Tyrosine Hydroxylase Inhibitors and Dopamine Receptor Agonists Combination Therapy for Parkinson's Disease.

There are currently no disease-modifying therapies for Parkinson's disease (PD), a progressive neurodegenerative disorder associated with dopaminergic neuronal loss. There is increasing evidence that endogenous dopamine (DA) can be a pathological factor in neurodegeneration in PD. Tyrosine hydroxylase (TH) is the key rate-limiting enzyme for DA generation. Drugs that inhibit TH, such as alpha-methyltyrosine (α-MT), have recently been shown to protect against neurodegeneration in various PD models. DA receptor agonists can activate post-synaptic DA receptors to alleviate DA-deficiency-induced PD symptoms. However, DA receptor agonists have no therapeutic effects against neurodegeneration. Thus, a combination therapy with DA receptor agonists plus TH inhibitors may be an attractive therapeutic approach. TH inhibitors can protect and promote the survival of remaining dopaminergic neurons in PD patients' brains, whereas DA receptor agonists activate post-synaptic DA receptors to alleviate PD symptoms. Additionally, other PD drugs, such as N-acetylcysteine (NAC) and anticholinergic drugs, may be used as adjunctive medications to improve therapeutic effects. This multi-drug cocktail may represent a novel strategy to protect against progressive dopaminergic neurodegeneration and alleviate PD disease progression.

GCH1 Deficiency Activates Brain Innate Immune Response and Impairs Tyrosine Hydroxylase Homeostasis.

The Parkinson's disease (PD) risk gene GTP cyclohydrolase 1 (GCH1) catalyzes the rate-limiting step in tetrahydrobiopterin (BH4) synthesis, an essential cofactor in the synthesis of monoaminergic neurotransmitters. To investigate the mechanisms by which GCH1 deficiency may contribute to PD, we generated a loss of function zebrafish gch1 mutant (gch1-/-), using CRISPR/Cas technology. gch1-/- zebrafish develop marked monoaminergic neurotransmitter deficiencies by 5 d postfertilization (dpf), movement deficits by 8 dpf and lethality by 12 dpf. Tyrosine hydroxylase (Th) protein levels were markedly reduced without loss of ascending dopaminergic (DAergic) neurons. L-DOPA treatment of gch1-/- larvae improved survival without ameliorating the motor phenotype. RNAseq of gch1-/- larval brain tissue identified highly upregulated transcripts involved in innate immune response. Subsequent experiments provided morphologic and functional evidence of microglial activation in gch1-/- The results of our study suggest that GCH1 deficiency may unmask early, subclinical parkinsonism and only indirectly contribute to neuronal cell death via immune-mediated mechanisms. Our work highlights the importance of functional validation for genome-wide association studies (GWAS) risk factors and further emphasizes the important role of inflammation in the pathogenesis of PD.SIGNIFICANCE STATEMENT Genome-wide association studies have now identified at least 90 genetic risk factors for sporadic Parkinson's disease (PD). Zebrafish are an ideal tool to determine the mechanistic role of genome-wide association studies (GWAS) risk genes in a vertebrate animal model. The discovery of GTP cyclohydrolase 1 (GCH1) as a genetic risk factor for PD was counterintuitive, GCH1 is the rate-limiting enzyme in the synthesis of dopamine (DA), mutations had previously been described in the non-neurodegenerative movement disorder dopa-responsive dystonia (DRD). Rather than causing DAergic cell death (as previously hypothesized by others), we now demonstrate that GCH1 impairs tyrosine hydroxylase (Th) homeostasis and activates innate immune mechanisms in the brain and provide evidence of microglial activation and phagocytic activity.

Coumarin-Based Compounds as Inhibitors of Tyrosinase/Tyrosine Hydroxylase: Synthesis, Kinetic Studies, and In Silico Approaches.

