Tyrosine Hydroxylase mRNA Levels Research Articles

Psychosocial stress-related changes in gene expression of norepinephrine biosynthetic enzymes in stellate ganglia of adult rats

In this study we investigated the changes in norepinephrine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) gene expression in the stellate ganglia of naive controls and long-term socially isolated (12 weeks) adult rats and the response of these animals to additional immobilization stress. Psychosocial stress produced a significant increase of both TH mRNA and DBH mRNA levels in stellate ganglia. Additional immobilization of long-term psychosocially stressed rats expressed no effect on gene expression of these enzymes. The results presented here suggest that psychosocial stress-induced increase in gene expression of norepinephrine biosynthetic enzymes in stellate ganglia may be connected to the increased risk of cardiovascular disease.

Interaction between Nicotine and Estradiol in Regulation of Gene Expression in Substantia Nigra (SN) in Animal Model of Parkinson's Disease (PD)

The incidence of PD is two fold higher in men than in women. Nicotine (N), which reduces the risk of PD, exhibits sex specific effects. We found that injections of estradiol benzoate (EB) to ovariectomized (OVX) rats change the sensitivity of GTP cyclohydrolase I (GTPCH), the rate‐limiting enzyme for biosynthesis of essential co‐factor for tyrosine hydroxylase (TH), to N. We hypothesize that N and EB have interactive effects on physiology of SN in rats with PD at the level of regulation of GTPCH gene.PD rats were generated by infusion of 6‐OHDA in striatum of OVX rats. The control group received ascorbic acid. 6‐OHDA rats were injected with EB or vehicle (V) once daily for 13 days. EB or V treated rats were divided into two subgroups and from 14th day, in addition to EB or V, were administered saline or N every 12 hrs. Rats with 6‐OHDA lesions developed forelimb and sensory‐motor deficits while those with EB itself, and in combination with N, improved apomorphine induced rotation behavior. TH and GTPCH mRNA levels were lower in the SN of 6‐OHDA rats compared to controls. N restored both mRNAs in SN only in EB treated rats, with significant interactive effects for GTPCH mRNA. The improvement might be mediated by regulation of nAChR subtype expression. Significant interactive effects of N and EB were found on mRNA levels of α3, α6, α7 subunits of nAChR in SN of the PD rats.Supported by American Parkinson Disease Association.

Regulation of catecholamine biosynthetic enzymes by nitric oxide

The neurotransmitters/neurohormones catecholamines (CA) are involved in the sympathetic control of arterial blood pressure and cardiac function. Recent studies show that nitric oxide (NO) can regulate the release of CA from the adrenal medulla. The in vitro PC12 cell line was used to examine the effect of NO on the regulation of the CA biosynthetic enzymes: tyrosine hydroxylase (TH), dopamine‐β‐hydroxylase (DBH) and phenylethanolamine N‐methyltransferase (PNMT). Results obtained from drug treatments with the NO donor, sodium nitroprusside (SNP) constitute an assessment of its effects in PC12 cells. Treatment of cells with SNP for 6 hours significantly increased TH and PNMT mRNA levels. Combination drug studies with SNP and intracellular kinase activators (forskolin, PMA, 8‐Br‐cGMP) and inhibitors for PKA (H‐89), cGMP (6‐anilinoquinone) PKG (DT‐2) and PKC (GF109203X) were also conducted. Increases in transcript levels for TH and PNMT were obtained under combination drug treatments of SNP and activators of PKA and PKG (p<0.05). mRNA transcript levels of TH, DBH and PNMT showed significant decreases when cells were pre‐treated with PKA, PKC and PKG inhibitors. Furthermore, preliminary transfections showed activation of the PNMT promoter by SNP treatment. Results from this study suggest that NO is capable of regulating CA biosynthetic enzymes, TH and PNMT via activation of the PKA and PKG pathways.

