Variable Response of Norepinephrine Transporter to Traumatic Stress and Relationship to Hyperarousal.

The noradrenergic systems play a key role in stress triggered disorders such as post-traumatic stress disorder (PTSD). We hypothesized that traumatic stress will alter expression of norepinephrine transporter (NET) in locus coeruleus (LC) and its target brain regions which could be related to hyperarousal. Male Sprague-Dawley rats were subjected to single prolonged stress (SPS) and several weeks later the LC was isolated. NET mRNA levels in LC, determined by RT-PCR, displayed variable response with high and low responsive subgroups. In different cohort, acoustic startle response (ASR) was measured 2 weeks after SPS and levels of NET mRNA and protein in LC determined. The high NET responsive subgroup had greater hyperarousal. Nevertheless, NET protein levels, as determined by western blots, were lower than unstressed controls in LC, ventral hippocampus and medial prefrontal cortex and displayed considerable variability. Hypermethylation of specific CpG region in promoter of SLC6A2 gene, encoding NET, was present in the low, but not high, NET mRNA responsive subgroup. Taken together, the results demonstrate variability in stress elicited changes in NET gene expression and involvement of epigenetic changes. This may underlie mechanisms of susceptibility and resilience to traumatic stress triggered neuropsychiatric symptoms, especially hyperarousal.

Effect of intranasal administration of neuropeptide Y and single prolonged stress on food consumption and body weight in male rats.

Emerging evidence indicates that intranasal delivery of neuropeptide Y (NPY) to the brain has therapeutic potential for management of stress-triggered neuropsychiatric disorders. Here we aimed to determine how intranasal administration of NPY, either before or immediately after, traumatic stress in single prolonged stress (SPS) rodent model of Post-traumatic stress disorder (PTSD) impacts food consumption and body weight. SPS stressors suppressed food consumption for at least two days in the vehicle-treated animals. When given prior to SPS stressors, intranasal NPY prevented the SPS-elicited reduction in food intake only for several hours afterwards. When given after the SPS stressors, under conditions shown to prevent behavioral and biochemical impairments, intranasal NPY had no effect on food intake. Although all groups showed circadian variation, the SPS-exposed rats ate less than unstressed animals during the dark (active) phase. Seven days after exposure to SPS stressors, there were no differences in food intake, although body weight was still lower than unstressed controls in all the experimental groups. Thus, traumatic stress has pronounced effect on food consumption during the rodent's active phase, and a prolonged effect on body weight. Single intranasal infusion of NPY, which was previously shown to prevent development of several PTSD associated behavioral and neuroendocrine impairments, did not elicit prolonged changes in stress triggered food consumption nor regulation of body weight.

Activation of NPY receptor subtype 1 by [D-His26]NPY is sufficient to prevent development of anxiety and depressive like effects in the single prolonged stress rodent model of PTSD.

The neuropeptide Y (NPY) system plays an important role in mediating resilience to the harmful effect of stress in post-traumatic stress disorder (PTSD). It can mediate its effects via several G-protein coupled receptors: Y1R, Y2R, Y4R and Y5R. To investigate the role of individual NPY receptors in the resilience effects of NPY to traumatic stress, intranasal infusion of either Y1R agonists [D-His26]NPY, [Leu31Pro34]NPY, Y2R agonist NPY (3-36) or NPY were administered to male Sprague-Dawley rats immediately following the last stressor of the single prolonged stress (SPS) protocol, a widely used PTSD animal model. After 7 or 14 days, effects of the treatments were measured on the elevated plus maze (EPM) for anxiety, in forced swim test (FST) for development of depressive-like or re-experiencing behavior, in social interaction (SI) test for impaired social behavior, and acoustic startle response (ASR) for hyperarousal. [D-His26]NPY, but not [Leu31Pro34]NPY nor NPY (3-36) Y2R, was effective in preventing the SPS-elicited development of anxiety. Y1R, but not Y2R agonists prevented development of depressive- feature on FST, with [D-His26]NPY superior to NPY. The results demonstrate that [D-His26]NPY was sufficient to prevent development of anxiety, social impairment and depressive symptoms, and has promise as an early intervention therapy following traumatic stress.

