AIMTo observe the dynamic changes of heme oxygenase-1 (HO-1) mRNA and protein express in subfornical organ in rats with experimental allergic encephalomyelitis (EAE) to confirm that SFO is one of the sites for blood-bearing signaling molecules entering into brain.

METHODSEAE was induced by CFA-GPSCH on Wistar rats, we observed the levels of HO-1 mRNA and its protein expression with immunohistochemistry and in situ hybridization technology on 1 d, 7 d, 14 d, and 21 d after EAE induction in SFO of rats. The relationship between HO-1 and symptoms of EAE was also investigated.

RESULTSThe expression levels of HO-1 mRNA and its protein expression were very low in the brains of the control group, whereas they were enhanced gradually with pathological course in the brain and onsets of symptoms, signs of EAE. On 1 d after induction of EAE, positive cells of HO-1 mRNA and its protein expression were observed at SFO, but the labeled cells were rarely seen in the other brain regions. On 7 d, the positive cells increased markedly. On 14 d the levels of HO-1 mRNA and its protein expression in the brains reached the peak, the positive cells of HO-1 were mainly located at the choroid plexuses and SFO, as well as the regions around "sleeve-like" lesion foci, all of which were coincident with the locations of lesions of EAE. The changes of incidence, symptom, reduction of the body weight, and pathology lesions of EAE in rat brains were the most significant. On 21 d, the levels of HO-1 mRNA and its protein expression reduced gradually, which was in parallel with remitted symptoms of EAE. When a specific inhibitor of HO-1, Snpp9, was applied, the symptoms and pathological lesions of EAE in brains were mitigated markedly.

CONCLUSIONSFO may be one of the earliest sites for blood-bearing signaling molecules entering into brain. The dynamic changes of HO-1 mRNA and its protein expression are in parallel with the changes of symptoms and pathological lesions of EAE in the brains. Application of some inhibitors of HO-1 may be one of potential therapeutic methods for prevention and treatment of EAE.

Animals ; Encephalomyelitis, Autoimmune, Experimental ; metabolism ; Female ; Heme Oxygenase (Decyclizing) ; genetics ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; Subfornical Organ ; metabolism

The present study was designed to investigate the effect of administration of adrenomedullin (ADM) into subfornical organ (SFO) on renal tubular Na(+),K(+)-ATPase activity in rats. Rats under anesthesia were injected with ADM 0.1 mL (20 ng/mL) via an implanted cannula into SFO (n=6). Plasma ADM and serum endogenous digitalis-like factor (EDLF) levels were assayed with radioimmunoassay, and urine samples were collected via a canoula intubated in bladder. Urinary sodium concentration was assayed with flame spectrophotometry. Single proximal renal tubule segments were obtained by hand under stereomicroscope and its Na(+),K(+)-ATPase activity was measured by liquid scintillation counting. In addition, single proximal renal tubule segments from normal rats (n=6) were incubated with serum from animals administered with ADM into SFO, and then the Na(+),K(+)-ATPase activity was determined. The results showed that both urinary volume and sodium excretion amounted to the peak value at 30 min after ADM administration, and sustained a significant high level at 60 min (P<0.01). At 30 min after ADM administration, there was a significant increase in serum EDLF and a decrease in Na(+),K(+)-ATPase activity of proximal tubule (P<0.01, respectively), but not in plasma ADM level. Na(+),K(+)-ATPase activity was decreased significantly in single proximal renal tubule segments from normal rats incubated with serum from rats administered with ADM into SFO (P<0.01). These results suggest that the diuretic and natriuretic responses following administration of ADM into SFO are associated with the inhibition of renal tubule Na(+),K(+)-ATPase activity. The inhibition of renal tubule Na(+),K(+)-ATPase activity is related to the increase in the serum level of EDLF.
Adrenomedullin ; pharmacology ; Animals ; Kidney Tubules, Proximal ; drug effects ; enzymology ; Rats ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Subfornical Organ

AIMTo investigate the effect of micro-injection Ang II into the subfornical organ (SFO) on the proximal tubules (PT) Na+, K(+)-ATPase activity in rats and its mechanism.

METHODSSFO in SD rats was administrated respectively with Ang II (20 ng), or losartan (5 microg) and AngII (20 ng) successively. The levels of serum EDLS and plasm AngII were assessed with radioimmunoassay (RIA). The PT segments were microdissected freehand and their Na+, K(+)-ATPase activities were assessed by liquid scintillation counter (LSC).

