Estrogen increases nociception-evoked brain-derived neurotrophic factor gene expression in the female rat

Chronic pain induces plastic changes in nociceptive sensory pathways, and is often accompanied and exacerbated by depression. Estrogen can influence nociceptive sensory processing, but the molecular mechanisms underlying sex differences in pain remain unclear. Brain-derived neurotrophic factor (BDNF) may orchestrate changes occurring during persistent pain or depression by increasing spinal nociceptive signaling and altering neuronal growth in higher brain structures. This study addressed whether estrogen regulates BDNF gene expression in central systems associated with nociceptive processing and/or affective state, which may in turn influence sex differences in pain sensitivity. Thus, BDNF gene expression was quantified in intact female rats in proestrus and diestrus, and in ovariectomized (OVX) rats with or without 17beta-estradiol (E2) replacement following intraplantar injection of dilute formalin as an inflammatory nociceptive stimulus. Twenty-four hours after formalin injection, central nervous system (CNS) tissues were removed and solution hybridization-nuclease protection assays used to quantify BDNF mRNA levels. Results demonstrated that estrogen replacement increased BDNF mRNA levels in the hippocampus, cortex and spinal cord. Cortical BDNF mRNA levels were significantly decreased by nociception, in the hippocampus this decrease was only evident in estrogen-treated rats. Spinal BDNF expression was robustly increased by nociception. The pain-evoked up-regulation of spinal BDNF gene expression was significantly potentiated by concomitant estrogen treatment. Results demonstrate that BDNF gene expression in certain brain structures is inhibited by inflammatory pain, yet estrogen may enhance central nervous system sensitization associated with sensory processing. Since alterations in BDNF gene expression in higher brain centers may be relevant to cognitive changes that occur in recurrent depression, these results may provide insights into the coincidence of chronic pain and depression.     

Repeated stress increases the density of angiotensin II binding sites in rat paraventricular nucleus and subfornical organ

We have studied the properties of angiotensin II binding sites in the paraventricular nucleus, subfornical organ and anterior pituitary lobe of rats subjected to repeated immobilization stress. This treatment produced significant increase in the density of angiotensin II binding sites in these two nuclei without any significant alteration in binding affinity. Repeated stress did not alter angiotensin II binding properties in the anterior pituitary lobe. Our results suggest that brain angiotensin binding sites may have a role in regulation of the stress response.     

Neuropeptide-Y-immunoreactive innervation of thyrotropin-releasing hormone-synthesizing neurons in the rat hypothalamic paraventricular nucleus

The association of neuropeptide-Y (NPY)-immunoreactive (IR) axon terminals with TRH-synthesizing neurons in the rat hypothalamic paraventricular nucleus (PVN) has been studied. Immunocytochemical single and double labeling studies were performed at both light and electron microscopic levels using antiserum to NPY and, as a marker of TRH-containing neurons, antisera recognizing the N-terminal flanking peptides of the TRH prohormone, prepro-TRH-(25-50) and prepro-TRH-(53-74). At the light microscopic level, a diffuse group of TRH-IR cell bodies were observed in the anterior parvocellular subdivision of the PVN and became more numerous and densely clustered in the medial and periventricular parvocellular subdivisions. NPY-IR fibers were observed to innervate all subdivisions of the PVN, but were particularly dense in the anterior, medial, and periventricular parvocellular subdivisions of the nucleus, where they appeared to contact TRH-synthesizing perikarya and neuronal processes. At the ultrastructural level, numerous NPY-IR axon terminals containing labeled vesicles were either tightly juxtaposed to TRH-producing neurons or seen to establish both symmetric and asymmetric synaptic contacts with TRH-containing cell bodies and dendrites. Some NPY-IR axon terminals also established synaptic contacts with unlabeled PVN perikarya and processes or were found in close apposition to blood vessels. These data provide a morphological basis to suggest NPY-mediated neuroendocrine regulation over the biosynthesis and/or secretion of TRH in the PVN. Reports of the colocalization of NPY and catecholamines in the same axon terminals raises the possibility of a potential interaction between NPY and catecholamines to influence TRH neurons in the PVN. Morphological evidence for synaptic interactions between NPY-IR axon terminals and non-TRH-containing neurons in the PVN further suggests that this peptide may influence other neuroendocrine systems.     