Preclinical findings on the potential of intranasal neuropeptide Y for treating hyperarousal features of PTSD.

Acoustic startle response (ASR) assesses hyperarousal, a core symptom of posttraumatic stress disorder (PTSD). Intranasal neuropeptide Y (NPY) administration was shown to prevent hyperarousal in single prolonged stress (SPS) rodent PTSD model. However, it is unclear how ASR itself alters responses to stress. Rats (A-S-A) were exposed to acoustic startle (AS) 1 day before SPS (ASR1) and 2 weeks afterward (ASR2). Other groups were exposed in parallel to either AS (A-A) or SPS or neither. SPS enhanced ASR2. In relevant brain areas, mRNA levels were determined by qRT-PCR. In mediobasal hypothalamus, AS or SPS each increased CRH mRNA levels without an additive effect. Exposure to AS appeared to dampen some responses to SPS. The SPS-triggered reduction of GR and FKBP5 gene expression was not observed in A-S-A group. In locus coeruleus, SPS increased CRHR1 and reduced Y2R mRNAs, but not in A-S-A group. In both regions, AS altered NPY receptor gene expression, which may mediate dampening responses to SPS. In second experiment, intranasal NPY administered 2 weeks after SPS reversed hyperarousal symptoms for at least 7 days. This study reveals important effects of AS on the NPY system and demonstrates that intranasal NPY elicits long-lasting reversal of traumatic stress-triggered hyperarousal.

Single prolonged stress PTSD model triggers progressive severity of anxiety, altered gene expression in locus coeruleus and hypothalamus and effected sensitivity to NPY.

PTSD is heterogeneous disorder that can be long lasting and often has delayed onset following exposure to a traumatic event. Therefore, it is important to take a staging approach to evaluate progression of biological mechanisms of the disease. Here, we begin to evaluate the temporal trajectory of changes following exposure to traumatic stressors in the SPS rat PTSD model. The percent of animals displaying severe anxiety on EPM increased from 17.5% at one week to 57.1% two weeks after SPS stressors, indicating delayed onset or progressive worsening of the symptoms. The LC displayed prolonged activation, and dysbalance of the CRH/NPY systems, with enhanced CRHR1 gene expression, coupled with reduced mRNAs for NPY and Y2R. In the mediobasal hypothalamus, increased CRH mRNA levels were sustained, but there was a flip in alterations of HPA regulatory molecules, GR and FKBP5 and Y5 receptor at two weeks compared to one week. Two weeks after SPS, intranasal NPY at 300 µg/rat, but not 150 µg which was effective after one week, reversed SPS triggered elevated anxiety. It also reversed SPS elicited depressive/despair symptoms and hyperarousal. Overall, the results reveal time-dependent progression in development of anxiety symptoms and molecular impairments in gene expression for CRH and NPY systems in LC and mediobasal hypothalamus by SPS. With longer time afterwards only a higher dose of NPY was effective in reversing behavioral impairments triggered by SPS, indicating that therapeutic approaches should be adjusted according to the degree of biological progression of the disorder.

Single Prolonged Stress as a Prospective Model for Posttraumatic Stress Disorder in Females.

