Restoration of Noradrenergic Function in Parkinson's Disease Model Mice.

Dysfunction of the central noradrenergic and dopaminergic systems is the primary neurobiological characteristic of Parkinson's disease (PD). Importantly, neuronal loss in the locus coeruleus (LC) that occurs in early stages of PD may accelerate progressive loss of dopaminergic neurons. Therefore, restoring the activity and function of the deficient noradrenergic system may be an important therapeutic strategy for early PD. In the present study, the lentiviral constructions of transcription factors Phox2a/2b, Hand2 and Gata3, either alone or in combination, were microinjected into the LC region of the PD model VMAT2 Lo mice at 12 and 18 month age. Biochemical analysis showed that microinjection of lentiviral expression cassettes into the LC significantly increased mRNA levels of Phox2a, and Phox2b, which were accompanied by parallel increases of mRNA and proteins of dopamine ß-hydroxylase (DBH) and tyrosine hydroxylase (TH) in the LC. Furthermore, there was considerable enhancement of DBH protein levels in the frontal cortex and hippocampus, as well as enhanced TH protein levels in the striatum and substantia nigra. Moreover, these manipulations profoundly increased norepinephrine and dopamine concentrations in the striatum, which was followed by a remarkable improvement of the spatial memory and locomotor behavior. These results reveal that over-expression of these transcription factors in the LC improves noradrenergic and dopaminergic activities and functions in this rodent model of PD. It provides the necessary groundwork for the development of gene therapies of PD, and expands our understanding of the link between the LC-norepinephrine and dopamine systems during the progression of PD.

Clioquinol inhibits dopamine-ß-hydroxylase secretion and noradrenaline synthesis by affecting the redox status of ATOX1 and copper transport in human neuroblastoma SH-SY5Y cells.

Clioquinol (5-chloro-7-indo-8-quinolinol), a chelator and ionophore of copper/zinc, was extensively used as an amebicide to treat indigestion and diarrhea in the mid-1900s. However, it was withdrawn from the market in Japan because its use was epidemiologically linked to an increase in the incidence of subacute myelo-optic neuropathy (SMON). SMON is characterized by the subacute onset of sensory and motor disturbances in the lower extremities with occasional visual impairments, which are preceded by abdominal symptoms. Although pathological studies demonstrated axonopathy of the spinal cord and optic nerves, the underlying mechanisms of clioquinol toxicity have not been elucidated in detail. In the present study, a reporter assay revealed that clioquinol (20-50 µM) activated metal response element-dependent transcription in human neuroblastoma SH-SY5Y cells. Clioquinol significantly increased the cellular level of zinc within 1 h, suggesting zinc influx due to its ionophore effects. On the other hand, clioquinol (20-50 µM) significantly increased the cellular level of copper within 24 h. Clioquinol (50 µM) induced the oxidation of the copper chaperone antioxidant 1 (ATOX1), suggesting its inactivation and inhibition of copper transport. The secretion of dopamine-ß-hydroxylase (DBH) and lysyl oxidase, both of which are copper-dependent enzymes, was altered by clioquinol (20-50 µM). Noradrenaline levels were reduced by clioquinol (20-50 µM). Disruption of the ATOX1 gene suppressed the secretion of DBH. This study suggested that the disturbance of cellular copper transport by the inactivation of ATOX1 is one of the mechanisms involved in clioquinol-induced neurotoxicity in SMON.

The Role of the Brain in the Regulation of Peripheral Organs-Noradrenaline Sources in Neonatal Rats: Noradrenaline Synthesis Enzyme Activity.

