Melatonin modulate the expression of α1- and ß2-adrenoceptors in the hippocampus of rats subjected to unpredictable chronic mild stress.

OBJECTIVE: This study investigated the effects of chronic melatonin treatment on gene expression of α1-, α2-, ß1- and ß2-adrenoceptors in the hippocampus of rats subjected to chronic unpredictable mild stress (CUMS). BACKGROUND: Preclinical studies have also shown that melatonin prevented short- and long-term memory impairments and exhibited antidepressant-like actions. METHODS: For this study, we used 24 animals, which were divided into four groups, and the experiment lasted 4 weeks. We quantified the changes in mRNA and protein levels of α1-, α2-, ß1- and ß2-adrenoceptors in the hippocampus after melatonin treatment. RESULTS: Our results demonstrated a decreased gene expression of α1-, α2- and ß2-adrenoceptors in the hippocampus of rats subjected to unpredictable chronic mild stress, while there was no change in gene expression of ß1-adrenoceptors. Melatonin treatment in the CUMS rats prevented the stress-induced decrease in mRNA and protein levels of α1-and ß2-adrenoceptors, whereas did not affect either on mRNA or protein level of ß1-and α2-adrenoceptors. CONCLUSION: Our data suggest that melatonin, by increasing reduced levels of α1- and ß2-adrenoceptors mRNA and protein in the hippocampus of chronic stressed rats, may be beneficial in conditions such as chronic stress and provides an experimental opportunity to probe into further molecular mechanisms underlying the regulation of these receptor subtype (Fig. 2, Ref. 28).

Anxiety and Hyperlocomotion Induced by Chronic Unpredictable Mild Stress Can Be Moderated with Melatonin Treatment.

Preclinical studies have shown that melatonin exercised antidepressant-like and anxiolyticlike effects in animal models of anxiety. The aim of the present study was to correlate the changes in behaviour induced by melatonin treatment with the activity of the dopaminergic system in the hippocampus of Wistar rats exposed to chronic, unpredictable, mild stress (CUMS). Male Wistar rats, 11 weeks old, were subjected to chronic stress for 28 successive days. Separate groups of control and stressed rats were intraperitoneally injected daily either with melatonin (10 mg/kg/day, i.p.) or placebo (5% ethanol). The open-field and elevated plus-maze tests were used to assess locomotor activities and anxiety levels. The content of dopamine (DA) in the hippocampal tissues was determined using radioenzymatic assay, while changes in tyrosine hydroxylase (TH) mRNA and protein levels in the hippocampus were determined using real-time RT-PCR and Western immunoblotting. Chronic stress led to reduction in the hippocampal dopaminergic content without affecting the levels of TH protein. These changes were accompanied by increased locomotor activity and higher anxiety levels in the open-field test. Administration of melatonin for 28 days resulted in an increase in the hippocampal DA content as a result of elevated TH protein levels. Melatonin showed an improvement in anxiety-like behaviour along with significantly reduced exploration. We could conclude that melatonin may stimulate dopaminergic synthesis in the hippocampus in order to suppress stress-induced behaviour.

Differential expression of tyrosine hydroxylase and transporters in the right and left stellate ganglion of socially isolated rats.

Chronic isolation stress of adult rat males acted increasing gene expression of tyrosine hydroxylase (TH) and neuronal norepinephrine transporter (NET) in the right stellate ganglia, while vesicular monoamine transporter 2 (VMAT2) level remained unchanged. The stress decreased protein level of TH, as well as mRNA levels for NET and VMAT2 in the left stellate ganglia, but expressed no effect on protein levels of these two transporters. These results demonstrate asymmetry in noradrenergic genes in the right and left stellate ganglia during stress and provide molecular evidence to help explain the difference in response to the stress.

Peripheral oxytocin treatment affects the rat adreno-medullary catecholamine content modulating expression of vesicular monoamine transporter 2.

