Tyrosine Hydroxylase mRNA Research Articles

LC3-II may mediate ATR-induced mitophagy in dopaminergic neurons through SQSTM1/p62 pathway.

Atrazine (2-chloro-4-ethylamino-6-isopropylamine-1,3,5-triazine; ATR) has been demonstrated to regulate autophagy- and apoptosis-related proteins in doparminergic neuronal damage. In our study, we investigated the role of LC3-II in ATR-induced degeneration of dopaminergic neurons. In vivo dopaminergic neuron degeneration model was set up with ATR treatment and confirmed by the behavioral responses and pathological analysis. Dopaminergic neuron cells were transfected with LC3-II siRNA and treated with ATR to observe cell survival and reactive oxygen species release. The process of mitochondrial autophagy and the neurotoxic effects of mitochondrial autophagy were detected by immunofluorescence assay, immunohistochemical analysis, real-time PCR, and western blot analysis. Results showed that after ATR treatment, the grip strength of Wistar rats was significantly decreased, and behavioral signs of anxiety were clearly observed. The mRNA and protein levels of tyrosine hydroxylase, LC3-II, PINK1, and Parkin were significantly decreased in ATR-induced rat dopaminergic neurons and PC-12 cells, while the mRNA expression and protein levels of SQSTM1/p62 and Parl were increased. Exposure to ATR also led to accumulation of autophagic lysosomes and autophagic bodies along with significantly decreased levels of dopaminergic neurons and alterations in mitochondrial homeostasis, which was reversed by LC3-II siRNA. Our results suggest that ATR affects the mitochondria-mediated dopaminergic neuronal death, which may be mediated by LC3-II and other autophagy markers in vivo and in vitro through SQSTM1/p62 signaling pathway.

Oxytocin modulates the expression of norepinephrine transporter, β3-adrenoceptors and muscarinic M2 receptors in the hearts of socially isolated rats

Social stress produces behavioral alterations, and autonomic and cardiac dysfunction in animals. In addition to the well-known roles of oxytocin on birth and maternal bonding, recent evidence shows that this neuropeptide possesses cardio-protective properties. However less is known about its role in the regulation of the autonomic nervous system. The direct influence of oxytocin on the cardiac catecholamine synthesizing enzyme, transport beta-adrenoceptors and muscarinic receptors in animals exposed to chronic social isolation stress has not yet been studied. In this study, we examined the influence of peripheral chronic oxytocin treatment on anxiety-related behavior, the morphology and content of epinephrine and norepinephrine, mRNA and protein levels of tyrosine hydroxylase (TH), norepinephrine transporter (NET) and receptors <beta> 3 (β3-AR) and muscarinic 2 (M2 MR) in the right and left cardiac atrium and ventricle of chronically socially isolated male rats. Our results show that oxytocin treatment exhibits an anxiolytic effect, decreases the heart/body weight ratio and prevents the hypertrophy of cardiomyocytes in the wall of the left ventricle of stressed rats. Epinephrine and TH protein levels were unchanged after prolonged oxytocin treatment. Peripheral oxytocin administration led to the enhancement of gene expression of β3-AR in both atria, NET protein in the left ventricle and gene expression of M2 MR in the right atrium and the left ventricle of chronically socially isolated rats. The study provides evidence that oxytocin treatment in chronically socially isolated animals enhances norepinephrine uptake and expression of cardio-inhibitory receptors in cardiac tissues, which could have a beneficial effect on the cardiovascular system under the increased activity of the sympathoneural system.

HIV-1 Tat regulation of dopamine transmission and microglial reactivity is brain region specific.

The transactivator of transcription protein, HIV-1 Tat, is linked to neuroAIDS, where degeneration of dopamine neurons occurs. Using a mouse model expressing GFAP-driven Tat protein under doxycycline (Dox) regulation, we investigated microglial-neuronal interactions in the rostral substantia nigra pars compacta (SNc). Immunohistochemistry for microglia and tyrosine hydroxylase (TH) showed that the ratio of microglia to dopamine neurons is smaller in the SNc than in the ventral tegmental area (VTA) and that this difference is maintained following 7-day Dox exposure in wild type animals. Administration of Dox to wild types had no effect on microglial densities. In addressing the sensitivity of neurons to potentially adverse effects of HIV-1 Tat, we found that HIV-1 Tat exposure in vivo selectively decreased TH immunoreactivity in the SNc but not in the VTA, while levels of TH mRNA in the SNc remained unchanged. HIV-1 Tat induction in vivo did not alter the total number of neurons in these brain regions. Application of Tat (5 ng) into dopamine neurons with whole-cell patch pipette decreased spontaneous firing activity. Tat induction also produced a decline in microglial cell numbers, but no microglial activation. Thus, disappearance of dopaminergic phenotype is due to a loss of TH immunoreactivity rather than to neuronal death, which would have triggered microglial activation. We conclude that adverse effects of HIV-1 Tat produce a hypodopamine state by decreasing TH immunoreactivity and firing activity of dopamine neurons. Reduced microglial numbers after Tat exposure in vivo suggest impaired microglial functions and altered bidirectional interactions between dopamine neurons and microglia.