This work is aimed at studying the mechanisms of reciprocal humoral regulation of noradrenaline-producing organs in rats in the perinatal period of development. The activity of noradrenaline synthesis enzymes tyrosine hydroxylase and dopamine-beta-hydroxylase was measured in the brain and adrenal glands 48 and 72 h after the injection of immunotoxin (anti-dopamine-beta-hydroxylase-saporin) into the rat brain ventricles. It was shown that, 48 h after the immunotoxin injection into the brain, the activity of tyrosine hydroxylase in the brain decreased; however, 72 h after the injection it reached the control levels. This fact indicates that noradrenaline synthesis in the survived neurons increases. In the adrenal glands, 72 h after the immunotoxin injection into the brain, the activity of dopamine-beta-hydroxylase increased. This points to a compensatory increase in the rate of noradrenaline synthesis in the adrenal glands when the synthesis of noradrenaline in the brain is inhibited.

Maternal Treatment With Captopril Persistently Alters Gut-Brain Communication and Attenuates Hypertension of Male Offspring.

Maternal-fetal crosstalk has been implicated in long-term control of the health of offspring, including transgenerational hypertension. However, current knowledge is limited regarding maternal influences on the gut and its microbiome in blood pressure control in offspring. Therefore, the current study was designed to test the hypothesis that maternal factors influence the gut-brain axis impacting hypertension in offspring. We elected to use captopril, an antihypertensive angiotensin-converting enzyme inhibitor that possesses antibacterial properties, for the study. Pregnant female spontaneously hypertensive rats and normotensive Wistar Kyoto rats were treated with captopril water (100 mg/[kg·day]) or sterile water throughout pregnancy and lactation. At weaning, the pups from dams drinking sterile water were continued with sterile water until 12 weeks of age. The male pups from dams drinking captopril water were divided at weaning into 2 groups: offspring drinking captopril water and offspring withdrawn from captopril water, then drinking sterile water until 12 weeks of age. Captopril changed gut microbiota of spontaneously hypertensive rat dams, and some of these changes were reflected in their 12-week-old male offspring. These 12-week-old spontaneously hypertensive rat male offspring exposed to captopril via dams demonstrated persistently decreased systolic blood pressure, decreased number of activated microglia and neuroinflammation, as well as improvement of gut inflammation and permeability. Therefore, maternal captopril treatment improves the dysregulated gut-brain axis in spontaneously hypertensive rat male offspring, providing conceptual support that targeting the gut-brain axis via the mother may be a viable strategy for control of hypertension in the offspring.

Differences in the expression of catecholamine-synthesizing enzymes between vesicular monoamine transporter 1- and 2-immunoreactive glomus cells in the rat carotid body.

Vesicular monoamine transporters (VMAT) 1 and 2 are responsible for monoamine transportation into secretary vesicles and are tissue-specifically expressed in central and peripheral monoaminergic tissues, including the carotid body (CB). The aim of the present study was to examine the expression of catecholamine-synthesizing enzymes in VMAT1- and VMAT2-immunoreactive glomus cells in the rat CB using multiple immunolabeling. The expression of VMAT1 and VMAT2 mRNA in the CB was confirmed by RT-PCR. Immunohistochemistry revealed that VMAT1 immunoreactivity was predominant in glomus cells rather than VMAT2 immunoreactivity. Glomus cells with VMAT1 immunoreactivity exhibited weak/negative VMAT2 immunoreactivity, and vice versa. Immunoreactivities for VMAT1 and tyrosine hydroxylase, the rate-limiting enzyme for catecholamine biosynthesis, were co-localized in the same glomus cells and a positive correlation was confirmed between the two immunoreactivities (Spearman's coefficient = 0.82; p <  0.05). Although some glomus cells showed co-localization of VMAT2 and dopamine ß-hydroxylase immunoreactivity, the biosynthetic enzyme for noradrenaline, VMAT2 immunoreactivity appeared to be less associated with both catecholamine-synthesizing enzymes as indicated by a correlation analysis (TH: Spearman's coefficient = 0.38, DBH: Spearman's coefficient = 0.26). These results indicate that heterogeneity on functional role would exist among glomus cells in terms of VMAT isoform and catecholamine-synthesizing enzymes expression.

Co-Administration of Curcumin and Artepillin C Induces Development of Brown-Like Adipocytes in Association with Local Norepinephrine Production by Alternatively Activated Macrophages in Mice.