Dexamethasone inhibits corticotropin-releasing factor gene expression in the rat paraventricular nucleus

The effect of glucocorticoids on corticotropin-releasing factor (CRF) gene expression was studied by combination of in situ hybridization histochemistry and steroid implantation. Dexamethasone micropellets, implanted around the hypothalamic paraventricular nucleus (PVN), caused total inhibition of the hybridizable CRF mRNA signal above the parvocellular neurons of the PVN. Unilateral implantation of dexamethasone around the PVN resulted in a decrease of hybridizable CRF mRNA at the dexamethasone-implanted side. Dexamethasone implants into the cerebral cortex, dorsal hippocampus, ventral subiculum, lateral septum or amygdala were without any effect on the CRF expression in the PVN. Corticosterone did not result in any significant change in CRF mRNA, when implanted into the paraventricular region, dorsal hippocampus or ventral subiculum. When it was placed into the amygdala however, in a few cases it slightly inhibited the CRF mRNA levels in the ipsilateral PVN.     

Estrogen in the paraventricular nucleus attenuates L-glutamate-induced increases in mean arterial pressure through estrogen receptor beta and NO

Estrogen (E2) acts in the brain to decrease blood pressure (BP) responses to psychological stress. A likely site for the effects of E2 is the hypothalamic paraventricular nucleus (PVN), an important regulator of autonomic functions. We studied the effects of E2 in the PVN on BP and heart rate (HR) responses to l-glutamate injections into the PVN of male urethane-anesthetized rats. Microinjections of l-glutamate (50 nmol) into the PVN increased BP by 14+/-2.5 mm Hg and HR by 30+/-5.6 bpm. Microinjections of E2 (0.1, 1, and 10 pmol) into the PVN 30 minutes before l-glutamate dose-dependently attenuated the pressor response by 25%, 34%, and 59%, respectively, but did not affect HR. We determined that E2 receptor (ER) beta mediates the effect of E2, because activation of ERbeta with diarylpropionitrile (50 pmol) attenuated the response by 57%, whereas activation of ERalpha with propyl-pyrazole-triol (20 pmol) had no effect. Furthermore, inhibition of ERbeta with R,R-tetrahydrochrysene (50 pmol) blocked the effect of E2, but inhibition of ERalpha with methyl-piperidino-pyrazole (1 nmol) did not. Finally, we found that the effect of E2 is mediated by NO, because the NO synthase (NOS) inhibitor, N(G)-nitro-l-arginine methyl ester (2 nmol), the neuronal NOS inhibitor, 7-nitroindazole sodium salt (0.1 pmol), and the endothelial NOS inhibitor, N5-(1-iminoethyl)-l-ornithine (200 pmol) blocked the effect of E2. The effect was partially blocked with the gamma-aminobutyric acid(A) receptor inhibitor bicuculline. Our results demonstrate that E2 in the PVN attenuates the l-glutamate-induced pressor response and that this effect is mediated by ERbeta, NO produced by neuronal NO synthase and eNOS, and partly by gamma-aminobutyric acid.     

Hypothyroidism increases vasoactive intestinal polypeptide (VIP) immunoreactivity and gene expression in the rat hypothalamic paraventricular nucleus.

Vasoactive intestinal polypeptide (VIP) is produced by neurons in the rat hypothalamic paraventricular nucleus (PVN) and may have an important role as a prolactin-releasing factor. Recent work from our laboratories has shown that thyroid hormone regulates the content of VIP and VIP mRNA in the rat anterior pituitary, but its effect on VIP in the PVN is not known. To determine whether thyroid hormone alters VIP biosynthesis in the PVN, we studied the effect of hypothyroidism on the content of immunoreactive (IR)-VIP and VIP mRNA in PVN neurons using histochemical techniques. By immunocytochemistry, only scattered IR-VIP fibers were present in the PVN of control animals whereas IR-VIP perikarya and fibers were present in hypothyroid rats. By in situ hybridization histochemistry, no labeled neurons were recognized in the PVN in control animals whereas PVN neurons were labeled in hypothyroid rats. These findings raise the possibility that hypothyroidism exerts negative feedback regulation on VIP-producing neurons in the PVN and suggest that this may be important to modulate the stimulatory effects of VIP on anterior and/or posterior pituitary function.     