Probiotic Kefir Antihypertensive Effects in Spontaneously Hypertensive Rats Involves Central and Peripheral Mechanisms

Gut microbiota dysbiosis has been associated with hypertension. Long‐term use of probiotics have been shown to decrease blood pressure, improve baroreflex, and ameliorate endothelial dysfunction in Spontaneously Hypertensive Rats (SHR). Yet, the mechanisms are not well understood. We aimed to evaluate peripheral and centrally mediated mechanisms by which the probiotic kefir promotes beneficial effects in SHRs. We used Wistar Kyoto (WKY) and SHR male rats, divided into: WKY and SHR treated with vehicle, and SHR treated with milk fermented by the kefir grains (5%; SHR‐Kefir; oral gavage, daily/9 weeks). We evaluated blood pressure by tail cuff method weekly. The effects of kefir were evaluated on gut anatomy (histological analysis of the small intestine), gut microbiota composition (microbiological analysis of the feces), and endotoxin levels (Lipopolysaccharide (LPS) content analyzed in serum). Within the paraventricular nucleus of the hypothalamus (PVN), we evaluated microglia activation and tyrosine hydroxylase (TH) protein expression via immunofluorescence. TH mRNA levels were analyzed via real time PCR. SHR‐Kefir presented a lower mean blood pressure (151.9±2.9mmHg) compared to SHR (173.1±2.41mmHg, P&lt;0.0001 n=6). Basal gut microbiota composition in SHRs differed from that observed for WKY, suggesting dysbiosis. Treatment with kefir restored the normal gut microbiota composition profile of Lactobacillus, Staphylococcus, Brucella and Bifidobacterium spp. The number of Paneth cells/crypt was decreased in SHR (1.66±0.06) compared to WKY (2.40±0.08 p&lt;0.0004), an effect which was normalized with kefir treatment (SHR‐Kefir:2.23±0.15). An increase in LPS serum content in SHR (0.71±0.02) compared to WKY (0.54±0.04 EU/mL p&lt;0.01), suggesting increased intestinal permeability during hypertension, was reversed in SHR‐Kefir (0.54±0.01). Within the PVN, microglia density was used as an indication of microglia activation. We found increased microglia activation in SHR (9.47±0.61) compared to WKY (6.87±0.68 p&lt;0.07), which was normalized in SHR‐Kefir rats (4.77±0.55, arbitrary units [AU]). TH protein overexpression observed in SHRs was reduced with treatment of probiotic (WKY: 5.25±0.58; SHR: 13.68±0.81 and SHR‐Kefir: 8.42±0.55, AU p&lt;0.0001). The mRNA expression of TH within the PVN (punches) was increased in SHR by 47%. Kefir treatment decreased mRNA TH expression (WKY: 1.21±0.45; SHR: 1.79±0.50; SHR‐Kefir: 1.30±0.27 fold change). Our data shows that kefir treatment in SHRs restores gut microbiota composition and intestinal structure, diminishes levels of serum endotoxin, reduces neuroinflammation, and balances levels of tyrosine hydroxylase within the hypothalamus. Altogether, our data suggests that kefir antihypertensive‐associated mechanisms involves gut microbiota–brain axis communication during hypertension.Support or Funding InformationThis work was funded by CAPES PDSE 88881.133261/2016‐01 to MAS. VCB is funded by AHA 14SDG20400015.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of ghrelin on the motor deficit caused by the ablation of nigrostriatal dopaminergic cells or the inhibition of striatal dopamine receptors

