Dopamine Metabolite 3,4-dihydroxyphenylacetic Acid Levels Research Articles

Inhibition of phosphorylated tyrosine hydroxylase attenuates ethanol-induced hyperactivity in adult zebrafish (Danio rerio)

Zebrafish have been successfully employed in the study of the behavioural and biological effects of ethanol. Like in mammals, low to moderate doses of ethanol induce motor hyperactivity in zebrafish, an effect that has been attributed to the activation of the dopaminergic system. Acute ethanol exposure increases dopamine (DA) in the zebrafish brain, and it has been suggested that tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis, may be activated in response to ethanol via phosphorylation. The current study employed tetrahydropapaveroline (THP), a selective inhibitor of phosphorylated tyrosine hydroxylase, for the first time, in zebrafish. We treated zebrafish with a THP dose that did not alter baseline motor responses to examine whether it can attenuate or abolish the effects of acute exposure to alcohol (ethanol) on motor activity, on levels of DA, and on levels of dopamine's metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). We found that 60-minute exposure to 1% alcohol induced motor hyperactivity and an increase in brain DA. Both of these effects were attenuated by pre-treatment with THP. However, no differences in DOPAC levels were found among the treatment groups. These findings suggest that tyrosine hydroxylase is activated via phosphorylation to increase DA synthesis during alcohol exposure in zebrafish, and this partially mediates alcohol's locomotor stimulant effects. Future studies will investigate other potential candidates in the molecular pathway to further decipher the neurobiological mechanism that underlies the stimulatory properties of this popular psychoactive drug.

Isotopic reinforcement of essential polyunsaturated fatty acids diminishes nigrostriatal degeneration in a mouse model of Parkinson's disease

Oxidative damage of membrane polyunsaturated fatty acids (PUFA) is thought to play a major role in mitochondrial dysfunction related to Parkinson's disease (PD). The toxic products formed by PUFA oxidation inflict further damage on cellular components and contribute to neuronal degeneration. Here, we tested the hypothesis that isotopic reinforcement, by deuteration of the bisallylic sites most susceptible to oxidation in PUFA may provide at least partial protection against nigrostriatal injury in a mouse model of oxidative stress and cell death, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model. Mice were fed a fat-free diet supplemented with saturated acids, oleic acid and essential PUFA: either normal, hydrogenated linoleic (LA, 18:2n−6) and α-linolenic (ALA, 18:3n−3) or deuterated 11,11-D2-LA and 11,11,14,14-D4-ALA in a ratio of 1:1 (to a total of 10% mass fat) for 6 days; each group was divided into two cohorts receiving either MPTP or saline and then continued on respective diets for 6 days. Brain homogenates from mice receiving deuterated PUFA (D-PUFA) vs. hydrogenated PUFA (H-PUFA) demonstrated a significant incorporation of deuterium as measured by isotope ratio mass-spectrometry. Following MPTP exposure, mice fed H-PUFA revealed 78.7% striatal dopamine (DA) depletion compared to a 46.8% reduction in the D-PUFA cohort (as compared to their respective saline-treated controls), indicating a significant improvement in DA concentration with D-PUFA. Similarly, higher levels of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were detected in MPTP-exposure mice administered D-PUFA; however, saline-treated mice revealed no change in DA or DOPAC levels. Western blot analyses of tyrosine hydroxylase (TH) confirmed neuroprotection with D-PUFA, as striatal homogenates showed higher levels of TH immunoreactivity in D-PUFA (88.5% control) vs. H-PUFA (50.4% control) in the MPTP-treated cohorts. In the substantia nigra, a significant improvement was noted in the number of nigral dopaminergic neurons following MPTP exposure in the D-PUFA (79.5% control) vs. H-PUFA (58.8% control) mice using unbiased stereological cell counting. Taken together, these findings indicate that dietary isotopic reinforcement with D-PUFA partially protects against nigrostriatal damage from oxidative injury elicited by MPTP in mice.