Cancer represents the main cause of morbidity and mortality worldwide, constituting a serious health problem. In this context, melanoma represents the most aggressive and fatal type of skin cancer, with death rates increasing every year. Scientific efforts have been addressed to the development of inhibitors targeting the tyrosinase enzyme as potential anti-melanoma agents due to the importance of this enzyme in melanogenesis biosynthesis. Coumarin-based compounds have shown potential activity as anti-melanoma agents and tyrosinase inhibitors. In this study, coumarin-based derivatives were designed, synthesized, and experimentally evaluated upon tyrosinase. Compound FN-19, a coumarin-thiosemicarbazone analog, exhibited potent anti-tyrosinase activity, with an IC50 value of 42.16 ± 5.16 µM, being more active than ascorbic acid and kojic acid, both reference inhibitors. The kinetic study showed that FN-19 acts as a mixed inhibitor. Still, for this compound, molecular dynamics (MD) simulations were performed to determine the stability of the complex with tyrosinase, generating RMSD, RMSF, and interaction plots. Additionally, docking studies were performed to elucidate the binding pose at the tyrosinase, suggesting that the hydroxyl group of coumarin derivative performs coordinate bonds (bidentate) with the copper(II) ions at distances ranging from 2.09 to 2.61 Å. Then, MM/PBSA calculations revealed that van der Waals interactions are the most relevant intermolecular forces for complex stabilization. Furthermore, it was observed that FN-19 has a binding energy (ΔEMM) value similar to tropolone, a tyrosinase inhibitor. Therefore, the data obtained in this study will be useful for designing and developing novel coumarin-based analogs targeting the tyrosinase enzyme.

Effect of levodopa/carbidopa on stress response in zebrafish.

The dopaminergic system of zebrafish is complex and the numerous pathways and receptors in the central nervous system (CNS) are being extensively studied. A critical factor for the synthesis, activation and release of catecholamines (CAs) is the presence of tyrosine hydroxylase, an enzyme which converts L-tyrosine into levodopa. Levodopa thus is the intermediary in the synthesis of dopamine (DA) and norepinephrine (NE) and promotes its release; therefore, CAs play an important role in the CNS with hormonal functions. Here, we use levodopa/carbidopa to clarify the involvement of the dopaminergic pathway in the stress response in zebrafish submitted to an acute stress challenge. Acute stress was induced by chasing fish with a net for 2 min and assessed by measuring whole-body cortisol levels. Two experiments were carried out, the first with exposure to levodopa/carbidopa and the second with exposure to AMPT and levodopa/carbidopa. Levodopa/carbidopa balances the stress response through its action on the zebrafish hypothalamic-pituitary-adrenal (HPA) axis. Changes in cortisol levels suggest that DA was related to the balance of the stress response and that NE decreased this response. These effects were specific to stress since levodopa/carbidopa did not induce changes in cortisol in non-stressed fish.

ß-Methylphenylalanine exerts neuroprotective effects in a Parkinson's disease model by protecting against tyrosine hydroxylase depletion.

We evaluated the neuroprotective effects of ß-methylphenylalanine in an experimental model of rotenone-induced Parkinson's disease (PD) in SH-SY5Y cells and rats. Cells were pre-treated with rotenone (2.5 µg/mL) for 24 hours followed by ß-methylphenylalanine (1, 10 and 100 mg/L) for 72 hours. Cell viability, reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), mitochondrial fragmentation, apoptosis, and mRNA and protein levels of tyrosine hydroxylase were determined. In a rat model of PD, dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels, bradykinesia and tyrosine hydroxylase expression were determined. In rotenone-pre-treated cells, ß-methylphenylalanine significantly increased cell viability and MMP, whereas ROS levels, apoptosis and fragmented mitochondria were reduced. ß-Methylphenylalanine significantly increased the mRNA and protein levels of tyrosine hydroxylase in SH-SY5Y cells. In the rotenone-induced rat model of PD, oral administration of ß-methylphenylalanine recovered DA and DOPAC levels and bradykinesia. ß-Methylphenylalanine significantly increased the protein expression of tyrosine hydroxylase in the striatum and substantia nigra of rats. In addition, in silico molecular docking confirmed binding between tyrosine hydroxylase and ß-methylphenylalanine. Our experimental results show neuroprotective effects of ß-methylphenylalanine via the recovery of mitochondrial damage and protection against the depletion of tyrosine hydroxylase. We propose that ß-methylphenylalanine may be useful in the treatment of PD.