Classical brown adipocytes, characterized by interscapular depots, have multilocular fat depots and are known to release excess energy. Recent studies have shown that induction of brown-like adipocytes, also referred to as beige or brite cells, in white adipose tissue (WAT) results in the release of excess energy through mitochondrial heat production via uncoupling protein 1. This has potential a therapeutic strategy for obesity and related diseases as well as classical brown adipocytes. In our previous studies, we found that artepillin C (ArtC, 10 mg/kg body weight), a characteristic constituent of Brazilian propolis, significantly induced the development of brown-like adipocytes in inguinal WAT (iWAT) of mice. Furthermore, we recently demonstrated that curcumin (Cur, 4.5 mg/kg) also significantly induced the development of brown-like adipocytes in mice. The combined administration of several food-derived factors can enhance their bioactivity and reduce their required functional doses. In this study, we showed that co-administration of Cur and ArtC at lower doses (Cur, 1.5 mg/kg; ArtC, 5 mg/kg) additively induce brown-like adipocyte development in mouse iWAT. Moreover, this induction is associated with the localized production of norepinephrine following accumulation of alternatively activated macrophages in iWAT. These findings suggest that co-administration of Cur and ArtC is significantly effective to reduce the dose and enhance the formation of brown-like adipocyte via a unique molecular mechanism.

The Role of the Brain in the Regulation of Peripheral Noradrenaline-producing Organs in Rats During Morphogenesis.

This work represents one part of our research project, in which we attempted to prove that a humoral regulation between noradrenaline-producing organs exist in the perinatal period. In this study, we used a rat model that allowed blocking the synthesis of noradrenalin in the brain and evaluated gene expression and protein levels of noradrenaline key synthesis enzymes such as tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) in peripheral noradrenaline-producing organs. As a result, we showed an increased gene expression of TH and DBH in adrenal glands. These data indicate that, if neonatal rat brain lacks the ability to produce noradrenaline, then the synthesis of noradrenaline in adrenal glands increased as a compensatory process, so that the concentration levels in blood are maintained at normal levels. This indicates that there is a humoral regulation between brain and adrenal glands, which is not fully understood yet.

Conversion of Astrocytes and Fibroblasts into Functional Noradrenergic Neurons.

Dysfunction of noradrenergic (NA) neurons is associated with a number of neuronal disorders. Diverse neuronal subtypes can be generated by direct reprogramming. However, it is still unknown how to convert non-neuronal cells into NA neurons. Here, we show that seven transcription factors (TFs) (Ascl1, Phox2b, AP-2α, Gata3, Hand2, Nurr1, and Phox2a) are able to convert astrocytes and fibroblasts into induced NA (iNA) neurons. These iNA neurons express the genes required for the biosynthesis, release, and re-uptake of noradrenaline. Moreover, iNA neurons fire action potentials, receive synaptic inputs, and control the beating rate of co-cultured ventricular myocytes. Furthermore, iNA neurons survive and integrate into neural circuits after transplantation. Last, human fibroblasts can be converted into functional iNA neurons as well. Together, iNA neurons are generated by direct reprogramming, and they could be potentially useful for disease modeling and cell-based therapies.

Angiotensin II mediates the axonal trafficking of tyrosine hydroxylase and dopamine ß-hydroxylase mRNAs and enhances norepinephrine synthesis in primary sympathetic neurons.