Alterations in amphetamine‐stimulated dopamine overflow due to the Nurr1‐null heterozygous genotype and postweaning isolation

The nuclear receptor Nurr1 functions to regulate dopamine neurotransmission, as Nurr1-null heterozygous (+/-) mice have alterations in dopamine function and, when raised in isolation immediately after weaning, have disruptions in sensorimotor gaiting, a behavior altered in schizophrenia and modulated by dopamine neurotransmission. The goal of this study was to determine nigrostriatal and mesoaccumbens dopamine neurotransmission using microdialysis in +/- and wild-type (+/+) mice raised in groups or isolation. In the striatum, isolation significantly reduced amphetamine-stimulated dopamine overflow and levels of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). The +/- genotype alone caused a small, nonsignificant reduction in basal dopamine levels but a significant reduction in basal DOPAC levels. In the nucleus accumbens shell, the +/- genotype elevated basal dopamine levels. Isolation had genotype specific effects, causing an elevation in amphetamine-stimulated dopamine overflow in +/- mice but a reduction in +/+ mice, resulting in a large difference in stimulated dopamine overflow when comparing the +/+ and +/- isolated mice. These data indicate that a deletion of a single allele of Nurr1, which produces only subtle changes alone, when coupled with a developmental stressor, can dramatically alter mesoaccumbens dopamine neurotransmission. These observations demonstrate how the combination of genetic predisposition and an environmental insult during development can cause dysfunction of dopamine neurotransmission and could contribute to diseases such as schizophrenia or attention deficit hyperactivity disorder.

High Susceptibility to Experimental Myopia in a Mouse Model with a Retinal ON Pathway Defect

Nob mice share the same mutation in the Nyx gene that is found in humans with complete congenital stationary night blindness (CSNB1). Nob mutant mice were studied to determine whether this defect resulted in myopia, as it does in humans. Refractive development was measured in unmanipulated wild-type C57BL/6J (WT) and nob mice from 4 to 12 weeks of age by using an infrared photorefractor. The right eye was form deprived by means of a skull-mounted goggling apparatus at 4 weeks of age. Refractive errors were recorded every 2 weeks after goggling. The content of dopamine and the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were measured by HPLC with electrochemical detection (HPLC-ECD) in retinas of nob and WT mice under light- and dark-adapted conditions. The nob mice had greater hyperopic refractive errors than did the WT mice under normal visual conditions, until 12 weeks of age when both strains had similar refractions. At 6 weeks of age, refractions became less hyperopic in the nob mice but continued to become more hyperopic in the WT mice. After 2 weeks of form deprivation (6 weeks of age), the nob mice displayed a significant myopic shift (~4 D) in refractive error relative to the opposite and control eyes, whereas WT mice required 6 weeks of goggling to elicit a similar response. As expected with loss of ON pathway transmission, light exposure did not alter DOPAC levels in the nob mice. However, dopamine and DOPAC levels were significantly lower in the nob mice compared with WT. Under normal laboratory visual conditions, only minor differences in refractive development were observed between the nob and WT mice. The largest myopic shift in the nob mice resulted after form deprivation, suggesting that visual pathways dependent on nyctalopin and/or abnormally low dopaminergic activity play a role in regulating refractive development. These findings demonstrate an interaction of genetics and environment in refractive development.

Cerebrospinal fluid 3,4-dihydroxyphenylacetic acid level after tolcapone administration as an indicator of nigrostriatal degeneration

The development of reliable biological markers of nigrostriatal degeneration has important implications from both experimental and clinical viewpoints, since such biomarkers could be used for diagnostic and monitoring purposes in models of parkinsonism as well as in Parkinson’s disease patients. In this study, levels of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were measured in the cerebrospinal fluid (CSF) of normal and parkinsonian squirrel monkeys in order to assess their reliability as indicators of nigrostriatal injury. In particular, we tested the hypothesis that these measurements may become more accurate by inhibiting catecholamine-O-methyltransferase (COMT) activity and therefore blocking the conversion of DOPAC to homovanillic acid. Oral administration of the COMT inhibitor tolcapone (2 doses of 15 mg/kg each with a 4-h interval) significantly reduced enzyme activity in the monkey brain. Tolcapone treatment enhanced CSF DOPAC concentrations in unlesioned animals (by approximately four times) as well as monkeys rendered parkinsonian after severe nigrostriatal dopaminergic injury caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Importantly, however, COMT inhibition greatly magnified the differences in CSF DOPAC levels between control and parkinsonian monkeys, since MPTP-induced DOPAC depletion was 35% in the absence vs >60% in the presence of tolcapone. Thus, tolcapone administration enhances the detection of DOPAC in the CSF and, by doing so, improves the reliability of CSF DOPAC as a marker of nigrostriatal degeneration.