The neuropeptide oxytocin has been shown to influence on neuroendocrine function. The aim of the present study was to investigate the effect of peripheral oxytocin treatment on the synthesis, uptake and content of adreno-medullary catecholamine. For this purpose oxytocin (3.6µg/100g body weight, s.c) was administrated to male rats once a day over 14 days. In order to assess the effect of peripheral oxytocin treatment on adreno-medullary catecholamine we measured epinephrine and norepinephrine content and gene expression of tyrosine hydroxylase (TH), norepinephrine transporter (NET) and vesicular monoamine transporter 2 (VMAT2) in the adrenal medulla. Our results show a significant increase of epinephrine (1.7-fold, p<0.05) and norepinephrine (1.5-fold, p<0.05) content in oxytocin treated animals compared to saline treated ones. Oxytocin treatment had no effect either on mRNA or protein level of TH and NET. Under oxytocin treatment the increase in VMAT2 mRNA level was not statistically significant, but it caused a significant increase in protein level of VMAT2 (3.7-fold, p<0.001). These findings indicate that oxytocin treatment increases catecholamine content in the rat adrenal medulla modulating VMAT2 expression.

Effect of chronic forced running on gene expression of catecholamine biosynthetic enzymes in stellate ganglia of rats.

The sympathoneural system has a profound influence on the heart function. Sympathetic neurons are the major contributors to the huge rise of circulating noradrenaline (NA) level in response to stressful stimuli. Treadmill training in rats is forced exercise which has the propensity to induce both psychological and physical stress. The aim of this study is to examine how chronic forced running (CFR) affects the expression of catecholamine biosynthetic enzymes (tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT)) and cAMP response element-binding (CREB) in stellate ganglia, as well as the concentrations of catecholamines, adrenocorticotropic hormone (ACTH) and corticosterone (CORT) in the plasma of rats. Also, we investigated how the additional acute immobilization stress changes the mentioned parameters. The rat training program consisted of 12 weeks running on a treadmill (20 m/min, 20 min/day). We found that CFR increases TH and DBH mRNA and protein levels in stellate ganglia, which is followed by increased NA concentration in the plasma. CFR reduces the level of PNMT mRNA, while the level of PNMT protein remains unchanged in stellate ganglia. The increased expression of TH and DBH genes positively correlates with the expression of CREB in stellate ganglia and with plasma ACTH level, while reduced level of PNMT mRNA in stellate ganglia correlates with reduced plasma CORT level. The additional acute immobilization stress increased gene expression of catecholamine biosynthetic enzymes in stellate ganglia, as well as catecholamines, ACTH and CORT levels in the plasma. The results presented here suggest that the continuous increase of the noradrenaline biosynthetic enzyme expression in stellate ganglia due to CFR may play a role in growing risk of cardiovascular diseases.

Flouxetine treatment acts selectively increasing myocardial beta1-adrenoceptor mRNA expression in stress-induced depression.

Changes in gene expression of beta1- and beta2-adrenoceptors (beta1 - and beta2-AR) in right and left atria and ventricles after fluoxetine treatment in stress-induced depression of adult rat males were studied. Elevated beta1-AR mRNA levels in the left atria and significantly higher levels of beta2-AR mRNA in the left atria and ventricles were observed in stress-induced depression in comparison with those of unstressed controls. Fluoxetine treatment led to increasing expression of beta1-AR mRNA in the right atria and left ventricles, while the level of beta2-AR mRNA remained unchanged. These findings suggest that fluoxetine therapy plays an important role in cardiac beta-adrenergic subsensitivity and gene regulation of beta-AR in animals with heightened sympathetic nervous activity.

Regulation of catecholamine-synthesising enzymes and beta-adrenoceptors gene expression in ventricles of stressed rats.