In the sympatho-adrenal system, angiotensin II (Ang II) acts as a key neuromodulatory component. At sympathetic nerve terminals, Ang II influences sympathetic transmission by enhancing norepinephrine (NE) synthesis, facilitating NE release and inhibiting NE uptake. Previously, it was demonstrated that tyrosine hydroxylase (TH) mRNA is trafficked to the distal axons of primary superior cervical ganglia (SCG) neurons, directed by a cis-acting regulatory element (i.e. zipcode) located in the 3'UTR of the transcript. Results of metabolic labeling studies established that the mRNA is locally translated. It was further shown that the axonal trafficking of the mRNA encoding the enzyme plays an important role in mediating dopamine (DA) and NE synthesis and may facilitate the maintenance of axonal catecholamine levels. In the present study, the hypothesis was tested that Ang II induces NE synthesis in rat primary SCG neurons via the modulation of the trafficking of the mRNAs encoding the catecholamine synthesizing enzymes TH and dopamine ß-hydroxylase (DBH). Treatment of SCG neurons with the Ang II receptor type 1 (AT1R) agonist, L-162,313, increases the axonal levels of TH and DBH mRNA and protein and results in elevated NE levels. Conversely, treatment of rat SCG neurons with the AT1R antagonist, Eprosartan, abolished the L-162,313-mediated increase in axonal levels of TH and DBH mRNA and protein. In a first attempt to identify the proteins involved in the Ang II-mediated axonal transport of TH mRNA, we used a biotinylated 50-nucleotide TH RNA zipcode as bait in the affinity purification of TH zipcode-associated proteins. Mass spectrometric analysis of the TH zipcode ribonucleoprotein (RNP) complex immune-purified from SCG neurons led to the identification of 163 somal and 127 axonal proteins functionally involved in binding nucleic acids, the translational machinery or acting as subunits of cytoskeletal and motor proteins. Surprisingly, immune-purification of the TH axonal trafficking complex, results in the acquisition of DBH mRNA, suggesting that these mRNAs maybe transported to the axon together, possibly in the same RNP complex. Taken together, our results point to a novel mechanism by which Ang II participates in the regulation of axonal synthesis of NE by modulating the local trafficking and expression of TH and DBH, two key enzymes involved in the catecholamine biosynthetic pathway.

The Presynaptic Regulation of Dopamine and Norepinephrine Synthesis Has Dissociable Effects on Different Kinds of Cognitive Conflicts.

Goal-directed behavior requires the ability to resolve subliminally or consciously induced response conflicts, both of which may benefit from catecholamine-induced increases in gain control. We investigated the effects of presynaptic differences in dopamine and norepinephrine synthesis with the help of the tyrosine hydroxylase (TH) rs10770141 and the dopamine-ß-hydroxylase (DBH) rs1611115, rs6271, and rs1611122 polymorphisms. Conscious and subliminal response conflicts were induced with flanker and prime distractors in (n = 207) healthy young participants while neurophysiological data (EEG) was recorded. The results demonstrated that the increased presynaptic catecholamine synthesis associated with the TH rs10770141 TT genotype improves cognitive control in case of consciously perceived (flanker) conflicts, but not in case of subliminally processed (prime) conflicts. Only norepinephrine seemed to also modulate subliminal conflict processing, as evidenced by better performance of the DBH rs1611122 CC genotype in case of high subliminal conflict load. Better performance was linked to larger conflict-induced modulations in post-response alpha band power arising from parietal and inferior frontal regions, which likely helps to suppress the processing of distracting information. In summary, presynaptic catecholamine synthesis benefits consciously perceived conflicts by improving the suppression of distracting information following a conflict. Subliminal conflicts were modulated via the same mechanism, but only by norepinephrine.

Tannins from Terminalia chebula fruits attenuates GABA antagonist-induced anxiety-like behaviour via modulation of neurotransmitters.

OBJECTIVES: This article investigates the anxiolytic activity of Terminalia chebula tannin-rich extract against picrotoxin (PTX; GABA antagonist)-induced anxiety in mice model. METHODS: Anxiolytic activity was studied by elevated plus maze (EPM), open field test (OFT), light/dark box test (LDT) and Vogel's conflict test (VCT). Electroencephalogram (EEG) was performed to know the changes in brain activity instigated by GABA antagonist. 5-hydroxytryptamine (5-HT), dopamine and norepinephrine levels in brain tissues were estimated by HPLC. The mRNA (CREB, BDNF, GABA, and 5-HT1A ) and protein expression (CREB, p-CREB, BDNF, ERK ½, p-ERK ½, GABAA Rα1, 5-HT1A and GAPDH) levels in brain tissue were determined by RT-PCR and Western blot analysis, respectively. KEY FINDINGS: Terminalia chebula tannin-rich extract (TCHE) supplementation increased locomotion in mice towards open arm (EPM), time spent in illuminated area (LDT), rearing frequency (OFT) and number of shocks (VCT) compared to PTX (P < 0.05). Furthermore, TCHE down-regulated serum cortisol levels and showed increased levels of 5-HT, DA and NE. Gene expressions such as BDNF, CREB, GABAA and 5-HT1A were up-regulated by TCHE treatment compared to PTX. CONCLUSIONS: Terminalia chebula tannin-rich extract showed significant anxiolytic activity against picrotoxin and could be used as natural therapy in neurodegenerative disorders.

