Expression Of Fos-related Antigens Research Articles

Exogenous adenosine activates A2A adenosine receptor to inhibit RANKL-induced osteoclastogenesis via AP-1 pathway to facilitate bone repair

Adenosine is a purine nucleoside involved in regulating bone homeostasis through binding to A1, A2A, A2B, and A3 adenosine receptors (A1R, A2AR, A2BR, and A3R, respectively). However, the underlying mechanisms by which adenosine and receptor subtypes regulate osteoclast differentiation remain uncertain. This study aims to assess the role of exogenous adenosine and receptor subtypes in receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation and explore the underlying molecular mechanisms. The nanofibrous mats incorporated with adenosine exhibited robust ability to facilitate rat critical-size calvarial defect healing with decreased number of osteoclasts. Moreover, exogenous adenosine substantially enhanced the expression of A2AR and suppressed tartrate-resistant acid phosphatase-positive osteoclast formation and expression of osteoclast-related genes Ctsk, NFATc1, MMP9, and ACP5. This enhancement and suppression could be reversed by adding an A2AR antagonist, ZM241385, in RAW264.7 cells. Finally, RNA sequencing showed that the expression of Fos-related antigen 2 (Fra2) was distinctly downregulated through stimulation of adenosine in RAW264.7 cells treated with RANKL. This downregulation was reversed by ZM241385 according to real-time PCR, Western blot, and immunofluorescence analyses. These findings demonstrated that exogenous adenosine binding to A2AR attenuated osteoclast differentiation via the inhibition of activating protein-1 (AP-1, including Fra2 subunit) pathway both in vitro and in vivo.

FRA-1 suppresses apoptosis of Helicobacter pylori infected MGC-803 cells.

Previous research has demonstrated a correlation between elevated expression of Fos-related antigen 1 (FRA-1) and malignancies. Nevertheless, the role of FRA-1 in Helicobacter pylori infected gastric cancer cells remains vague. Our study aims to investigate whether FRA-1 plays a role in the apoptosis of MGC-803 induced by H. pylori and possible mechanisms. MGC-803 cells were used in vitro to establish a cell model of H. pylori infection. After stimulation with H. pylori, the expression of FRA-1 was increased in MGC-803 cells. H. pylori infection promoted the apoptosis of MGC-803 cells, and led to cell cycle arrest and increased oxidative stress levels. Furthermore, the knockdown of FRA-1 reinforced these changes. H. pylori decreased the expression of Bcl2, Caspase3 and Caspase9, while increased the level of BAX, Cleaved-Caspase3 and Cleaved-Caspase9; in addition, it led to the decrease of major proteins in Ras/Erk and PI3K/AKT signaling pathway. As expected, these changes were augmented by FRA-1 knockdown. Our results demonstrated that high expression of FRA-1 induced by H. pylori suppresses apoptosis in MGC-803 cells which may be regulated by oxidative stress and cycle arrest through caspase family, Ras/Erk and PI3K/AKT signaling pathway.

Neuro-behavioral effects after systemic administration of MK-801 and disinhibition of the anterior thalamic nucleus in rats: Potential relevance in schizophrenia

Non-competitive N-methyl-d-aspartate receptor (NMDA-R) antagonists have been suggested to evoke psychotomimetic-like behaviors by selectively targeting GABAergic elements in cortical and thalamic circuits. In previous studies, we had reported the involvement of the reticular and anterior thalamic nuclei (ATN) in the MK-801-evoked hyperactivity and other motor alterations. Consistent with the possibility that these responses were mediated by thalamic disinhibition, we examined the participation of cortical and hippocampal areas innervated by ATN in the responses elicited by the systemic administration of MK-801 (0.2 mg/kg) and compared them to the effects produced by the microinjection of a subconvulsive dose of bicuculline (GABAA receptor antagonist) in the ATN. We used the expression of Fos related antigen 2 (Fra-2) as a neuronal activity marker in the ATN and its projection areas such as hippocampus (HPC), retrosplenial cortex (RS), entorhinal cortex (EC) and medial prefrontal cortex (mPFC). Dorsal (caudate-putamen, CPu) and ventral striatum (nucleus accumbens, core and shell, NAc,co and NAc,sh) were also studied.Behavioral and brain activation results suggest a partial overlap after the effect of MK-801 administration and ATN disinhibition. MK-801 and ATN disinhibition increases locomotor activity and disorganized movements, while ATN disinhibition also reduces rearing behavior. A significant increase in Fra-2 immunoreactivity (Fra-2-IR) in the ATN, mPFC (prelimbic area, PrL) and NAc,sh was observed after MK-801, while a different pattern of Fra-2-IR was detected following ATN disinhibition (e.g., increase in DG and NAc,sh, and decrease in PrL cortex). Overall, our data may contribute to the understanding of dysfunctional neural circuits involved in schizophrenia.