Sex plays an important role in susceptibility to stress triggered disorders. Posttraumatic Stress disorder (PTSD), a debilitating psychiatric disorder developed after exposure to a traumatic event, is two times more prevalent in women than men. However, the vast majority of animal models of PTSD, including single prolonged stress (SPS), were performed mostly with males. Here, we evaluated SPS as an appropriate PTSD model for females in terms of anxiety, depressive symptoms and changes in gene expression in the noradrenergic system in the brain. In addition, we examined intranasal neuropeptide Y (NPY) as a possible treatment in females. Female rats were subjected to SPS and given either intranasal NPY or vehicle in two separate experiments. In the first experiment, stressed females were compared to unstressed controls on forced swim test (FST) and for levels of expression of several genes in the locus coeruleus (LC) 12 days after SPS exposure. Using a separate cohort of animals, experiment two examined stressed females and unstressed controls on the elevated plus maze (EPM) and LC gene expression 7 days after SPS stressors. SPS led to increased anxiety-like behavior on EPM and depressive-like behavior on FST. Following FST, the rats displayed elevated tyrosine hydroxylase (TH), CRHR1 and Y1R mRNA levels in the LC, consistent with increased activation of the noradrenergic system. The expression level of these mRNAs was unchanged following EPM, except Y1R. Intranasal NPY at the doses shown to be effective in males, did not prevent development of depressive or anxiety-like behavior or molecular changes in the LC. The results indicate that while SPS could be an appropriate PTSD model for females, sex differences, such as response to NPY, are important to consider.

Glucocorticoid withdrawal affects stress-induced changes of urocortin 2 gene expression in rat adrenal medulla and brain.

Corticotropin-releasing factor is well known activator of the hypothalamic-pituitary-adrenocortical axis, that represents crucial system participating on stress response of the organism. Urocortins are members of the corticotropin-releasing factor family of peptides with proposed effects on neuroendocrine and behavioral stress response mechanisms. Urocortin 2, one of three known urocortins, is present in central and peripheral stress response system and its expression can be augmented by glucocorticoids. In the present study we have examined how glucocorticoid withdrawal affects urocortin 2 gene expression after acute immobilization in the adrenal medulla and selected brain areas in rats. We used pharmacological adrenalectomy to block synthesis of corticosterone. Our results show that the immobilization-induced rise in urocortin 2 mRNA levels in rat adrenal medulla was not inhibited by glucocorticoid withdrawal. On the other hand, observed changes in the brain indicate that the effect of stress and pharmacological adrenalectomy on urocortin 2 gene expression is site-specific. While in the paraventricular nucleus and locus coeruleus the immobilization induced rise of urocortin 2 was not inhibited by pharmacological adrenalectomy in the arcuate nucleus and central amygdala it was. Moreover, we have seen a significant depletion of urocortin 2 plasma levels after immobilization. The immobilization induced rise of urocortin 2 gene expression in rat adrenal medulla and brain areas regulating stress response pathways and preservation of its induction after adrenalectomy suggests a role of urocortin 2 in the neuroendocrine stress response of an organism. This article is protected by copyright. All rights reserved.

Potential of Intranasal Neuropeptide Y (NPY) and/or Melanocortin 4 Receptor (MC4R) Antagonists for Preventing or Treating PTSD.

There is a great need for effective treatment options for post-traumatic stress disorder (PTSD). Neuropeptide Y (NPY) is associated with resilience to traumatic stress. MC4R antagonists, such as HS014, also reduce response to stress. Both regulate stress-responsive systems - the hypothalamic-pituitary-axis (HPA) and the noradrenergic nervous system and their associated behaviors. Therefore, we examined if their intranasal delivery to brain could attenuate development of PTSD-related symptoms in single prolonged stress (SPS) rodent PTSD model. Three regimens were used: (1) prophylactic treatment 30 min before SPS stressors, (2) early intervention right after SPS stressors, (3) therapeutic treatment when PTSD behaviors are manifested 1 wk or more after the traumatic stress. NPY delivered by regimen 1 or 2 prevented SPS-triggered elevation in anxiety, depressive-like behavior, and hyperarousal and reduced dysregulation of HPA axis. Hypothalamic CRH mRNA and GR in ventral hippocampus were significantly induced in vehicle- but not NPY-treated group. NPY also prevented hypersensitivity of LC/NE system to novel mild stressor and induction of CRH in amygdala. Some of these impairments were also reduced with HS014, alone or together with NPY. When given after symptoms were manifested (regiment 3), NPY attenuated anxiety and depressive behaviors. This demonstrates strong preclinical proof of concept for intranasal NPY, and perhaps MC4R antagonists, for non-invasive early pharmacological interventions for PTSD and comorbid disorders and possibly also as therapeutic strategy.

