Lidia Serova

Estradiol Stimulates Gene Expression of Norepinephrine Biosynthetic Enzymes in Rat Locus coeruleus

Gender-specific differences in susceptibility to a number of disorders related to catecholaminergic systems, including depression and hypertension, have been postulated to be mediated, at least in part, by estrogens. In this study, we examined if estrogens may regulate gene expression of norepinephrine biosynthetic enzymes. Administration of five injections of 15 or 40 µg/kg estradiol benzoate to ovariectomized (OVX) female rats elicited a dose-dependent elevation in mRNA levels of tyrosine hydroxylase (TH) in locus coeruleus, to as great as 3-fold over control. Dopamine β-hydroxylase (DBH) mRNA levels were also similarly increased. To examine the mechanism, PC12 cells were cotransfected with luciferase reporter constructs under control of DBH or TH promoters [pDBH/Luc(–2,236/+21) or pTH/Luc(–272/+27 or –773/+27)] with an expression vector for estradiol receptor α. The cells were treated with 17β-estradiol (E2) for 12–36 h. E2 triggered a several fold increase in luciferase activity under control of the DBH promoter in a dose-dependent fashion. Omission of estrogen receptor α or addition of the estrogen receptor antagonist ICI 182,780 prevented the DBH promoter-driven increase in luciferase. When E2 was given with 0.2 mM CPT-cAMP, reporter activity with pDBH/Luc(–2,236/+21) was increased greater than with either treatment alone. In contrast, addition of E2 to cells transfected with pTH/Luc(–272/+27) elicited no change in basal luciferase activity nor in the response to 0.2 mM CPT-cAMP. These findings are the first to reveal that estrogen can stimulate DBH gene expression. Differing mechanisms may underlie the regulation of TH and DBH gene expression by estrogens.

Single intranasal neuropeptide Y infusion attenuates development of PTSD-like symptoms to traumatic stress in rats

Exposure to severe stress leads to development of neuropsychiatric disorders, including depression and Post-Traumatic Stress Disorder (PTSD) in at-risk individuals. Neuropeptide Y (NPY) is associated with resilience or improved recovery. Therefore exogenous administration to the brain has therapeutic potential although peripheral administration can trigger undesirable side effects. Here, we established conditions with intranasal (IN) NPY infusion to rats to obtain CSF concentrations in the proposed anxiolytic range without significant change in plasma NPY. Rats were pretreated with IN NPY or vehicle before exposure to single prolonged stress (SPS) animal model of PTSD and compared to untreated controls. The IN NPY appeared to lessen the perceived severity of stress, as these animals displayed less time immobile in forced swim part of the SPS. Thirty minutes after SPS the elevation of plasma adrenocorticotropic hormone (ACTH) and corticosterone was not as pronounced in NPY-infused rats and the induction of tyrosine hydroxylase (TH) in locus coeruleus (LC) was attenuated. Seven days after SPS, they displayed lower depressive-like behavior on Forced Swim Test and reduced anxiety-like behavior on Elevated Plus Maze. The prolonged effect of SPS on Acoustic Startle Response was also lower in NPY-infused rats. Plasma ACTH, corticosterone, and hippocampal glucocorticoid receptor levels were significantly above controls only in the vehicle - but not IN NPY-treated group 1week after SPS. Baseline TH mRNA levels in LC did not differ among groups, but increased with forced swim in the vehicle - but not NPY-pretreated animals. Administration of IN NPY after exposure to SPS led to similar, but not identical, reduction in development of anxiety, depressive-like behavior and hyperarousal. The results show that single IN NPY can alter stress-triggered dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis and activation of central noradrenergic activity. These findings provide proof of concept for potential of IN NPY for non-invasive prophylactic treatment or early intervention in response to traumatic stress.

