Mobilization of Storage Pool Dopamine and Late Ipsilateral Augmentation of Striatal Dopamine Synthesis in the Trained Circling Rat

Rats were infused intraventricularly with [3H]tyrosine over a 20-min period during various times while circling. 3,4-Dihydroxyphenylethylamine (dopamine) and dihydroxyphenylacetic acid (DOPAC) levels were measured using HPLC with electrochemical detection and fractions were collected for tritium monitoring. During the first 20 min of circling, the specific activity of dopamine was increased by 290% in striatum contralateral to the circling direction whereas DOPAC specific activity was increased 50% on the same side. This differential change in relative specific activity suggests that unlabeled storage pool dopamine was mobilized to DOPAC during circling. Synthesis of dopamine and DOPAC in contralateral striatum returned to baseline levels as turning slowed (50-70 min). When turning ceased, there was an increase in ipsilateral striatal dopamine synthesis during the 20-min period following circling. We hypothesize that this ipsilateral increase represents either a "stop" signal following circling or a release of inhibition of ipsilateral nigral neurons.     

Normal rats trained to circle show asymmetric caudate dopamine release

Caudate catecholamine release was monitored by bilateral $\text{in}$$\text{vivo}$ electrochemical electrodes in male Sprague-Dawley rats trained to circle for sucrose/water reward. Baseline release of dopamine was equal from both sides of caudate. When reinforced circling began, 33 ± 4 percent greater catechol release occured from the caudate contralateral to the circling direction. As turning subsided, differential release returned to basal levels. Further evidence that the catecholamine metabolism was affected by turning was obtained by direct measurement of caudate dopamine and DOPAC at selected time points. Concentration data showed relative increases in dopamine and DOPAC in the contralateral caudate. These data provide evidence that dopamine is released asymmetrically from caudate in unlesioned rats during voluntary behavior.     

Influence of Calcium on Dopamine Synthesis and Tyrosine Hydroxylase Activity in Rat Striatum

Abstract: We have investigated three aspects of the relationship between calcium and tyrosine hydroxylase activity in rat striatum. In the first series of experiments, we examined the hypothesis that the rise in dopamine synthesis during increased impulse flow results from a calcium-induced activation of tyrosine hydroxylase. Calcium (12.5–200 μ M ) had no effect when added to crude enzyme or enzyme partially purified by gel filtration. Moreover, incubation of synaptosomes with excess calcium (up to 3.5 m M ) had little or no effect on dopamine synthesis. Incubation with the depolarizing alkaloid veratridine (75 μ M ) did increase dopamine synthesis, but did not alter the activity of tyrosine hydroxylase subsequently prepared from the synaptosomes, despite the presumed rise in intracellular calcium. In the second series we examined the hypothesis that increased dopamine synthesis after axotomy results from activation of tyrosine hydroxylase owing to a decrease in intracellular calcium. Addition of the calcium chelator EGTA (100 μ M ) to crude or partially purified enzyme was without effect, whereas incubation of synaptosomes with EGTA (500 μM ) decreased cell-free enzyme activity. In the third experimental series we examined the relationship between calcium and activation of tyrosine hydroxylase by dibutyryl cyclic AMP. EGTA failed to alter the increase in the activity of tyrosine hydroxylase prepared from synaptosomes incubated with dibutyryl cyclic AMP. However, it blocked the increase in synaptosomal dopamine synthesis and dopamine content normally produced by the cyclic AMP analogue. Thus, tyrosine hydroxylase does not appear to be activated by either increases or decreases in calcium availability. However, calcium may be important for the maintenance of basal tyrosine hydroxylase activity, and may play an indirect role in the expression of tyrosine hydroxylase activation produced by other means.     

