Natasa Spasojevic

Chronic Individual Housing-Induced Stress Decreased Expression of Catecholamine Biosynthetic Enzyme Genes and Proteins in Spleen of Adult Rats

Objective: Social isolation is regarded as one of the most relevant causes of diseases in mammalian species. The activation of the sympathoneural system represents one of the key components of the stress response. The sympathetic nervous system is one of the major pathways involved in immune-neuroendocrine interactions. The aim of the present study was to determine plasma epinephrine and norepinephrine in individually housed rats, as well as to find out whether splenic gene expression of catecholamine synthesizing enzymes and their protein levels are affected by chronic psychosocial stress. Methods: Tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) mRNA levels were quantified by quantitative real-time RT-PCR. The TH, DBH and PNMT immunoproteins were assayed by Western blot. Results: Chronic social isolation of adult male rats produced a significant increase in plasma catecholamine levels and a decrease in splenic TH mRNA, DBH mRNA and PNMT mRNA. Protein levels of TH, DBH and PNMT were also reduced. Conclusion: These results suggest that increased plasma catecholamines and decreased gene expression and protein levels of catecholamine biosynthetic enzymes in the spleen of chronically individually housed animals might reduce catecholamine synthesis, thus leaving the immunocompetent tissues depleted of catecholamines and consequently leading to an impairment of immune response.

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-β-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.

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

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

Novel stressors affected catecholamine stores in socially isolated normotensive and spontaneously hypertensive rats

Catecholamines in some central (hypothalamus and hippocampus) and peripheral tissues (adrenal glands and heart auricles) of long-term socially isolated normotensive and spontaneously hypertensive rats exposed to novel immobilization stress were determined by a simultaneous single isotope radioenzymatic assay. Long-term isolation (21 days) produced depletion of hypothalamic norepinephrine (NE) stores and hippocampal dopamine (DA) stores in both normotensive and spontaneously hypertensive rats. Acute immobilization stress (2 h) significantly decreased NE and DA stores in hypothalamus and hippocampus of naive normotensive and spontaneously hypertensive rats controls. However, novel immobilization stress applied to normotensive rats previously subjected to long-term isolation produced no changes in catecholamine levels in hypothalamus, while resulting in somewhat higher depletion of NE stores in hypothalamus of spontaneously hypertensive rats treated in the same way. Novel immobilization stress decreased NE and DA stores in hippocampus of normotensive but was without effect on NE and DA stores of spontaneously hypertensive rats. Social isolation did not affect catecholamine stores in peripheral tissues but novel immobilization stress produced a significant decrease in catecholamine content. The results suggest that some central and peripherals tissues of spontaneously hypertensive rats and normotensive rats differ with regard to catecholamine content and that there are certain differences in their responsiveness to stress.

Behavioral and endocrine responses of socially isolated rats to long-term diazepam treatment

The effects of diazepam (0.2 mg/kg/ during 21 days, i.p.) on behavior, pituitary-adrenocortical and sympatho-adrenomedullary system of socially isolated and group-housed adult male rats additionally exposed to immobilization, were studied. Social isolation led to a shorter duration of grooming and longer latency to start grooming. Diazepam in social isolated rats reduced incorrect transitions percentage, but the number of grooming bouts, duration and latency to start grooming remained unchanged. Long-term isolation significantly elevated plasma ACTH and corticosterone, while not affecting noradrenaline and adrenaline. Diazepam decreased only plasma ACTH. Social isolation and immobilization significantly elevated all examined hormones. Immobilization of diazepam-treated isolated rats enhanced plasma ACTH , the increase being significantly lower, comparing to isolated vehicle-treated rats. Immobilization significantly increased plasma adrenaline, noradrenaline and corticosterone of diazepam- or vehicle-treated socially isolated rats. No differences in adrenaline, noradrenaline and corticosterone level between these two groups were observed. This indicates that chronic diazepam treatment of socially isolated rats changes some grooming behavior parameters, but insignificantly affects stress-related adrenomedullary and adrenocortical alterations.

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.