Response of tyrosine hydroxylase and GTP cyclohydrolase I gene expression to estrogen in brain catecholaminergic regions varies with mode of administration

The effect of different dose, mode and duration of estradiol administration was examined in the different brain catecholaminergic areas in ovariectomized (OVX) female rats. We determined changes in mRNA levels of tyrosine hydroxylase (TH), rate-limiting enzyme in catecholamine (CA) biosynthesis of GTP cyclohydrolase I (GTPCH), rate-limiting enzyme in biosynthesis as well as of tetrahydrobiopterin (BH4), and concentration of BH4, which is an essential cofactor for TH, tryptophan hydroxylase and nitric oxide synthase. Short-term administration of estradiol benzoate (EB) by five injections of 15 or 40 microg/kg 12 h apart led to increase in TH and GTPCH mRNA levels in dopaminergic and noradrenergic cell bodies of the ventral tegmental area (VTA), substantia nigra (SN), locus coeruleus (LC) and the nucleus of solitary tract (NTS) depending on dose of administration. Estrogen-elicited alterations in BH4 concentrations were mostly correlated with changes in GTPCH mRNA levels, except in SN. Long-term administration of estradiol by injections (EB: 25 microg/kg, 16 injections 26 h apart; 50 microg/kg, 16 injections 48 h apart) or pellets (0.1 mg 17 beta-estradiol, 14 days) were not very effective in modulating mRNA levels for both genes in most locations except the NTS. Long-term injections of EB elevated GTPCH mRNA levels throughout the NTS and in microvessels. Administration of estradiol by pellets led to decline of TH mRNA in rostral-medial and elevation in caudal parts of the NTS. Thus, estradiol has a complex and differential effect on TH and GTPCH gene expression in a tissue specific manner and depends on the mode of administration.

Immobilization Stress elevates tryptophan hydroxylase mRNA and protein in the rat raphe nuclei

BACKGROUND Stress triggers adaptive and maladaptive changes in the central nervous system, including activation of the hypothalamic-pituitary-adrenal axis, and can trigger mood disorders and posttraumatic stress disorder. We examined the effect of immobilization stress (IMO) on gene expression of tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin biosynthesis, and the role of cortisol in that response. METHODS Regular and adrenalectomized Sprague-Dawley rats were exposed to various repetitions of IMO. Tryptophan hydroxylase messenger ribonucleic acid (mRNA) was determined by competitive reverse transcriptase polymerase chain reaction, and TPH protein was examined by immunoblot and immunocytochemistry. RESULTS Elevation of TPH mRNA by IMO was tissue-specific and dose-dependent. A single IMO elicited a threefold rise in TPH mRNA in median raphe nucleus (MRN), but repeated (3x) IMOs were needed for similar response in dorsal raphe nucleus (DRN). Repeated daily IMO, up to 7 days, triggered a robust induction (6-10-fold) in TPH mRNA, accompanied by corresponding rise in TPH protein levels in raphe nuclei but not in the pineal gland. The rise in TPH immunoreactivity was widespread throughout the DRN and MRN. Bilateral adrenalectomy did not prevent the IMO-triggered increase in TPH immunoreactive protein in the raphe nuclei. CONCLUSIONS This study reveals adrenal glucocorticoid-independent induction of TPH gene expression in raphe nuclei in response to immobilization stress.

Tryptophan hydroxylase mRNA levels are elevated by repeated immobilization stress in rat raphe nuclei but not in pineal gland

Repeated stress triggers a wide range of adaptive changes in the central nervous system including the elevation of serotonin (5-HT) metabolism and an increased susceptibility to affective disorders. To begin to examine whether these changes are mediated by alterations in gene expression for tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis, we quantitated its mRNA levels by competitive reverse transcription-polymerase chain reaction (RT-PCR). Repeated immobilization stress (2 h, 7 days) elicited a six- or ten-fold rise in TPH mRNA in median raphe nucleus (MRN) and dorsal raphe nucleus (DRN), respectively, without significantly altering TPH mRNA levels in the pineal gland. In contrast, there was little change in mRNA levels for GTP cyclohydrolase I (GTPCH), the rate limiting enzyme in synthesis of the tetrahydrobiopterin (BH4), the obligate cofactor for TPH. This is the first study to reveal stress-elicited activation of TPH gene expression.