Tyrosine Hydroxylase Regulation: Apparent Kinetic Alterations Following Incubation of Brain Slices in a Sodium-free Medium

In an attempt to determine if alterations in intraneuronal Ca2+ may regulate tyrosine hydroxylase activity, brain slices were subjected to experimental manipulations known to increase the intraneuronal concentration of free Ca2+ ions. Incubation of either striatal or olfactory tubercle slices in a Na+-free medium for 15 min at 37 degrees resulted in a marked increase in the activity of tyrosine hydroxylase present in the 20,000 g supernatant fraction of homogenates prepared from the slices. Tyrosine hydroxylase isolated from slices previously incubated in a Na+-free, choline-enriched medium or in a Na+-free, sucrose-enriched medium exhibited maximal activities when assayed at pH 6.0 and 7.0, respectively. However, the percentage stimulation of enzyme activity induced by incubation of the slices in a Na+-free medium was maximal when the enzyme assays were performed at pH 7.0. The observed increase in enzyme activity seems to be mediated by a decrease in the apparent Km of the enzyme for pteridine cofactor, regardless of whether the kinetic enzyme analyses were conducted at pH 6.0 or 7.0, and by an increase in the Ki of the enzyme for end-product inhibitor dopamine. The apparent kinetic changes in the enzyme do not seem to result from alterations in the endogenous dopamine content of the slices, and they are independent of any increase in dopamine release that might have occurred as a response to the augmented intraneuronal Ca2+ concentration. Furthermore, the activation of tyrosine hydroxylase produced by incubating slices in a Na+-free medium is observed even in slices depleted of dopamine by pretreatment of rats with reserpine 90 min before preparation of brain slices. The activation of tyrosine hydroxylase observed under these experimental conditions does not seem to be mediated by cAMP or by a cAMP-dependent phosphorylation process. It is suggested that the changes in tyrosine hydroxylase reported are mediated primarily by a rise in the free Ca2+ concentration within the nerve tissue. These observations are consistent with the hypothesis that the kinetic activation of tyrosine hydroxylase produced after depolarization of central dopaminergic neurons may occur through a Ca2+-dependent even other than transmitter release.     

Regulation of tyrosine hydroxylase activity and phosphorylation at Ser(19) and Ser(40) via activation of glutamate NMDA receptors in rat striatum

The activity of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by phosphorylation. In this study, we examined the effects of activation of NMDA receptors on the state of phosphorylation and activity of tyrosine hydroxylase in rat striatal slices. NMDA produced a time-and concentration-dependent increase in the levels of phospho-Ser(19)-tyrosine hydroxylase in nigrostriatal nerve terminals. This increase was not associated with any changes in the basal activity of tyrosine hydroxylase, measured as DOPA accumulation. Forskolin, an activator of adenylyl cyclase, stimulated tyrosine hydroxylase phosphorylation at Ser(40) and caused a significant increase in DOPA accumulation. NMDA reduced forskolin-mediated increases in both Ser(40) phosphorylation and DOPA accumulation. In addition, NMDA reduced the increase in phospho-Ser(40)-tyrosine hydroxylase produced by okadaic acid, an inhibitor of protein phosphatase 1 and 2A, but not by a cyclic AMP analogue, 8-bromo-cyclic AMP. These results indicate that, in the striatum, glutamate decreases tyrosine hydroxylase phosphorylation at Ser(40) via activation of NMDA receptors by reducing cyclic AMP production. They also provide a mechanism for the demonstrated ability of NMDA to decrease tyrosine hydroxylase activity and dopamine synthesis.     

Reversal of amphetamine-induced circling preference in trained circling rats

Others have shown that amphetamine given to normal rats causes turning in a particular, preferred direction in most animals. We have studied the effect of training on amphetamine-induced behavioral and biochemical asymmetries in male Sprague-Dawley rats. Water deprived animals were trained to circle either in the same or opposite direction to their intrinsic bias using a sucrose water reward. Acquisition of the learned circling behavior was independent of turning preference and all animals were able to make the operant association. After training, animals given amphetamine turned in the trained direction regardless of their previous circling preference. Amphetamine-induced circling also led to increased dopamine concentrations in caudate contralateral to the trained circling direction. Therefore, intrinsic striatal is not resistant to behavioral modification and both the behavioral and biochemical asymmetries can be resersed by circling training.     