Effects of scoparone on dopamine biosynthesis and L‐DOPA‐induced cytotoxicity in PC12 cells

The effects of scoparone on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. PC12 cells treated with scoparone at concentrations of 100-200 microM showed a 128-136% increase in dopamine levels over the course of 24 hr. Scoparone significantly increased the secretion of dopamine into the culture medium. Under the same conditions, the activities of tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) were enhanced by treatment with 200 microM scoparone for 6-48 hr, but the activity of TH was regulated for a longer period than that of AADC. The intracellular levels of cyclic AMP and Ca(2+) were increased by treatment with 200 microM scoparone. The levels of TH mRNA and the phosphorylation of cyclic AMP-response element-binding protein (CREB) were also significantly increased by treatment with 200 microM scoparone. In addition, scoparone at a concentration of 200 microM stimulated the activities of cyclic AMP-dependent protein kinase (PKA), protein kinase C (PKC), and Ca(2+)/calmodulin kinase II (CaMK II). Finally, pretreatment with 200 microM scoparone reduced the cytotoxicity induced by L-DOPA (20-100 microM) at 24 hr. These results suggest that scoparone enhances dopamine biosynthesis by regulating TH activity and TH gene expression, which is mediated by the PKA, CREB, PKC, and CaMK II pathways, and protects cells from L-DOPA-induced cytotoxicity by inducing cyclic AMP-PKA systems in PC12 cells.

Minimal Residual Disease in Peripheral Blood Stem Cell Harvests From High-risk Neuroblastoma Patients

We investigated whether detection of minimal residual disease (MRD) in peripheral blood stem cells (PBSC) by using tyrosine hydroxylase (TH) expression could predict outcome of patients with advanced neuroblastoma. Quantitative real time polymerase chain reaction was performed for the detection of tumor contamination using TH messenger ribonucleic acid (mRNA) and correlated to clinical parameters and outcome in 45 high-risk neuroblastoma patients. High TH expression was detected in PBSC harvests obtained from 26 out of 45 (58%) patients. We did not find a significant correlation between MYCN status, DNA index or primary tumor site, and the TH mRNA level. Patients harboring high TH expression had reduced progression-free survival (PFS) (23%) versus those with low/negative TH expression (43%), although these results were not statistically significant. No significant correlation was observed between TH expression and overall survival. The correlation between an unfavorable outcome and high TH expression in patients' PBSC harvests was not significant. This indicates a need to increase sample size in a long-term clinical outcome study to clarify the importance of TH mRNA contamination in PBSC.

Regulation of Gene Expression of Catecholamine Biosynthetic Enzymes in Dopamine‐β‐Hydroxylase‐ and CRH‐Knockout Mice Exposed to Stress

Norepinephrine-deficient mice harbor a disruption of the gene for dopamine-beta-hydroxylase (DBH-KO). Corticotropin-releasing hormone knockout mice (CRH-KO) have markedly reduced HPA activity. The aim of the present work was to study how deficiency of DBH and CRH would affect tyrosine hydroxylase (TH), DBH, and phenylethanolamine N-methyltransferase (PNMT) gene expression and protein levels in the adrenal medulla (AM) and stellate ganglia (SG) of control and stressed mice. Both in AM and SG, single immobilization significantly increased TH and DBH mRNA and protein levels both in wild-type (WT) and CRH-KO mice. On the other hand, the stress-triggered increase in PNMT mRNA and protein levels seen in WT mice was absent in CRH-KO mice. DBH-KO mice are more sensitive to stress but survive a single 2 h restraint stress in a tube. The increase in TH mRNA levels induced by restraint stress in WT was not observed in DBH-KO mice. PNMT mRNA and especially PNMT protein levels were significantly elevated in AM of DBH-KO mice. In SG of DBH-KO mice, TH mRNA levels were not affected; however, PNMT gene expression was highly elevated. Thus, disruption of the DBH gene surprisingly blocks the stress-induced elevation of TH mRNA levels in AM but increases PNMT gene expression in both AM and SG. Our data indicate that adrenergic signaling is required for stress-induced increase in TH mRNA and that this signaling restrains stress-induced increase in PNMT mRNA. They also confirm that the HPA system plays a crucial role in the stress-induced regulation of PNMT gene expression.