INVOLVEMENT OF CATECHOLAMINES IN THE MYOCARDIUM OF RATS SUBMITTED TO EXPERIMENTAL MODEL OF PORTAL HYPERTENSION.

BACKGROUND: The role of autonomic nervous system in the development and maintenance of portal hypertension is not fully elucidated. It is known that the gene expression of norepinephrine in the superior mesenteric artery varies with time, and it may contribute for splanchnic vasodilation and its consequent hemodynamic repercussions. It is still not known exactly how the adrenergic expression behaves at the heart level in the initial stages of this process. AIM: To evaluate the immunohistochemical expression of the enzyme tyrosine hydroxylase (tyrosine 3-monooxygenase), involved in the synthesis of norepinephrine, in the myocardium of rats submitted to partial ligation of the portal vein. METHODS: Twenty-four Wistar rats were divided into two groups: Sham Operated and Portal Hypertension. The partial ligation was performed in the Portal Hypertension group, and after 1/6/24 h and 3/5/14 days the animals were euthanized. Immunohistochemical analysis was performed to quantify the expression of the stained enzyme using the ImageJ program. RESULTS: The Portal Hypertension group expressed percentages between 4.6-6% of the marked area, while the Sham Operated group varied between 4-5%. Although there was no statistical significance, the percentage stained in the Portal Hypertension group followed an increasing pattern in the first 6 h and a decreasing pattern after 24 h, which was not observed in the Sham Operated group. CONCLUSION: The expression of noradrenaline in rat myocardium during the first two weeks after partial ligation of the portal vein, with tyrosine hydroxylase as marker, did not show differences between groups over time.

The Cholinergic and Adrenergic Autocrine Signaling Pathway Mediates Immunomodulation in Oyster Crassostrea gigas.

It is becoming increasingly clear that neurotransmitters impose direct influence on regulation of the immune process. Recently, a simple but sophisticated neuroendocrine-immune (NEI) system was identified in oyster, which modulated neural immune response via a "nervous-hemocyte"-mediated neuroendocrine immunomodulatory axis (NIA)-like pathway. In the present study, the de novo synthesis of neurotransmitters and their immunomodulation in the hemocytes of oyster Crassostrea gigas were investigated to understand the autocrine/paracrine pathway independent of the nervous system. After hemocytes were exposed to lipopolysaccharide (LPS) stimulation, acetylcholine (ACh), and norepinephrine (NE) in the cell supernatants, both increased to a significantly higher level (2.71- and 2.40-fold, p < 0.05) comparing with that in the control group. The mRNA expression levels and protein activities of choline O-acetyltransferase and dopamine ß-hydroxylase in hemocytes which were involved in the synthesis of ACh and NE were significantly elevated at 1 h after LPS stimulation, while the activities of acetylcholinesterase and monoamine oxidase, two enzymes essential in the metabolic inactivation of ACh and NE, were inhibited. These results demonstrated the existence of the sophisticated intracellular machinery for the generation, release and inactivation of ACh and NE in oyster hemocytes. Moreover, the hemocyte-derived neurotransmitters could in turn regulate the mRNA expressions of tumor necrosis factor (TNF) genes, the activities of superoxide dismutase, catalase and lysosome, and hemocyte phagocytosis. The phagocytic activities of hemocytes, the mRNA expressions of TNF and the activities of key immune-related enzymes were significantly changed after the block of ACh and NE receptors with different kinds of antagonists, suggesting that autocrine/paracrine self-regulation was mediated by transmembrane receptors on hemocyte. The present study proved that oyster hemocyte could de novo synthesize and release cholinergic and adrenergic neurotransmitters, and the hemocyte-derived ACh/NE could then execute a negative regulation on hemocyte phagocytosis and synthesis of immune effectors with similar autocrine/paracrine signaling pathway identified in vertebrate macrophages. Findings in the present study demonstrated that the immune and neuroendocrine system evolved from a common origin and enriched our knowledge on the evolution of NEI system.