Stress exposure activates the sympathoneural system, resulting in catecholamine release. Chronic stress is associated with development of numerous disorders, including cardiovascular diseases. Here we investigated the expression of mRNAs for catecholamine biosynthetic enzymes tyrosine-hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine N-methyl-transferase, and for beta(1)- and beta(2)-adrenoceptors in the right and left ventricles of rats exposed to chronic unpredictable mild stress. The tyrosine-hydroxylase and dopamine-beta-hydroxylase mRNA levels were not affected by stress, whereas the phenylethanolamine N-methyltransferase mRNA levels significantly increased in both right and left ventricles. No changes in beta(1)-adrenoceptor mRNA levels in either right or left ventricles were observed. At the same time, stress produced a significant increase of beta(2)-adrenoceptor mRNA levels in left ventricles. These results suggest that elevated expression of phenylethanolamine N-methyltransferase in both ventricules and beta(2)-adrenoceptor genes in left ventricles could provide a molecular mechanism that leads to altered physiological response, which is important for the organism coping with stress.

Effects of repeated maprotiline and fluoxetine treatment on gene expression of catecholamine synthesizing enzymes in adrenal medulla of unstressed and stressed rats.

1 Repeated maprotiline (a noradrenaline reuptake inhibitor) and fluoxetine (a serotonin reuptake inhibitor) treatment on gene expression of catecholamine biosynthetic enzymes were examined in adrenal medulla of unstressed control and chronic unpredictable mild stressed rats. 2 Maprotiline did not change gene expression of catecholamine biosynthetic enzymes in control and stressed rats. 3 Fluoxetine increased gene expression of tyrosine hydroxylase (TH) and dopamine-ß-hydroxylase (DBH), but did not phenylethanolamine N-methyltransferase in both unstressed and chronic unpredictable mild stressed animals. 4 In conclusion, we have demonstrated that repeated administration of fluoxetine enhanced gene transcription of TH and DBH and subsequently stimulates noradrenaline synthesis in adrenal medulla of control and stressed rats.

Effect of immobilization stress on gene expression of catecholamine biosynthetic enzymes in heart auricles of socially isolated rats

Chronic stress is associated with the development of cardiovascular diseases. The sympathoneural system plays an important role in the regulation of cardiac function both in health and disease. In the present study, the changes in gene expression of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine-â-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) and protein levels in the right and left heart auricles of naive control and long-term (12 weeks) socially isolated rats were investigated by Taqman RT-PCR and Western blot analysis. The response of these animals to additional immobilization stress (2 h) was also examined. Long-term social isolation produced a decrease in TH mRNA level in left auricles (about 70 percent) compared to the corresponding control. Expression of the DBH gene was markedly decreased both in the right (about 62 percent) and left (about 81 percent) auricles compared to the corresponding control, group-maintained rats, whereas PNMT mRNA levels remained unchanged. Exposure of group-housed rats to acute immobilization for 2 h led to a significant increase of mRNA levels of TH (about 267 percent), DBH (about 37 percent) and PNMT (about 60 percent) only in the right auricles. Additional 2-h immobilization of individually housed rats did not affect gene expression of these enzymes in either the right or left auricle. Protein levels of TH, DBH and PNMT in left and right heart auricles were unchanged either in both individually housed and immobilized rats. The unchanged mRNA levels of the enzymes examined after short-term immobilization suggest that the catecholaminergic system of the heart auricles of animals previously exposed to chronic psychosocial stress was adapted to maintain appropriate cardiovascular homeostasis.

Effect of immobilization stress on gene expression of catecholamine biosynthetic enzymes in heart auricles of socially isolated rats.

Chronic stress is associated with the development of cardiovascular diseases. The sympathoneural system plays an important role in the regulation of cardiac function both in health and disease. In the present study, the changes in gene expression of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) and protein levels in the right and left heart auricles of naive control and long-term (12 weeks) socially isolated rats were investigated by Taqman RT-PCR and Western blot analysis. The response of these animals to additional immobilization stress (2 h) was also examined. Long-term social isolation produced a decrease in TH mRNA level in left auricles (about 70%) compared to the corresponding control. Expression of the DBH gene was markedly decreased both in the right (about 62%) and left (about 81%) auricles compared to the corresponding control, group-maintained rats, whereas PNMT mRNA levels remained unchanged. Exposure of group-housed rats to acute immobilization for 2 h led to a significant increase of mRNA levels of TH (about 267%), DBH (about 37%) and PNMT (about 60%) only in the right auricles. Additional 2-h immobilization of individually housed rats did not affect gene expression of these enzymes in either the right or left auricle. Protein levels of TH, DBH and PNMT in left and right heart auricles were unchanged either in both individually housed and immobilized rats. The unchanged mRNA levels of the enzymes examined after short-term immobilization suggest that the catecholaminergic system of the heart auricles of animals previously exposed to chronic psychosocial stress was adapted to maintain appropriate cardiovascular homeostasis.