Subfornical organ efferents influence the excitability of neurohypophyseal and tuberoinfundibular paraventricular nucleus neurons in the rat

Electrical stimulation in the subfornical organ (SFO) alters the excitability of antidromically identified paraventricular nucleus neurons. Extracellular recordings demonstrate that the dominant effect of single stimuli delivered to the SFO on neurohypophyseal oxytocin and vasopressin containing neurons is an increase in excitability. In 35% of cells tested, this excitation showed a long latency (44.3 +/- 3.4 ms) prolonged duration (208.7 +/- 23.5 ms), while in 16% of the neurons the excitation observed may be described as short latency (24.7 +/- 1.8 ms) short duration (11.6 +/- 1.4 ms). Of the remaining cells antidromically identified as projecting to the posterior pituitary, 12% showed initial decreases in excitability following SFO stimulation while the remaining 37% were unaffected. Evidence is presented demonstrating that stimulation in the region of the SFO results in short latency (27.9 +/- 2.4 ms) short duration (7.8 +/- 0.7 ms) increases in excitability in 22% of antidromically identified PVN tuberoinfundibular neurons tested. These data provide electrophysiological evidence in support of the proposed role of the subfornical organ in the control of posterior and anterior pituitary function.     

Peptide YY directly inhibits ghrelin-activated neurons of the arcuate nucleus and reverses fasting-induced c-Fos expression

The hypothalamic arcuate nucleus (Arc) monitors and integrates hormonal and metabolic signals involved in the maintenance of energy homeostasis. The orexigenic peptide ghrelin is secreted from the stomach during negative status of energy intake and directly activates neurons of the medial arcuate nucleus (ArcM) in rats. In contrast to ghrelin, peptide YY (PYY) is released postprandially from the gut and reduces food intake when applied peripherally. Neurons in the ArcM express ghrelin receptors and neuropeptide Y receptors. Thus, PYY may inhibit feeding by acting on ghrelin-sensitive Arc neurons. Using extracellular recordings, we (1) characterized the effects of PYY on the electrical activity of ghrelin-sensitive neurons in the ArcM of rats. In order to correlate the effect of PYY on neuronal activity with the energy status, we (2) investigated the ability of PYY to reverse fasting-induced c-Fos expression in Arc neurons of mice. In addition, we (3) sought to confirm that PYY reduces food intake under our experimental conditions. Superfusion of PYY reversibly inhibited 94% of all ArcM neurons by a direct postsynaptic mechanism. The PYY-induced inhibition was dose-dependent and occurred at a threshold concentration of 10(-8)M. Consistent with the opposite effects of ghrelin and PYY on food intake, a high percentage (50%) of Arc neurons was activated by ghrelin and inhibited by PYY. In line with this inhibitory action, peripherally injected PYY partly reversed the fasting-induced c-Fos expression in Arc neurons of mice. Similarly, refeeding of food-deprived mice reversed the fasting-induced activation in the Arc. Furthermore, peripherally injected PYY reduced food intake in 12-hour fasted mice. Thus the activity of Arc neurons correlated with the feeding status and was not only reduced by feeding but also by administration of PYY in non-refed mice. In conclusion, our current observations suggest that PYY may contribute to signaling a positive status of energy intake by inhibiting Arc neurons, which are activated under a negative status of energy intake by signals such as ghrelin.     