Ghrelin plays roles in a wide range of central functions by activating the growth hormone secretagogue receptor (GHSR). This receptor has recently been found in the substantia nigra (SN) to control dopamine (DA)-related physiological functions. The dysregulation of DA neurons in the SN pars compacta (SNc) and the consequent depletion of striatal DA are known to underlie the motor deficits observed in Parkinson's disease (PD). In the present study, we further investigated the role of the SN-ghrelin system in motor function under the stereotaxic injection of AAV-CMV-FLEX-diphtheria toxin A (DTA) into the SN of dopamine transporter (DAT)-Cre (DATSN::DTA) mice to expunge DA neurons of the SNc. First, we confirmed the dominant expression of GHSR1a, which is a functional GHSR, in tyrosine hydroxylase (TH)-positive DA neurons in the SNc of control mice. In DATSN::DTA mice, we clearly observed motor dysfunction using several behavioral tests. An immunohistochemical study revealed a dramatic loss of TH-positive DA neurons in the SNc and DAT-labeled axon terminals in the striatum, and an absence of mRNAs for TH and DAT in the SN of DATSN::DTA mice. The mRNA level of GHSR1a was drastically decreased in the SN of these mice. In normal mice, we also found the mRNA expression of GHSR1a within GABAergic neurons in the SN pars reticulata (SNr). Under these conditions, a single injection of ghrelin into the SN failed to improve the motor deficits caused by ablation of the nigrostriatal DA network using DATSN::DTA mice, whereas intra-SN injection of ghrelin suppressed the motor dysfunction caused by the administration of haloperidol, which is associated with the transient inhibition of DA transmission. These findings suggest that phasic activation of the SNc-ghrelin system could improve the dysregulation of nigrostriatal DA transmission related to the initial stage of PD, but not the motor deficits under the depletion of nigrostriatal DA. Although GHSRs are found in non-DA cells of the SNr, GHSRs on DA neurons in the SNc may play a crucial role in motor function.

Tyrosine Hydroxylase, Vesicular Monoamine Transporter and Dopamine Transporter mRNA Expression in Nigrostriatal Tissue of Rats with Pedunculopontine Neurotoxic Lesion.

Background: The degeneration of the pedunculopontine nucleus (PPN) precedes the degeneration of the nigral cells in the pre-symptomatic stages of Parkinson’s disease (PD). Although the literature recognizes that a lesion of the PPN increases the vulnerability of dopaminergic cells, it is unknown if this risk is associated with the loss of capability of handling the dopaminergic function. Methods: In this paper, the effects of a unilateral neurotoxic lesion of the PPN in tyrosine hydroxylase (TH), vesicular monoamine transporter 2 (VMAT2) and dopamine transporter (DAT) mRNA expression in nigrostriatal tissue were evaluated. Three experimental groups were organized: non-treated rats, NMDA-lesioned rats and Sham-operated rats. Results: Seven days after the PPN lesion, in nigral tissue, TH mRNA expression was higher in comparison with control groups (p < 0.05); in contrast, VMAT2 mRNA expression showed a significant decrease (p < 0.01). DAT mRNA expression showed a significant decrease (p < 0.001) in the striatal tissue. Comparing nigral neuronal density of injured and control rats revealed no significant difference seven days post-PPN injury. Conclusions: Findings suggest that the PPN lesion modifies the mRNA expression of the proteins associated with dopaminergic homeostasis at nigrostriatal level. It could represent vulnerability signals for nigral dopaminergic cells and further increase the risk of degeneration of these cells.

Safranal-promoted differentiation and survival of dopaminergic neurons in an animal model of Parkinson’s disease

Context: Safranal (SAF) is verified to have potential effects in promoting nerve growth.Objectives: This study verifies the role of SAF in promoting dopaminergic neurons growth in vitro and in vivo.Material and methods: Rat neural stem cells (NSC) were treated with 1, 20, or 100 ng/mL of SAF, and the expression levels of tyrosine hydroxylase (TH) and dopamine transporter (DAT) were assayed by flow cytometry and real-time PCR and the secretion of dopamine (DA) was assayed by ELISA. Then, 2 × 106 cells of SAF-treated NSC was administrated into PD rat models induced by 6-OHDA. The differentiation and survival of dopaminergic neurons was identified by fluorescence microscope and TH+ cells by immunostaining and DA secretion by ELISA at week 2 and week 4, respectively.Results: After being treated with SAF at 20 and 100 ng/mL for 1 week, TH and DAT positive rates increased 1.4- and 1.7-fold (p < 0.01, respectively). TH and DAT mRNA also increased 8.05- and 4.41-fold, respectively. And the release of DA statistically increased 1.5-fold (p < 0.01). In vivo, the number of rotations decreased to 4.33 ± 0.97 rpm (p < 0.01) and the survival rates increased to 77.66 ± 7.87% (p < 0.05) at week 4 after transplantation of SAF-treated NSC. Moreover, the transplanted cells increased three-fold, TH fluorescence density increased four-fold and DA releases increased 1.4-fold (p < 0.01) at week 4 after transplantation.Conclusions: SAF promoted the production of functional DA cells and alleviated PD, which may contribute to a new therapy for PD patients.