Liver glucocorticoid receptor and heat shock protein 70 levels in rats exposed to different stress models.

The aim of the present study was to define the stress-induced pattern of cytosolic glucocorticoid receptor (GR) and Hsp70 protein in the liver of male Wistar rats exposed to different stress models: acute (2 h/day) immobilization or cold (4 degrees C); chronic (21 days) isolation, crowding, swimming or isolation plus swimming and combined (chronic plus acute stress). Changes in plasma levels of corticosterone were studied by radioimmunoassay (RIA). The results obtained by Western immunoblotting showed that both acute stressors led to a significant decrease in cytosolic GR and Hsp70 levels. Compared to acute stress effects, only a weak decrease in the levels of GR and Hsp70 was demonstrated in chronic stress models. Chronically stressed rats, which were subsequently exposed to novel acute stressors (immobilization or cold), showed a lower extent of GR down-regulation when compared to acute stress. The exception was swimming, which partially restores this down-regulation. The observed changes in the levels of these major stress-related cellular proteins in liver cytosol lead to the conclusion that chronic stressors compromise intracellular GR down-regulation in the liver.

Effects of stress on catecholamine stores in central and peripheral tissues of long-term socially isolated rats.

Both the peripheral sympatho-adrenomedullary and central catecholaminergic systems are activated by various psycho-social and physical stressors. Catecholamine stores in the hypothalamus, hippocampus, adrenal glands, and heart auricles of long-term socially isolated (21 days) and control 3-month-old male Wistar rats, as well as their response to immobilization of all 4 limbs and head fixed for 2 h and cold stress (4 degrees C, 2 h), were studied. A simultaneous single isotope radioenzymatic assay based on the conversion of catecholamines to the corresponding O-methylated derivatives by catechol-O-methyl-transferase in the presence of S-adenosyl-l-(3H-methyl)-methionine was used. The O-methylated derivatives were oxidized to 3H-vanilline and the radioactivity measured. Social isolation produced depletion of hypothalamic norepinephrine (about 18%) and hippocampal dopamine (about 20%) stores and no changes in peripheral tissues. Immobilization decreased catecholamine stores (approximately 39%) in central and peripheral tissues of control animals. However, in socially isolated rats, these reductions were observed only in the hippocampus and peripheral tissues. Cold did not affect hypothalamic catecholamine stores but reduced hippocampal dopamine (about 20%) as well as norepinephrine stores in peripheral tissues both in control and socially isolated rats, while epinephrine levels were unchanged. Thus, immobilization was more efficient in reducing catecholamine stores in control and chronically isolated rats compared to cold stress. The differences in rearing conditions appear to influence the response of adult animals to additional stress. In addition, the influence of previous exposure to a stressor on catecholaminergic activity in the brainstem depends on both the particular catecholaminergic area studied and the properties of additional acute stress. Therefore, the sensitivity of the catecholaminergic system to habituation appears to be tissue-specific.