PS02.200: A COMPREHENSIVE SCREENING OF THE FRA-1 REGULATORY GENES IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract Background The expression of Fos-related antigen 1 (Fra-1) affects tumor progression, migration and invasion. We previously reported that a high Fra-1 expression level is associated with lymph node metastasis and a poor prognosis in patients with esophageal squamous cell carcinoma (ESCC). In this study, we identified the genes regulated by Fra-1 in ESCC. Methods We constructed Fra-1 knockdown models via the transfection of small interfering RNA (siRNA) into ESCC cell lines (TE10, TE11). The expression levels of the genes in the knockdown models were analyzed using a microarray experiment and Biobase Upstream Analysis (Cytoline Solutions, Tokyo, Japan), and candidate genes regulated by Fra-1 in the ESCC cell lines were detected. The actual connection of Fra-1 to the promoter region was identified in a ChIP-PCR experiment. The expression levels of the candidate genes regulated by Fra-1 in the primary tumors of surgical specimens obtained from ESCC patients (n = 135) were compared to those observed in normal tissues using real-time PCR and immunohistochemical staining, and the clinicopathological features were analyzed. Results The results of the Biobase Upstream Analysis and ChIP-PCR showed high mobility group protein 1 (HMGA1) and hyaluronan mediated motility receptor (HMMR) to be significant genes regulated by Fra-1. The expression levels of both HMGA1 and HMMR were found to closely correlate with the Fra-1 expression in the clinical specimens. The patients with a positive HMGA1 expression had a poorer prognosis than those with a negative expression (P = 0.017) and the patients with positive HMMR expression also had a poorer prognosis (P = 0.018). Moreover, a multivariate analysis demonstrated a positive HMGA1 expression to be a significant independent prognostic factor in the ESCC patients. Conclusion HMGA1 and HMMR are regulated by Fra-1 in the setting of ESCC and the HMGA1 expression is significantly associated with a poor prognosis in ESCC patients. Downregulating the HMGA1 and HMMR expression may become a practical treatment strategy against ESCC in the future. Disclosure All authors have declared no conflicts of interest.

Fra-1 Regulates theExpression of HMGA1,Which is Associated with a Poor Prognosis in Human Esophageal Squamous Cell Carcinoma.

The expression of Fos-related antigen 1 (Fra-1) affects tumor progression, migration, and invasion. In this study, we identified the genes regulated by Fra-1 in esophageal squamous cell carcinoma (ESCC). We constructed Fra-1 knockdown models via the transfection of small interfering RNA (siRNA) into ESCC cell lines (TE10, TE11). The expression levels of the genes in the knockdown models were analyzed using a microarray and a Biobase Upstream Analysis, while the expression levels of the candidate genes in the primary tumors of surgical specimens obtained from ESCC patients were determined using real-time polymerase chain reaction (PCR) and immunohistochemical staining. The clinicopathological features were then analyzed. The Biobase Upstream Analysis showed the high-mobility-group protein-1 (HMGA1) to be a significant gene regulated by Fra-1. Actual binding of Fra-1 to the promotor region of HMGA1 was revealed in subsequent chromatin immunoprecipitation PCR experiments. Patients with a positive HMGA1 expression had a poor prognosis, and a multivariate analysis demonstrated a positive HMGA1 expression to be a significant independent prognostic factor. HMGA1 is regulated by Fra-1 in ESCC, and the HMGA1 expression is significantly associated with a poor prognosis in ESCC patients. Downregulation of the HMGA1 expression may become a practical treatment strategy against ESCC in the future.