Splicing Pattern of Gsα mRNA in Human and Rat Brain

The enzyme tyrosine hydroxylase catalyzes the first step in the biosynthesis of dopamine, norepinephrine, and epinephrine. Tyrosine hydroxylase is a substrate for cyclic AMP-dependent protein kinase as well as other protein kinases. We determined the Km and Vmax of rat pheochromocytoma tyrosine hydroxylase for cyclic AMP-dependent protein kinase and obtained values of 136 microM and 7.1 mumol/min/mg of catalytic subunit, respectively. These values were not appreciably affected by the substrates for tyrosine hydroxylase (tyrosine and tetrahydrobiopterin) or by feedback inhibitors (dopamine and norepinephrine). The high Km of tyrosine hydroxylase correlates with the high content of tyrosine hydroxylase in catecholaminergic cells. We also determined the kinetic constants for peptides modeled after actual or potential tyrosine hydroxylase phosphorylation sites. We found that the best substrates for cyclic AMP-dependent protein kinase were those peptides corresponding to serine 40. Tyrosine hydroxylase (36-46), for example, exhibited a Km of 108 microM and a Vmax of 6.93 mumol/min/mg of catalytic subunit. The next best substrate was the peptide corresponding to serine 153. The peptide containing the sequence conforming to serine 19 was a very poor substrate, and that conforming to serine 172 was not phosphorylated to any significant extent. The primary structure of the actual or potential phosphorylation sites is sufficient to explain the substrate behavior of the native enzyme.     

The effect of dopaminergic nigrostriatal system on sleep deprivation in rats

The analysis of the electrophysiological features of sleep-wakefulness cycle in Wistar rats for 9h after a 6h sleep deprivation was carried out. The delay of sleep rebound (since 2.5-3 h after deprivation) was found in the form of moderate increasing of slow-wave sleep and fast-wave sleep phases. According to these sleep-wakefulness cycle changes, a quantitative immunohistochemical study of tyrosine hydroxylase: a key enzyme of dopamine synthesis--and D1 and D2 receptors in nigro-striatal projections has been performed. After sleep, an elevation of D1 receptors immunoreactivity in caudate nucleus and reduction of tyrosine hydroxylase immunoreactivity in compact part of substancia nigra was found. After postdeprivation sleep, a decrease of D1 receptors immunoreactivity and increase of D2 receptors immunoreactivity in caudate nucleus together some increase of tyrosine hydroxilase immunoreactivity in substancia nigra compacta has been observed. These data can testify about active role of dopaminergic nigrostriatal system which provide at the same time with another neurotransmitters of the central nervous system the telencephalo-diencephalic interaction in sleep-wakefulness-sleep cycle.     

Dopamine and sensory tissue development in Drosophila melanogaster

Dopamine is an important signaling molecule in the nervous system; it also plays a vital role in the development of diverse non-neuronal tissues in the fruit fly Drosophila melanogaster. The current study demonstrates that males depleted of dopamine as third instar larvae (via inhibition of the biosynthetic enzyme tyrosine hydroxylase) demonstrated abnormalities in courtship behavior as adults. These defects were suggestive of abnormalities in sensory perception and/or processing. Electroretinograms (ERGs) of eyes from adults depleted of dopamine for 1 day as third instar larvae revealed diminished or absent on- and off-transients. These sensory defects were rescued by the addition of L-DOPA in conjunction with tyrosine hydroxylase inhibition during the larval stage. Depletion of dopamine in the first or second larval instar was lethal, but this was not due to a general inhibition of proliferative cells. To establish that dopamine was synthesized in tissues destined to become part of the adult sensory apparatus, transgenic lines were generated containing 1 or 4 kb of 5' upstream sequences from the Drosophila tyrosine hydroxylase gene (DTH) fused to the E. coli beta-galactosidase reporter. The DTH promoters directed expression of the reporter gene in discrete and consistent patterns within the imaginal discs, in addition to the expected expression in gonadal, brain, and cuticular tissues. The beta-galactosidase expression colocalized with tyrosine hydroxylase protein. These results are consistent with a developmental requirement for dopamine in the normal physiology of adult sensory tissues.     