Changes in dopamine, norepinephrine and serotonin levels in the pituitary, telencephalon and hypothalamus during gonadal development of male Lutjanus argentiventris (Teleostei)

Biogenic amines are important in the regulation of certain aspects of teleost reproduction, but relatively little is known about variations in their levels in the nervous system during the reproductive cycle. The aim of this study was to investigate changes in the levels of dopamine (DA), norepinephrine (NE), serotonin (5-HT), the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) and the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the pituitary and brain (hypothalamus and telencephalon) of the adult male of the tropical marine yellow snapper Lutjanus argentiventris during the reproductive cycle (quiescent, prespawning and spawning periods). Gonad weight, gonad/body weight ratio and plasma-testosterone levels increased steadily throughout gonadal development, but 17β-estradiol levels did not vary significantly. Pituitary DA and DOPAC levels and the DOPAC/DA ratio declined with gonadal development, whereas hypothalamic DOPAC level and DOPAC/DA ratio increased. In the telencephalon, DA and DOPAC levels and DOPAC/DA ratio all peaked in the prespawning period; however, DA level was lowest in the spawning period, whereas DOPAC level and DOPAC/DA ratio were both lowest in the quiescent period. Telencephalic NE, 5-HT and 5-HIAA levels likewise all peaked in the prespawning period, and were lowest in the spawning period. Telencephalic 5-HIAA/5-HT ratio peaked in the quiescent period and was lowest in the spawning period. No significant variation was observed either in pituitary NE level or in hypothalamic NE, 5-HT and 5-HIAA levels. The present results indicate that males of L. argentiventris show a decline in pituitary DA activity during gonadal development. We discuss the possible involvement of biogenic amines in the regulation of GTH II secretion, through direct action on the pituitary or indirect modulation of gonadotrophin-releasing hormone neuron activity.

Neurochemical predisposition to self-administer morphine in rats

Using in vivo microdialysis, this study attempted to determine whether a neurochemical predisposition to self-administer morphine could be identified. Extracellular levels of dopamine and its metabolites were measured bilaterally in the mesocorticolimbic and nigrostriatal systems of naive rats that were subsequently trained to self-administer morphine intravenously. There were several significant relationships between dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels and rates of morphine self-administration during both acquisition and asymptotic phases of testing. DOPAC and HVA levels in the nucleus accumbens were positively correlated with self-administration rates during both phases. Higher DOPAC and HVA levels in the striatum were inversely correlated with self-administration rates during the asymptotic phase whereas hemispheric asymmetries in striatal metabolite levels were inversely correlated with self-administration during the acquisition phase. DOPAC and HVA levels in the right but not in the left side of the medial prefrontal cortex were positively correlated with self-administration rates during the acquisition phase; right/left asymmetries in cortical metabolite levels were also corelated with acquisition rates. There were no significant relationships between neurochemical indices and rates of bar-pressing for water. These results suggest that the normal variability in drug seeking behavior is at least in part attributable to individual differences in the organization and activity of brain dopamine systems. Furthermore, different mechanisms appear to be responsible for the initiation and maintenance of morphine intake: DA release in the nucleus accumbens appears to be a critical component of both mechanisms; DA release in the striatum appears to modulate maintenance and, in relationship to striatal lateralization, modulate initiation; DA release in the right but not in the left medial prefrontal cortex appears to be an important predictor of initiation.

Different effects of the calcium antagonists nimodipine and flunarizine on dopamine metabolism in the rat brain.

The effect of two calcium antagonists, nimodipine and flunarizine, on striatal dopamine (DA) metabolism in rats was compared. Flunarizine (5-20 mg/kg i.p.) caused a dose-dependent increase in the DA metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC) in the caudate nucleus. Following the 20 mg/kg dose, DOPAC levels were maximally elevated by about 50% from 2 to 12 hrs after treatment. On the contrary, nimodipine at the dose of 20 mg/kg i.p. produced a modest decrease in DOPAC levels. Neither calcium antagonist modified DA content. However, both nimodipine and flunarizine, at the dose of 20 mg/kg, markedly reduced the accumulation of DOPAC in the caudate nucleus induced by haloperidol (1 mg/kg). It is suggested that flunarizine, but not nimodipine, has a neuroleptic-like action, whereas the two calcium antagonists have in common the ability to attenuate the hyperactivity of DA neurons.