Role of catalpol in ameliorating the pathogenesis of experimental autoimmune encephalomyelitis by increasing the level of noradrenaline in the locus coeruleus.

The endogenous neurotransmitter, noradrenaline, exerts anti-inflammatory and neuroprotective effects in vivo and in vitro. Reduced noradrenaline levels results in increased inflammation and neuronal damage. The primary source of noradrenaline in the central nervous system is tyrosine hydroxylase (TH)­positive neurons, located in the locus coeruleus (LC). TH is the rate­limiting enzyme for noradrenaline synthesis; therefore, regulation of TH protein expression and intrinsic enzyme activity represents the central means for controlling the synthesis of noradrenaline. Catalpol is an iridoid glycoside purified from Rehmannia glutinosa Libosch, which exerts a neuroprotective effect in multiple sclerosis (MS). The present study used an experimental mouse model of autoimmune encephalomyelitis to verify the neuroprotective effects of catalpol. Significant improvements in the clinical scores were observed in catalpol­treated mice. Furthermore, catalpol increased TH expression and increased noradrenaline levels in the spinal cord. In primary cultures, catalpol exerted a neuroprotective effect in rat LC neurons by increasing the noradrenaline output. These results suggested that drugs targeting LC survival and function, including catalpol, may be able to benefit patients with MS.

Involvement of catecholamines in the myocardium of rats submitted to experimental model of portal hypertension / Envolvimento das catecolaminas no miocárdio de ratos submetidos a modelo experimental de hipertensão portal

ABSTRACT Background: The role of autonomic nervous system in the development and maintenance of portal hypertension is not fully elucidated. It is known that the gene expression of norepinephrine in the superior mesenteric artery varies with time, and it may contribute for splanchnic vasodilation and its consequent hemodynamic repercussions. It is still not known exactly how the adrenergic expression behaves at the heart level in the initial stages of this process. Aim: To evaluate the immunohistochemical expression of the enzyme tyrosine hydroxylase (tyrosine 3-monooxygenase), involved in the synthesis of norepinephrine, in the myocardium of rats submitted to partial ligation of the portal vein. Methods: Twenty-four Wistar rats were divided into two groups: Sham Operated and Portal Hypertension. The partial ligation was performed in the Portal Hypertension group, and after 1/6/24 h and 3/5/14 days the animals were euthanized. Immunohistochemical analysis was performed to quantify the expression of the stained enzyme using the ImageJ program. Results: The Portal Hypertension group expressed percentages between 4.6-6% of the marked area, while the Sham Operated group varied between 4-5%. Although there was no statistical significance, the percentage stained in the Portal Hypertension group followed an increasing pattern in the first 6 h and a decreasing pattern after 24 h, which was not observed in the Sham Operated group. Conclusion: The expression of noradrenaline in rat myocardium during the first two weeks after partial ligation of the portal vein, with tyrosine hydroxylase as marker, did not show differences between groups over time.