Gene expression profiling of rat brain neurons reveals angiotensin II-induced regulation of calmodulin and synapsin I: possible role in neuromodulation

Angiotensin (Ang II) activates neuronal AT(1) receptors located in the hypothalamus and the brainstem and stimulates noradrenergic neurons that are involved in the control of blood pressure and fluid intake. In this study we used complementary DNA microarrays for high throughput gene expression profiling to reveal unique genes that are linked to the neuromodulatory actions of Ang II in neuronal cultures from newborn rat hypothalamus and brainstem. Of several genes that were regulated, we focused on calmodulin and synapsin I. Ang II (100 nM; 1-24 h) elicited respective increases and decreases in the levels of calmodulin and synapsin I messenger RNAs, effects mediated by AT(1) receptors. This was associated with similar changes in calmodulin and synapsin protein expression. The actions of Ang II on calmodulin expression involve an intracellular pathway that includes activation of phospholipase C, increased intracellular calcium, and stimulation of protein kinase C. Taken together with studies that link calmodulin and synapsin I to axonal transport and exocytotic processes, the data suggest that Ang II regulates these two proteins via a Ca(2+)-dependent pathway, and that this may contribute to longer term or slower neuromodulatory actions of this peptide.     

Substance P targets sympathetic control neurons in the paraventricular nucleus

The paraventricular nucleus (PVN) contains spinally-projecting neurons implicated in fine-tuning the cardiovascular system. In vivo activity of "presympathetic" parvocellular neurons is suppressed by tonic inhibition from GABA-ergic inputs, inhibition of which increases sympathetic pressor activity and heart rate. Targeting of this specific neuronal population could potentially limit elevations of heart rate and blood pressure associated with disease. Here we show, for the first time, that "presympathetic" PVN neurons are disinhibited by the neuropeptide substance P (SP) acting via tachykinin NK1 receptor inhibition of GABA(A) currents. Application of SP to the paraventricular nucleus of rats increases heart rate and blood pressure. In in vitro brain slice experiments, in the presence of GABA, 1 micromol/L SP increased action current frequency by a factor of 2.7+/-0.6 (n=5, P< or =0.05, ANOVA). Furthermore, 1 micromol/L SP inhibited GABA(A) currents by 70+/-8% (n=8, P< or =0.005 paired t test). These effects were abolished by NK1 antagonists, but not NK2 and NK3 antagonists. GABA(A) inhibition was not reproduced by NK2 or NK3 agonists. The inhibition of parvocellular GABA(A) currents by SP was also abolished by a protein kinase C (PKC) inhibitor peptide and mimicked by application of phorbol-12-myristate-13-acetate (PMA), implicating a PKC-dependent mechanism. Single-channel analysis indicates that SP acts through reduction of channel mean open-time (cmot): GABA(A) cmot being reduced by approximately 60% by SP (P< or =0.05 ANOVA, Bonferroni). These data suggest that tachykinins mediate their pressor activity by increasing the excitability of spinally-projecting neurons and identifies NK1 receptors as potential targets for therapeutic modulation of the cardiovascular system.     

c-Fos mediates angiotensin II-induced aldosterone production and protein synthesis in bovine adrenal glomerulosa cells

Angiotensin II (AngII), potassium ion, and ACTH are the main factors controlling aldosterone biosynthesis in adrenal glomerulosa cells. AP-1 response elements for the immediate early gene products, c-Fos and c-Jun, have been identified, among others, in the promoter of the steroidogenic acute regulatory (StAR) protein gene, whose expression is acutely regulated by activators of aldosterone production. In bovine glomerulosa cells, AngII treatment led to a rapid and transient increase in c-fos mRNA expression, c-Fos protein expression, and c-Fos phosphorylation. Inhibition of the ERK1/2 MAPK pathway abolished the effect of AngII on c-fos mRNA, protein, and phosphorylation. EMSA and chromatin immunoprecipitation experiments demonstrated that c-Fos binds with c-Jun to the proximal StAR promoter and that AngII treatment increases the amount of c-Fos bound to the promoter. Overexpression of a dominant-negative form of c-Fos with adenoviral vectors inhibited StAR mRNA and StAR protein expression as well as aldosterone biosynthesis in response to AngII. The dominant-negative c-Fos also prevented the increase in protein synthesis induced by AngII in glomerulosa cells, as assessed by [(3)H]leucine incorporation. These results indicate that AngII rapidly induces c-Fos expression and posttranslational modifications. Furthermore, a heterodimeric c-Fos/c-Jun complex binds to the proximal StAR promoter in glomerulosa cells, thus activating StAR gene expression and acute aldosterone biosynthesis. Finally, c-Fos also contributes to other functional responses to the hormone, such as protein synthesis.     