Update on Pheochromocytoma and Paraganglioma from the SSO Endocrine and Head and Neck Disease Site Working Group, Part 2 of 2: Perioperative Management and Outcomes of Pheochromocytoma and Paraganglioma.

This is the second part of a two-part review on pheochromocytoma and paragangliomas (PPGLs). In this part, perioperative management, including preoperative preparation, intraoperative, and postoperative interventions are reviewed. Current data on outcomes following resection are presented, including outcomes after cortical-sparing adrenalectomy for bilateral adrenal disease. In addition, pathological features of malignancy, surveillance considerations, and the management of advanced disease are also discussed.

Temporary Anosmia in Mice Following Nasal Lavage With Dilute Detergent Solution.

Olfactory sensory deprivation induces anosmia and reduces tyrosine hydroxylase and dopamine levels in the olfactory bulb. The behavioral consequences specific to the loss of olfactory bulb dopamine are difficult to determine because sensory deprivation protocols are either confounded by side effects or leave the animal anosmic. A new method to both induce sensory deprivation and to measure the behavioral and circuit consequences is needed. We developed a novel, recoverable anosmia protocol using nasal lavage with a dilute detergent solution. Detergent treatment did not damage the olfactory epithelium as measured by scanning electron microscopy, alcian blue histology, and acetylated tubulin immunohistochemistry. One treatment-induced anosmia that lasted 24 to 48 h. Three treatments over 5 days reduced olfactory bulb tyrosine hydroxylase and dopamine levels indicating that anosmia persists between treatments. Importantly, even with multiple treatments, olfactory ability recovered within 48 h. This is the first report of a sensory deprivation protocol that induces recoverable anosmia and can be paired with biochemical, histological, and behavioral investigations of olfaction.

Methylmercury causes epigenetic suppression of the tyrosine hydroxylase gene in an in vitro neuronal differentiation model.

Methylmercury (MeHg) is the causative substance of Minamata disease, which is associated with various neurological disorders such as sensory disturbance and ataxia. It has been suggested low-level dietary intake of MeHg from MeHg-containing fish during gestation adversely affects the fetus. In our study, we investigated the toxicological effects of MeHg exposure on neuronal differentiation focusing on epigenetics. We used human fetal brain-derived immortalized cells (LUHMES cells) as a human neuronal differentiation model. Cell viability, neuronal, and catecholamine markers in LUHMES cells were assessed after exposure to MeHg (0-1000 nM) for 6 days (from day 2 to day 8 of neuronal differentiation). Cell viability on day 8 was not affected by exposure to 1 nM MeHg for 6 days. mRNA levels of AADC, DBH, TUJ1, and SYN1 also were unaffected by MeHg exposure. In contrast, levels of TH, the rate-limiting enzyme for dopamine synthesis, were significantly decreased after MeHg exposure. Acetylated histone H3, acetylated histone H3 lysine 9, and tri-methyl histone H3 lysine 9 levels at the TH gene promoter were not altered by MeHg exposure. However, tri-methylation of histone H3 lysine 27 levels, related to transcriptional repression, were significantly increased at the TH gene promotor after MeHg exposure. In summary, MeHg exposure during neuronal differentiation led to epigenetic changes that repressed TH gene expression. This study provides useful insights into the toxicological mechanisms underlying the effects of developmental MeHg exposure during neuronal differentiation.

Conjugation with Dihydrolipoic Acid Imparts Caffeic Acid Ester Potent Inhibitory Effect on Dopa Oxidase Activity of Human Tyrosinase.