Changes in Gene Expression in the Locus Coeruleus-Amygdala Circuitry in Inhibitory Avoidance PTSD Model.

The locus coeruleus (LC)-amygdala circuit is implicated in playing a key role in responses to emotionally arousing stimuli and in the manifestation of post-traumatic stress disorder (PTSD). Here, we examined changes in gene expression of a number of important mediators of the LC-amygdala circuitry in the inhibition avoidance model of PTSD. After testing for basal acoustic startle response (ASR), rats were exposed to a severe footshock (1.5 mA for 10 s) in the inhibitory avoidance apparatus. They were given contextual situational reminders every 5 day for 25 days. Controls were treated identically but with the footshock inactivated. Animals were re-tested on second ASR and decapitated 1 h later. The shock group had enhanced hyperarousal and several changes in gene expression compared to controls. In the LC, mRNA levels of norepinephrine (NE) biosynthetic enzymes (TH, DBH), NE transporter (NET), NPY receptors (Y1R, Y2R), and CB1 receptor of endocannabinoid system were elevated. In the basolateral amygdala (BLA), there were marked reductions in gene expression for CB1, and especially Y1R, with rise for corticotropin-releasing hormone (CRH) system (CRH, CRH receptor 1), and no significant changes in the central amygdala. Our results suggest a fast forward mechanism in the LC-amygdala circuitry in the shock group.

Cardiovascular responses to intranasal neuropeptide Y in single prolonged stress rodent model of post-traumatic stress disorder.

Delivery of neuropeptide Y (NPY) to the brain by intranasal administration shows promise as non-invasive means for preventing or treating PTSD symptoms. Here, radiotelemetry and echocardiography were used to determine effects of intranasal NPY on cardiovascular functions in absence and presence of stress. Male adult Sprague Dawley rats were implanted with radiotelemetric probes, and subjected to single prolonged stress (SPS), followed by intranasal vehicle (V) or NPY (150µg) under conditions shown to prevent development of many of the behavioral neuroendocrine and biochemical impairments. In both groups, mean arterial pressure (MAP) rose rapidly peaking at about 125mmHg, remaining near maximal levels for 1h. SPS also elicited robust rise in heart rate (HR) which was mitigated by intranasal NPY, and significantly lower than V-treated rats 12-50min after exposure to SPS stressors. In the first hr. after SPS, locomotor activity was elevated but only in the V-treated group. By 3h, MAP returned to pre-stress levels in both groups with no further change when monitored for 6days. HR remained elevated during the 6h remaining light phase after SPS. Subsequently HR was at pre-SPS levels during the remaining days. However dark phase HR was low following SPS, gradually recovered over 6days and was associated with reduced activity. When administered in the absence of further stress, intranasal NPY or V elicited similar much smaller, short-lived rises in MAP and HR. Echocardiography revealed no change in HR, stroke volume (SV) or cardiac output (Q) with intranasal NPY in the absence of stress. SPS led to reduced SV and Q but was not affected by NPY. Overall the results demonstrate no major cardiovascular effects of intranasal NPY and indicate possible benefit from transient amelioration of HR response in line with its translational potential to combat PTSD and comorbid impairments.

Potential of neuropeptide Y for preventing or treating post-traumatic stress disorder.

There is extensive evidence that NPY in the brain can modulate the responses to stress and play a critical role in resistance to, or recovery from, harmful effects of stress. Development of PTSD and comorbid depression following exposure to traumatic stress are associated with low NPY. This review discusses putative mechanisms for NPY's anti-stress actions. Recent preclinical data indicating potential for intranasal delivery of NPY to brain as a promising non-invasive strategy to prevent a variety of neuroendocrine, molecular and behavioral impairments in PTSD model are summarized.

