 5-HT do not appear to be mediated by 5-HT(2) receptors.