Rat Area Postrema Research Articles

Three-plane description of astroglial architecture and gliovascular connections of area postrema in rat: Long tanycyte connections to other parts of brainstem.

The study demonstrates the astroglial and gliovascular structures of the area postrema (AP) in three planes, and compares them to our former findings on the subfornical organ (SFO) and the organon vasculosum laminae terminalis (OVLT). The results revealed long glial processes interconnecting the AP with deeper areas of brain stem. The laminin and β-dystroglycan immunolabeling altered along the vessels indicating alterations of the gliovascular relations. These and the distributions of glial markers displayed similarities to the SFO and OVLT. In every organ, there was a central area with vimentin- and nestin-immunopositive glia, whereas GFAP and the water-channel aquaporin 4 were found at the periphery. This separation supports different functions of the two regions. The presence of nestin may indicate stem cell capabilities, whereas aquaporin 4 has been suggested by other studies to be a possible participant of osmoperception. Numerous S100-immunopositive glial cells were found approximately evenly distributed in both parts of the AP. Frequency of glutamine synthetase-immunoreactive cells was similar in the surrounding brain tissue in contrast to that found in the OVLT and SFO. Our findings on the three sensory circumventricular organs (AP, OVLT, and SFO) are compared in parallel.

Dynamics of cachexia-associated inflammatory changes in the brain accompanying intra-abdominal fibrosarcoma growth in Wistar rats

Accumulated data indicate that inflammation affecting brain structures participates in the development of cancer-related cachexia. However, the mechanisms responsible for the induction and progression of cancer-related neuroinflammation are still not fully understood. Therefore, we studied the time-course of neuroinflammation in selected brain structures and cachexia development in tumor-bearing rats. After tumor cells inoculation, specifically on the 7th, 14th, 21st, and 28th day of tumor growth, we assessed the presence of cancer-associated cachexia in rats. Changes in gene expression of inflammatory factors were studied in selected regions of the hypothalamus, brain stem, and circumventricular organs. We showed that the initial stages of cancer growth (7th and 14th day after tumor cells inoculation), are not associated with cachexia, or increased expression of inflammatory molecules in the brain. Even when we did not detect cachexia in tumor-bearing rats by the 21st day of the experiment, the inflammatory brain reaction had already started, as we found elevated levels of interleukin 1 beta, interleukin 6, tumor necrosis factor alpha, and glial fibrillary acidic protein mRNA levels in the nucleus of the solitary tract. Furthermore, we found increased interleukin 1 beta expression in the locus coeruleus and higher allograft inflammatory factor 1 expression in the vascular organ of lamina terminalis. Ultimately, the most pronounced manifestations of tumor growth were present on the 28th day post-inoculation of tumor cells. In these animals, we detected cancer-related cachexia and significant increases in interleukin 1 beta expression in all brain areas studied. We also observed significantly decreased expression of the glial cell activation markers allograft inflammatory factor 1 and glial fibrillary acidic protein in most brain areas of cachectic rats. In addition, we showed increased expression of cluster of differentiation 163 and cyclooxygenase 2 mRNA in the hypothalamic paraventricular nucleus, A1/C1 neurons, and area postrema of cachectic rats. Our data indicate that cancer-related cachexia is associated with complex neuroinflammatory changes in the brain. These changes can be found in both hypothalamic as well as extrahypothalamic structures, while their extent and character depend on the stage of tumor growth.

