Evidence for the role of brain biogenic amines in depressed motor activity seen in chemically thyroidectomized rats.

Abstract— The effects of exposure to an antithyroid drug, methimazole, on brain tyrosine hydroxylase and tryptophan hydroxylase activity, as well as the levels of norepinephrine, dopamine, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid have been investigated in maturing brain. Daily treatment of neonatal rats with methimazole for 30 days induced chemical thyroidectomy as evidenced by significant impairment of body and brain growth. The activities or brain tyrosine hydroxylase and tryptophan hydroxylase and the levels of norepinephrine, dopamine and 5-hydroxytryptamine were markedly altered in a dose- and time-dependent manner in methimazole-treated rats. Conversely, the concentration of brain 5-hydroxyindoleacetic acid was elevated (46%) by methimazole administration. Treatment with the antithyroid drug failed to exert any significant effect on the endogenous levels of brain tryptophan, as well as on the activity of the deaminating enzyme, monoamine oxidase. Administration of triiodothyronine (25 or 100 μg/100 g) to hypothyroid rats for 30 days did not produce any appreciable effect upon the neurochemical parameters related to either norepinephrine or 5-hydroxytryptamine mctabolism. However, increasing the dose of triiodothyronine to 250 μg/100 g significantly elevated the levels of norepinephrine and 5-hydroxytryplamine as well as the activities of the two synthesizing enzymes, tyrosine hydroxylase and tryptophan hydroxylase. Brain 5-hydroxyindoleacetic acid levels were restored to normal values in thyroid hormone-deficient rats treated with this higher dose of triiodothyronine. Evidencc also was obtained to show that chemical thyroidectomy suppressed the spontancous locomotor activity in neonatal rats; the changes being apparent at 15 days of age. Our data support the view that thyroid hormone in neonatal life displays an important regulatory effect on the metabolism of norepinephrine, dopamine and 5-hydroxytryptamine. Since certain amines have been known to be implicated as the neurochemical substrates for behavioural arousal, it is conceivable that the observed hypoactivity in methimazolc-treated rats may, at least in part, be related to impaired maturation of norepinephrine and dopamine-synthesizing systems in brains of cretinous rats.     

Chronic Cocaine Administration Increases CNS Tyrosine Hydroxylase Enzyme Activity and mRNA Levels and Tryptophan Hydroxylase Enzyme Activity Levels

Cocaine is an inhibitor of dopamine and serotonin reuptake by synaptic terminals and has potent reinforcing effects that lead to its abuse. Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate-limiting steps in dopamine and serotonin biosynthesis, respectively, and are the subject of dynamic regulatory mechanisms that could be sensitive to the actions of cocaine. This study assessed the effects of chronic cocaine on brain TH and TPH activities. Cocaine was administered (0.33 mg/infusion, i.v.) to rats for 7 days every 8 min for 6 h per day. This administration schedule is similar to patterns of self-administration by rats when given ad libitum access to this dose. This chronic, response-independent administration increased TH enzyme activity in the substantia nigra (30%) and ventral tegmental area (43%). Moreover, TH mRNA levels were also increased (45 and 50%, respectively). In contrast to the enzymatic and molecular biological changes in the cell bodies, TH activity was unchanged in the terminal fields (corpus striaturn and nucleus accumbens). Similarly, TPH activity was increased by 50% in the raphe nucleus (serotonergic cell bodies). In summary, the chronic response-independent administration of cocaine produces increases in the expression of TH mRNA and activity in both the cell bodies of motor (nigrostriatal) and reinforcement (mesolimbic) dopamine pathways. These increases are not manifested in the terminal fields of these pathways.     

The catecholamine system in rat fetal brain with a disrupted corticosteroid balance]

The disturbance of corticosteroids balance of female rats on the 16 and 18 days of pregnancy by injections of exogenous corticosterone or methopyrone--blocker of endogenous hormone formation--decreased both body weight and activity of the rate-limiting catecholamine synthesising enzyme--tyrosine hydroxylase (TH) in the stem half of the 21 day fetal brain. Concomitantly with inhibitory action, which may be caused by general retardation of the organism development, corticosteroids stimulated TH activity during prenatal ontogenesis. Fetuses developed under elevated corticosteroid level had lower body weight but higher TH activity in comparison with fetuses endured the deficit of these hormones. Besides, corticosterone injection to the females on the 20th day of gestation increased in 6 hours TH activity in stem half of their fetus brain. The data obtained suggested the prominent role of corticosteroids in the prenatal development of brain catecholaminergic system.     