Inhibition of H3K27 histone trimethylation activates fibroblasts and induces fibrosis

ObjectivesEpigenetic modifications such as DNA methylation and histone acetylation have been implicated in the pathogenesis of systemic sclerosis. However, histone methylation has not been investigated so far. We therefore aimed...

Estrogen-induced Expression of Fos-related Antigen 1 (FRA-1) Regulates Uterine Stromal Differentiation and Remodeling

Concerted actions of estrogen and progesterone via their cognate receptors orchestrate changes in the uterine tissue, regulating implantation during early pregnancy. The uterine stromal cells undergo steroid-dependent differentiation into morphologically and functionally distinct decidual cells, which support embryonic growth and survival. The hormone-regulated pathways underlying this unique cellular transformation are not fully understood. Previous studies in the mouse revealed that, following embryo attachment, de novo synthesis of estrogen by the decidual cells is critical for stromal differentiation. In this study we report that Fos-related antigen 1 (FRA-1), a member of the Fos family of transcription factors, is a downstream target of regulation by intrauterine estrogen. FRA-1 expression was localized in the differentiating uterine stromal cells surrounding the implanted embryo. Attenuation of estrogen receptor α (Esr1) expression by siRNA mediated silencing in primary uterine stromal cells suppressed FRA-1 expression. Furthermore, chromatin immunoprecipitation demonstrated direct recruitment of ESR1 to an estrogen response element in the Fra-1 promoter. Down-regulation of Fra-1 expression during in vitro decidualization blocked stromal differentiation and resulted in a marked decrease in stromal cell migration. Interestingly, FRA-1 controls the expression of matrix metalloproteinases MMP9 and MMP13, which are critical modulators of stromal extracellular matrix remodeling. Collectively, these results suggest that FRA-1, induced in response to estrogen signaling via ESR1, is a key regulator of stromal differentiation and remodeling during early pregnancy.

Dextromethorphan-induced psychotoxic behaviors cause sexual dysfunction in male mice via stimulation of σ-1 receptors

Dextromethorphan (DM) is a well-known antitussive dextrorotatory morphinan. We and others have demonstrated that sigma (σ) receptors may be important for DM-mediated neuromodulation. Because an earlier report suggested that DM might affect sexual function and that σ receptor ligands affect signaling pathways in the periphery, we examined whether DM-induced psychotoxic burden affected male reproductive function. We observed that DM had a high affinity at σ-1 receptors in the brain and testis but relatively low affinity at σ-2 receptors. Prolonged treatment with DM resulted in conditioned place preference and hyperlocomotion, followed by an increase in Fos-related antigen expression in the nucleus accumbens in male mice. Simultaneously, DM induced significant reductions in gonadotropin-releasing-hormone immunoreactivity in the hypothalamus. Moreover, we observed that DM induced increased sperm abnormalities and decreased sperm viability and sexual behavior. These phenomena were significantly attenuated by combined treatment with BD1047, a σ-1 receptor antagonist, but not by SM-21, a σ-2 receptor antagonist. Thus, these results suggest that DM psychotoxicity might lead to reproductive stress in male mice by activating σ-1 receptors.

Exposure to Extremely Low Frequency Magnetic Fields Induces Fos-Related Antigen-Immunoreactivity Via Activation of Dopaminergic D1 Receptor

We previously demonstrated that repeated exposure to extremely low frequency magnetic fields (ELF-MF) increases locomotor activity via stimulation of dopaminergic D1 receptor (J. Pharmacol. Sci., 2007;105:367-371). Since it has been demonstrated that activator protein-1 (AP-1) transcription factors, especially 35-kDa fos-related antigen (FRA), play a key role in the neuronal and behavioral adaptation in response to various stimuli, we examined whether repeated ELF-MF exposure induces FRA-immunoreactivity (FRA-IR) in the striatum and nucleus accumbens (striatal complex) of the mice. Repeated exposure to ELF-MF (0.3 or 2.4 mT, 1 h/day, for consecutive fourteen days) significantly induced hyperlocomotor activity and FRA-IR in the striatal complex in a field intensity-dependent manner. ELF-MF-induced FRA-IR lasted for at least 1 year, while locomotor activity returned near control level 3 months after the final exposure to ELF-MF. Pretreatment with SCH23390, a dopaminergic D1 receptor antagonist, but not with sulpiride, a dopaminergic D2 receptor antagonist, significantly attenuated hyperlocomotor activity and FRA-IR induced by ELF-MF. Our results suggest that repeated exposure to ELF-MF leads to prolonged locomotor stimulation and long-term expression of FRA in the striatal complex of the mice via stimulation of dopaminergic D1 receptor.