Nest box exploration may stimulate breeding physiology and alter mRNA expression in the medial preoptic area of female European starlings.

Environmental resources are proposed to fine-tune the timing of breeding, yet how they may do so remains unclear. In female European starlings (Sturnus vulgaris), nest cavities are limited resources that are necessary for breeding. Females that explore nest cavities, compared with those that do not, readily perform sexually motivated behaviors. We assigned female starlings to aviaries with: (1) no nest boxes, (2) nest boxes, or (3) nest boxes, plants, flowing water, insects and berries to test the hypothesis that environmental resources alter neural systems to stimulate mating behavior. Compared with other females, females that were housed with and explored nest boxes had higher estradiol, higher preproenkephalin (PENK) mRNA and lower levels of D1 and D2 dopamine receptor mRNA in the medial preoptic area (mPOA); a region in which opioids and dopamine modify female sexual behaviors and sexual motivation. Additionally, in the mPOA, PENK and tyrosine hydroxylase mRNA positively predicted, whereas estrogen receptor beta mRNA negatively predicted, nest box exploration. In the ventromedial hypothalamus (a region in which estradiol acts to stimulate sexual behavior), estrogen receptor alpha mRNA was highest in females that had access to but did not explore nest cavities. It is likely that seasonal increases in estradiol modify mRNA in the mPOA to facilitate nest cavity exploration. It is also possible that nest cavity exploration further alters gene expression in the mPOA, functioning to coordinate mating with resource availability. Thus, nest cavity exploration may be a form of self-stimulation that alters neural systems to fine-tune sexual behavior.

Imidacloprid, a neonicotinoid insecticide, facilitates tyrosine hydroxylase transcription and phenylethanolamine N-methyltransferase mRNA expression to enhance catecholamine synthesis and its nicotine-evoked elevation in PC12D cells

Imidacloprid is a neonicotinoid insecticide acting as an agonist of nicotinic acetylcholine receptors (nAChRs) in the target insects. However, questions about the safety to mammals, including human have emerged. Overactivation of mammalian peripheral catecholaminergic systems leads to onset of tachycardia, hypertension, vomiting, etc., which have been observed in acutely imidacloprid-poisoned patients as well. Physiological activation of the nAChRs is known to drive catecholamine biosynthesis and secretion in mammalian adrenal chromaffin cells. Yet, the impacts of imidacloprid on the catecholaminergic function of the chromaffin cells remain to be evaluated. In this study using PC12D cells, a catecholaminergic cell line derived from the medulla chromaffin-cell tumors of rat adrenal gland, we examined whether imidacloprid itself could impact the catecholamine-synthesizing ability. Imidacloprid alone did facilitate tyrosine hydroxylase (TH) transcription via activation of α3β4 nAChR and the α7 subunit-comprising receptor. The insecticide showed the TH transcription-facilitating ability at the concentrations of 3 and 30 μM, at which acetylcholine is known to produce physiological responses, including catecholamine secretion through the nAChRs in adrenal chromaffin cells. The insecticide-facilitated TH transcription was also dependent on PKA- and RhoA-mediated signaling pathways. The insecticide coincidentally raised levels of TH and phenylethanolamine N-methyltransferase (PNMT) mRNA, and as a consequence, increased catecholamine production, although the efficacy of the neonicotinoid was lesser than that of nicotine, indicating its partial agonist-like action. Intriguingly, in cultured rat adrenal chromaffin cells, imidacloprid did increase levels of TH and PNMT protein. When the chromaffin cells were treated with nicotine in the presence of the insecticide, nicotine-elevated adrenaline production was enhanced due to facilitation of nicotine-increased TH and PNMT protein expression, and simultaneous enhancement of nicotine-elevated adrenaline secretion also took place. These findings thus suggest that imidacloprid may facilitate the physiological functions of adrenal glands in mammals.