Alterations in norepinephrine,serotonin, c-AMP,and transsynaptic induction of tyrosine hydroxylase after spinal cord transection in the rat

Tyrosine hydroxylase (TH) activity and the concentrations of norepinephrine (NE), serotonin (5-HT), and cyclic adenosine 3, 5-monophosphate (c-AMP) were measured in the heart, adrenals, and brain stem of paraplegic rats. Following spinal cord transection NE concentration in the heart dropped to 30% within 24 hours and that of 5-HT decreased to 60% of control. Tyrosine hydroxylase activity and c-AMP levels in the brain stem were elevated while NE concentration remained low. At seven days, however, NE and 5-HT levels were higher than in controls while TH activity and c-AMP concentration dropped to control levels. The increase in TH activity in the brain stem may be due to curtailed end-product feedback inhibition and to reduced receptor activation. The sustained induction of the adrenal TH is probably a consequence of a continual stimulation of splanchnic nerves.     

Effects of hydrocortisone and electric footshock on mouse brain tyrosine hydroxylase activity and tyrosine levels

Tyrosine hydroxylase (TH) activity and levels of tyrosine were measured in whole brains from mice subjected to brief electric foot shock or to single or multiple injections of hydrocortisone acetate (HCA; 20 mg/kg). Adult brain tyrosine levels showed a rapid increase after either foot shock or a single injection of HCA; TH activity was also rapidly increased by foot shock but not by HCA. Multiple injections of HCA over three days increased brain TH activity in neonatal mice, but had no effect in adults. These results suggest that glucocorticoid hormone may have a regulatory influence on brain TH during the neonatal period, and that the hormone may also affect brain tyrosine. The acute effect of foot shock stress on brain TH activity is not a glucocorticoid-mediated event, but can be interpreted as enzyme activation due to neural stimulation.     

Activity of enzymes involved in norepinephrine biosynthesis in the brown adipose tissue of the developing rat. Influence of hypothyroidism

The activities of tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) were measured in vitro in the brown adipose tissue (BAT) of control and hypothyroid developing rats. Neonatal hypothyroidism slows the development of TH activity, as manifest in lower BAT TH activity, relative to controls, up to 20 days. This effect is more pronounced when the onset of hypothyroidism is induced prenatally. No clear effect of hypothyroidism on DBH activity was evident.     

Brain biogenic amines and altered thyroid function.

Evidence has been presented that alterations in thyroidal status produce marked changes in the metabolism of several biogenic amines in developing brain. Neonatal hypothyroidism induced either by ¹³¹I or by an anti-thyroid agent, methimazole, markedly decreased the concentrations of norepinephrine, dopamine and 5-hydroxytryptamine and the activity of their rate-limiting enzymes, tyrosine hydroxylase and tryptophan hydroxylase. However, the levels of 5-hydroxyindoleacetic acid, the chief metabolite of 5-hydroxytryptamine were elevated in several regions of the brain. Whereas thyroid deficiency in early life produced no appreciable change in whole brain monoamine oxidase activity, it was increased in mid brain and decreased in the hypothalamus. Brain acetylcholine levels were significantly elevated and the activity of acetylcholinesterase remained unchanged in rats made hypothyroid at 1 day of age. Delaying thyroidectomy for 20 days after birth produced less appreciable changes in norepinephrine and 5-hydroxytryptamine metabolism. Thyroid deficiency suppressed the ontogenesis of behavioural arousal and spontaneous locomotor activity. The administration of L-triiodothyronine to hypothyroid animals in early life restored the metabolism of various neurohumors virtually to the normal limits. However, when the replacement therapy was postponed until adulthood, L-triiodothyronine failed to produce any restorative effects, suggesting that a critical period exists in early life during which thyroid hormone must be present to permit normal developmental pattern of central amines. Data also have been obtained demonstrating that neonatal hyperthyroidism induced by daily administration of L-triiodothyronine results in an increased turnover of norepinephrine and 5-hydroxytryptamine. These amine changes were accompanied by a marked rise in the spontaneous locomotor activity in hyperthyroid rats. Finally, chronic treatment with lithium, an antimanic drug, also known to suppress thyroid hormone production, significantly decreased not only the spontaneous locomotor activity, but also changes in the turnover of 5-hydroxytryptamine and norepinephrine in neonatally hyperthyroid rats.     