Initiating egg production in turkey breeder hens: Thyroid hormone involvement

The role of thyroid hormones in the expression of photosensitivity-photorefractoriness in female turkeys was investigated through the use of an antithyroidal agent, 6-n-propyl-2-thiouracil (PTU). In experiment 1, females held continuously from hatch on long day lengths (16L:8D; LD) and fed 0.1% PTU from 0 to 16 wk, began laying eggs at 26 wk of age, peaking at 75% hen-day egg production by 29 wk, whereas controls initiated lay 3 wk earlier but only achieved less than 50% hen-day egg production. In experiment 2, PTU treatment from 10 to 18 wk severely suppressed plasma triiodothyronine and thyroxine, as confirmed by RIA. Egg production of PTU and control hens held on LD from hatch began by 23 wk, with PTU hens reaching a substantially greater rate of lay than controls. Eggs were smaller initially in both treatments but exceeded 75 g by 28 wk. In experiment 3, recycled hens on short day lengths (8L:16D) received PTU for 2 wk before LD and 12 wk thereafter; a subset of these hens was killed after 48 h of LD for immunohistochemical analysis of fos-related antigen (FRA) expression in the tuberal hypothalamus as a marker of photoinduced neuronal activity. The PTU treatment completely forestalled egg production until its withdrawal; egg production then rose sharply to control levels before resuming, along with controls, a typical seasonal decline. The PTU treatment did not impair photoinduced FRA expression. Together, these results demonstrate the following: 1) that a period of pharmacological suppression of triiodothyronine and thyroxine can substitute for short day exposure in conferring photosensitivity on juvenile-aged turkeys (and is actually superior to short day exposure), 2) that reproductive development does not limit egg production of turkey hens photostimulated as young as approximately 20 wk of age, and 3) that effects of thyroid suppression on photostimulation lie downstream of photoinduced FRA expression. Taken together, these results suggest that there is ample physiological potential to substantially advance the age of photoinduced egg production in commercial flocks.

Activity of Hypothalamic Dopaminergic Neurones During the Day of Oestrus: Involvement in Prolactin Secretion

A secretory surge of prolactin occurs on the afternoon of oestrus in cycling rats. Pituitary prolactin is inhibited by dopamine. We evaluated the activity of the neuroendocrine dopaminergic neurones during oestrus and dioestrus, as determined by dopaminergic activity in the median eminence and neurointermediate lobe of the pituitary, as well as Fos-related antigen expression in tyrosine hydroxylase (TH)-immunoreactive (ir) neurones of the arcuate nucleus (ARC) and periventricular nucleus (Pe). During oestrus, the 4-dihydroxyphenylacetic acid/dopamine ratio in the median eminence decreased at 16.00 h, coinciding with the increase in plasma prolactin levels. Similarly, the expression of Fos-related antigen in TH-ir neurones of Pe and rostral-, dorsomedial- and caudal-ARC also decreased at 16.00 h. On dioestrus, 4-dihydroxyphenylacetic acid/dopamine ratio in the median eminence and Fos-related antigen expression in TH-ir neurones of Pe and rostral-ARC decreased at 18.00 h, whereas prolactin levels were unaltered. No variation in dopaminergic activity was found in the neurointermediate lobe of the pituitary on either oestrus or dioestrus. The number of TH-ir neurones in the ARC and parameters of dopaminergic activity were found to be generally lower on oestrus compared to dioestrus. The transitory decrease in the activity of neuroendocrine dopaminergic neurones temporally associated with the prolactin surge on the afternoon of oestrus suggests a role for dopamine in the generation of the oestrous prolactin surge.