Changes in Levels of Dopamine and Tyramine in the Rat Caudate Nucleus Following Alterations of Impulse Flow in the Nigrostriatal Pathway

Following electric stimulation of the substantia nigra for 1 h there was a substantial increase in dopamine (DA) turnover in the rat caudate nucleus evidenced by an increase in its acid metabolite homovanillic acid (HVA). Concurrently there was an increase in striatal m-tyramine (mTA) and a substantial decrease in p-tyramine (pTA). Lesioning the substantia nigra to decrease impulse flow resulted in a buildup of striatal DA and mTA, but again a decrease in pTA. Following pretreatment with a tyrosine hydroxylase inhibitor, the effects of stimulation of the nigra on mTA were reversed, there being a significant decrease in this amine. The decrease of pTA in response was partially prevented by tyrosine hydroxylase inhibition. The effects of stimulation or substantia nigra lesions on pTA levels were reversed, however, by tyrosine hydroxylase inhibition, a significant increase in this amine being recorded. mTA and DA levels were largely unaffected by a combination of lesion and tyrosine hydroxylase inhibition. The results provide insight into the possible biosynthetic interrelationships between DA and the tyramine isomers in the rat caudate nucleus.     

Direct binding of GTP cyclohydrolase and tyrosine hydroxylase: regulatory interactions between key enzymes in dopamine biosynthesis

The signaling functions of dopamine require a finely tuned regulatory network for rapid induction and suppression of output. A key target of regulation is the enzyme tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, which is activated by phosphorylation and modulated by the availability of its cofactor, tetrahydrobiopterin. The first enzyme in the cofactor synthesis pathway, GTP cyclohydrolase I, is activated by phosphorylation and inhibited by tetrahydrobiopterin. We previously reported that deficits in GTP cyclohydrolase activity in Drosophila heterozygous for mutant alleles of the gene encoding this enzyme led to tightly corresponding diminution of in vivo tyrosine hydroxylase activity that could not be rescued by exogenous cofactor. We also found that the two enzymes could be coimmunoprecipitated from tissue extracts and proposed functional interactions between the enzymes that extended beyond provision of cofactor by one pathway for another. Here, we confirm the physical association of these enzymes, identifying interacting regions in both, and we demonstrate that their association can be regulated by phosphorylation. The functional consequences of the interaction include an increase in GTP cyclohydrolase activity, with concomitant protection from end-product feedback inhibition. In vivo, this effect would in turn provide sufficient cofactor when demand for catecholamine synthesis is greatest. The activity of tyrosine hydroxylase is also increased by this interaction, in excess of the stimulation resulting from phosphorylation alone. Vmax is elevated, with no change in Km. These results demonstrate that these enzymes engage in mutual positive regulation.     

Participation of agouti-related peptide in the regulation of the wakefulness-sleep cycle

Agouti-related peptide is expressed in the hypothalamic neurons in humans and animals. Immunohistochemical studies in Wistar rats shows significant changes in the optical density of agouti-related peptide in the neurons of the arcuate hypothalamic nucleus, as well as in their processes in the hypothalamus and nucleus accumbens after 6 h of sleep deprivation (an increase) and after 2 h of post-deprivative sleep (a decrease). Comparison of these findings with the earlier results shows the opposite trends in the changes in the optical density of agouti-related peptide and the speed of the limiting enzyme of dopamine synthesis, tyrosine hydroxylase, in the hypothalamus and in the striatonigral system. An increase in the agouti-related peptide level was accompanied by a decrease in tyrosine hydroxylase, while a decrease in agouti-related peptide, on the contrary, was accompanied by an increase in the tyrosine hydroxylase activity. Our data show the role played by agouti-related peptide as a modulator of the functional activity of the dopaminergic brain neurons. The interrelation between various functions of the body, such as food behavior, sleep, and stress, is considered to be mediated by the participation of the same neurotransmitter systems in their regulation.     