Stress‐induced Changes in Tyrosine Hydroxylase Gene Expression in Rat Hypothalamic Paraventricular, Periventricular, and Dorsomedial Nuclei

Particular hypothalamic nuclei--the paraventricular (PVN), periventricular (PeV), and dorsomedial (DMN)--are important centers regulating neuroendocrine and autonomic systems during stress. The present study investigated gene expression of catecholamines (CA)-synthesizing enzymes and protein levels, especially of tyrosine hydroxylase (TH), in response to acute (120 min) and repeated (7 x 120 min) immobilization (IMO) stress. Immunohistochemical staining revealed many TH-positive perikarya in the PeV, less in the DMN, and a few in the PVN. Analysis of microdissected tissue samples obtained from frozen brain sections by quantitative competitive PCR method revealed TH mRNA in the PVN, periventricular subdivision of the PVN (PePVN), and DMN. TH mRNA and TH protein levels were increased in all the nuclei investigated, in both acute and repeated stress conditions. Interruption of ascending CA pathways from the brain stem areas abolished TH gene expression in the PVN and DMN in response to stress. The present data suggest that cell groups expressing TH gene are present in all three hypothalamic nuclei studied and are highly activated by stress. The regulation of the majority of these TH-positive cells during stress is under modulatory control by brain stem or spinal cord areas.

Ethanol and Stress Activate Catecholamine Synthesis in the Adrenal

Ethanol consumption and mental stress activate the sympathetic nervous system, which can adversely affect bone. We compared six groups of 10 young adult rats, three with and three without 2 h daily restraint stress. Two groups consumed food and water ad libitum, two received food and 6% (w/v) ethanol as drinking water, and two received the amount of food consumed by ethanol rats the previous day plus water ad libitum (pairfed). After 6 weeks, rats were killed. Plasma, femurs, lumbar vertebrae, and adrenals were harvested. Femoral dimensions were measured and biomechanical properties were tested by three-point bending. Plasma osteocalcin, vertebral osteocalcin mRNA levels, and adrenomedullary tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyl transferase (PNMT) mRNA levels were quantified. Daily restraint decreased weight gain and femoral length compared to dietary controls, and appeared to partially preserve bone strength, especially in calorie-restricted pairfed rats. Femoral strength was significantly affected by treatment in that bones of pairfed controls were weakest, ethanol drinkers were intermediate, and ad libitum restrained were strongest. Femoral yield load, displacement, and work at yield load were negatively correlated with TH and DBH mRNA levels, but not PNMT, suggesting a negative influence of norepinephrine. Plasma osteocalcin and dry weight of lumbar 3-5 vertebrae were unaffected; however, osteocalcin mRNA in second lumbar vertebrae was positively correlated with TH, DBH, and PNMT levels. Ethanol consumption at this level had little effect on femur morphology or strength. In contrast, the data suggested possible stimulation rather than inhibition of vertebral bone formation.

Amphetamine‐induced locomotion and gene expression are altered in BDNF heterozygous mice

Administration of amphetamine overstimulates medium spiny neurons (MSNs) by releasing dopamine and glutamate from afferents in the striatum. However, these afferents also release brain-derived neurotrophic factor (BDNF) that protects striatal MSNs from overstimulation. Intriguingly, all three neurochemicals increase opioid gene expression in MSNs. In contrast, striatal opioid expression is less in naive BDNF heterozygous (BDNF(+/-)) vs. wild-type (WT) mice. This study was designed to determine whether partial genetic depletion of BDNF influences the behavioral and molecular response to an acute amphetamine injection. An acute injection of amphetamine [5 mg/kg, intraperitoneal (i.p.)] or saline was administered to WT and BDNF(+/-) mice. WT and BDNF(+/-) mice exhibited similar locomotor activity during habituation, whereas BDNF(+/-) mice exhibited more prolonged locomotor activation during the third hour after injection of amphetamine. Three hours after amphetamine injection, there was an increase of preprodynorphin mRNA in the caudate putamen and nucleus accumbens (Acb) and dopamine D(3) receptor mRNA levels were increased in the Acb of BDNF(+/-) and WT mice. Striatal/cortical trkB and BDNF, and mesencephalic tyrosine hydroxylase mRNA levels were only increased in WT mice. These results indicate that BDNF modifies the locomotor responses of mice to acute amphetamine and differentially regulates amphetamine-induced gene expression.