Kisspeptin Regulates Prolactin Release through Hypothalamic Dopaminergic Neurons

Prolactin (PRL) is tonically inhibited by dopamine (DA) released from neurons in the arcuate and periventricular nuclei. Kisspeptin plays a pivotal role in LH regulation. In rodents, kisspeptin neurons are found mostly in the anteroventral periventricular and arcuate nuclei, but the physiology of arcuate kisspeptin neurons is not completely understood. We investigated the role of kisspeptin in the control of hypothalamic DA and pituitary PRL secretion in adult rats. Intracerebroventricular kisspeptin-10 (Kp-10) elicited PRL release in a dose-dependent manner in estradiol (E2)-treated ovariectomized rats (OVX+E2), whereas no effect was found in oil-treated ovariectomized rats (OVX). Kp-10 increased PRL release in males and proestrous but not diestrous females. Associated with the increase in PRL release, intracerebroventricular Kp-10 reduced Fos-related antigen expression in tyrosine hydroxylase-immunoreactive (ir) neurons of arcuate and periventricular nuclei in OVX+E2 rats, with no effect in OVX rats. Kp-10 also decreased 3,4-dihydroxyphenylacetic acid concentration and 3,4-dihydroxyphenylacetic acid-DA ratio in the median eminence but not striatum in OVX+E2 rats. Double-label immunofluorescence combined with confocal microscopy revealed kisspeptin-ir fibers in close apposition to and in contact with tyrosine hydroxylase-ir perikarya in the arcuate. In addition, Kp-10 was not found to alter PRL release from anterior pituitary cell cultures regardless of E2 treatment. We provide herein evidence that kisspeptin regulates PRL release through inhibition of hypothalamic dopaminergic neurons, and that this mechanism is E2 dependent in females. These findings suggest a new role for central kisspeptin with possible implications for reproductive physiology.

Daily Immediate Early Gene Expression in the Suprachiasmatic Nucleus of Male and Female Octodon degus

Diurnal and nocturnal species have different patterns of general activity, sleep-wake rhythms, and endocrine rhythms, but the mechanisms underlying these differences are not clear. Here, we tested the hypothesis that rhythms in immediate early gene (IEG) products in the suprachiasmatic nucleus (SCN) of a diurnal rodent (Octodon degus) reflect their diurnal chronotype. We also compared male and female degus with respect to temporal patterns of expression of one of these gene products, Fos-related Antigen (FRA). Animals were killed across the light:dark cycle, brains were collected, and sections through the SCN were stained for FRA (females and males) or c-Fos and Calbindin (males only) using immunocytochemistry. An additional set of females placed in constant darkness was pulsed with light (or not) during the subjective day or night. Labeled cells in the SCN were counted in all animals. In males, c-Fos/FRA positive (+) cell counts in the dorsomedial SCN were low after lights-on and peaked around the time of lights-off (ZT 13, ZT 0=lights-on). Effect size analyses indicated that females have a 24 h rhythm in FRA expression that is shifted relative to that of males. Light pulses in the subjective day and night produced area-specific changes in IEG expression in females that differs from that reported for males. Overall, these studies reveal the pattern of immediate early gene expression in the SCN of degus is not the same in males and females, and that it differs from the one seen in other species, both nocturnal and diurnal.

Photostimulated Expression of Type 2 Iodothyronine Deiodinase mRNA is Greatly Attenuated in the Rostral Tuberal Hypothalamus of the Photorefractory Turkey Hen