Ontogenesis of Monoamine-Synthesizing Enzyme Activities and Biopterin Levels in Rat Brain or Salivary Glands, and the Effect of Thyroxine Administration

Neonatal changes in the activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TrpH) and in the content of the co-factor, biopterin, were studied in rat midbrain for the first 20 days after birth. Changes in TH activity in the parotid and submandibular glands were also examined. Changes in TH activity per unit weight in the developing rat brain were briefly similar to those in the salivary glands; the activity increased from day 2 or 4 to day 9 after birth, and remained constant or slightly decreased at day 12, then rapidly increased on day 16. TrpH activity in the midbrain increased about twofold up to day 16. The biopterin concentration in the brain increased, reached a maximum level on day 12 after birth, and thereafter decreased. The effect of hyperthyroidism in rats given 0.2 mg/kg i.p. of thyroxine every 2 days postnatally was studied on the activity of TH in rat salivary glands at 12-day-old rats. In parotid or submandibular gland of hyperthyroid rats, TH activity increased at day 12 postnatally. In comparison with the effect on TH activity in the salivary glands, TH activity in the midbrain on day 20 postnatally was not induced by hyperthyroidism. Furthermore, increase of the TrpH activity and biopterin and catecholamine levels in the midbrain of hyperthyroid rats was not found on day 20 after birth in comparison with the corresponding controls. From these data, we suppose that postnatal hyperthyroidism may cause precocious induction of TH in rat salivary gland, but may not increase the activity of TH or TrpH, and the level of their co-factor, biopterin, in rat midbrain.     

Tyrosine hydroxylase activity and expression of its gene in mice with contrasting capacity to dominate in social stress]

Changes of tyrosine hydroxylase (TH) activity and level of mRNA of TH gene in PT and CBA/Lac mouse strains, which are contrast by ability to dominate in heterogenous populations, were investigated. It was established, that the activity of TH both in dominate PT and subordinate CBA/Lac mice in hypothalamus, hippocampus and brain stem elevated in one hour after forming of micropopulations. But the appearance of this increase was different: activation of TH in hypothalamus and brain stem of PT mice was stronger then one in CBA/Lac mice. Moreover, the beginning of the reaction in brain stem of PT mice was earlier then that of CBA/Lac mice. MRNA level of TH gene in hypothalamus and brain stem in one hour was elevated only in PT mice for 50% and 200%, respectively. No changing in expression TH gene was found in hippocampus. In conclusion, it was suggested that the activation of catecholamine biosynthesis under social stress in hypothalamus and brain stem of male mice was due to the TH activation and increase of its gene expression.     

Biochemical Evidence for Alteration of Neostriatal Dopaminergic Function by 5,7-Dihydroxytryptamine

Unilateral injection of 5,7-dihydroxytryptamine (DHT) into the rat neostriatum markedly reduced not only striatal tryptophan hydroxylase (TPH) activity but also striatal tyrosine hydroxylase (TH) activity and dopamine (DA) concentration measured 10--15 days later. The decrease in striatal TH activity was dose related over the range of 8--32 micrograms of DHT; a dose of 16 micrograms reduced striatal TH activity to 40--50% of control, DA concentration to 38% of control, and TPH activity to 5--20% of control. Intrastriatal injection of 16 micrograms of DHT reduced TH activity in the ipsilateral substantia nigra to 51% of control. Pretreatment with amfonelic acid, a potent DA uptake inhibitor, significantly reduced the effect of DHT on striatal and nigral TH activity and striatal DA concentration without affecting the DHT-induced decrease in striatal TPH activity. Desmethylimipramine (5 and 25 mg/kg) had no effect on the DHT-induced decrease in striatal TH activity. Striatal choline acetyltransferase and glutamic acid decarboxylase activities were not decreased by 16 micrograms of DHT. The results indicate that DHT can alter dopaminergic function in the rat neostriatum through a direct effect of the drug on DA neurons.     

Region - specific alterations in tyrosine hydroxylase activity in rats exposed to lead

Previous studies from our laboratory showed that subchronic exposure to low levels of Pb resulted in significant decrease in dopamine (DA) content, attenuation of stimulus-induced release of DA in the dopaminergic projection area of nucleus accumbens (NA), and alterations in tyrosine hydroxylase (TH) activity in rat whole brain homogenates. The present study reported here was conducted to assess the functional integrity of DA synthesis in different brain regions of rats subchronically (90-days) exposed to 50 ppm Pb by measuring the activity of the rate limiting enzyme, tyrosine hydroxylase, in seven brain regions. In Pb-exposed rats, TH activity was reduced in two of the seven brain regions investigated, i.e., nucleus accumbens (42% reduction) and frontal cortex (61% reduction) when compared to controls. In contrast, Pb exposure did not affect the TH activity in cerebellum, brainstem, hippocampus, hypothalamus and striatum. The changes in TH activity in nucleus accumbens (NA) and frontal cortex (FC) in Pb-exposed rats were further confirmed by Western blot analysis using TH polyclonal antibody. Collectively, these results indicate that low level subchronic Pb exposure may affect TH protein in these brain regions.     