Valproic acid regulates catecholaminergic pathways by concentration-dependent threshold effects on TH mRNA synthesis and degradation

The spectrum of neurological conditions and psychiatric disorders affected by valproic acid (VPA) ranges from control of seizure and mood disorders to migraine, neuropathic pain, and even congenital malformations and autism. While widely used clinically, the mechanism(s) of action of VPA is not completely understood. Emerging evidence indicates that brain noradrenergic systems contribute to the symptoms of mood disorders and may involve regulation of tyrosine hydroxylase (TH) expression, the rate-limiting enzyme in the biosynthesis of dopamine, norepinephrine and epinephrine. We previously showed that the structurally related short chain fatty acid sodium butyrate (SB) induces TH transcription and alters TH mRNA stability in PC12 cells. The present study was undertaken to determine whether the branched short chain fatty acid VPA could also regulate TH gene expression in vitro. Similar to SB, VPA induced TH transcription at all concentrations tested. VPA-stimulated transcription was significantly attenuated by introducing point mutations in either the canonical cAMP- or in the butyrate-response elements of the TH promoter; or by co-expression of dominant-negative forms of CREB. As with SB, increasing concentrations of VPA demonstrated opposing effects on TH mRNA and protein abundance: elevation of both at low (0.1 mM) but attenuation at concentrations higher than 0.5 mM. This concentration-dependence is consistent with a novel and previously unrecognized cellular/molecular drug regulatory step at the level of TH mRNA stability. Thus, the therapeutic efficacy of VPA might be related to its ability to regulate TH mRNA and protein levels, and thereby central catecholaminergic-dependent behavioral pathways.

Effects of asimilobine on dopamine biosynthesis and l-DOPA-induced cytotoxicity in PC12 cells

The effects of asimilobine, an aporphine isoquinoline alkaloid, on dopamine biosynthesis and l-DOPA-induced cytotoxicity in PC12 cells were investigated. Asimilobine at concentration ranges of 0.05–0.2 μM showed a significant inhibition of intracellular dopamine levels for 24 h in a concentration-dependent manner with an IC50 value of 0.13 μM. Asimilobine at 0.15 μM inhibited tyrosine hydroxylase (TH) and aromatic l-amino acid decarboxylase (AADC) activities at 24 h (73.2% inhibition of TH activity): the inhibition of TH activity was stronger and longer than that of AADC activity. Asimilobine also decreased TH mRNA levels and intracellular cyclic AMP levels, but not the basal Ca2 + concentrations. In addition, asimilobine at 0.05–5.0 μM, but not 10 μM, did not alter cell viability toward PC12 cells. A non-cytotoxic asimilobine (0.15 μM) associated with l-DOPA (20, 50, and 100 μM) for 24 h inhibited l-DOPA-induced increases in dopamine levels and enhanced l-DOPA-induced cell death when compared with l-DOPA alone. These results suggest that asimilobine inhibits dopamine biosynthesis by mainly reducing the TH activity and TH mRNA expression, and enhances l-DOPA-induced cytotoxicity in PC12 cells.

The Harman and Norharman Reduced Dopamine Content and Induced Cytotoxicity in PC12 Cells

The effects of harman and norharman on dopamine content and L-DOPA-induced cytotoxicity were investigated in PC12 cells. Harman and norharman decreased the intracellular dopamine content for 24 h. The <TEX>$IC_{50}$</TEX> values of harman and norharman were 20.4 <TEX>${\mu}M$</TEX> and 95.8 <TEX>${\mu}M$</TEX>, respectively. Tyrosine hydroxylase (TH) activity and TH mRNA levels were also decreased by 20 <TEX>${\mu}M$</TEX> harman and 100 <TEX>${\mu}M$</TEX> norharman. Under the same conditions, the intracellular cyclic AMP levels were decreased by harman and norharman. In addition, harman and norharman at concentrations higher than 80 <TEX>${\mu}M$</TEX> and 150 <TEX>${\mu}M$</TEX> caused cytotoxicity at 24 h in PC12 cells. Non-cytotoxic ranges of 10-30 <TEX>${\mu}M$</TEX> harman and 50-150 <TEX>${\mu}M$</TEX> norharman inhibited L-DOPA (20-50 <TEX>${\mu}M$</TEX>)-induced increases of dopamine content at 24 h. Harman at 20-150 <TEX>${\mu}M$</TEX> and norharman at 100-300 <TEX>${\mu}M$</TEX> also enhanced LDOPA (20-100 <TEX>${\mu}M$</TEX>)-induced cytotoxicity at 24 h. These results suggest that harman and norharman decrease dopamine content by reducing TH activity and aggravate L-DOPA-induced cytotoxicity in PC12 cells.