PCB95 and PCB153 change dopamine levels and turn-over in PC12 cells

Polychlorinated biphenyls (PCB) exposure at low chronic levels is a significant public health concern. Animal and epidemiological studies indicate that low PCB body burden may cause neurotoxicity and be a risk factor for neurodegenerative diseases. In the current study, we measured the ability of two non-dioxin like PCBs, 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) and 2,2′3,5′,6-pentachlorobiphenyl (PCB95), to alter dopamine (DA) levels and metabolism using the dopaminergic PC12 cell line. Our hypothesis is that treatment of PC12 cells with non-toxic concentrations of PCB153 or PCB95 for 12 and 24 h will have different effects due to different congener structures. Levels of DA and of 3,4-dihydroxyphenylacetaldehyde (DOPAL), 3, 4-dihyroxylphenylethanol (DOPET), and 3,4-dihyroxylphenylacetic acid (DOPAC) metabolite, gene expression of the dopamine synthesis enzyme tyrosine hydroxylase (TH) and the vesicular monoamine transporter (VMAT2), and gene expression of the anti-oxidant enzymes Cu/Zn and Mn superoxide oxidase (Cu/ZnSOD, MnSOD), glutathione peroxidase (GPx) and catalase were determined. PCB153 decreased intracellular and extracellular levels of DA after 12 h exposure and this was consistent with an increase in DA metabolites. After 24 h, the level of DA in medium increased compared to the control. In contrast, PCB95 exposure increased the intracellular DA level and decreased DA in medium consistent with a down-regulation of VMAT2 expression at 12 h. After 24 h exposure, PCB95 increased DA levels in media. Expression of TH mRNA increased slightly following 12 h but not at 24 h exposure. MnSOD mRNA increased up to 6–7 fold and Cu/ZnSOD increased less than two-fold after treatment with both congeners. Catalase expression was up-regulated following 24 h exposure to PCB153 and PCB95, but GPx expression was down-regulated after 12 h exposure to PCB95 only. These results suggest that PCB153 and PCB95 are neurotoxic and affect DA turnover with structure-dependent differences between these two congeners.

Finasteride inhibited brain dopaminergic system and open-field behaviors in adolescent male rats.

Finasteride inhibits the conversion of testosterone to dihydrotestosterone. Because androgen regulates dopaminergic system in the brain, it could be hypothesized that finasteride may inhibit dopaminergic system. The present study therefore investigates the effects of finasteride in adolescent and early developmental rats on dopaminergic system, including contents of dopamine and its metabolites (dihydroxy phenyl acetic acid and homovanillic acid) and tyrosine hydroxylase expressions both at gene and protein levels. Meanwhile, open-field behaviors of the rats are examined because of the regulatory effect of dopaminergic system on the behaviors. Open-field behaviors were evaluated by exploratory and motor behaviors. Dopamine and its metabolites were assayed by liquid chromatography-mass spectrometry. Tyrosine hydroxylase mRNA and protein expressions were determined by real-time qRT-PCR and western blot, respectively. It was found that in adolescent male rats, administration of finasteride at doses of 25 and 50mg/kg for 14days dose dependently inhibited open-field behaviors, reduced contents of dopamine and its metabolites in frontal cortex, hippocampus, caudate putamen, nucleus accumbens, and down-regulated tyrosine hydroxylase mRNA and protein expressions in substantia nigra and ventral tegmental area. However, there was no significant change of these parameters in early developmental rats after finasteride treatment. These results suggest that finasteride inhibits dopaminergic system and open-field behaviors in adolescent male rats by inhibiting the conversion of testosterone to dihydrotestosterone, and imply finasteride as a potential therapeutic option for neuropsychiatric disorders associated with hyperactivities of dopaminergic system and androgen.

Long lasting effects of chronic WIN55,212-2 treatment on mesostriatal dopaminergic and cannabinoid systems in the rat brain

Cannabinoid administration modulates dopamine transmission via an indirect, multisynaptic mechanism that includes the activation of cannabinoid type-1 receptor (CB1R). The present study evaluated in rodents, the effects of acute and chronic (20 days) WIN55,212-2 administration, a non-selective CB1R agonist, on dopamine uptake and synthesis in the mesolimbic and nigrostriatal dopaminergic pathways and associate them to its effects on the endocannabinoid system. The effect of spontaneous withdrawal, after different abstinence periods (7 days, 20 days), was also assessed. Acute and chronic administration of WIN55,212-2 decreased dopamine transporter (DAT) binding and mRNA levels, as well as tyrosine hydroxylase (TH) mRNA expression in the substantia nigra (SN) and ventral tegmental area (VTA). In the striatum, chronic WIN55,212-2 administration led to decreased protein expression of DAT and TH, whereas no alterations were observed after acute administration, suggesting a diminished dopamine uptake and synthesis after chronic agonist treatment. Furthermore, after chronic agonist treatment, we observed reduced CB1R binding and mRNA levels in SN and striatum, providing evidence for a possible regulatory role of the endocannabinoid system on dopaminergic function. Seven days after WIN55,212-2 cessation, we observed a rebound increase in mRNA, binding and total protein levels of DAT and TH in VTA, SN and striatum proposing the existence of a biphasic expression pattern, which was also observed in CB1R binding levels. Within the 20-day period of abstinence, TH mRNA and protein levels and CB1R binding levels remain increased. The above results indicate that chronic CB1R agonist treatment induces long-lasting control of the mesostriatal dopaminergic activity.