Differential Regional Effects of Methamphetamine on the Activities of Tryptophan and Tyrosine Hydroxylase

Abstract : Administration of high doses of methamphetamine (METH) produces both short- and long-term enzymatic deficits in central monoaminergic systems. To determine whether a correlative relationship exists between these acute and long-term consequences of METH treatment, in the present study we examined the regional effects of METH on tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH) activities in various regions of the caudate nucleus, nucleus accumbens, and globus pallidus. A single METH administration decreased TPH activity 1 h after treatment in the globus pallidus, in the nucleus accumbens, and throughout the caudate ; in the anterior caudate, the ventral-medial was more affected than the dorsal-lateral region. In contrast, TH activity was not decreased in either the caudate or the globus pallidus after a single METH administration ; however, it was altered in the nucleus accumbens. Seven days after multiple METH administrations, TH and TPH activities were decreased in most caudate regions but not in the nucleus accumbens or globus pallidus. These data demonstrate that (1) the effects of METH on TPH and TH vary regionally ; and (2) the short-term and long-term regional responses of TPH to METH in the caudate and globus pallidus correlated. In contrast, METH-induced acute TH responses did not predict the long-term changes in TH activity.     

Effects of nitric oxide synthase inhibitor on tyrosine hydroxylase mRNA in the adrenal medulla of spontaneously hypertensive and Wistar Kyoto rats.

We studied the effects of N(G)-nitro-l-arginine methyl ester (L-NAME) on catecholamine levels, tyrosine hydroxylase (TH) activity, and TH mRNA levels in the adrenal medulla of spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). L-NAME (100 mg/L in drinking water) and atropine (10 mg/L in drinking water) were administered for 2 weeks. Epinephrine and norepinephrine levels, TH activity, and TH mRNA levels in the adrenal medulla of L-NAME-treated WKY were significantly decreased. These parameters were not significantly altered in the adrenal medulla of L-NAME-treated SHR. Nitrite/nitrate levels in the adrenal medulla of L-NAME-treated WKY were significantly decreased; however, no significant change in L-NAME-treated SHR was observed. Ca(2+)-dependent nitric oxide synthase (NOS) activity in the adrenal medulla of SHR was significantly decreased compared to that of WKY. TH mRNA levels in L-NAME + atropine-treated and L-NAME-treated WKY were significantly lower than TH mRNA levels in control WKY. These results suggest that nitric oxide in the adrenal medulla may enhance the catecholamine biosynthetic pathway via increased TH mRNA expression. Our results also suggest that this effect is suppressed in SHR due to decreased NOS activity in the adrenal medulla.     

p-ethynylphenylalanine: a potent inhibitor of tryptophan hydroxylase

Tryptophan hydroxylase (TPH) is the initial and rate-limiting enzyme in serotonin biosynthesis. The enzyme activity is dependent on molecular oxygen, a tetrahydropterin cosubstrate, and ferrous iron. The present study demonstrates that TPH is inhibited by a novel compound, p-ethynylphenylalanine (pEPA), produced by the Heck reaction of trimethylsilylacetylene with N-tertbutyloxycarbonyl-4-iodo-L-phenylalanine methyl ester. pEPA is a more potent and specific inhibitor of TPH than p-chlorophenylalanine (pCPA). In the present study, pEPA was demonstrated to inhibit competitively and reversibly TPH in vitro (Ki = 32.6 +/- 6.2 microM vs. tryptophan). pEPA displayed little inhibitory activity toward tyrosine hydroxylase (EC 1.14.16.2), the initial and rate-limiting enzyme for catecholamine biosynthesis, and no inhibition of phenylalanine hydroxylase or tyrosinase. In addition, pEPA was a poor ligand for the serotonin transporter and several serotonin receptors. Administration of pEPA (30 mg/kg) to rats produced a 95 +/- 5% decrease in TPH activity in brain homogenates and a concomitant decrease in serotonin and 5-hydroxyindole-3-acetic acid levels (85%) at 24 h after injection. In contrast, pCPA produced a similar effect (87 +/- 5% decrease in TPH activity) only at 10 times the concentration (300 mg/kg). These results suggest that pEPA is a selective, reversible, and potent inhibitor of TPH both in vitro and in vivo. The potential for pEPA to inhibit selectively and reversibly the biosynthesis of serotonin may contribute to the characterization of the role of serotonin in behavioral and physiological activities.     