Dopamine synthesis and metabolism in rhesus monkey retina: development, aging, and the effects of monocular visual deprivation.

The normal postnatal development, the influence of age, and the effects of visual deprivation on the dopamine system in the retina of rhesus monkeys were examined. The lowest level of retinal dopamine was found at birth. By 3-4 weeks of age, the dopamine concentration had more than doubled. This level remained relatively constant in the retinas of older infants and of adult monkeys up to 34 yr of age. The level of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) and the activity of tyrosine hydroxylase did not significantly change as a function of age during the postnatal life span. Monocular occlusion of newborn or infant monkeys for 1-15 months with opaque contact lenses resulted in decreases in the retinal concentrations of dopamine and DOPAC relative to the concentrations in the same animals' unoccluded eyes. Occlusion also resulted in a lower level of tyrosine hydroxylase activity in the retina. Monocular eyelid suture from birth to 15 months of age resulted in less consistent alterations of retinal dopamine and DOPAC levels; tyrosine hydroxylase activity, however, was consistently reduced by lid suture. Thus, dopamine synthesis and metabolism, and the ontogenetic increase of the retinal dopamine level of rhesus monkey are reduced by light deprivation.

Hypothalamic monoamines associated with the cephalic phase insulin response.

This study elucidated the hypothalamic monoamine systems associated with the cephalic phase insulin release. Male Wistar rats conditioned to drink a glucose solution were killed 2 min after the onset of their scheduled feeding; control rats were killed at the same time. The ventromedial (VMH) and lateral (LH) portions of the hypothalamus were analyzed for the monoamines and their principal metabolites. Serum was assayed for insulin and glucose. The results showed that the experimental animals had significantly higher serum insulin levels than did the control animals, although glucose levels were unchanged. In the LH, there were increased levels of norepinephrine, its metabolite dihydroxyphenylethylene glycol (DHPG), and serotonin [(5-hydroxytryptamine (5-HT)]. In the VMH, 5-HT, its metabolite 5-hydroxyindoleacetic acid, and the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were all higher in the conditioned animals. 5-HT levels in the LH and DOPAC levels in the VMH were closely associated with serum insulin (r = 0.80 and 0.71, respectively). This study has defined, in the normal animal, changes in monoamines in specific areas of the hypothalamus associated with the cephalic phase insulin release.

In vivo measurement of extracellular dopamine and DOPAC in rat striatum after various dopamine-releasing drugs implications for the origin of extracellular DOPAC

In order to further examine the likely origin of the dopamine (DA) metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), certain drugs known to release DA from different intraneuronal pools were tested for their effects on extracellular striatal DA and DOPAC levels by means of brain microdialysis in the halothane-anaesthetized rat. Amphetamine (10 −6 and 10 −5 M), nomifensine (10 −5 M), potassium chloride (30 and 60 mM), methylphenidate (10 −5 and 10 −4 M) and tyramine (10 −5 M), when added to the perfusion medium and administered locally into the striatum via the dialysis membrane, increased the level of DA in striatal perfusates during the 20 min of application. In comparison, the level of DOPAC in the perfusates was decreased by both amphetamine (10 −5 M) and potassium chloride (60 mM), but was not significantly changed by nomifensine, methylphenidate or tyramine. The effect of amphetamine (10 −6 M) and nomifensine (10 −5 M) on DA and DOPAC levels was further studied by administering the drugs over a longer period of time (3×20 min). Although both of these treatments produced a similar increase of DA, only amphetamine reduced the levels of DOPAC. DA (10 −4 but not 10 −5 M) increased the levels of DOPAC but this effect was also seen in DA-denervated animals. These data indicate that when the DA nerve terminal is exposed to drugs which release newly synthesized DA, DOPAC declines possible because intraneuronal monoamine oxidase is deprived of its main substrate. We suggest that these findings support the hypothesis that a major portion of the DA metabolite, DOPAC, is derived from an intraneuronal pool of newly synthesized DA.