Thyroidectomy induces Fos-like immunoreactivity within thyrotropin-releasing hormone-expressing neurons located in the paraventricular nucleus of the adult rat hypothalamus

Effects of thyroidectomy on Fos-like immunoreactivity (IR) in the rat brain were examined using single and double-label immunocytochemical techniques. In particular, the possibility that Fos might be involved in thyroid hormone regulation of thyrotropin releasing hormone (TRH)-containing neurons located in the parvocellular region of the paraventricular nucleus of the hypothalamus (pPVN) was examined. Adult, male, Sprague-Dawley rats were used and all animals received either surgical removal of the thyroid gland or sham surgery. Two experiments were performed. In the first experiment, animals were killed 1, 3, or 6 days after surgery and numbers of Fos-like IR cells in the parvocellular (pPVN) and magnocellular (mPVN) regions of the PVN, the anterior hypothalamic nucleus (AH), the lateral hypothalamic nucleus (LH), and the pyriform cortex were determined. In the second experiment, animals received an intracerebroventricular injection of colchicine 5 days after surgery. The next day, animals were killed and numbers of Fos-like IR cells double-labeled for either TRH, corticotropin releasing factor (CRF), or methionine-enkephalin (met-Enk) were determined. Six days after thyroidectomy there was a significant increase in the number of Fos-like IR cells detected in the pPVN. No induction in the pPVN was observed 1 and 3 days after thyroidectomy, and no effects attributable specifically to thyroidectomy (as opposed to stress) on Fos expression in the mPVN, AH, LH, or pyriform cortex were observed. In addition, a rapid, stress-related, induction of Fos-like IR was detected in the mPVN, AH, and LH and was easily distinguished from Fos expression induced in the pPVN as a function of thyroidectomy. The time course for the effect of thyroidectomy on Fos expression in the pPVN paralleled increased plasma TSH concentration. A significant correlation between numbers of Fos-like IR cells in the pPVN and plasma TSH concentration following thyroidectomy was also observed, suggesting that plasma levels of TSH correlate directly with the number of activated TRH-containing neurons located in the pPVN. Double staining for Fos and TRH, CRF, or met-Enk revealed that thyroidectomy induced Fos-like IR specifically within TRH-, but not within CRF-, or met-Enk, expressing neurons in the pPVN. Taken together, the data suggest that Fos-like IR is induced within TRH-expressing neurons in the pPVN as a consequence of decreasing levels of circulating thyroid hormone (TH). Whether this reflects a direct effect of decreasing TH on Fos expression is not yet known; however, the data are consistent with the hypothesis that Fos is involved in TH-associated regulation of TRH production and release.     

Differential effects of refeeding on melanocortin-responsive neurons in the hypothalamic paraventricular nucleus

To explore the effect of refeeding on recovery of TRH gene expression in the hypothalamic paraventricular nucleus (PVN) and its correlation with the feeding-related neuropeptides in the arcuate nucleus (ARC), c-fos immunoreactivity (IR) in the PVN and ARC 2 h after refeeding and hypothalamic TRH, neuropeptide Y (NPY) and agouti-related protein (AGRP) mRNA levels 4, 12, and 24 h after refeeding were studied in Sprague-Dawley rats subjected to prolonged fasting. Despite rapid reactivation of proopiomelanocortin neurons by refeeding as demonstrated by c-fos IR in ARC alpha-MSH-IR neurons and ventral parvocellular subdivision PVN neurons, c-fos IR was present in only 9.7 +/- 1.1% hypophysiotropic TRH neurons. Serum TSH levels remained suppressed 4 and 12 h after the start of refeeding, returning to fed levels after 24 h. Fasting reduced TRH mRNA compared with fed animals, and similar to TSH, remained suppressed at 4 and 12 h after refeeding, returning toward normal at 24 h. AGRP and NPY gene expression in the ARC were markedly elevated in fasting rats, AGRP mRNA returning to baseline levels 12 h after refeeding and NPY mRNA remaining persistently elevated even at 24 h. These data raise the possibility that refeeding-induced activation of melanocortin signaling exerts differential actions on its target neurons in the PVN, an early action directed at neurons that may be involved in satiety, and a later action on hypophysiotropic TRH neurons involved in energy expenditure, potentially mediated by sustained elevations in AGRP and NPY. This response may be an important homeostatic mechanism to allow replenishment of depleted energy stores associated with fasting.     