Heightened transcription for enzymes involved in norepinephrine biosynthesis in the rat locus coeruleus by immobilization stress.

BACKGROUND The locus coeruleus (LC), a target for CRH neurons, is critically involved in responses to stress. Various physiological stresses increase norepinephrine turnover, tyrosine hydroxylase (TH) enzymatic activity, protein and mRNA levels in LC cell bodies and terminals; however, the effect of stress on other enzymes involved in norepinephrine biosynthesis in the LC is unknown. METHODS Rats were exposed to single (2 hour) or repeated (2 hour daily) immobilization stress (IMO). Recombinant rat dopamine b-hydroxylase (DBH) cDNA was expressed in E. coli and used to generate antisera for immunohistochemistry and immunoblots in LC. Northern blots were used to assess changes in mRNA levels for TH, DBH, and GTP cyclohydrolase I (GTPCH) in the LC in response to the stress. Conditions were found to isolate nuclei from LC and to use them for run-on assays of transcription. RESULTS Repeated stress elevated the DBH immunoreactive protein levels in LC. Parallel increases in TH, DBH and GTPCH mRNA levels of about 300% to 400% over control levels were observed with single IMO, and remained at similar levels after repeated IMO. This effect was transcriptionally mediated, and even 30 min of a single IMO significantly increased the relative rate of transcription. CONCLUSIONS This study is the first to reveal transcriptional activation of the genes encoding catecholamine biosynthetic enzymes in the LC by stress. In addition to TH, changes in DBH and GTPCH gene expression may also contribute to the development of stress-triggered affective disorders.

Transcriptional regulation of tyrosine hydroxylase by estrogen : opposite effects with estrogen receptors α and β and interactions with cyclic AMP

Reported effects of estrogen administration on tyrosine hydroxylase (TH) gene expression are confusing. Therefore, we studied the mechanism of regulation of TH transcription by estrogen with different estradiol receptor (ER) subtypes. PC12 cells, transiently co‐transfected with expression vector for ERα or ERβ, and luciferase gene under control of the TH promoter, were treated with 17 β‐estradiol (E2). E2 doubled luciferase activity with ERα; however, it was decreased with ERβ. Mapping the TH promoter showed that the putative half estrogen response element (ERE) motif at − 675, as well as the activation protein 1 motif at − 205, were not required for response to E2 with either ER. The specificity protein 1/early growth response gene 1 (Egr 1) motif was required for the E2‐elicited response with ERβ, but not with ERα. Deletion of the cyclic AMP/Ca2+ response element (CRE/CaRE) nearly abolished E2‐triggered responses with either ER. Further analysis revealed an imperfect canonical putative ERE overlapping with CRE/CaRE and Nurr1 response element. Oligonucleotides spanning this ERE displayed binding to ER, Cyclic AMP Response Element Binding Protein (CREB) and other proteins. Moreover, E2 attenuated the increase in TH transcription seen with cyclic AMP analogs. Thus, TH is transcriptionally regulated by estradiol in opposite directions depending on ER subtype. The overlapping ERE and CRE/CaRE may integrate interactions elicited by various regulators of TH transcription including cAMP and estrogens.

Single and repeated immobilization stress differentially trigger induction and phosphorylation of several transcription factors and mitogen-activated protein kinases in the rat locus coeruleus.

The locus coeruleus (LC) is a critical stress‐responsive location that mediates many of the responses to stress. We used immunoblotting and immunohistochemistry to investigate changes in induction and phosphorylation of several transcription factors and kinases in the LC that may mediate the stress‐triggered induction of tyrosine hydroxylase (TH) transcription. Rats were exposed to single or repeated immobilization stress (IMO) for brief (5 min), intermediate (30 min) or sustained (2 h) duration. Single IMO elicited rapid induction of c‐Fos and phosphorylation of cyclic AMP response element‐binding protein (CREB) without changing the expression of early growth response (Egr)1, Fos‐related antigen (Fra)‐2 or phosphorylated activating transcription factor‐2. Repeated IMO triggered increased phosphorylation and levels of CREB along with transient induction of c‐Fos and increased Fra‐2 expression. Several mitogen‐activated protein kinases were activated by repeated IMO, shown by increased phosphorylation of p38, c‐Jun N‐terminal kinase (JNK)1/2/3 and extracellular signal‐regulated kinase (ERK1/2). ERK1 was the major isoform expressed, and ERK2 the predominant isoform phosphorylated. Repeated IMO elicited hyperphosphorylation of ERK1/2 selectively in TH immunoreactive neurons, with substantial nuclear localization. These distinct alterations in transcriptional pathways following repeated compared with single stress may be involved in mediating long‐lasting neuronal remodeling and are implicated in the mechanisms by which acute beneficial responses to stress are converted into prolonged adaptive or maladaptive responses.