Locus coeruleus response to single-prolonged stress and early intervention with intranasal neuropeptide Y.

Dysregulation of the central noradrenergic system is a core feature of post-traumatic stress disorder (PTSD). Here, we examined molecular changes in locus coeruleus (LC) triggered by single-prolonged stress (SPS) PTSD model at a time when behavioral symptoms are manifested, and the effect of early intervention with intranasal neuropeptide Y (NPY). Immediately following SPS stressors, male SD rats were administered intranasal NPY (SPS/NPY) or vehicle (SPS/V). Seven days later, TH protein, but not mRNA, was elevated in LC only of the SPS/V group. Although 90% of TH positive cells expressed GR, its levels were unaltered. Compared to unstressed controls, LC of SPS/V, but not SPS/NPY, expressed less Y2 receptor mRNA with more CRHR1 mRNA in subset of animals, and elevated corticotropin-releasing hormone (CRH) in central nucleus of amygdala. Following testing for anxiety on elevated plus maze (EPM), there were significantly increased TH, DBH and NPY mRNAs in LC of SPS-treated, but not previously unstressed animals. Their levels highly correlated with each other but not with behavioral features on EPM. Thus, SPS triggers long-term noradrenergic activation and higher sensitivity to mild stressors, perhaps mediated by the up-regulation influence of amygdalar CRH input and down-regulation of Y2R presynaptic inhibition in LC. Results also demonstrate the therapeutic potential of early intervention with intranasal NPY for traumatic stress-elicited noradrenergic impairments. Single-prolonged stress (SPS)-triggered long-term changes in the locus coeruleus/norepinephrine (LC/NE) system with increased tyrosine hydroxylase (TH) protein and CRH receptor 1(CRHR1) mRNA and lower neuropeptide Y receptor 2 (Y2R) mRNA levels as well as elevated corticotropin-releasing hormone (CRH) in the central nucleus of amygdala (CeA) that were prevented by early intervention with intranasal neuropeptide Y (NPY). SPS treatment led to increased sensitivity of LC to mild stress of elevated plus maze (EPM), with elevated mRNA for NE biosynthetic enzymes in subset of animals.

Comparative effects of intranasal neuropeptide Y and HS014 in preventing anxiety and depressive-like behavior elicited by single prolonged stress.

Stress triggered neuropsychiatric disorders are a serious societal problem. Prophylactic treatment or early intervention has great potential in increasing resilience to traumatic stress and reducing its harmful impact. Previously, we demonstrated proof of concept that intranasal administration of neuropeptide Y (NPY) or the melanocortin receptor four (MC4R) antagonist, HS014, prior to single prolonged stress (SPS) rodent post-traumatic stress disorder (PTSD) model, can prevent or attenuate many PTSD associated impairments. Here, we compare effects of NPY or HS014 given 30 min before or immediately after SPS stressors on development of anxiety, depressive-like behavior and associated biochemical abnormalities. SPS triggered anxiety on elevated plus maze (EPM) was reduced by intranasal administration of 100 µg NPY and to even greater extent HS014 (3.5 ng or 100 µg). The SPS-elicited depressive-like behavior on forced swim test was prevented with 100 µg NPY or the high dose HS014. Combined administration of low HS014 and NPY, ineffective by themselves, prevented development of depressive-like behavior. Reductions in stress triggered activation of locus coeruleus/noradrenergic system and HPA axis were observed with both HS014 and NPY. In contrast to NPY which has been showed earlier, infusion of HS014 immediately after SPS did not prevent the development of anxiogenic behavior on EPM. However, HS014 given after SPS stressors effectively even at very low dose, prevented development of depressive-like behavior. Thus, both MC4R antagonist and NPY, alone or combined, have potential for prophylactic treatment against traumatic stress triggered anxiety or depressive-like behaviors, while NPY has more widespread potential for early intervention.

Blockage of melanocortin-4 receptors by intranasal HS014 attenuates single prolonged stress-triggered changes in several brain regions.