Single nuclei RNA sequencing of the rat AP and NTS following GDF15 treatment

ObjectiveGrowth differentiation factor 15 (GDF15) is known to play a role in feeding, nausea, and body weight, with action through the GFRAL-RET receptor complex in the area postrema (AP) and nucleus tractus solitarius (NTS). To further elucidate the underlying cell type-specific molecular mechanisms downstream of GDF15 signaling, we used a single nuclei RNA sequencing (snRNAseq) approach to profile AP and NTS cellular subtype-specific transcriptomes after systemic GDF15 treatment. MethodsAP and NTS micropunches were used for snRNAseq from Sprague Dawley rats 6 h following GDF15 or saline injection, and Seurat was used to identify cellular subtypes and cell type-specific alterations in gene expression that were due to the direct and secondary effects of systemic GDF15 treatment. ResultsUsing the transcriptome profile of ∼35,000 individual AP/NTS nuclei, we identified 19 transcriptomically distinct cellular subtypes, including a single population Gfral and Ret positive excitatory neurons, representing the primary site of action for GDF15. A total of ∼600 cell type-specific differential expression events were identified in neurons and glia, including the identification of transcriptome alterations specific to the direct effects of GDF15 in the Gfral-Ret positive excitatory neurons and shared transcriptome alterations across neuronal and glial cell types. Downstream analyses identified shared and cell type-specific alterations in signaling pathways and upstream regulatory mechanisms of the observed transcriptome alterations. ConclusionsThese data provide a considerable advance in our understanding of AP and NTS cell type-specific molecular mechanisms associated with GDF15 signaling. The identified cellular subtype-specific regulatory mechanism and signaling pathways likely represent important targets for future pharmacotherapies.

Increased neuronal activation in sympathoregulatory regions of the brain and spinal cord in type 2 diabetic rats.

Increased cardiac sympathetic nerve activity in type 2 diabetes mellitus (DM) suggests impaired autonomic control of the heart. However, the central regions that contribute to the autonomic cardiac pathologies in type 2 DM are unknown. Therefore, we tested the hypothesis that neuronal activation would be increased in central sympathoregulatory areas in a pre-clinical type 2 DM animal model. Immunohistochemistry in 20-week-old male Zucker diabetic fatty (ZDF) rats revealed an increased number of neurones expressing ΔFosB (a marker of chronic neuronal activation) in the intermediolateral column (IML) of the spinal cord in DM compared to non-diabetic (non-DM) rats (P<0.05). Rostral ventrolateral medulla (RVLM) neurones activate IML neurones and receive inputs from the hypothalamic paraventricular nucleus (PVN), as well as the nucleus tractus solitarius (NTS) and area postrema (AP), in the brainstem. We observed more ΔFosB-positive noradrenergic RVLM neurones (P<0.001) and corticotrophin-releasing hormone PVN neurones (P<0.05) in DM compared to non-DM rats. More ΔFosB-positive neurones were also observed in the NTS (P<0.05) and AP (P<0.01) of DM rats compared to non-DM rats. Finally, because DM ZDF rats are obese, we also expected increased activation of pro-opiomelanocortin (POMC) arcuate nucleus (ARC) neurones in DM rats; however, fewer ΔFosB-positive POMC ARC neurones were observed in DM compared to non-DM rats (P<0.01). In conclusion, increased neuronal activation in the IML of type 2 DM ZDF rats might be driven by RVLM neurones that are possibly activated by PVN, NTS and AP inputs. Elucidating the contribution of central sympathoexcitatory drive in type 2 DM might improve the effectiveness of pharmacotherapies for diabetic heart disease.

Hypoglycemia attenuates acute amylin-induced reduction of food intake in male rats