大鼠脑缺血-再灌注后下丘脑室旁核促肾上腺皮质激素的表达

目的 探讨大鼠脑缺血-再灌注后，不同时期中枢下丘脑室旁核促肾上腺皮质激素样阳性免疫物的表达。方法 取雄性Wislar大鼠70只，随机分成对照组（10只）、假手术对照组（30只）及脑缺血-再灌注组（30只）以颈动脉引流法行全脑缺血-再灌注造模，术后分6、24、72h3个时间段取脑，釆用免疫细胞化学技术，用图像分析系统行下丘脑室旁核促肾上腺皮质激素样阳性免疫反应面积、平均吸光度值检测。结果 显微镜下观察示，促肾上腺皮质激素样阳性免疫物呈深棕色，胞核深染，并广泛分布于中枢各脑区，核仁清晰可辨、在丘脑及下丘脑区域有散在分布“串珠”状阳性纤维。图像分析结果显示，假手术对照组、脑缺血-再灌注组大鼠的阳性免疫面积和平均吸光度值呈同步变化：从术后6h开始，假手术对照组、脑缺血-再灌注组大鼠均较正常对照组显着升高，术后24h达高峰（P〈0．05），然后逐渐回落。在3个不同时间段，3组间差异均有显着意义（P〈0．05），术后6及24h为：脑缺血-再灌注组，假手术对照组，正常对照组；术后72h为：假手术对照组〉正常对照组〉脑缺血-再灌注组。结论 在脑缺血-再灌注不同时间段，下丘脑室旁核神经元促肾上腺皮质激素样阳性免疫物表达呈现明显的时间规律；促肾上腺皮质激素可能在神经元功能调控，中枢内环境控制及脑缺血-再灌注损伤过程中发挥着重要作用。     

Involvement of reactive oxygen species in angiotensin II-induced endothelin-1 gene expression in rat cardiac fibroblasts

OBJECTIVES: The aim of this study was to investigate the effects of angiotensin II (Ang II) on fibroblast proliferation and endothelin-1 (ET-1) gene induction, focusing especially on reactive oxygen species (ROS)-mediated signaling in cardiac fibroblasts. BACKGROUND: Angiotensin II increases ET-1 expression, which plays an important role in Ang II-induced fibroblast proliferation. Angiotensin II also stimulates ROS generation in cardiac fibroblasts. However, whether ROS are involved in Ang II-induced proliferation and ET-1 expression remains unknown. METHODS: Cultured neonatal rat cardiac fibroblasts were stimulated with Ang II, and then [(3)H]thymidine incorporation and the ET-1 gene expression were examined. We also examined the effects of antioxidants on Ang II-induced proliferation and mitogen-activated protein kinase (MAPK) phosphorylation to elucidate the redox-sensitive pathway in fibroblast proliferation and ET-1 gene expression. RESULTS: Both AT(1) receptor antagonist (losartan) and ET(A) receptor antagonist (BQ485) inhibited Ang II-increased DNA synthesis. Endothelin-1 gene was induced with Ang II as revealed by Northern blotting and promoter activity assay. Angiotensin II increased intracellular ROS levels, which were inhibited with losartan and antioxidants. Antioxidants further suppressed Ang II-induced ET-1 gene expression, DNA synthesis, and MAPK phosphorylation. PD98059, but not SB203580, fully inhibited Ang II-induced ET-1 expression. Truncation and mutational analysis of the ET-1 gene promoter showed that AP-1 binding site was an important cis-element in Ang II-induced ET-1 gene expression. CONCLUSIONS: Our data suggest that ROS are involved in Ang II-induced proliferation and ET-1 gene expression. Our findings imply that the combination of AT(I) and ET(A) receptor antagonists plus antioxidants may be beneficial in preventing the formation of excessive cardiac fibrosis.