Melanocortin receptor four (MC4R) is implicated in regulation of stress-related functions. We previously demonstrated that intranasal infusion of MC4R antagonist HS014, shortly before single prolonged stress (SPS) animal model of post-traumatic stress disorder, lessened the development of anxiety- and depression-like behavior depending on the dose. Here, we evaluated effects of HS014 on SPS-elicited changes in hypothalamic-pituitary-adrenal axis and expression of several genes of interest in mediobasal hypothalamus, hippocampus, and locus coeruleus. Rats were given intranasal infusion of HS014 (3.5 ng or 100 µg) and 30 min later subjected to SPS stressors. Short-term responses of HS014 rats in comparison with vehicle-treated, evident 30 min following SPS stressors, included smaller rise in plasma corticosterone (100 µg HS014), absence of induction of corticotrophin-releasing hormone mRNA in mediobasal hypothalamus and of mRNA for tyrosine hydroxylase and dopamine-ß hydroxylase in locus coeruleus. Long-term responses found 7 days after SPS stressors, included lower induction corticotrophin-releasing hormone mRNA levels in the mediobasal hypothalamus without effect on mRNAs for the glucocorticoid receptor (GR) and FK506-binding protein 51 (FKBP5), a component of GR co-chaperone complex; and no induction of GR protein in ventral hippocampus. Thus, antagonism of MC4R prior to SPS attenuates development of several abnormalities in gene expression in regions implicated in post-traumatic stress disorder. Blockade of brain melanocortine receptor 4 (MC4R) with intranasal infusion of the MC4R antagonist HS014 to rats prior to single prolonged stress (SPS) leads to faster termination of stress responses (30 min later) and prevents or attenuates SPS-triggered abnormal gene expression related to post-traumatic stress disorder (7 days later). Targeting of brain MC4R is a promising strategy to protect HPA axis, LC-NE (locus coeruleus-norepinephrine) systems and hippocampus from overstimulation.

Early intervention with intranasal NPY prevents single prolonged stress-triggered impairments in hypothalamus and ventral hippocampus in male rats.

Intranasal administration of neuropeptide Y (NPY) is a promising treatment strategy to reduce traumatic stress-induced neuropsychiatric symptoms of posttraumatic stress disorder (PTSD). We evaluated the potential of intranasal NPY to prevent dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis, a core neuroendocrine feature of PTSD. Rats were exposed to single prolonged stress (SPS), a PTSD animal model, and infused intranasally with vehicle or NPY immediately after SPS stressors. After 7 days undisturbed, hypothalamus and hippocampus, 2 structures regulating the HPA axis activity, were examined for changes in glucocorticoid receptor (GR) and CRH expression. Plasma ACTH and corticosterone, and hypothalamic CRH mRNA, were significantly higher in the vehicle but not NPY-treated group, compared with unstressed controls. Although total GR levels were not altered in hypothalamus, a significant decrease of GR phosphorylated on Ser232 and increased FK506-binding protein 5 mRNA were observed with the vehicle but not in animals infused with intranasal NPY. In contrast, in the ventral hippocampus, only vehicle-treated animals demonstrated elevated GR protein expression and increased GR phosphorylation on Ser232, specifically in the nuclear fraction. Additionally, SPS-induced increase of CRH mRNA in the ventral hippocampus was accompanied by apparent decrease of CRH peptide particularly in the CA3 subfield, both prevented by NPY. The results show that early intervention with intranasal NPY can prevent traumatic stress-triggered dysregulation of the HPA axis likely by restoring HPA axis proper negative feedback inhibition via GR. Thus, intranasal NPY has a potential as a noninvasive therapy to prevent negative effects of traumatic stress.

Intranasal infusion of melanocortin receptor four (MC4R) antagonist to rats ameliorates development of depression and anxiety related symptoms induced by single prolonged stress.