The ability of amylin to inhibit gastric emptying and glucagon secretion in rats is reduced under hypoglycemic conditions. These effects are considered part of a fail-safe mechanism that prevents amylin from further decreasing nutrient supply when blood glucose levels are low. Because these actions and amylin-induced satiation are mediated by the area postrema (AP), it is plausible that these phenomena are based on the co-sensitivity of AP neurons to amylin and glucose. Using hyperinsulinemic glucose clamps in unrestrained and freely-feeding rats, we investigated whether amylin's ability to inhibit food intake is also reduced by hypoglycemia (HYPO). Following an 18 h fast, rats were infused with insulin and glucose for 45 min to clamp blood glucose at baseline levels (between 90 and 100 mg/dL). HYPO (approximately 55 mg/dL) was induced between 45 and 60 min and then maintained for the remainder of the clamp. Rats were injected with amylin (20 µg/kg) or saline and offered normal chow at 85 min. Food intake was measured at 30 and 60 min after amylin. Control hyperinsulinemic/euglycemic (EU) rats were maintained at approximately 150 mg/dL (which is a physiological periprandial glucose level) before and after amylin injection. Terminal experiments tested the effect of amylin to induce the phosphorylation of ERK, a marker of amylin action in the AP, in EU and HYPO conditions. Amylin significantly reduced 30- and 60-min food intake in EU rats, but the effect at 60-min was attenuated in HYPO rats. Interestingly, glucose infusion rate had to be dramatically reduced at meal onset in saline-treated, but not in amylin-treated, EU or HYPO rats; this suggests that meal-related glucose appearance in the blood was inhibited by amylin under both EU and HYPO. Finally, amylin induced a similar pERK response in the AP in EU and HYPO rats. We conclude that amylin's action to decrease eating is blunted in hypoglycemia, and this effect seems to be downstream from amylin-induced pERK in AP neurons. These data allow us to extend the idea of a hypoglycemic brake on amylin's actions to its food intake-reducing effect, but also demonstrate that amylin can buffer meal-induced glucose appearance at EU and HYPO levels.

Effect of different diets on left ventricle and area postrema in adult male albino rats predisposed to prolonged intake of high salt

Background Excess dietary salt is the most common environmental factor that contributes to the pathogenesis of hypertension. High-sugar and high-fat intakes in hypertension accelerate left ventricular hypertrophy and heart failure and increase the incidence of strokes and infarctions. Aim This work aimed to assess the correlation of long-term high-salt intake with hypertension in adult male albino rats and to evaluate the effect of different dietary regimens on left ventricle and area postrema of those rats. Materials and methods A total of 24 adult male albino rats were used in this study. Rats were divided into group I (control), which included six rats that received standard diet, and group II (experimental), which included 18 rats that received high-salt diet for 8 weeks, which was then classified as subgroup IIA (reversal), IIB (high sugar), and IIC (high fat). Systolic blood pressure was measured at baseline, after 8 weeks, and after 12 weeks from the start of the experiment. Specimens from left ventricle of the heart and area postrema were subjected to histological and immunohistochemical examinations. Data of the body weight, heart weight, and thickness of the left ventricle were analyzed statistically. Results The current study revealed increased weight gain, heart weight, left ventricular thickness, and systolic blood pressure in different experimental groups. These groups showed disturbed histological architecture of left ventricle of the heart and area postrema, but in subgroup IIA, partial improvement of the histological findings was found. Conclusion There is a correlation between high-salt diet and hypertension. Salt, sugar, and fat in high doses have hazardous effects on the histological architecture of the left ventricle and area postrema of adult male albino rats. Restriction of salt in diet leads to some improvement of the adverse effects.

Isoflurane induces c-Fos expression in the area postrema of the rat.

Volatile anesthetics are speculated to cause postoperative nausea and vomiting via stimulation of the chemoreceptor trigger zone (CTZ). However, the precise mechanism underlying the emetic action of these drugs is not well understood. In this study, we assessed whether isoflurane induced the expression of c-Fos, a neuronal activation marker, in the area postrema (AP), the locus of the CTZ, in rats, which do not have vomiting action. Male rats were exposed to 1.3% isoflurane for 0-240min, or to various concentrations of isoflurane (0, 1.3%, or 2.6%) for 120min. Finally, the rats were exposed to 1.3% isoflurane for 120min after ondansetron administration. After the treatments, immunohistochemistry of the rat AP was performed using c-Fos antibody staining. One-way analysis of variance showed that isoflurane exposure significantly increased c-Fos expression in the AP; however, the rats pretreated with 4mg/kg ondansetron showed significantly decreased c-Fos expression. Moreover, we evaluated the effect of the anesthetic on inducing pica in the rats, and found that kaolin intake was not influenced by isoflurane exposure. Overall, these results suggest that isoflurane activates AP neurons and may be involved in the emetic mechanism of isoflurane. This study further suggests the feasibility of using rats as a model for studying emetic mechanisms of drugs, despite their lack of vomit action.