Glial Cell Line-derived Neurotrophic Factor Reverses Ethanol-mediated Increases in Tyrosine Hydroxylase Immunoreactivity via Altering the Activity of Heat Shock Protein 90

We previously found that glial cell line-derived neurotrophic factor (GDNF) in the midbrain ventral tegmental area (VTA) negatively regulates alcohol drinking (He, D. Y., McGough, N. N., Ravindranathan, A., Jeanblanc, J., Logrip, M. L., Phamluong, K., Janak, P. H., and Ron, D. (2005) J. Neurosci. 25, 619-628). Several studies suggest a role for GDNF in the regulation of tyrosine hydroxylase (TH) levels in the midbrain (Georgievska, B., Kirik, D., and Bjorklund, A. (2004) J. Neurosci. 24, 6437-6445). Up-regulation of TH levels has been reported as a hallmark of biochemical adaptations to in vivo chronic exposure to drugs of abuse, including ethanol (Ortiz, J., Fitzgerald, L. W., Charlton, M., Lane, S., Trevisan, L., Guitart, X., Shoemaker, W., Duman, R. S., and Nestler, E. J. (1995) Synapse 21, 289-298). We hypothesized that GDNF plays an important role in regulating prolonged ethanol-mediated increases in TH protein levels. Using the SH-SY5Y dopaminergic-like cell line, we found that the increase in TH levels in the presence of ethanol required the activation of the cAMP/PKA pathway and was reversed by GDNF. Ethanol treatment did not alter the mRNA level or protein translation of TH, but enhanced the stability of the protein that was decreased by GDNF. Interestingly, we observed that ethanol treatment resulted in an increase in TH association with the chaperone heat shock protein (HSP90) that was mediated by the cAMP/PKA pathway and inhibited by GDNF. Taken together, these data suggest that prolonged ethanol exposure leads to increased association of TH and HSP90 via the cAMP/PKA pathway, resulting in the stabilization and subsequent accumulation of TH. GDNF reverses this ethanol-mediated adaptation by inhibiting the interaction of TH with HSP90.

Effect of age on angiotensin II-mediated downregulation of adrenomedullary catecholamine biosynthetic enzymes

Expression of catecholamine biosynthesizing enzymes, tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DβH) increase with age in the adrenal medulla, however, the underlying mechanisms are unclear. In the present study, we examined the effect of peripheral angiotensin II (AngII) on the expression of TH and DβH, in the adrenal medulla of young (6 mo) and old (23 mo) Fischer-344 rats. Saline or AngII (230 ng/kg/min sc) was infused for 3 days using osmotic minipumps. Adrenomedullary TH and DβH mRNA levels increased significantly with age, and while AngII reduced the expression of these enzymes in young animals, it had no such effect in the old animals. Neuropeptide Y (NPY), which is co-released with catecholamines in the adrenal medulla and stimulates the synthesis of TH and DβH, was also upregulated with age and downregulated in response to AngII in young rats. However, in the old animals, the already elevated NPY expression remained unchanged following AngII treatment. This data indicate that the hypertensive effect of peripheral AngII is compensated by an inhibition of adrenomedullary catecholamine biosynthesis in young animals, but this mechanism is impaired in senescence, potentially contributing to the age-related increase in catecholamine biosynthesis.