Effect of Wnt1 and Wnt5a on the development of dopaminergic neurons, and toxicity induced by combined exposure to paraquat and maneb during gestation and lactation.

Paraquat (PQ) and maneb (MB) are widely used herbicides. Wingless (Wnt) proteins serve a role in the development and differentiation of dopaminergic neurons. Previous studies demonstrated that combined exposure to PQ and MB damages dopaminergic neurons in the midbrain. Effects of PQ and MB exposure on midbrain Wnt proteins have also been previously reported. In the present study, from the 5th day of gestation to weaning of the offspring, pregnant Sprague‑Dawley rats were administrated saline, or PQ and MB at two different doses: high, 15mg/kg body weight PQ+45mg/kg body weight MB; or low, 10mg/kg body weight PQ+30mg/kg body weight MB. Dopamine content in the striatum was examined by high performance liquid chromatography with a fluorescence detector and mRNA and protein expression of Wnt1, Wnt5a, nuclear receptor related factor 1 (Nurr1) and tyrosine hydroxylase (TH) in the midbrain was examined by reverse transcription‑quantitative polymerase chain reaction and western blotting. Combined exposure to PQ and MB during development decreased mRNA and protein expression of Wnt1, TH and Nurr1 and increased expression of Wnt5a in the offspring.

The expression and significance of tyrosine hydroxylase in the brain tissue of Parkinsons disease rats.

The expression and significance of tyrosine hydroxylase (TH) in brain tissue of rats with Parkinson's disease (PD) were explored and analyzed. A total of 120 clean-grade and healthy adult Wistar rats weighing 180–240 g were randomly divided equally into four groups according to the random number table method. Rats were sacrificed before and after the model establishment for 3, 6 or 8 weeks. The number of revolutions in rats was observed and the relative expression of TH mRNA in brain tissue was measured. The relative expression of TH mRNA in left and right brain tissue of normal rats was respectively 1.039±0.112 and 0.956±0.120. There was no significant difference in the expression of TH mRNA in either side of the brain tissue (p>0.05). With the extension of modeling time, the success rate of model establishment was significantly improved (p<0.05). After the model establishment for 6 weeks, the success rate peaked and there was no significant difference with 8 weeks (p>0.05). The relative expression of TH mRNA in the right brain was 0.053±0.082, which was significantly lower than that in the left brain tissue with 1.056±0.094 (p<0.05). After 6 weeks, the relative expression of TH mRNA in the left and right brain of PD rats was respectively 1.004±0.034 and 0.316±0.012, the expression of TH mRNA in right brain tissue was also significantly lower than that in the left (p<0.05). Similarly, after 8 weeks of the model establishment, the relative expression of TH mRNA in the right brain was significantly lower than that in the left brain tissue, with 0.395±0.041 and 1.021±0.057 (p<0.05). Compared with the control group, the expression of TH mRNA in both sides of the brain tissue decreased, and the expression of TH mRNA in the injured brain tissue was significantly lower than that in normal rats (p<0.05). The expression of TH mRNA in brain tissue of PD rats was lower than that in normal brain tissue, which may be related to the occurrence and development of PD.