Effects of stress on catecholamine stores in central and peripheral tissues of long-term socially isolated rats

Both the peripheral sympatho-adrenomedullary and central catecholaminergic systems are activated by various psycho-social and physical stressors. Catecholamine stores in the hypothalamus, hippocampus, adrenal glands, and heart auricles of long-term socially isolated (21 days) and control 3-month-old male Wistar rats, as well as their response to immobilization of all 4 limbs and head fixed for 2 h and cold stress (4°C, 2 h), were studied. A simultaneous single isotope radioenzymatic assay based on the conversion of catecholamines to the corresponding O-methylated derivatives by catechol-O-methyl-transferase in the presence of S-adenosyl-l-(³H-methyl)-methionine was used. The O-methylated derivatives were oxidized to ³H-vanilline and the radioactivity measured. Social isolation produced depletion of hypothalamic norepinephrine (about 18 percent) and hippocampal dopamine (about 20 percent) stores and no changes in peripheral tissues. Immobilization decreased catecholamine stores (approximately 39 percent) in central and peripheral tissues of control animals. However, in socially isolated rats, these reductions were observed only in the hippocampus and peripheral tissues. Cold did not affect hypothalamic catecholamine stores but reduced hippocampal dopamine (about 20 percent) as well as norepinephrine stores in peripheral tissues both in control and socially isolated rats, while epinephrine levels were unchanged. Thus, immobilization was more efficient in reducing catecholamine stores in control and chronically isolated rats compared to cold stress. The differences in rearing conditions appear to influence the response of adult animals to additional stress. In addition, the influence of previous exposure to a stressor on catecholaminergic activity in the brainstem depends on both the particular catecholaminergic area studied and the properties of additional acute stress. Therefore, the sensitivity of the catecholaminergic system to habituation appears to be tissue-specific.

Alterations in hippocampal antioxidant enzyme activities and sympatho-adrenomedullary system of rats in response to different stress models.

The study deals with activity of three antioxidant enzymes, copper, zinc-superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), catalase (CAT) in hippocampus of rats, following the exposure to single chronic (individual housing or forced swimming) and acute (immobilization or cold) stress, as well as to combined chronic/acute stress. In addition, plasma noradrenaline (NA) and adrenaline (A) concentrations were measured in the same stress conditions, because their autooxidation can add to the oxidative stress. We observed that i) long-term social isolation and repeated forced swimming had minor effects on plasma catecholamines, but in the long-term pretreated groups, acute stressors caused profound elevation NA and A levels, ii) chronic stressors activate antioxidant enzymes, iii) acute stressors decrease catalase activity, their effects on CuZnSOD appear to be stressor-dependent, whereas MnSOD is not affected by acute stressors, and iv) pre-exposure to chronic stress affects the antioxidant-related effects of acute stressors, but this effect depends to a large extent on the type of the chronic stressor. Based on both metabolic and neuroendocrine data, long-term isolation appears to be a robust psychological stressor and to induce a "priming" effect specifically on the CuZnSOD and CAT activity.

Effect of novel stressors on tyrosine hydroxylase gene expression in the adrenal medulla of repeatedly immobilized rats.

The activity of the sympathetic-adrenomedullary system in rats submitted to novel stressors after prior repeated or chronic stress exposure is poorly understood. The purpose of the present work was to investigate changes in adrenomedullary (AM) tyrosine hydroxylase (TH) gene expression after a single or long-term repeated exposure of rats to immobilization stress (IMMO; 42 times), as well as in repeatedly immobilized rats (41 times) exposed once to various novel heterotypic stressors. Cold exposure for 5 h, administration of insulin (INS, 51U), or 2-deoxyglucose (2DG, 500 mg/kg) were used as novel stressors. A single exposure to cold, INS, or 2DG produced transient increases in TH mRNA levels in AM. Animals exposed to repeated homotypic IMMO stress showed permanently increased TH mRNA levels, TH activity, and protein levels; however, an exposure of such animals to heterotypic novel stressors did not induce any further changes. Thus the observed differences in TH mRNA levels in the AM of control rats and long-term repeatedly IMMO rats suggest that an adaptation to this stressor is displayed by a permanently increased TH gene expression, TH activity, and protein level. The exposure of repeatedly IMMO rats to a single episode of novel stressor does not induce exaggerated responses in TH gene expression, as some other stressors do. The mechanism of this finding could involve a central regulation and/or adrenomedullary signaling pathway(s), leading to additional modifications or accumulation of transcription factors. The precise mechanism(s) of this phenomenon remains to be elucidated.