Intranasal neuropeptide Y reverses anxiety and depressive-like behavior impaired by single prolonged stress PTSD model

PTSD is a debilitating neuropsychiatric disorder and many patients do not respond sufficiently to current treatments. Neuropeptide Y (NPY) is suggested to provide resilience to the development of PTSD and co-morbid depression. Injections of NPY to the rodent brain are anxiolytic. Recently we showed that intranasal delivery of NPY to rats before or immediately after exposure to single prolonged stress (SPS) animal model of PTSD prevented development of many biochemical and behavioral symptoms of PTSD, indicating its prophylactic potential. Here, we investigated whether intranasal NPY might provide benefits once symptoms have already developed. One week after exposure to SPS stressors, animals were given intranasal NPY or vehicle and tested on elevated plus maze 2h or 2 days later. The NPY treated rats had lower anxiety-like behavior than vehicle treated rats as indicated by more entries into open arms and fewer into closed arms, lower anxiety index, higher risk assessment and unprotected head dips and reduced grooming time. Their anxiety index was similar to that of unstressed controls. On most of these variables there was no effect of time interval and rats displayed similar overall changes 2h or 2 days after the infusion. Moreover, intranasal NPY led to reduced depressive-like behavior, assessed by forced swim test. Thus, intranasal NPY reversed several behavioral impairments triggered by the traumatic stress of SPS and has potential for non-invasive PTSD therapeutic intervention.

Gene expression of catecholamine biosynthetic enzymes following exercise: modulation by age

Both age and exercise training are associated with tissue specific alterations in the catecholaminergic system. We examined the effect of short-term exercise training on tyrosine hydroxylase and dopamine beta-hydroxylase gene expression in adrenals and specific brain regions with aging. In addition, we examined activator protein-1 and cyclic AMP response element transcription factor binding activity in the adrenal medulla. Male, six- and 24-month-old F-344 rats were exercised by treadmill running for five consecutive days. One group was killed immediately and a second group was killed 2h after the last training session. Exercise significantly elevated tyrosine hydroxylase messenger RNA equally in adrenals of both young and old rats. Training had no effect on dopamine beta-hydroxylase messenger RNA in adrenals of young, but levels were elevated in old rats. Binding activities of both activator protein-1 and cyclic AMP response element binding protein were diminished with age in the adrenal medulla. Exercise training had no significant effect on the binding activity of cyclic AMP response element binding protein in either young or old animals, whereas activator protein-1 binding activity increased equally in young and old animals. Exercise training revealed divergent changes in tyrosine hydroxylase messenger RNA in brain catecholaminergic neurons. In the locus coeruleus and the ventral tegmental areas, training elevated tyrosine hydroxylase messenger RNA levels only in young rats. In the substantia nigra, there was no change in young, but a 45% increase in tyrosine hydroxylase messenger RNA in old rats. In the ventral tegmental area, training increased tyrosine hydroxylase gene expression 80% in young but not in old rats. These results indicate that short-term exercise training increases tyrosine hydroxylase messenger RNA levels in young animals in the adrenals, the locus coeruleus and the ventral tegmental area. The responses for exercise training of aged animals differed from the young in brain noradrenergic and dopaminergic nuclei, especially in the substantia nigra, and to some extent in the locus coeruleus and the ventral tegmental area.