Involvement of Amylin and Leptin in the Development of Projections from the Area Postrema to the Nucleus of the Solitary Tract.

The area postrema (AP) and the nucleus of the solitary tract (NTS) are important hindbrain centers involved in the control of energy homeostasis. The AP mediates the anorectic action and the inhibitory effect on gastric emptying induced by the pancreatic hormone amylin. Amylin’s target cells in the AP project to the NTS, an integrative relay center for enteroceptive signals. Perinatal hormonal and metabolic factors influence brain development. A postnatal surge of the adipocyte-derived hormone leptin represents a developmental signal for the maturation of projections between hypothalamic nuclei controlling energy balance. Amylin appears to promote neurogenesis in the AP in adult rats. Here, we examined whether amylin and leptin are required for the development of projections from the AP to the NTS in postnatal and adult mice by conducting neuronal tracing studies with DiI in amylin- (IAPP−/−) and leptin-deficient (ob/ob) mice. Compared to wild-type littermates, postnatal (P10) and adult (P60) IAPP−/− mice showed a significantly reduced density of AP-NTS projections. While AP projections were also reduced in postnatal (P14) ob/ob mice, AP-NTS fiber density did not differ between adult ob/ob and wild-type animals. Our findings suggest a crucial function of amylin for the maturation of neuronal brainstem pathways controlling energy balance and gastrointestinal function. The impaired postnatal development of neuronal AP-NTS projections in ob/ob mice appears to be compensated in this experimental model during later brain maturation. It remains to be elucidated whether an amylin- and leptin-dependent modulation in neuronal development translates into altered AP/NTS-mediated functions.

The metabolic effects of GDF15 are mediated by the orphan receptor GFRAL

Growth/differentiation factor 15 (GDF15), also known as MIC-1, is a distant member of the transforming growth factor-β (TGF-β) superfamily and has been implicated in various biological functions, including cancer cachexia, renal and heart failure, atherosclerosis and metabolism. A connection between GDF15 and body-weight regulation was initially suggested on the basis of an observation that increasing GDF15 levels in serum correlated with weight loss in individuals with advanced prostate cancer. In animal models, overexpression of GDF15 leads to a lean phenotype, hypophagia and other improvements in metabolic parameters, suggesting that recombinant GDF15 protein could potentially be used in the treatment of obesity and type 2 diabetes. However, the signaling and mechanism of action of GDF15 are poorly understood owing to the absence of a clearly identified cognate receptor. Here we report that GDNF-family receptor α-like (GFRAL), an orphan member of the GFR-α family, is a high-affinity receptor for GDF15. GFRAL binds to GDF15 in vitro and is required for the metabolic actions of GDF15 with respect to body weight and food intake in vivo in mice. Gfral-/- mice were refractory to the effects of recombinant human GDF15 on body-weight, food-intake and glucose parameters. Blocking the interaction between GDF15 and GFRAL with a monoclonal antibody prevented the metabolic effects of GDF15 in rats. Gfral mRNA is highly expressed in the area postrema of mouse, rat and monkey, in accordance with previous reports implicating this region of the brain in the metabolic actions of GDF15 (refs. 4,5,6). Together, our data demonstrate that GFRAL is a receptor for GDF15 that mediates the metabolic effects of GDF15.

The use of siRNA as a pharmacological tool to assess a role for the transcription factor NF-IL6 in the brain under in vitro and in vivo conditions during LPS-induced inflammatory stimulation.