Caffeic acid derivatives represent promising lead compounds in the search for tyrosinase inhibitors to be used in the treatment of skin local hyperpigmentation associated to an overproduction or accumulation of melanin. We recently reported the marked inhibitory activity of a conjugate of caffeic acid with dihydrolipoic acid, 2-S-lipoylcaffeic acid (LCA), on the tyrosine hydroxylase (TH) and dopa oxidase (DO) activities of mushroom tyrosinase. In the present study, we evaluated a more lipophilic derivative, 2-S-lipoyl caffeic acid methyl ester (LCAME), as an inhibitor of tyrosinase from human melanoma cells. Preliminary analysis of the effects of LCAME on mushroom tyrosinase indicated more potent inhibitory effects on either enzyme activities (IC50 = 0.05 ± 0.01 µM for DO and 0.83 ± 0.09 µM for TH) compared with LCA and the reference compound kojic acid. The inhibition of DO of human tyrosinase was effective (Ki = 34.7 ± 1.1 µM) as well, while the action on TH was weaker. Lineweaver⁻Burk analyses indicated a competitive inhibitor mechanism. LCAME was not substrate of tyrosinase and proved nontoxic at concentrations up to 50 µM. No alteration of basal tyrosinase expression was observed after 24 h treatment of human melanoma cells with the inhibitor, but preliminary evidence suggested LCAME might impair the induction of tyrosinase expression in cells stimulated with α-melanocyte-stimulating hormone. All these data point to this compound as a valuable candidate for further trials toward its use as a skin depigmenting agent. They also highlight the differential effects of tyrosinase inhibitors on the human and mushroom enzymes.

Borna disease virus phosphoprotein impairs the developmental program controlling neurogenesis and reduces human GABAergic neurogenesis.

It is well established that persistent viral infection may impair cellular function of specialized cells without overt damage. This concept, when applied to neurotropic viruses, may help to understand certain neurologic and neuropsychiatric diseases. Borna disease virus (BDV) is an excellent example of a persistent virus that targets the brain, impairs neural functions without cell lysis, and ultimately results in neurobehavioral disturbances. Recently, we have shown that BDV infects human neural progenitor cells (hNPCs) and impairs neurogenesis, revealing a new mechanism by which BDV may interfere with brain function. Here, we sought to identify the viral proteins and molecular pathways that are involved. Using lentiviral vectors for expression of the bdv-p and bdv-x viral genes, we demonstrate that the phosphoprotein P, but not the X protein, diminishes human neurogenesis and, more particularly, GABAergic neurogenesis. We further reveal a decrease in pro-neuronal factors known to be involved in neuronal differentiation (ApoE, Noggin, TH and Scg10/Stathmin2), demonstrating that cellular dysfunction is associated with impairment of specific components of the molecular program that controls neurogenesis. Our findings thus provide the first evidence that a viral protein impairs GABAergic human neurogenesis, a process that is dysregulated in several neuropsychiatric disorders. They improve our understanding of the mechanisms by which a persistent virus may interfere with brain development and function in the adult.

α-Methyltyrosine, a tyrosine hydroxylase inhibitor, decreases stress response in zebrafish (Danio rerio).

In this article, we show that the tyrosine hydroxylase inhibitor α-Methyl-l-tyrosine (AMPT) decreased the responsiveness of the zebrafish stress axis to an acute stressful challenge. These effects were specific for responses to stimulation, since unstimulated (basal) cortisol levels were not altered by AMPT. Moreover, AMPT decreased the stress response 15min after stimulation, but not after that time period. To our knowledge, this is the first report about the effects of AMPT on the neuroendocrine axis of adult zebrafish in acute stress responses. Overall, these results suggest a mechanism of catecholamine-glucocorticoid interplay in neuroendocrine responses of fish, pointing an interesting avenue for physiological research, as well as an important endpoint that can be disrupted by environmental contamination. Further experiments will unravel the mechanisms by which AMPT blocked the cortisol response.

A Novel Model of Dexamethasone-Induced Hypertension: Use in Investigating the Role of Tyrosine Hydroxylase.

Our objective was to study hypertension induced by chronic administration of synthetic glucocorticoid, dexamethasone (DEX), under nonstressful conditions and examine the role of catecholamine biosynthesis. To achieve this, we did the following: 1) used radiotelemetry to record mean arterial pressure (MAP) and heart rate (HR) in freely moving rats, and 2) administered different doses of DEX in drinking water. To evaluate the involvement of tyrosine hydroxylase (TH), the rate-limiting step in catecholamine biosynthesis, we treated rats with the TH inhibitor, α-methyl-para-tyrosine (α-MPT), for 3 days prior to administration of DEX and assessed TH mRNA and protein expression by quantitative real-time polymerase chain reaction and Western blot in the adrenal medulla. We observed a dose-dependent elevation in blood pressure with a DEX dose of 0.3 mg/kg administered for 10 days, significantly increasing MAP by +15.0 ± 1.1 mm Hg, while concomitantly reducing HR. Although this DEX treatment also significantly decreased body weight, pair-fed animals that showed similar decreases in body weight due to lowered food intake were not hypertensive, suggesting that body weight changes may not account for DEX-induced hypertension. Chronic DEX treatment significantly increased the TH mRNA and protein levels in the adrenal medulla, and α-MPT administration not only reduced DEX pressor effects, but also inhibited TH (serine(40)) phosphorylation. Our study thus validates a novel model to study hypertension induced by chronic intake of DEX in freely moving rats not subject to the confounding factors of previous models and establishes its dependence on concomitant activation of peripheral catecholamine biosynthesis.