Subsequent stress increases gene expression of catecholamine synthetic enzymes in cardiac ventricles of chronic-stressed rats

Since previous experience of stressful situation profoundly affects response to a subsequent novel stressor, we examined changes in gene expression and protein levels of catecholamine biosynthetic enzymes in cardiac ventricles after exposure of chronic psychosocially isolated adult Wistar male rats to short-term immobilization stress. Chronic social isolation did not affect gene expression of tyrosine hydroxylase (TH) in either right or left ventricle. Subsequent immonilization of these animals produced an elevation of TH mRNA level in right and left ventricles. The levels of dopamine-β-hydroxylase (DBH) mRNA were detectable only after immobilization both in right and left ventricles of control and chronically isolated rats. Chronic isolation stress increased phenylethanolamine N-methyltransferase (PNMT) mRNA levels in the right ventricle. Immobilization led to an elevated PNMT mRNA level in right and left ventricles of both control and chronically stressed animals. Protein levels of TH, DBH, and PNMT in right and left ventricles of socially isolated rats were increased after subsequent immobilization. Taking into consideration the role of cardiac catecholamines in physiological and pathophysiological processes, it could be hypothesized that increased catecholamine synthesis in the ventricles after novel immobilization stress could point to the susceptibility of the heart to subsequent stress.

Melatonin mediated antidepressant-like effect in the hippocampus of chronic stress-induced depression rats: Regulating vesicular monoamine transporter 2 and monoamine oxidase A levels

The hippocampus is sensitive to stress which activates norepinephrine terminals deriving from the locus coeruleus. Melatonin exerts positive effects on the hippocampal neurogenic process and on depressive-like behaviour. Thus, in the present study, an examination was made of the effect of chronic melatonin treatment on norepinephrine content, synthesis, uptake, vesicular transport and degradation in the hippocampus of rats exposed to CUMS. This entailed quantifying the norephinephrine, mRNA and protein levels of DBH, NET, VMAT 2, MAO-A and COMT. The results show that CUMS evoked prolonged immobility. Melatonin treatment decreased immobility in comparison with the placebo group, reflecting an antidepressant-like effect. Compared with the placebo group, a dramatic decrease in norepinephrine content, decreased VMAT2 mRNA and protein and increased MAO-A protein levels in the hippocampus of the CUMS rats were observed. However, no significant differences in the levels of DBH, NET, COMT mRNA and protein and MAO-A mRNA levels between the placebo and the stressed groups were found. The results showed the restorative effects of melatonin on the stress-induced decline in the norepinephrine content of the hippocampus. It was observed that melatonin treatment in the CUMS rats prevented the stress-induced decrease in VMAT2 mRNA and protein levels, whereas it reduced the increase of the mRNA of COMT and protein levels of MAO-A. Chronic treatment with melatonin failed to alter the gene expression of DBH or NET in the hippocampus of the CUMS rats. Additionally, the results show that melatonin enhances VMAT2 expression and norepinephrine storage, whilst it reduces norepinephrine degrading enzymes.

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.

Molecular basis of chronic stress-induced hippocampal lateral asymmetry in rats and impact on learning and memory

Neurochemical lateralization has been demonstrated in the rat brain suggesting that such lateralization might contribute to behavior. Thus, the aim of the present study was to examine neurochemical asymmetry in the hippocampus, molecular basis of neurochemical lateralization and its impact on spatial learning and memory. Changes in noradrenaline content, tyrosine hydroxylase (TH) were studied in the right and left hippocampus of naive control and chronically isolated rats, by applying TaqMan RT-PCR and Western blot analysis. Hippocampal-based spatial learning and memory were evaluated using the Barnes maze. In control rats an asymmetrical right-left distribution of noradrenaline content and gene expression of catecholamine synthesizing enzyme was found. Chronic psychosocial stress further emphasized asymmetry. Isolation stress reduced noradrenaline content only in the right hippocampus. No changes were observed in gene expression and protein levels of TH in the right hippocampus, whereas expression of catecholamine synthesizing enzyme was elevated in the left hippocampus. Reduced noradrenaline content in the right hippocampus did not cause impairment in spatial learning and memory. Our findings suggest that chronic psychosocial stress reduces noradrenaline stores in the right hippocampus which may be caused by molecular asymmetry, but it does not affect spatial learning and memory.