Activation of the HMGB1-RAGE axis upregulates TH expression in dopaminergic neurons via JNK phosphorylation

The derangement of tyrosine hydroxylase (TH) activity reduces dopamine synthesis and is implicated in the pathogenesis of Parkinson's disease. However, the extracellular modulator and intracellular regulatory mechanisms of TH have yet to be identified. Recently, high-mobility group box 1 (HMGB1) was reported to be actively secreted from glial cells and is regarded as a mediator of dopaminergic neuronal loss. However, the mechanism for how HMGB1 affects TH expression, particularly through the receptor for advanced glycation endproducts (RAGE), has not yet been investigated. We found that recombinant HMGB1 (rHMGB1) upregulates TH mRNA expression via simultaneous activation of JNK phosphorylation, and this induction of TH expression is blocked by inhibitors of RAGE and JNK. To investigate how TH expression levels change through the HMGB1-RAGE axis as a result of MPP+ toxicity, we co-treated SN4741 dopaminergic cells with MPP+ and rHMGB1. rHMGB1 blocked the reduction of TH mRNA following MPP+ treatment without altering cell survival rates. Our results suggest that HMGB1 upregulates TH expression to maintain dopaminergic neuronal function via activating RAGE, which is dependent on JNK phosphorylation.

Changes in the secretory activity of organs producing noradrenaline upon inhibition of its synthesis in neonatal rat brain

The study is devoted to investigating the humoral interaction of the organs that are major sources of the morphogenetic factor noradrenaline in the general circulation system of rats in the neonatal period of development. The change in the synthesis of noradrenaline in the central and peripheral organs when its synthesis in the brains of neonatal rats is switched off was considered as one of the mechanisms of such interaction. For this purpose, the level of gene expression and the content of enzymes involved in the synthesis of noradrenaline—tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH)—was determined in the brain, adrenal glands, and the organ of Zuckerkandl. The synthesis of noradrenaline after destruction of noradrenergic neurons was switched off by injecting anti-DBH-saporin-antibodies against dopamine-β-hydroxylase conjugated to the cytotoxin saporin into the cerebral ventricles of neonatal rats. The content of TH and DBH mRNA in organs was determined by realtime PCR, and the protein content of enzymes was determined by Western blotting. It is shown that, 48 h after switching off the synthesis of noradrenaline in the brain of 2-day-old animals, a sharp increase in TH and DBH mRNA content in adrenal glands and the organ of Zuckerkandl was observed, although the content of both enzymes was retained at the control level. These data indicate a compensatory enhancement of expression of the genes encoding noradrenaline synthesis enzymes at the transcriptional level as a result of humoral interaction of central and peripheral sources of noradrenaline in the neonatal period of ontogeny.

The efficacy of (+)-Naltrexone on alcohol preference and seeking behaviour is dependent on light-cycle

Circadian rhythm affects drug-induced reward behaviour and the innate immune system. Peaks in reward-associated behaviour and immune responses typically occur during the active (dark) phase of rodents. While the role of the immune system, specifically, Toll-like receptor 4 (TLR4, an innate immune receptor) in drug-induced reward is becoming increasingly appreciated, it is unclear whether its effects vary according to light-cycle. Therefore, the aim of this study was to characterise the effects of the phase of the light-cycle and the state of the innate immune system on alcohol reward behaviour and subsequently determine whether the efficacy of targeting the immune component of drug reward depends upon the light-cycle.This study demonstrates that mice exhibit greater alcohol-induced conditioned place preference and alcohol two-bottle choice preference during the dark cycle. This effect overlapped with elevations in reward-, thirst- and immune-related genes. Administration of (+)-Naltrexone, a TLR4 antagonist, reduced immune-related gene mRNA expression and alcohol preference with its effects most pronounced during the dark cycle. However, (+)-Naltrexone, like other TLR4 antagonists exhibited off-target side effects, with a significant reduction in overall saccharin intake – an effect likely attributable to a reduction in tyrosine hydroxylase (Th) mRNA expression levels. Collectively, the study highlights a link between a time-of-day dependent influence of TLR4 on natural and alcohol reward-like behaviour in mice.

Prior Repeated Stress Attenuates Cold-Induced Immunomodulation Associated with "Browning" in Mesenteric Fat of Rats.