Effects of harman and norharman on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells

The effects of harman and norharman on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Harman and norharman at a concentration of 20 μM and 100 μM showed 49.4% and 49.5% inhibition of dopamine content for 48 h, respectively. The IC 50 values of harman and norharman were 21.2 μM and 103.3 μM. Dopamine content, tyrosine hydroxylase (TH) activity and TH mRNA levels were decreased during the first 6 h, maintained for up to 48 h and then gradually recovered at 72 h after exposure to 20 μM harman and 100 μM norharman. Under the same conditions, the intracellular cyclic AMP levels and Ca 2+ concentrations were also decreased by harman and norharman. In addition, harman and norharman at concentrations higher than 80 μM and 150 μM caused cytotoxicity at 48 h in PC12 cells. Non-cytotoxic ranges of 10–30 μM harman and 50–150 μM norharman inhibited L-DOPA (20–50 μM)-induced increases in dopamine content at 48 h. Harman at 20–150 μM and norharman at 100–300 μM also enhanced L-DOPA (20–100 μM)-induced cytotoxicity at 48 h with an apoptotic process. These results suggest that harman and norharman inhibit dopamine biosynthesis by reducing TH activity and enhance L-DOPA-induced cytotoxicity in PC12 cells.

Silencing α-Synuclein Gene Expression Enhances Tyrosine Hydroxylase Activity in MN9D Cells

α-Synuclein has been implicated in the pathogenesis of Parkinson’s disease (PD). Previous studies have shown that α-synuclein is involved in the regulation of dopamine (DA) metabolism, possibly by down-regulating the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in DA biosynthesis. In this study, we constructed α-synuclein stably silenced MN9D/α-SYN− cells by vector mediated RNA interference and examined its effects on DA metabolism. We found that there were no significant differences in TH protein and mRNA levels between MN9D, MN9D/α-SYN− and MN9D/CON cells, suggesting that silencing α-synuclein expression does not affect TH gene expression. However, significant increases in phosphorylated TH, cytosolic 3, 4-dihydroxyphenylalanine (l-DOPA) and DA levels were observed in MN9D/α-SYN− cells. Our data show that TH activity and DA biosynthesis were enhanced by down-regulation of α-synuclein, suggesting that α-synuclein may act as a negative regulator of cytosolic DA. With respect to PD pathology, a loss of functional α-synuclein may result in increased DA levels in neurons that may lead to cell injury or even death.

Activation of Tyrosine Hydroxylase mRNA Translation by cAMP in Midbrain Dopaminergic Neurons

During prolonged stress or chronic treatment with neurotoxins, robust compensatory mechanisms occur that maintain sufficient levels of catecholamine neurotransmitters in terminal regions. One of these mechanisms is the up-regulation of tyrosine hydroxylase (TH), the enzyme that controls catecholamine biosynthesis. In neurons of the periphery and locus coeruleus, this up-regulation is associated with an initial induction of TH mRNA. In contrast, this induction either does not occur or it is nominal in mesencephalic dopamine neurons. The reasons for this lack of compensatory TH mRNA induction remain obscure, because so little is known about the regulation of TH expression in these neurons. In this study, we test whether activation of the cAMP signaling pathway regulates TH gene expression in two rodent models of midbrain dopamine neurons, ventral midbrain organotypic slice cultures and MN9D cells. Our results demonstrate that elevation of cAMP leads to induction of TH protein and TH activity in both model systems; however, TH mRNA levels are not up-regulated by cAMP. The induction of TH protein is the result of a novel post-transcriptional mechanism that activates TH mRNA translation. This translational activation is mediated by sequences within the 3' untranslated region (UTR) of TH mRNA. Our results support a model in which cAMP induces or activates trans-factors that interact with the TH mRNA 3'UTR to increase TH protein synthesis. An understanding of this novel regulatory mechanism may help to explain the control of TH gene expression and consequently dopamine biosynthesis in midbrain neurons under different physiological and pathological conditions.

Estrogen Dependent Response to Nicotine in Substantia Nigra and Ventral Tegmental Area of OVX female rats

The incidence of Parkinson's disease is higher in men than women and nicotine (Nic) reduces the risk. To study the interaction between Nic and estrogen (E), OVX rats were treated for 16 days with estradiol benzoate (EB) or vehicle and received Nic injections on days 13–16 (twice daily). Tyrosine hydroxylase (TH) and GTP cyclohydrolase (GTPCH) mRNA levels were determined by qRT‐PCR in VTA and SN. The VTA of OVX rats was especially susceptible to the lowest dose of Nic. In contrast, with EB treatment, only the highest dose of Nic was effective. In SN, both doses of Nic did not change TH mRNA levels in OVX rats with or without EB injections, but EB treatment increased sensitivity of GTPCH gene to Nic. To determine whether E can modulate Nic triggered transcriptional regulation of TH and GTPCH genes, dopaminergic MN9D cells were co‐transfected with pGTPCH/Luc and expression vector for ERa or ERβ. The results revealed that Nic and E elevated GTPCH promoter activity with both ERa and ERβ. The response was reduced with Nic and E combined. The findings provide insight for specificity of Nic regulation of catecholamine and tetrahydrobiopterin biosynthesis in DA neurons in gender specific fashion.