Studies with NF-IL6-deficient mice indicate that this transcription factor plays a dual role during systemic inflammation with pro- and anti-inflammatory capacities. Here, we aimed to characterize the role of NF-IL6 specifically within the brain. In this study, we tested the capacity of short interfering (si) RNA to silence the inflammatory transcription factor nuclear factor-interleukin 6 (NF-IL6) in brain cells under in vitro and in vivo conditions. In cells of a mixed neuronal and glial primary culture from the rat area postrema (AP), short interfering RNA (siRNA) directed against NF-IL6 strongly reduced basal and lipopolysaccharide (LPS)-induced nuclear immunoreactivity of this transcription factor, with the strongest effect on astrocytes. The siRNA did not exert inflammatory effects in the primary culture as confirmed by unaltered levels of IL-6 in supernatants. In vivo, intracerebroventricular (i.c.v.) injections of fluorochrome labelled siRNA caused its appearance in relevant brain structures for fever induction pathways such as the vascular organ of lamina terminalis, the subfornical organ, the median preoptic nucleus (MnPO) and the AP in several cell types, including microglial cells. However, i.c.v. injections of siRNA per se caused signs of fever, anorexia and reduced locomotor activity, i.e. sickness behavior. This approach was, thus, not suitable to characterize the role NF-IL6 in the brain in vivo, namely during experimentally induced systemic inflammation.

Role of the area postrema in the hypophagic effects of oleoylethanolamide

The satiety-promoting action of oleoylethanolamide (OEA) has been associated to the indirect activation of selected brain areas, such as the nucleus of the solitary tract (NST) in the brainstem and the tuberomammillary (TMN) and paraventricular (PVN) nuclei in the hypothalamus, where noradrenergic, histaminergic and oxytocinergic neurons play a necessary role. Visceral ascending fibers were hypothesized to mediate such effects. However, our previous findings demonstrated that the hypophagic action of peripherally administered OEA does not require intact vagal afferents and is associated to a strong activation of the area postrema (AP). Therefore, we hypothesized that OEA may exert its central effects through the direct activation of this circumventricular organ. To test this hypothesis, we subjected rats to the surgical ablation of the AP (APX rats) and evaluated the effects of OEA (10mgkg−1 i.p.) on food intake, Fos expression, hypothalamic oxytocin (OXY) immunoreactivity and on the expression of dopamine beta hydroxylase (DBH) in the brainstem and hypothalamus. We found that the AP lesion completely prevented OEA’s behavioral and neurochemical effects in the brainstem and the hypothalamus. Moreover OEA increased DBH expression in AP and NST neurons of SHAM rats while the effect in the NST was absent in APX rats, thus suggesting the possible involvement of noradrenergic AP neurons. These results support the hypothesis of a necessary role of the AP in mediating OEA’s central effects that sustain its pro-satiety action.

The satiating hormone amylin enhances neurogenesis in the area postrema of adult rats

ObjectiveAdult neurogenesis in the subgranular zone and subventricular zone is generally accepted, but its existence in other brain areas is still controversial. Circumventricular organs, such as the area postrema (AP) have recently been described as potential neurogenic niches in the adult brain. The AP is the major site of action of the satiating hormone amylin. Amylin has been shown to promote the formation of neuronal projections originating from the AP in neonatal rodents but the role of amylin in adult neurogenesis remains unknown. MethodsTo test this, we first performed an RNA-sequencing of the AP of adult rats acutely injected with either amylin (20 μg/kg), amylin plus the amylin receptor antagonist AC187 (500 μg/kg) or vehicle. Second, animals were subcutaneously equipped with minipumps releasing either amylin (50 μg/kg/day) or vehicle for 3 weeks to assess cell proliferation and differentiation with the 5′-bromo-2-deoxyuridine (BrdU) technique. ResultsAcute amylin injections affected genes involved in pathways and processes that control adult neurogenesis. Amylin consistently upregulated NeuroD1 transcript and protein in the adult AP, and this effect was blocked by the co-administration of AC187. Further, chronic amylin treatment increased the number of newly proliferated AP-cells and significantly promoted their differentiation into neurons rather than astrocytes. ConclusionOur findings revealed a novel role of the satiating hormone amylin in promoting neurogenesis in the AP of adult rats.