Early Antipsychotic Treatment in Juvenile Rats Elicits Long-Term Alterations to the Dopamine Neurotransmitter System.

Prescription of antipsychotic drugs (APDs) to children has substantially increased in recent years. Whilst current investigations into potential long-term effects have uncovered some alterations to adult behaviours, further investigations into potential changes to neurotransmitter systems are required. The current study investigated potential long-term changes to the adult dopamine (DA) system following aripiprazole, olanzapine and risperidone treatment in female and male juvenile rats. Levels of tyrosine hydroxylase (TH), phosphorylated-TH (p-TH), dopamine active transporter (DAT), and D1 and D2 receptors were measured via Western blot and/or receptor autoradiography. Aripiprazole decreased TH and D1 receptor levels in the ventral tegmental area (VTA) and p-TH levels in the prefrontal cortex (PFC) of females, whilst TH levels decreased in the PFC of males. Olanzapine decreased PFC p-TH levels and increased D2 receptor expression in the PFC and nucleus accumbens (NAc) in females only. Additionally, risperidone treatment increased D1 receptor levels in the hippocampus of females, whilst, in males, p-TH levels increased in the PFC and hippocampus, D1 receptor expression decreased in the NAc, and DAT levels decreased in the caudate putamen (CPu), and elevated in the VTA. These results suggest that early treatment with various APDs can cause different long-term alterations in the adult brain, across both treatment groups and genders.

Failure of metyrosine therapy for preoperative management of pheochromocytoma: a case report.

PURPOSE: Pheochromocytomas (PHEOS) are rare catecholamine-secreting adrenal tumours requiring surgical resection. Preoperative alpha-adrenergic receptor blockade to prevent intraoperative hypertension has traditionally been achieved with phenoxybenzamine. Due to changes in the availability of phenoxybenzamine in Canada, alternate therapies are needed for patients. We report our first experience using metyrosine, a tyrosine hydroxylase inhibitor, for preoperative management in a symptomatic patient with a unilateral PHEO. CLINICAL FEATURES: A 50-yr-old male was referred to our centre with a history of symptoms suggestive of a catecholamine-secreting PHEO, including tachycardia, diaphoresis, nervousness, and tremor. Computerized tomography revealed a right adrenal mass, and additional positive imaging and elevated urine epinephrine levels supported a diagnosis of PHEO. The patient was admitted to hospital five days prior to surgery, and metyrosine therapy was initiated and titrated to 4 g daily over four days. Despite adequate blood pressure (BP) control leading up to the resection, the initial BP reading in the operating room was 191/106 mmHg, but it subsequently declined and was well controlled during induction (100-110 mmHg systolic BP). Significant hypertension (up to 201/110 mmHg) developed upon tumour manipulation and resolved with phentolamine administration and surgical isolation of the tumour. The patient's BP remained stable throughout the residual part of the procedure and in the recovery room and step-down unit. CONCLUSION: In the case of this patient's PHEO, the use of metyrosine was unsatisfactory in achieving sufficient inhibition of catecholamine synthesis as evidenced by significant intraoperative hypertension. Metyrosine could have a role in preoperative management of these patients, but it may not be optimal as monotherapy for some patients with actively secreting tumours.

Prefrontal D1 dopamine signaling is required for temporal control.