Continuous exposure to cold leads to activation of adaptive thermogenesis in brown adipose tissue but also to induction of brown/beige cell phenotype in white adipose tissue. The aim of this work was to investigate whether prior exposure to immobilization (IMO) stress may affect immune response associated with adipocyte "browning" in mesenteric adipose tissue (mWAT). In the first experiment, Sprague-Dawley rats were exposed to acute (3h) or prolonged (7days) cold exposure (4±1°C). 7-day cold stimulated gene expression of uncoupling protein 1 and other "browning"-associated factors. In the second experiment, rats were immobilized for 7days (2h daily) followed by exposure to continuous cold for 1 or 7days. Prior IMO exaggerated cold-induced sympathetic response manifested by elevated tyrosine hydroxylase (TH) protein and norepinephrine in mWAT. Induction of non-sympathetic catecholamine production demonstrated by elevated TH and PNMT (phenylethanolamine N-methyltransferase) mRNAs was observed after 7-day cold; however, prior IMO attenuated this response. 7-day cold-induced gene expression of anti-inflammatory mediators (IL-4, IL-13, IL-10, adiponectin), markers of M2 macrophages (Arg1, Retnlα), and eosinophil-associated molecules (eotaxin, IL-5), while inhibited expression of pro-inflammatory cytokines (IFNγ, IL-1b, IL-6, IL-17) and monocytes (MCP-1, Ly6C). This immune response was accompanied by elevated expression of uncoupling protein-1 and other thermogenic factors. Rats exposed to prior IMO exhibited inhibition of cold-induced immune and "browning"-related expression pattern. Overall, we demonstrated that 7-day cold-induced browning"-associated changes in rat mWAT, while prior history of repeated stress prevented this response.

Angiotensin-(1-7) attenuates atrial tachycardia-induced sympathetic nerve remodeling.

Introduction:The effect of Angiotensin-(1–7) (Ang-(1–7)) on atrial autonomic remodeling is still unknown. We hypothesized that Ang-(1–7) could inhibit sympathetic nerve remodeling in a canine model of chronic atrial tachycardia.Materials and methods:Eighteen dogs were randomly assigned to sham group, pacing group and Ang-(1–7) group. Rapid atrial pacing was maintained for 14 days in the pacing and Ang-(1–7) groups. Ang-(1–7) was administered intravenously in the Ang-(1–7) group. The atrial effective refractory period and atrial fibrillation inducibility level were measured at baseline and under sympathetic nerve stimulation after 14 days of measurement. The atrial sympathetic nerves labeled with tyrosine hydroxylase were detected using immunohistochemistry and Western blotting, and tyrosine hydroxylase and nerve growth factor mRNA levels were measured by reverse transcription polymerase chain reaction.Results:Pacing shortened the atrial effective refractory period and increased the atrial fibrillation inducibility level at baseline and under sympathetic nerve stimulation. Ang-(1–7) treatment attenuated the shortening of the atrial effective refractory period and the increase in the atrial fibrillation inducibility level. Immunohistochemistry and Western blotting showed sympathetic nerve hyperinnervation in the pacing group, while Ang-(1–7) attenuated sympathetic nerve proliferation. Ang-(1–7) alleviated the pacing-induced increases in tyrosine hydroxylase and nerve growth factor mRNA expression levels.Conclusion:Ang-(1–7) can attenuate pacing-induced atrial sympathetic hyperinnervation.

RT-qPCR for PHOX2B mRNA is a highly specific and sensitive method to assess neuroblastoma minimal residual disease in testicular tissue.

Neuroblastoma (NB) is the most common type of extracranial solid tumor in children with a high prevalence in toddlers. For childhood cancer survivors, preservation of reproductive potential is an important factor for quality of life. The optimization of NB minimal residual disease (MRD) detection in testicular tissue is crucial to evaluate the risk of malignant cell reintroduction. The first step in the present study was to assess the accuracy of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect tyrosine hydroxylase (TH), paired-like homeobox 2b (PHOX2B) and doublecortin (DCX) mRNA expression in frozen/thawed testicular tissues of patients with non-obstructive azoospermia (NOA) contaminated (in vitro model) with an increasing number of IMR-32 and SK-N-SH NB cells. Testicular tissues were frozen by slow or snap freezing. The second step was to determine the expression levels of these markers in testicular samples from 4 pre-pubertal males (2 with stage IV NB and 2 with non-NB malignancy). The yield of extracted RNA was similar in testicular samples frozen by slow or snap freezing. In the in vitro model, TH and DCX transcripts were detected in uncontaminated testicular tissues, whereas PHOX2B mRNA was not detected. There was a strong positive association between the number of NB cells used for contamination and PHOX2B transcript levels. For IMR-32 and SK-N-SH NB cell lines, specificity and sensitivity rates of detection were 100% for PHOX2B following in vitro contamination with 10 tumor cells. In testicular samples from pre-pubertal males with and without NB, PHOX2B mRNA expression was not observed, but high expression levels of TH and DCX mRNA were detected, which were similar to expression detected in the in vitro model. Among the markers used in blood and bone marrow for NB MRD studies, the detection of PHOX2B transcripts by RT-qPCR may provide an accurate assessment of NB cells in testicular tissues from males who require fertility preservation.