Amylin receptor components and the leptin receptor are co-expressed in single rat area postrema neurons.

Amylin is a pancreatic β-cell hormone that acts as a satiating signal to inhibit food intake by binding to amylin receptors (AMYs) and activating a specific neuronal population in the area postrema (AP). AMYs are heterodimers that include a calcitonin receptor (CTR) subunit [CTR isoform a or b (CTRa or CTRb)] and a member of the receptor activity-modifying proteins (RAMPs). Here, we used single-cell quantitative polymerase chain reaction to assess co-expression of AMY subunits in AP neurons from rats that were injected with amylin or vehicle. Because amylin interacts synergistically with the adipokine leptin to reduce body weight, we also assessed the co-expression of AMY and the leptin receptor isoform b (LepRb) in amylin-activated AP neurons. Single cells were collected from Wistar rats and from transgenic Fos-GFP rats that express green fluorescent protein (GFP) under the control of the Fos promoter. We found that the mRNAs of CTRa, RAMP1, RAMP2 and RAMP3 were all co-expressed in single AP neurons. Moreover, most of the CTRa+ cells co-expressed more than one of the RAMPs. Amylin down-regulated RAMP1 and RAMP3 but not CTR mRNAs in AMY+ neurons, suggesting a possible negative feedback mechanism of amylin at its own primary receptors. Interestingly, amylin up-regulated RAMP2 mRNA. We also found that a high percentage of single cells that co-expressed all components of a functional AMY expressed LepRb mRNA. Thus, single AP cells expressed both AMY and LepRb, which formed a population of first-order neurons that presumably can be directly activated by amylin and, at least in part, also by leptin.

High dietary fat intake influences the activation of specific hindbrain and hypothalamic nuclei by the satiety factor oleoylethanolamide

Chronic exposure to a diet rich in fats changes the gastrointestinal milieu and alters responses to several signals involved in the control of food intake. Oleoylethanolamide (OEA) is a gut-derived satiety signal released from enterocytes upon the ingestion of dietary fats. The anorexigenic effect of OEA, which requires intestinal PPAR-alpha receptors and is supposedly mediated by vagal afferents, is associated with the induction of c-fos in several brain areas involved in the control of food intake, such as the nucleus of the solitary tract (NST) and the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON). In the present study we investigated whether the exposure to a high fat diet (HFD) alters the hindbrain and hypothalamic responses to OEA. To this purpose we evaluated the effects of OEA at a dose that reliably inhibits eating (10mg/kg i.p.) on the induction of c-fos in the NST, area postrema (AP), PVN and SON in rats maintained either on standard chow or a HFD. We performed a detailed analysis of the different NST subnuclei activated by i.p. OEA and found that peripheral OEA strongly activates c-fos expression in the AP, NST and in the hypothalamus of both chow and HFD fed rats. The extent of c-fos expression was, however, markedly different between the two groups of rats, with a weaker activation of selected NST subnuclei and stronger activation of the PVN in HFD-fed than in chow-fed rats. HFD-fed rats were also more sensitive to the immediate hypophagic action of OEA than chow-fed rats. These effects may be due to a decreased sensitivity of vagal afferent fibers that might mediate OEA's actions on the brain and/or an altered sensitivity of brain structures to OEA.

5-HT neurons of the area postrema become c-Fos-activated after increases in plasma sodium levels and transmit interoceptive information to the nucleus accumbens.