For many temperate-zone avian species, termination of breeding occurs when individuals no longer respond to previously stimulatory day lengths, a condition called photorefractoriness. Long day lengths induce significantly greater expression of c-fos and fos-related antigens (FRAs) in the tuberal hypothalamus of the photosensitive hen than that of the photorefractory hen. The tuber is also a site of photoinducible glial expression of type 2 iodothyronine deiodinase (Dio2), which converts thyroxine into its active form, triiodothyronine (T3). T3 induces withdrawal of glial processes from gonadotrophin-releasing hormone (GnRH) I nerve terminals, which is believed to permit the efficient release of GnRH I into the associated portal vasculature. Using a riboprobe, we tested whether long days induce Dio2 mRNA expression in the turkey tuber and, if so, whether this expression is reduced in photorefractory hens. Long days significantly induced rostral and caudal tuberal hypothalamic Dio2 expression in photosensitive hens. Photorefractory hens had reduced expression of Dio2 with most subjects expressing no detectable mRNA in the rostral tuber and variably attenuated amounts throughout the medial and caudal tuber. We also performed double-label immunohistochemistry to identify co-localisation between FRAs and glial fibrillary acidic protein, a glial marker. FRAs were present in the nuclei of a few astrocytes in the median eminence and infundibular nucleus of the tuber. The temporal and spatial coincidence between FRA and Dio2 expression, their mutual association with glia, and the attenuation of their response during photorefractoriness suggests that the two events are linked and that photorefractoriness involves a reduced capacity for photo-inducible gene expression within glia of the tuberal hypothalamus.

Knockdown of clock genes in the suprachiasmatic nucleus blocks prolactin surges and alters FRA expression in the locus coeruleus of female rats

The nature of the circadian signal from the suprachiasmatic nucleus (SCN) required for prolactin (PRL) surges is unknown. Because the SCN neuronal circadian rhythm is determined by a feedback loop of Period (Per) 1, Per2, and circadian locomotor output cycles kaput (Clock) gene expressions, we investigated the effect of SCN rhythmicity on PRL surges by disrupting this loop. Because lesion of the locus coeruleus (LC) abolishes PRL surges and these neurons receive SCN projections, we investigated the role of SCN rhythmicity in the LC neuronal circadian rhythm as a possible component of the circadian mechanism regulating PRL surges. Cycling rats on proestrous day and estradiol-treated ovariectomized rats received injections of antisense or random-sequence deoxyoligonucleotide cocktails for clock genes (Per1, Per2, and Clock) in the SCN, and blood samples were taken for PRL measurements. The percentage of tyrosine hydroxylase-positive neurons immunoreactive to Fos-related antigen (FRA) was determined in ovariectomized rats submitted to the cocktail injections and in a 12:12-h light:dark (LD) or constant dark (DD) environment. The antisense cocktail abolished both the proestrous and the estradiol-induced PRL surges observed in the afternoon and the increase of FRA expression in the LC neurons at Zeitgeber time 14 in LD and at circadian time 14 in DD. Because SCN afferents and efferents were probably preserved, the SCN rhythmicity is essential for the magnitude of daily PRL surges in female rats as well as for LC neuronal circadian rhythm. SCN neurons therefore determine PRL secretory surges, possibly by modulating LC circadian neuronal activity.

Gene expression in rat vestibular and reticular structures during and after space flight

Space flight produces profound changes of neuronal activity in the mammalian vestibular and reticular systems, affecting postural and motor functions. These changes are compensated over time by plastic alterations in the brain. Immediate early genes (IEGs) are useful indicators of both activity changes and neuronal plasticity. We studied the expression of two IEG protein products [Fos and Fos-related antigens (FRAs)] with different cell persistence times (hours and days, respectively) to identify brainstem vestibular and reticular structures involved in adaptation to microgravity and readaptation to 1 G (gravity) during the NASA Neurolab Mission (STS-90). IEG protein expression in flight animals was compared to that of ground controls using Fisher 344 rats killed 1 and 12 days after launch and 1 and 14 days after landing. An increase in the number of Fos-protein-positive cells in vestibular (especially medial and spinal) regions was observed 1 day after launch and 1 day after landing. Fos-positive cell numbers were no different from controls 12 days after launch or 14 days after landing. No G-related changes in IEG expression were observed in the lateral vestibular nucleus. The pattern of FRA protein expression was generally similar to that of Fos, except at 1 day after landing, when FRA-expressing cells were observed throughout the whole spinal vestibular nucleus, but only in the caudal part of the medial vestibular nucleus. Fos expression was found throughout the entire medial vestibular nucleus at this time. While both Fos and FRA expression patterns may reflect the increased G force experienced during take-off and landing, the Fos pattern may additionally reflect recent rebound episodes of rapid eye movement (REM) sleep following forced wakefulness, especially after landing. Pontine activity sources producing rhythmic discharges of vestibulo-oculomotor neurons during REM sleep could substitute for labyrinthine signals after exposure to microgravity, contributing to activity-related plastic changes leading to G readaptation. Reticular structures exhibited a contrasting pattern of changes in the numbers of Fos- and FRA-positive cells suggestive of a major influence from proprioceptive inputs, and plastic re-weighting of inputs after landing. Asymmetric induction of Fos and FRAs observed in some vestibular nuclei 1 day after landing suggests that activity asymmetries between bilateral otolith organs, their primary labyrinthine afferents, and vestibular nuclei may become unmasked during flight.