Temporal control, or how organisms guide movements in time to achieve behavioral goals, depends on dopamine signaling. The medial prefrontal cortex controls many goal-directed behaviors and receives dopaminergic input primarily from the midbrain ventral tegmental area. However, this system has never been linked with temporal control. Here, we test the hypothesis that dopaminergic projections from the ventral tegmental area to the prefrontal cortex influence temporal control. Rodents were trained to perform a fixed-interval timing task with an interval of 20 s. We report several results: first, that decreasing dopaminergic neurotransmission using virally mediated RNA interference of tyrosine hydroxylase impaired temporal control, and second that pharmacological disruption of prefrontal D1 dopamine receptors, but not D2 dopamine receptors, impaired temporal control. We then used optogenetics to specifically and selectively manipulate prefrontal neurons expressing D1 dopamine receptors during fixed-interval timing performance. Selective inhibition of D1-expressing prefrontal neurons impaired fixed-interval timing, whereas stimulation made animals more efficient during task performance. These data provide evidence that ventral tegmental dopaminergic projections to the prefrontal cortex influence temporal control via D1 receptors. The results identify a critical circuit for temporal control of behavior that could serve as a target for the treatment of dopaminergic diseases.

Effects of methylmercury on dopamine release in MN9D neuronal cells.

Epidemiological evidence has shown associations between prevalence of Parkinson's disease (PD) and exposure to environmental pollutants, but the mechanisms of pathogensis are still unclear. The objective of this study is to investigate effects of methylmercury (MeHg) on a dopaminergic neuronal cell line, MN9D and compare that to 1-methyl-4-phenylpyridinium (MPP+), a well-established agent associated with pathogenesis of PD. MN9D cells were exposed to MeHg (1-10 µM) and MPP+ (10-400 µM) for 24 or 48 h. Our results showed that MeHg induced cell death dose-dependently. MeHg also decreased the release of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) similar to the effects of MPP+. There was an increase in DOPAC + HVA/DA ratio. At the same time, both MeHg and MPP+ decreased the synthesis of tyrosine hydroxylase and dopamine transporter at the mRNA and protein levels. Expression of the α-Synuclein (α-Syn), a hallmark neuropathological indicator of PD, was also up-regulated at the mRNA level but not at the protein level after both MeHg and MPP+ dosing. Monoamine oxidase-B activity was suppressed in all MeHg treatments and MPP+ (1 µM)-treated cells. These findings suggest that MeHg can disrupt the synthesis, the uptake of DA and the metabolism as well as alter the biology of α-Syn similar to MPP+. Exposure to MeHg may potentially be a risk factor for the development of PD.

Nitrate (NO3(-)) and nitrite (NO2(-)) are endocrine disruptors to downregulate expression of tyrosine hydroxylase and motor behavior through conversion to nitric oxide in early development of zebrafish.

With a view to consider the increasing concern over nitrogen pollution in the aquatic environment, we investigated effects of nitrate (NO3(-)) and nitrite (NO2(-)) on the activity of dopaminergic neuron in zebrafish embryos and larvae. Both nitrate and nitrite exposure decreased the expression of tyrosine hydroxylase (TH) in dopaminergic neurons at 48hpf. Only nitrite decreased the response to tactile stimulation at 72hpf, whereas both nitrate and nitrite decreased the swimming activity at 6dpf. When the embryos were exposed to nitrate or nitrite together with an estrogen receptor blocker (ICI 182,780), the decreases in TH expression and motor behavior caused by nitrate or nitrite alone were reversed suggesting the effects of nitrate and nitrite were mediated through estrogen receptor (ER). The result of co-incubation with an oxidoreductase inhibitor, diphenyleneiodonium, indicated the conversion to nitric oxide (NO) is likely to be responsible for the effects of nitrate and nitrite, which was further supported by the increased staining for NO after exposure. The present study demonstrates that nitrate and nitrite are neurotoxicants acting as an endocrine disruptor possibly through conversion to NO to downregulate the activity of dopaminergic neuron in early development of zebrafish.

An in vitro study on catecholamine modulation of ovarian steroidogenic activity in the catfish Heteropneustes fossilis.