Serotonergic (5-hydroxytryptamine, 5-HT) neurons of the area postrema (AP) represent one neuronal phenotype implicated in the regulation of salt appetite. Tryptophan hydroxylase (Tryp-OH, synthetic enzyme-producing 5-HT) immunoreactive neurons in the AP of rats become c-Fos-activated following conditions in which plasma sodium levels are elevated; these include intraperitoneal injections of hypertonic saline and sodium repletion. Non-Tryp-OH neurons also became c-Fos-activated. Sodium depletion, which induced an increase in plasma osmolality but caused no significant change in the plasma sodium concentration, had no effect on the c-Fos activity in the AP. Epithelial sodium channels are expressed in the Tryp-OH-immunoreactive AP neurons, possibly functioning in the detection of changes in plasma sodium levels. Since little is known about the neural circuitry of these neurons, we tested whether the AP contributes to a central pathway that innervates the reward center of the brain. Stereotaxic injections of pseudorabies virus were made in the nucleus accumbens (NAc), and after 4 days, this viral tracer produced retrograde transneuronal labeling in the Tryp-OH and non-Tryp-OH AP neurons. Both sets of neurons innervate the NAc via a multisynaptic pathway. Besides sensory information regarding plasma sodium levels, the AP→NAc pathway may also transmit other types of chemosensory information, such as those related to metabolic functions, food intake, and immune system to the subcortical structures of the reward system. Because these subcortical regions ultimately project to the medial prefrontal cortex, different types of chemical signals from visceral systems may influence affective functions.

The combined effects of midazolam and propofol sedation on muscle power

We performed a randomised, crossover study to investigate the effects of intravenous sedation on grip strength and bite force. Twenty male volunteers received a bolus intravenous injection of midazolam (0.02 mg.kg(-1)) together with a 30-min propofol infusion designed to achieve an effect-site concentration of 1.0 μg.ml(-1). Observed variables included bispectral index, observer's assessment of alertness/sedation, correct answer rate of Stroop colour-word test, grip strength and bite force. Grip strength decreased from a median (IQR [range]) of 483 (443-517 [380-586]) N to 358 (280-405 [108-580]) N (p < 0.001) during sedation and recovered following flumazenil administration, while bite force increased from 818 (593-1026 [405-1406]) N to 1377 (1243-1585 [836-2357]) N (p < 0.001) during sedation. Although bite force gradually returned to baseline following flumazenil administration, it remained increased throughout the experimental period. We conclude that bite force increased during intravenous sedation and that this may have clinical implications.

Sex differences in estrogen receptor promoter expression in the area postrema.

Estrogen receptor α is widely distributed in the rat brain, but the tissue- or target-specificity of the estrogen receptor α gene promoters remains unknown. In the present study, we used transgenic rats expressing enhanced green fluorescent protein under the control of the estrogen receptor α 0/B promoter to examine expression driven by this promoter in two significant nuclei that regulate cardiovascular activity, the area postrema and the nucleus tractus solitarius. Immunohistochemistry showed that enhanced green fluorescent protein-labeled cells were distributed in the area postrema and the nucleus tractus solitarius of both female and male transgenic rats, and a neural network of enhanced green fluorescent protein-positive fibers was seen between the area postrema and the nucleus tractus solitarius. The number of enhanced green fluorescent protein-labeled cells in the area postrema of female rats was significantly higher than in the males, but no significant difference was found in the number of enhanced green fluorescent protein-labeled cells in the nucleus tractus solitarius. The sex differences in the number of enhanced green fluorescent protein-labeled cells in the area postrema was not affected after ovariectomy or 17β-estradiol benzoate treatment in adult rats. Our results suggest that the effects of estrogen in the area postrema are related to the expression of estrogen receptor α under the control of the 0/B promoter, and changes in the sex hormone environment in the adult period do not affect estrogen receptor α expression in the area postrema or the nucleus tractus solitarius.

Hypertension effects on p73 expression in the rat circumventricular organs and cerebrospinal fluid