Fos and FRA protein expression in rat nucleus paragigantocellularis lateralis during different space flight conditions

The nucleus paragigantocellularis lateralis (LPGi) exerts a prominent excitatory influence over locus coeruleus (LC) neurons, which respond to gravity signals. We investigated whether adult albino rats exposed to different gravitational fields during the NASA Neurolab Mission (STS-90) showed changes in Fos and Fos-related antigen (FRA) protein expression in the LPGi and related cardiovascular, vasomotor, and respiratory areas. Fos and FRA proteins are induced rapidly by external stimuli and return to basal levels within hours (Fos) or days (FRA) after stimulation. Exposure to a light pulse (LP) 1h prior to sacrifice led to increased Fos expression in subjects maintained for 2 weeks in constant gravity (either at ∼0 or 1G). Within 24h of a gravitational change (launch or landing), the Fos response to LP was abolished. A significant Fos response was also induced by gravitational stimuli during landing, but not during launch. FRA responses to LP showed a mirror image pattern, with significant responses 24h after launch and landing, but no responses after 2 weeks at ∼0 or 1G. There were no direct FRA responses to gravity changes. The juxtafacial and retrofacial parts of the LPGi, which integrate somatosensory/acoustic and autonomic signals, respectively, also showed gravity-related increases in LP-induced FRA expression 24h after launch and landing. The neighboring nucleus ambiguus (Amb) showed completely different patterns of Fos and FRA expression, demonstrating the anatomical specificity of these results. Immediate early gene expression in the LPGi and related cardiovascular vasomotor and ventral respiratory areas may be directly regulated by excitatory afferents from vestibular gravity receptors. These structures could play an important role in shaping cardiovascular and respiratory function during adaptation to altered gravitational environments encountered during space flight and after return to earth.

Antagonism of vasoactive intestinal peptide mRNA in the suprachiasmatic nucleus disrupts the rhythm of FRAs expression in neuroendocrine dopaminergic neurons

This study was designed to determine whether there is a functional relationship between cfos expression in vasoactive intestinal peptide (VIP) -containing neurons of the suprachiasmatic nucleus (SCN) and Fos-related antigens (FRAs) expression in neuroendocrine dopaminergic neurons of the arcuate (ARN) and periventricular (PeVN) nuclei of the hypothalamus. Brains were obtained from ovariectomized (OVX) female rats killed at 12:00 AM, 7:00 AM, 9:00 AM, 12:00 PM, and 7:00 PM (12 hours illumination beginning 6:00 AM). Antibodies against FRAs and tyrosine hydroxylase (TH) identified activated neuroendocrine dopaminergic neurons. Antibodies against cfos and VIP identified activated VIP-immunoreactive (IR) neurons in the SCN. The proportion of neuroendocrine dopaminergic neurons in the ARN and PeVN expressing FRAs was greatest and equivalent at 7:00 AM, 9:00 AM, 12:00 PM, and 12:00 AM. At 7:00 PM, the proportion of neuroendocrine dopaminergic neurons expressing FRAs was significantly lower than all other time points. In the SCN, a subpopulation of VIP-IR neurons maximally expressed cfos at 7:00 AM, which decreased through 9:00 AM. cFos was not expressed at 7:00 PM and 12:00 AM in VIP-IR neurons. Antisense VIP oligonucleotides were injected into the SCN to determine whether attenuation of VIP expression disturbs rhythms in neuroendocrine dopaminergic neuronal activity. OVX rats were infused with either antisense VIP oligonucleotides or scrambled sequence oligonucleotides bilaterally (0.5 microg in 0.5 microl of saline per side) in the SCN. Animals were killed 34 hours (7:00 PM) and 46 hours (7:00 AM) after receiving infusions, and brains were recovered. Administration of antisense VIP oligonucleotides decreased VIP protein expression in the SCN and prevented the decrease in the percentage of neuroendocrine dopaminergic neurons expressing FRAs at 7:00 PM but did not affect FRAs expression at 7:00 AM when compared with animals receiving scrambled oligonucleotides. These data suggest that VIP fibers from the SCN may relay time-of-day information to neuroendocrine dopaminergic neurons to inhibit their activity and, thus, initiate prolactin release in the evening.