Brain melanocortinergic systems and specifically melanocortin receptor four (MC4R) are implicated in modulation of anxiety- and depressive-like behavior induced by mild or moderate stress. Here we examine whether blockage of central MC4Rs with HS014 before severe traumatic stress may protect against development of anxiety and depression co-morbid with post-traumatic stress disorder (PTSD). Male rats were treated intranasally (IN) with vehicle or varied doses of HS014, 30min prior to single prolonged stress (SPS) animal model of PTSD. IN administration of 100µg HS014 pre-SPS improved despair behavior in forced swim (FS) immediately after immobilization stress part of SPS protocol. During all 4 intervals of 20min FS these rats spent less time immobile than rats given vehicle or 3.5ng HS014. This dose of HS014 also had a long-term beneficial effect manifested as reduction of immobility time in forced swim test performed after SPS. However, both HS014 doses were effective in ameliorating development of anxiety-like behavior after traumatic stress. Thus, rats given IN HS014 prior to SPS exhibited less open arms (OA) visits in elevated plus maze (EPM), spent longer time in OA and less in closed arms, had lower anxiety index, higher risk assessment and more head dips over borders in OA. They also spent longer time in the center of the open field and defecated less. Reduced grooming behavior in EPM was observed with 100µg HS014. This is the first study revealing pronounced resilience effects of HS014 on development of behavioral symptoms co-morbid with PTSD.

Vesicular monoamine transporters (VMATs) in adrenal chromaffin cells: stress-triggered induction of VMAT2 and expression in epinephrine synthesizing cells.

Vesicular monoamine transporters (VMATs) mediate transmitter uptake into neurosecretory vesicles. There are two VMAT isoforms, VMAT1 and VMAT2, encoded by separate genes and displaying different cellular distributions and pharmacological properties. We examined the effect of immobilization stress (IMO) on expression of VMATs in the rat adrenal medulla. Under basal conditions, VMAT1 is widely expressed in all adrenal chromaffin cells, while VMAT2 is co-localized with tyrosine hydroxylase (TH) but not phenylethanolamine N-methyltransferase (PNMT), indicating its expression in norepinephrine (NE)-, but not epinephrine (Epi)-synthesizing chromaffin cells. After exposure to IMO, there was no change in levels of VMAT1 mRNA. However, VMAT2 mRNA was elevated after exposure of rats to 2 h IMO once (1× IMO) or daily for 6 days (6× IMO). The changes in VMAT2 mRNA were reflected by increased VMAT2 protein after the repeated IMO. Immunofluorescence revealed an increased number of cells expressing VMAT2 following repeated IMO and its colocalization with PNMT in many chromaffin cells. The findings suggest an adaptive mechanism in chromaffin cells whereby enhanced catecholamine storage capacity facilitates more efficient utilization of the well-characterized heightened catecholamine biosynthesis with repeated IMO stress.

Modulation of responses to stress by estradiol benzoate and selective estrogen receptor agonists.

Previously, pretreatment with estradiol benzoate (EB) was found to modulate the response of hypothalamic-pituitary-adrenal (HPA) axis and gene expression in several catecholaminergic neuronal locations in ovariectomized (OVX) rats exposed to single immobilization stress (IMO). Here, we investigated the role of estrogen receptor (ER) subtypes, using selective agonists for ERalpha (propyl pyrazole triol, PPT) or ERbeta (WAY-200070) in two major central noradrenergic systems and the HPA axis after exposure to single and repeated IMO. OVX female rats received 21 daily injections of either EB (25 mug/kg), PPT (10 mg/kg), WAY-200070 (10 mg/kg), or vehicle. Injections of EB and PPT, but not WAY-200070, elicited reduced body weight and increased uterine weight, showing their selectivity. Both EB and PPT increased corticosterone levels about two- to threefold, but prevented any further rise with either single or repeated IMO, indicating an ERalpha (ESR1)-, but not ERbeta (ESR2)-, mediated mechanism. In the locus coeruleus (LC), the rise in dopamine-beta-hydroxylase (Dbh) mRNA with both stress paradigms was abrogated in EB- or PPT-injected animals. However, WAY-200070 blocked the response of DBH mRNA to single IMO but not to repeated IMO. In the nucleus of the solitary tract (NTS), the rise in tyrosine hydroxylase and DBH mRNAs with both IMOs was absent, or greatly attenuated, in EB- or PPT-treated rats. In most cases, WAY-200070 inhibited the response to single IMO but not to repeated IMO. The results demonstrate that pretreatment with estradiol, or ER-selective agonists, modulates the stress-triggered induction of gene expression of norepinephrine biosynthetic enzymes in LC and NTS, with ER selectivity depending on duration of the stress.