It has been reported that spontaneously hypertensive rats (SHR) show ventricular dilation, changes in CSF proteins and variations in the circumventricular organs (CVO) such as: the subcommissural organ (SCO), the subfornical organ (SFO) and the area postrema (AP) which are located in the walls of the third and fourth ventricles. On the other hand, p73 proteins are present in cells of the central nervous system (CNS) such as circumventricular structures and the neuroepithelium which are altered in ventricular dilation. The purpose of the present work is to study the TAp73 isoform expression in the circumventricular organs (CVO) and their variations in ventricular dilatation and arterial hypertension. Brains and cerebrospinal fluid (CSF) from control Wistar-Kyoto rats (WKY) and SHR were used. The paraffin sections containing the CVO were immunohistochemically proc-essed with anti-TAp73 and by western blot, p73 bands in the CSF and circumventricular organ extract were also identified. The western blot study showed bands marked with p73 in the CSF and CVO, the p73 band expression was bigger in the SHR than in the WKY rats. We also found stronger markings in the SFO, SCO and AP of the hypertensive rats than in the WKY rats. It could be concluded that hypertension in the SHR produces altera-tions in the relationship between the p73 protein, circumventricular structures and CSF.

Chemical phenotypes of P2X2 purinoreceptor immunoreactive cell bodies in the area postrema

Purines such as adenosine 5'-triphosphate (ATP) act as extracellular messengers through specific purinergic receptors. Three different classes of purinergic receptors have been identified and termed P1, P2X, and P2Y. The purinergic receptor subunit P2X2 is a ligand-gated ion channel that is widely expressed by neurons in the CNS. In the brainstem medulla oblongata, the ionotropic P2X2 receptor (P2X2R) is enriched in the area postrema (AP). Two different antisera to P2X2R were used to determine the chemical nature of P2X2R immunoreactive cell bodies in the rat AP, an area lacking a blood-brain barrier. Subcellularly, P2X2R immunoreactivity was located to the periphery of individual cell bodies. The majority of P2X2R-immunoreactive cells were shown to contain tyrosine hydroxylase (TH) (63.5 ± 7.7%) and dopamine β-hydroxylase (61.5 ± 5.1%). Phenylethanolamine N-methyltransferase (PNMT)-containing cells were not detected in the AP, supporting a noradrenergic nature of P2X2R cells in the AP. There were no P2X2R-immunoreactive cells in the AP that contained the GABA-synthesizing enzyme glutamic acid decarboxylase 65. Only single vesicular glutamate transporter 2-immunoreactive cell bodies that were not P2X2R-positive were demonstrated in the AP. Some P2X2R-positive cells in the AP were immunoreactive for the neuropeptides substance P and pituitary adenylate cyclase-activating polypeptide, whereas dynorphin-, enkephalin-, or cholecystokinin-positive cells were not P2X2R-immunoreactive. Presence of P2X2R in a majority of noradrenergic cells of the AP implies that ATP may have a regulatory action on neuronal noradrenaline release from the AP, a circumventricular organ with a strategic position enabling interactions between circulating substances and the central nervous system.

The transcriptome of the medullary area postrema: the thirsty rat, the hungry rat and the hypertensive rat

The area postrema (AP) is a sensory circumventricular organ characterized by extensive fenestrated vasculature and neurons which are capable of detecting circulating signals of osmotic, cardiovascular, immune and metabolic status. The AP can communicate these messages via efferent projections to brainstem and hypothalamic structures that are able to orchestrate an appropriate response. We have used microarrays to profile the transcriptome of the AP in the Sprague-Dawley (SD) and Wistar-Kyoto rat and present here a comprehensive catalogue of gene expression, focusing specifically on the population of ion channels, receptors and G protein-coupled receptors expressed in this sensory tissue; of the G protein-coupled receptors expressed in the rat AP, we identified ∼36% that are orphans, having no established ligand. We have also looked at the ways in which the AP transcriptome responds to the physiological stressors of 72 h dehydration (DSD) and 48 h fasting (FSD) and have performed microarrays in these conditions. Comparison between the DSD and SD or between FSD and SD revealed only a modest number of AP genes that are regulated by these homeostatic challenges. The expression levels of a much larger number of genes are altered in the spontaneously hypertensive rat AP compared with the normotensive Wistar-Kyoto control rat, however. Finally, analysis of these 'hypertension-related' elements revealed genes that are involved in the regulation of both blood pressure and immune function and as such are excellent targets for further study.