In the present study, α-methylparatyrosine (α-MPT), a tyrosine hydroxylase inhibitor was used to impair ovarian catecholaminergic activity in vitro. The consequent effects on catecholamine (CA) levels were correlated with follicular steroid production. l-dihdroxyphenylalanine (l-DOPA, the precursor of CA) and human gonadotropin (hCG) were supplemented to reverse the effect of α-MPT. The experiments were conducted in two reproductive phases, namely preparatory and pre-spawning phases in female catfish Heteropneustes fossilis. The incubation with α-MPT inhibited ovarian l-DOPA, dopamine (DA), norepinephrine (NE) and epinephrine (EP) levels and the l-DOPA supplementation compensated the inhibitory effect. The level of tyramine (TR) was increased by the α-MPT treatment but inhibited by the l-DOPA supplementation. α-MPT produced stage-specific (seasonal) effects on ovarian estradiol-17ß (E2); in the preparatory phase, E2 was decreased significantly at both 12 and 24h and in the pre-spawning phase, the level was stimulated over the respective control groups. The changes were higher at 24h in both phases. l-DOPA and hCG increased the E2 level significantly in the preparatory phase and reversed the inhibitory effect of α-MPT in the co-incubation groups. In the pre-spawning phase, α-MPT-stimulated the E2 level compared to the control groups, which was reversed by l-DOPA, hCG, or by both, in co-incubations. In contrast, the α-MPT treatment decreased progesterone (P4), 17-hydroxyprogesterone and 17,20ß-dihydroxy-4-prenen-3-one (17,20ß-DP) in a duration-dependent manner while the co-incubations with l-DOPA, hCG, or by both, significantly reversed the inhibitory effect. These results suggest that ovarian CAs (DA, NE and EP) may exert differential and stage-specific effects on E2, inhibition in the preparative phase and stimulation in the pre-spawning phase. The progestin steroids appear to be stimulated by CAs. In conclusion, this study highlights a possible direct/causal functional interaction between CA activity and gonadotropin on steroidogenic activity, and that CAs may be involved in regulating temporal secretion of the hormones through causing the shift in steroidogenic pattern.

Peripheral injections of cholecystokinin, apelin, ghrelin and orexin in cavefish (Astyanax fasciatus mexicanus): effects on feeding and on the brain expression levels of tyrosine hydroxylase, mechanistic target of rapamycin and appetite-related hormones.

The effects of intraperitoneal injections of cholecystokinin (CCK), apelin, ghrelin, and orexin on food intake were examined in the blind cavefish Astyanax fasciatus mexicanus. CCK (50ng/g) induced a decrease in food intake whereas apelin (100ng/g), orexin (100ng/g), and ghrelin (100ng/g) induced an increase in food intake as compared to saline-injected control fish. In order to better understand the central mechanism by which these hormones act, we examined the effects of injections on the brain mRNA expression of two metabolic enzymes, tyrosine hydroxylase (TH), and mechanistic target of rapamycin (mTOR), and of appetite-regulating peptides, CCK, orexin, apelin and cocaine and amphetamine regulated transcript (CART). CCK injections induced a decrease in brain apelin injections, apelin injections induced an increase in TH, mTOR, and orexin brain expressions, orexin treatment increased brain TH expression and ghrelin injections induced an increase in mTOR and orexin brain expressions. CART expression was not affected by any of the injection treatments. Our results suggest that the enzymes TH and mTOR and the hormones CCK, apelin, orexin, and ghrelin all regulate food intake in cavefish through a complex network of interactions.

Chronic Effect of Aspartame on Ionic Homeostasis and Monoamine Neurotransmitters in the Rat Brain.

Aspartame is one of the most widely used artificial sweeteners globally. Data concerning acute neurotoxicity of aspartame is controversial, and knowledge on its chronic effect is limited. In the current study, we investigated the chronic effects of aspartame on ionic homeostasis and regional monoamine neurotransmitter concentrations in the brain. Our results showed that aspartame at high dose caused a disturbance in ionic homeostasis and induced apoptosis in the brain. We also investigated the effects of aspartame on brain regional monoamine synthesis, and the results revealed that there was a significant decrease of dopamine in corpus striatum and cerebral cortex and of serotonin in corpus striatum. Moreover, aspartame treatment significantly alters the tyrosine hydroxylase activity and amino acids levels in the brain. Our data suggest that chronic use of aspartame may affect electrolyte homeostasis and monoamine neurotransmitter synthesis dose dependently, and this might have a possible effect on cognitive functions.