Influence of lead exposure on catecholamine metabolism in discrete rat brain nuclei

1. Using the rat exposed both acutely and chronically to lead as a model of lead neurotoxicity, various parameters of catecholamine metabolism were investigated. 2. The steady-state concentrations of noradrenaline, adrenaline and dopamine together with the activities of tyrosine hydroxylase and phenylethanolamine N-methyl transferase were measured in discrete brain nuclei--periventricular, paraventricular, median eminence, posterior and anterior hypothalamus, caudate putamen and globus pallidus. 3. Lead exposure resulted in significant fall in the activity of the rate-limiting enzyme of catecholamine synthesis, tyrosine hydroxylase which was associated with alterations in concentrations of catecholamines in the median eminence, periventricular nucleus and anterior hypothalamus. 4. No other brain nuclei investigated exhibited any effect of lead on the catecholaminergic nervous system and, therefore, the effect of lead on rat brain can be considered to be regionally specific.     

Adaptations of neurotransmitter synthesis to chronic hypoxia in cell culture

Tyrosine hydroxylase and tryptophan hydroxylase are widely held to be rate-limiting for the synthesis of the catecholamines and serotonin, respectively. Both enzymes are oxygen-requiring and kinetic properties suggest that oxygen availability may limit synthesis of these neurotransmitters in the brain. Using pheochromocytoma cells as a cell culture model for catecholamine synthesis, and neuroblastoma cells as a model for serotonin synthesis, enzyme activity was measured under control and hypoxic conditions. Both tyrosine hydroxylase and tryptophan hydroxylase activity increased substantially with chronic exposure but not with acute exposure. In the case of tyrosine hydroxylase, increased enzyme content with hypoxia accounts for increased activity. This suggests a mechanism for the maintenance of neurotransmitter synthesis with chronic hypoxia. Measurement of intracellular metabolites revealed no change in dopamine or norepinephrine in hypoxic pheochromocytoma cells, consistent with a simple adaptive mechanism. However, in neuroblastoma cells, hypoxia was associated with an increase in serotonin concentration. The reasons for this are still unclear.     

The neurotropic parasite Toxoplasma gondii increases dopamine metabolism

The highly prevalent parasite Toxoplasma gondii manipulates its host's behavior. In infected rodents, the behavioral changes increase the likelihood that the parasite will be transmitted back to its definitive cat host, an essential step in completion of the parasite's life cycle. The mechanism(s) responsible for behavioral changes in the host is unknown but two lines of published evidence suggest that the parasite alters neurotransmitter signal transduction: the disruption of the parasite-induced behavioral changes with medications used to treat psychiatric disease (specifically dopamine antagonists) and identification of a tyrosine hydroxylase encoded in the parasite genome. In this study, infection of mammalian dopaminergic cells with T. gondii enhanced the levels of K+-induced release of dopamine several-fold, with a direct correlation between the number of infected cells and the quantity of dopamine released. Immunostaining brain sections of infected mice with dopamine antibody showed intense staining of encysted parasites. Based on these analyses, T. gondii orchestrates a significant increase in dopamine metabolism in neural cells. Tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, was also found in intracellular tissue cysts in brain tissue with antibodies specific for the parasite-encoded tyrosine hydroxylase. These observations provide a mechanism for parasite-induced behavioral changes. The observed effects on dopamine metabolism could also be relevant in interpreting reports of psychobehavioral changes in toxoplasmosis-infected humans.     