Footshock and conditioned stress increase 3, 4-dihydroxyphenylacetic acid (DOPAC) in the ventral tegmental area but not substantia nigra

The effects of stress on dopamine (DA) metabolism in the mesencephalic DA cell body areas and DA terminal field regions were examined. Both mild footshock stress and exposure to a neutral stimulus previously paired with footshock resulted in a selective increase in the levels of the DA metabolite 3, 4-dihydroxyphenylacetic acid (DOPAC) in the prefrontal cortex as has been previously reported. Footshock stress also resulted in a slight but significant increase in DOPAC levels in the olfactory tubercles. DOPAC levels were selectively increased in the A10 cell body area (ventral tegmental area) but not A9 region (substantia nigra) by both footshock and the conditioned stress paradigm. These data indicate that the cell bodies of origin of the mesocortical dopaminergic system are activated by stress in contrast to those DA neurons innervating the striatum. It appears that mesocortical dopaminergic neurons exhibit different regulatory features than mesolimbic or nigrostriatal neurons.

Transneuronal regulation of tyrosine hydroxylase expression in olfactory bulb of mouse and rat

Peripheral afferent input regulates the expression of dopaminergic properties in a population of local circuit intrinsic neurons of the rodent olfactory bulb. Lesions of the olfactory receptor neurons produced in the mouse by intranasal irrigation with either ZnSO4 or Triton X-100 and in the rat by surgical deafferentation or axotomy are associated with a decrease in the levels of dopamine (DA), the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), the activity of the enzyme tyrosine hydroxylase (TH), bulb weight and an increase in norepinephrine (NE) levels in the olfactory bulb. The anatomical correlates of the biochemical sequelae of deafferentation of olfactory bulb DA neurons were studied using immunohistochemical techniques to localize TH. Within 3 to 4 weeks all lesions produced a dramatic and uniform reduction in TH staining of the juxtaglomerular DA neurons and their processes which was paralleled by a reduction in DA and DOPAC levels and bulb weight. Seven weeks following reversible chemical lesions produced by Triton X-100, DA and DOPAC levels and tissue weight as well as TH staining in the juxtaglomerular neurons returned to control levels. These observations suggested that DA neurons remained present even when not demonstrable with TH antibodies. Additional evidence for the continued presence of the DA neurons was the ability of the olfactory bulbs from both lesioned mouse and rat to synthesize DA from exogenously administered L-3,4-dihydroxyphenylalanine (l-DOPA). These data suggested that the decrease in DA levels and TH staining in the olfactory bulb following lesions of the olfactory receptor neurons were produced by transneuronal mechanisms since there was no direct injury of the bulb. Furthermore, the demonstration that following reinnervation, catecholamine synthetic capacity is restored suggests that the juxtaglomerular dopamine neurons remain in the bulb and that afferent receptor input is required for expression of TH enzyme.

Subcellular distribution of dopamine metabolites and their elimination from the rat brain

A compartmentation of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) was found in the rat brain when its synthesis was inhibited by the monoamine oxidase inhibitor pargyline. Homovanillic acid (HVA) did not show such compartmentation after pargyline administration, but similar compartmentation was observed for HVA after treatment with tropolone which is an inhibitor of catechol- o-methyl transferase. The most rapid HVA compartment was eliminated after inhibition of the active transport with probenecid. No anatomical substrate could be found for these compartments. A relatively larger portion of DOPAC than of HVA was found to be associated with the crude mitochondrial/synaptosomal fraction when the subcellular distribution of these two compounds was investigated. Intraventricular injection of DOPAC in conjunction with either probenecid or tropolone resulted in increased DOPAC levels compared to intraventricular DOPAC alone, but no potentiating effect was observed when probenecid and tropolone were given in combination. It is concluded that the compartmentation of DOPAC is related to the HVA/DOPAC ratio in the various brain regions and the different metabolic pathways involved. The compartmentation of HVA shows that the acid is transported between different structures prior to removal from the brain. These results indicate that great care must be taken in estimating dopamine metabolic turnover on the basis of the elimination of metabolites subsequent to the inhibition of dopamine synthesis.