Membrane-initiated estradiol signaling increases tyrosine hydroxylase promoter activity with ER alpha in PC12 cells.

Tyrosine hydroxylase (TH) promoter activity is induced by 17beta-estradiol (E(2)) in PC12 cells expressing estradiol receptor-alpha (ERalpha) requiring a cAMP/calcium response element (CRE/CaRE) at -45. To examine whether membrane-initiated estradiol signaling is underlying this induction, cells co-transfected with TH reporter construct and ERalpha expression vector were exposed to membrane-impermeant estradiol conjugate (beta-estradiol-6-(O-carboxy-methyl) oxime-bovine serum albumin, E(2)BSA). TH promoter activity was elevated by E(2)BSA in dose- and time-dependent manner. E(2)BSA also elicited rapid phosphorylation of CRE binding protein (CREB) and increased CRE-driven promoter activity. Over-expression of dominant negative forms of CREB, with mutations in DNA binding or phosphorylation site, prevented TH promoter response to E(2)BSA. Pre-treatment with protein kinase A (PKA) and MEK inhibitors reduced E(2) dependent phosphorylation of CREB and ERK, and also decreased induction of TH promoter activity by E(2) or E(2)BSA. Blocking S-palmitoylation of ERalpha with C451A mutation and/or pre-treatment with 2-Bromopalmitate did not prevent but instead enhanced E(2) or E(2)BSA-elicited induction of TH promoter activity. These findings reveal, for the first time, that estradiol induction of TH gene transcription with ERalpha in PC12 cells involves membrane-initiated estradiol signaling, rapid activation of dual PKA/MEK signaling pathways, leading to CREB phosphorylation, acting at CRE/CaRE. The data demonstrate possible mechanism whereby estradiol affects catecholaminergic systems in vivo.

Divergent effects of estradiol on gene expression of catecholamine biosynthetic enzymes.

Within the catecholaminergic systems, there are contradictory findings regarding ability of estradiol to regulate expression of genes related to catecholamine biosynthesis. Several parameters important for effects of estradiol on the catecholamine (CA) related enzyme gene expression were examined in two CA regions. Ovariectomized (OVX) female rats were given prolonged estradiol treatments, either in a pulsatile fashion by injections or continuously by pellets. The mode affected the response of tyrosine hydroxylase (TH) and GTP cyclohydrolase I (GTPCH) mRNAs differentially in the nucleus of solitary tract (NTS) and the locus coeruleus (LC). In rostral-medial NTS, TH mRNA levels were increased with injections, but declined in rats administered estradiol by pellets. In LC, a significant change was only observed in GTPCH with injections. These differences may reflect activation of different estrogen receptors (ER). The response to estradiol in the presence of ERalpha and ER beta was examined in PC12 cell culture. Estradiol directly regulated promoter activity of TH, GTPCH and dopamine beta-hydroxylase (DBH) genes. With ERalpha, 17 beta-estradiol elevated TH promoter activity, while there was a decline with ERbeta. In contrast, both DBH and GTPCH promoters were enhanced by 17 beta-estradiol over a wide range of concentrations with either ER subtype. Thus, mode of administration, location examined and ER subtype expressed are important considerations in the overall response of catecholamine related enzymes to estradiol.