Distribution of tyrosine hydroxylase in human and animal brain

The activity of tyrosine hydroxylase (EC 1.10.3.1) when assayed under ideal conditions in young human brains, was comparable to that in brains of other species in level of activity and distribution. The highest levels of activity were in the putamen, caudate nucleus and substantia nigra, in keeping with data on other species. The caudate activity in human brain appeared to decrease substantially with increasing age. In both humans and baboons, the enzyme in the neostriatum was particle-bound and inhibited by the 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine cofactor system. In the substantia nigra it was soluble and stimulated by the 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine cofactor system. The data suggest that tyrosine hydroxylase may be produced in a soluble form in the cell bodies of the substantia nigra but become bound as it moves toward the nerve endings in the putamen and caudate nucleus. The bound form of the enzyme was unstable but the soluble form exhibited considerable stability.     

Circadian Variations in the Activity of Tyrosine Hydroxylase, Tyrosine Aminotransferase, and Tryptophan Hydroxylase: Relationship to Catecholamine Metabolism

Abstract— Circadian variations in the activity of tyrosine hydroxylase, tyrosine aminotransferase, and tryptophan hydroxylase were observed in the rat brain stem. Tyrosine hydroxylase exhibited a bimodal pattern with peaks occurring during both the light and dark phases of the circadian cycle. Tyrosine aminotransferase had one daily peak of activity occurring late in the light phase, whereas tryptophan hydroxylase activity was maximal late in the dark phase. Circadian fluctuations in tyrosine hydroxylase activity did not correlate well with circadian variations in the turnover rates of norepinephrine or dopamine nor with levels of these catecholamines. This supports the idea that although tyrosine hydroxylase is the rate-limiting enzyme in the synthesis of catecholamines, other factors must also be involved in the in vivo regulation of this process. Administration of α -methyl- p -tyrosine (AMT) methyl ester HC1 (100 mg/kg) had no effect on the activity of tryptophan hydroxylase, but effectively eliminated the peak of tyrosine hydroxylase activity that occurred during the light phase. AMT also lowered levels of tyrosine aminotransferase, but only at times near the daily light to dark transition. These chronotypic effects of AMT emphasize the importance of "time of day" as a factor that must be taken into account in evaluating the biochemical as well as the pharmacological and toxicological effects of drugs.     

An HSV-1 Vector Expressing Tyrosine Hydroxylase Causes Production and Release of l-DOPA from Cultured Rat Striatal Cells

Abstract: In this report we demonstrate that a defective herpes simplex virus type one (HSV-1) vector can express enzymatically active tyrosine hydroxylase in cultured striatal cells that are thereby converted into l -DOPA-producing cells. A human tyrosine hydroxylase cDNA (form II) was inserted into an HSV-1 vector (pHSVth) and packaged into virus particles using an HSV-1 strain 17 mutant in the immediate early 3 gene (either ts K or D30EBA) as helper virus. Cultured fibroblasts were infected with pHSVth and 1 day later tyrosine hydroxylase immunoreactivity and tyrosine hydroxylase enzyme activity were observed. The tyrosine hydroxylase enzyme activity directed the production of l -DOPA. pHSVth infection of striatal cells in dissociated cell culture resulted in expression of tyrosine hydroxylase RNA and tyrosine hydroxylase immunoreactivity. Release of l -DOPA and low levels of dopamine were observed from cells in pHSVth-infected striatal cultures. Expression of tyrosine hydroxylase and release of catecholamines were maintained for at least 1 week after infection.     

Mast cells express tyrosine hydroxylase and store dopamine in a serglycin-dependent manner

Here we show that mast cells contain dopamine and that mast cell activation causes dopamine depletion, indicating its presence within secretory granules. Dopamine storage increased during mast cell maturation from bone marrow precursors, and was dependent on the presence of serglycin. Moreover, the expression of tyrosine hydroxylase, the key enzyme in dopamine biosynthesis, was induced during mast cell maturation; histidine decarboxylase and tryptophan hydroxylase 1 were also induced. Mast cell activation caused a robust induction of histidine decarboxylase, but no stimulation of tyrosine hydroxylase or tryptophan hydroxylase 1 expression. The present study points toward a possible role of dopamine in mast cell function.