Nuclear translocation of STAT5 and increased expression of Fos related antigens (FRAs) in hypothalamic dopaminergic neurons after prolactin administration

Ample evidence indicates feedback relationships between pituitary prolactin and hypothalamic dopaminergic neurons. Since the presence of prolactin receptors was earlier demonstrated in hypothalamic dopaminergic neurons, our working hypothesis was that prolactin induced activation of prolactin receptor coupled signaling leads to increased neuronal activity in these neurons. The aim of this study was to correlate prolactin receptor mediated signaling and prolactin induced activation in hypothalamic dopaminergic neurons. We used nuclear translocation of STAT5 as a marker of prolactin receptor induced signaling and expression of Fos related antigens (FRAs) as an indicator of neuronal activation. We performed double label immunocytochemical studies to determine the time course of the presence of FRAs and STAT5 in the nuclei of hypothalamic dopaminergic neurons after ovine prolactin treatment. Exogenous ovine prolactin treatment of ovariectomized rats resulted in an increase in serum ovine prolactin levels and a decrease in endogenous serum prolactin levels, indicating that ovine prolactin activated mechanisms inhibited pituitary prolactin secretion. Indeed, ovine prolactin activated the prolactin receptors in most subpopulations of hypothalamic dopaminergic neurons, resulting in nuclear translocation of STAT5. Also, increased neuronal activity, indicated by expression of FRAs, was observed in the same neuron populations after ovine prolactin treatment. These results suggest that signal transduction mechanisms coupled to prolactin receptors in hypothalamic dopaminergic neurons resemble those observed in other tissues; and nuclear translocation of STAT5 can be used as a marker of prolactin receptor activation in hypothalamic dopaminergic neurons.

Decreased expression of fos-related antigens (FRAs) in the hypothalamic dopaminergic neurons after immunoneutralization of endogenous prolactin.

In our previous studies we found that administration of exogenous prolactin increased dopamine turnover in the terminal areas of the hypothalamic dopaminergic neurons controlling prolactin secretion from pituitary lactotrophs. In this study we investigated the effect of immunoneutralization of endogenous prolactin on the expression of FRAs in the tuberoinfundibular dopaminergic (TIDA), tuberohypophysial dopaminergic (THDA), and periventricular hypothalamic dopaminergic (PHDA) subpopulations of the hypothalamic dopaminergic neurons. Female rats were ovariectomized on d 0 of the experiment. At 1000 h of d 10, all animals were injected with 20 microg of 17-beta-estradiol sc to induce a proestrous-like surge of prolactin at 1700 h the next day. At 1000 h on d 11, half of the animals were injected with 200 microL of rabbit anti-rat prolactin antiserum ip, while the controls received normal rabbit serum. Groups of animals were sacrificed for immunocytochemistry in 2 h intervals between 1300 and 2100 h. Double-label immunocytochemistry for FRAs and tyrosine hydroxylase (TH) was performed and the results are presented as percentage of TH-immunoreactive neurons expressing FRAs. In the control animals, expression of FRAs decreased at 1500 h, gradually increased by 1900 h, but was lower than the basal levels by 2100 h. Expression of FRAs was significantly lower at 1900 h in the PHDA, THDA and TIDA neurons of prolactin antiserum treated rats than in the controls. These results indicate that elimination of endogenous prolactin from the circulation lowers the activity and/or prevents the reactivation of neuroendocrine dopaminergic neurons at the beginning of the dark phase.