Developmental role of tryptophan hydroxylase in the nervous system

The serotonin 5-hydroxytryptamine (5-HT) neurotransmitter system contributes to various physiological and pathological conditions. 5-HT is the first neurotransmitter for which a developmental role was suspected. Tryptophan hydroxylase (TPH) catalyzes the rate-limiting reaction in the biosynthesis of 5-HT. Both TPH1 and TPH2 have tryptophan hydroxylating activity. TPH2 is abundant in the brain, whereas TPH1 is mainly expressed in the pineal gland and the periphery. However, TPH1 was found to be expressed predominantly during the late developmental stage in the brain. Recent advances have shed light on the kinetic properties of each TPH isoform. TPH1 showed greater affinity for tryptophan and stronger enzymic activity than TPH2 under conditions reflecting those in the developing brain stem. Transient alterations in 5-HT homeostasis during development modify the fine wiring of brain connections and cause permanent changes to adult behavior. An increasing body of evidence suggests the involvement of developmental brain disturbances in psychiatric disorders. These findings have revived a long-standing interest in the developmental role of 5-HT-related molecules. This article summarizes our understanding of the kinetics and possible neuronal functions of each TPH during development and in the adult.     

GLUCOCORTICOIDS AS A REGULATORY FACTOR FOR BRAIN TRYPTOPHAN HYDROXYLASE

—The normal developmental rise of tryptophan hydroxylase levels in neonatal rat brain was blocked by adrenalectomy. Similarly, adrenalectomy prevented the rescrpine-induced elevation of tryptophan hydroxylase activity in brain stem of adult mice. In both cases, the effects of adrenalectomy could be reversed by replacement injections of corticosterone. Repeated injections of corticosterone (5 mg/kg daily) in fact induced a rise of brain tryptophan hydroxylase levels in neonatal brain. However, neither adrenalectomy nor repeated injections of large doses of the hormone (20 mg/kg, daily) was found to be effective in affecting the normal enzyme levels in adult brain. Apparent K_m of the enzyme for substrate was unchanged by corticosterone in vivo or in vitro. These results indicate that glucocorticoids have a significant role in the regulation of brain tryptophan hydroxylase: possibly as an inducing signal during neonatal development and as a permissive factor at adult age.     

Ciliary neurotrophic factor induces cholinergic differentiation of rat sympathetic neurons in culture

Ciliary neurotrophic factor (CNTF) influences the levels of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) in cultures of dissociated sympathetic neurons from newborn rats. In the presence of CNTF both the total and specific activity of ChAT was increased 7 d after culture by 15- and 18-fold, respectively, as compared to cultures kept in the absence of CNTF. Between 3 and 21 d in culture in the presence of CNTF the total ChAT activity increased by a factor of greater than 100. Immunotitration demonstrated that the elevated ChAT levels were due to an increased number of enzyme molecules. In contrast to the increase in ChAT levels, the total and specific activity levels of TH were decreased by 42 and 36%, respectively, after 7 d in culture. Half-maximal effects for both ChAT increase and TH decrease were obtained at CNTF concentrations of approximately 0.6 ng and maximal levels were reached at 1 ng of CNTF per milliliter of medium. The effect of CNTF on TH and ChAT levels were seen in serum-containing medium as well as in serum-free medium. CNTF was shown to have only a small effect on the long-term survival of rat sympathetic neurons. We therefore concluded that the effects of CNTF on ChAT and TH are not due to selective survival of cells that acquire cholinergic traits in vitro, but are rather due to the induction of cholinergic differentiation of noradrenergic sympathetic neurons.     

Influence of thyroid hormone deficiency on the citrate synthase activity in rat brain regions during early postnatal development

The developmental pattern of citrate synthase activity has been studied in the liver and several brain areas of hypothyroid rats during the 4 first weeks of life. While citrate synthase activity in the liver showed a rise during the 2 first weeks of life, different patterns of enzyme activity were found in the brain regions of euthyroid animals. Citrate synthase activity increased in the cerebellum, decreased in the cerebral cortex and did not change significantly in the brain stem during the period studied. In the liver and brain areas, too, a decrease in citrate synthase activity was observed during hypothyroidism. From the 2nd week of birth, the citrate synthase activity in the brain but not in the liver was found to have recovered. The newly elevated citrate synthase activity coincided with a slight increase in thyroid hormone serum levels.