P.1.30 Inflammation down-regulates β2-adrenoceptor expression on primary glia: reversal by the glucocorticoid dexamethasone

GARCÍA-GIL, L., J. A. RAMOS, T. RUBINO, D. PAROLARO AND J. J. FERNÁNDEZ-RUIZ. Perinatal Δ9-tetrahydrocannabinol exposure did not alter dopamine transporter and tyrosine hydroxylase mRNA levels in midbrain dopaminergic neurons of adult male and female rats. NEUROTOXICOL TERATOL 20(5) 549–553, 1998.—We have recently demonstrated that the magnitude of L-3,4-dihydroxyphenylacetic acid (DOPAC) lowering effect caused by amphetamine in midbrain dopaminergic neurons of adult rats was lesser in animals that had been perinatally exposed to Δ9-tetrahydrocannabinol (Δ9-THC) than controls. In the present study, we have examined whether this loss in the responsiveness to amphetamine might be due to changes at the level of dopamine transporter (DAT), the main molecular site for the action of amphetamine, following the perinatal exposure to Δ9-THC. To this end, we have analyzed DAT mRNA levels, by using in situ hybridization, in the substantia nigra and ventral tegmental area, the areas where cell bodies of DAT-containing midbrain neurons are located, of adult male and female rats that had been perinatally exposed to Δ9-THC. In addition, we also analyzed mRNA levels of tyrosine hydroxylase (TH), the rate-limiting enzyme in DA synthesis. Results were as follows. Both adult male and female rats that had been perinatally exposed to Δ9-THC exhibited similar mRNA levels to controls for both DAT and TH in the substantia nigra as well as in the ventral tegmental area. This observation makes it difficult to support the idea that the differences found in adulthood after pharmacological challenges were caused by irreversible changes at the level of gene expression for these two key proteins.

Duration of airborne-manganese exposure in rhesus monkeys is associated with brain regional changes in biomarkers of neurotoxicity

Juvenile (20–24-month-old) rhesus monkeys were exposed to airborne-manganese sulfate (MnSO 4) 1.5 mg Mn/m 3 (6 h/day, 5 days/week) for 15 or 33 days, or for 65 days followed by a 45 or 90 days post-exposure recovery period, or air. We assessed biochemical endpoints indicative of oxidative stress and excitotoxicity in the cerebellum, frontal cortex, caudate, globus pallidus, olfactory cortex, and putamen. Glutamine synthetase (GS), glutamate transporters (GLT-1 and GLAST) and tyrosine hydroxylase (TH) protein levels, metallothionein (MT), GLT-1, GLAST, TH and GS mRNA levels, and total glutathione (GSH) levels were determined for all brain regions. Exposure to Mn significantly decreased MT mRNA in the caudate (vs. air-exposed controls). This depression persisted at least 90 days post-exposure. In contrast, putamen MT mRNA levels were unaffected by Mn exposure. GLT-1 and GLAST were relatively unaffected by short term Mn exposure, except in the globus pallidus where exposure for 33 days led to decreased protein levels, which persisted after 45 days of recovery for both proteins and 90 days of recovery in the case of GLAST. Exposure to 1.5 mg Mn/m 3 caused a significant decrease in GSH levels in the caudate and increased GSH levels in the putamen of monkey exposed for 15 and 33 days with both effects persisting at least 90 days post-exposure. Finally, TH protein levels were significantly lowered in the globus pallidus of the monkeys exposed for 33 days but mRNA levels were significantly increased in this same region. Overall, the nonhuman primate brain responds to airborne Mn in a heterogeneous manner and most alterations in these biomarkers of neurotoxicity are reversible upon cessation of Mn exposure.