RESUMO Racional: O papel do sistema nervoso autônomo na hipertensão portal não está completamente elucidado. Sabe-se que, nessa condição, a expressão gênica da norepinefrina na artéria mesentérica superior modifica-se com o tempo, podendo ser importante contribuinte para a vasodilatação esplâncnica e suas repercussões hemodinâmicas. Apesar dos estudos sobre as repercussões cardiovasculares na hipertensão portal, ainda não se sabe como a expressão adrenérgica se comporta a nível cardíaco nas etapas iniciais desse processo. Objetivo: Avaliar a expressão imunoistoquímica da enzima tirosina hidroxilase (tirosina 3-mono-oxigenase), relacionada à síntese da norepinefrina, no miocárdio de ratos submetidos à ligadura parcial da veia porta. Métodos: Foram utilizados 24 ratos, distribuídos em dois grupos: Sham Operated e Hipertensão Portal. A ligadura parcial da veia porta foi realizada apenas no grupo Hipertensão Portal e, após 1/6/24 h e 3/5/14 dias, os animais foram eutanasiados. Foi feita a análise imunoistoquímica para quantificar a expressão da enzima corada, utilizando o programa ImageJ. Resultados: No grupo Hipertensão Portal, o miocárdio expressou percentuais entre 4,6-6% de área marcada, enquanto que no grupo Sham Operated variou entre 4-5%, sem significância estatística. Apenas no grupo Hipertensão Portal, a porcentagem corada pela enzima seguiu padrão crescente nas primeiras 6 h e decrescente após 24 h. Conclusão: A expressão da noradrenalina no miocárdio de ratos durante as primeiras duas semanas após a ligadura parcial da veia porta, tendo como marcador a enzima tirosina hidroxilase, não apresentou diferenças entre grupos ao longo do tempo.

Gene expression and content of enzymes of noradrenaline synthesis in the rat organ of Zuckerkandl at the critical period of morphogenesis.

Gene expression and content of the key enzymes involved in the synthesis of noradrenaline-tyrosine hydroxylase and dopamine beta-hydroxylase-was evaluated in the organ of Zuckerkandl of rats in the critical period of morphogenesis. High levels of mRNA and protein of both enzymes in the perinatal period of development and their sharp decline on day 30 of postnatal development were detected. These data indicate that the synthesis of noradrenaline in the organ of Zuckerkandl is maximum during the critical period of morphogenesis and decreases during the involution of this paraganglion.

Disruption of the Axonal Trafficking of Tyrosine Hydroxylase mRNA Impairs Catecholamine Biosynthesis in the Axons of Sympathetic Neurons.

Tyrosine hydroxylase (TH) is the enzyme that catalyzes the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. In a previous communication, evidence was provided that TH mRNA is trafficked to the axon, where it is locally translated. In addition, a 50-bp sequence element in the 3'untranslated region (3'UTR) of TH mRNA was identified that directs TH mRNA to distal axons (i.e., zip-code). In the present study, the hypothesis was tested that local translation of TH plays an important role in the biosynthesis of the catecholamine neurotransmitters in the axon and/or presynaptic nerve terminal. Toward this end, a targeted deletion of the axonal transport sequence element was developed, using the lentiviral delivery of the CRISPR/Cas9 system, and two guide RNA (gRNA) sequences flanking the 50-bp cis-acting regulatory element in rat superior cervical ganglion (SCG) neurons. Deletion of the axonal transport element reduced TH mRNA levels in the distal axons and reduced the axonal protein levels of TH and TH activity as measured by phosphorylation of SER40 in SCG neurons. Moreover, deletion of the zip-code diminished the axonal levels of dopamine (DA) and norepinephrine (NE). Conversely, the local translation of exogenous TH mRNA in the distal axon enhanced TH levels and activity, and elevated axonal NE levels. Taken together, these results provide direct evidence to support the hypothesis that TH mRNA trafficking and local synthesis of TH play an important role in the synthesis of catecholamines in the axon and presynaptic terminal.

Amitraz changes NE, DA and 5-HT biosynthesis and metabolism mediated by alterations in estradiol content in CNS of male rats.

Amitraz is a formamidine insecticide/acaricide that alters different neurotransmitters levels, among other neurotoxic effects. Oral amitraz exposure (20, 50 and 80 mg/kg bw, 5 days) has been reported to increase serotonin (5-HT), norepinephrine (NE) and dopamine (DA) content and to decrease their metabolites and turnover rates in the male rat brain, particularly in the striatum, prefrontal cortex, and hippocampus. However, the mechanisms by which these alterations are produced are not completely understood. One possibility is that amitraz monoamine oxidase (MAO) inhibition could mediate these effects. Alternatively, it alters serum concentrations of sex steroids that regulate the enzymes responsible for these neurotransmitters synthesis and metabolism. Thus, alterations in sex steroids in the brain could also mediate the observed effects. To test these hypothesis regarding possible mechanisms, we treated male rats with 20, 50 and 80 mg/kg bw for 5 days and then isolated tissue from striatum, prefrontal cortex, and hippocampus. We then measured tissue levels of expression and/or activity of MAO, catechol-O-metyltransferase (COMT), dopamine-ß-hydroxylase (DBH), tyrosine hydroxylase (TH) and tryptophan hydroxylase (TRH) as well as estradiol levels in these regions. Our results show that amitraz did not inhibit MAO activity at these doses, but altered MAO, COMT, DBH, TH and TRH gene expression, as well as TH and TRH activity and estradiol levels. The alteration of these enzymes was partially mediated by dysregulation of estradiol levels. Our present results provide new understanding of the mechanisms contributing to the harmful effects of amitraz.

Soy peptide ingestion augments the synthesis and metabolism of noradrenaline in the mouse brain.

To examine whether edible peptide intake affects neurotransmitter metabolism in the brain, we evaluated the effect of peptides derived from soy proteins or fish collagen on free amino acids and monoamines in the mouse brain. Ingestion of soy peptides led to markedly higher levels of tyrosine, a catecholamine precursor, in the serum, and cerebral cortex compared to those following ingestion of vehicle alone or collagen peptides. Soy peptide ingestion also effectively increased 3-methoxy-4-hydroxyphenylethyleneglycol and normetanephrine, the principal metabolites of noradrenaline, in the cerebral cortex, hippocampus, and brainstem, whereas collagen peptides did not exert such effects. Further, soy peptide ingestion led to a significant increase in noradrenaline itself in the brainstem, where noradrenergic neurons are present. Noradrenergic turnover was also markedly stimulated in these regions after soy peptide ingestion. These in vivo observations suggest that soy peptide ingestion can maintain and promote the synthesis and metabolism of noradrenaline in the brain.

Glutamate Promotes Contraction of the Rat Ductus Arteriosus.

BACKGROUND: Extremely preterm infants frequently have patent ductus arteriosus (PDA). Recent recommendations include immediately beginning amino acid supplementation in extremely preterm infants. However, the effect of amino acids on closure of the ductus arteriosus (DA) remains unknown.Methods and Results:Aminogram results in human neonates at day 2 revealed that the plasma glutamate concentration was significantly lower in extremely preterm infants (<28 weeks' gestation) with PDA than in those without PDA and relatively mature preterm infants (28-29 weeks gestation). To investigate the effect of glutamate on DA closure, glutamate receptor expression in fetal rats was examined and it was found that the glutamate inotropic receptor, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type subunit 1 (GluR1), mRNA was highly expressed in the DA compared to the aorta on gestational day 19 (preterm) and gestational day 21 (term). GluR1 proteins were co-localized with tyrosine hydroxylase-positive autonomic nerve terminals in the rat and human DA. Intraperitoneal administration of glutamate increased noradrenaline production in the rat DA. A whole-body freezing method demonstrated that glutamate administration induced DA contraction in both preterm (gestational day 20) and term rat fetuses. Glutamate-induced DA contraction was attenuated by the calcium-sensitive GluR receptor antagonist, NASPM, or the adrenergic receptor α1 blocker, prazosin. CONCLUSIONS: These data suggest that glutamate induces DA contraction through GluR-mediated noradrenaline production. Supplementation of glutamate might help to prevent PDA in extremely preterm infants. (Circ J 2016; 80: 2388-2396).