RESULTSThe serum EDLS levels increased significantly compared with a CSF group after SFO administration with Ang II; The Na+, K(+)-ATPase activities in PT segments decreased significantly at 30 min and 60 min after SFO administration with Ang II. There was a negative linear correlation between serum EDLS level and the Na+, K(+)-ATPase activity of PT segments in rats administrated with Ang II (r = -0.938).

CONCLUSIONInhibition of the Na+, K(+)-ATPase activity in PT as a result of administration of Ang II in SFO is mediated by AT1 receptors. The increase in EDLS release may play an important role in this inhibition.

Angiotensin II ; administration & dosage ; pharmacology ; Animals ; Kidney Tubules, Proximal ; drug effects ; enzymology ; Male ; Rats ; Rats, Sprague-Dawley ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Subfornical Organ

AIMTo compare the drinking behavior and c-fos expression induced by chemical or electrical stimulation of subfornical organ (SFO) in rat brain.

METHODSL-glutamic acid microinjection and constant electrical current were used as chemical and electrical stimulation of SFO, respectively. The water intake over 1 h was recorded and Fos expression was examined immunohistochemically.

RESULTSA similar volume of water intake and Fos expression pattern were induced by both methods of stimulation of SFO. These include 11 forebrain areas (organum vasculosum of the lamina terminalis, median preoptic nucleus, hypothalamic paraventricular nucleus, supraoptic nucleus and lateral hypothalamic area, paraventricular nucleus, reunions nucleus and central medial nucleus of thalamus, bed nucleus of the stria terminalis, perifornical dorsal area and substantia innominata) and 4 areas of hindbrain (area postrema, nucleus solitary tract, lateral parabrachial nucleus and dorsal raphe nucleus).

CONCLUSIONThe drinking behavior and Fos expression in brain induced by SFO stimulation are the results of activation of the neuronal bodies in SFO.

Animals ; Brain ; drug effects ; metabolism ; Drinking Behavior ; Electric Stimulation ; Glutamic Acid ; pharmacology ; Male ; Proto-Oncogene Proteins c-fos ; metabolism ; Rats ; Rats, Sprague-Dawley ; Subfornical Organ ; metabolism ; physiology

Blood-borne angiotensin-II (Ang-II) has profound effects in the brain. We tested the hypothesis that Ang-II-dependent hypertension involves differential Ang-II type I (AT1) receptors expression in the subfornical organ (SFO) and the rostral ventrolateral medulla (RVLM). Male Wistar rats were implanted with 14-day osmotic minipump filled with Ang-II (150 ng/kg/min) or saline. AT1 receptor mRNA levels were detected in the SFO and RVLM by reverse transcription-polymerase chain reaction (RT-PCR). Ang-II caused hypertension (134 +/- 10 mmHg vs. 98 +/- 9 mmHg, n = 9, p < 0.05). RT-PCR revealed that Ang-II infusion induced increased AT1 receptor mRNA levels in RVLM and decreased in SFO. Our data suggest that Ang-II-induced hypertension involves differential expression of brain AT1 receptors.
Angiotensin II/*metabolism ; Animals ; Hypertension/chemically induced/*metabolism ; Male ; Medulla Oblongata/*metabolism ; RNA, Messenger/genetics ; Rats ; Rats, Wistar ; Receptor, Angiotensin, Type 1/*genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Subfornical Organ/*metabolism

In order to investigate the role of heme oxygenase-1 (HO-1) in the molecular mechanism of experimental allergic encephalomyelitis (EAE), which was induced by guinea pig spinal cord homogenate + complete freund adjuvant on Wistar rats, we observed the gene of HO-1 and its protein expression with reverse transcriptase polymerase chain reaction(RT-PCR) and immunohistochemistry 1, 7, 14, and 21 d after EAE induction in rats. The relationship between HO-1 and the symptoms of EAE was also observed. The results showed that the levels of HO-1 mRNA and its protein expression were very low in the brains of the control group, whereas they were enhanced gradually with pathological course in the brain and onsets of symptoms, signs of EAE. On day 7, the level of HO-1 mRNA reached the peak, but the expression level of HO-1 protein in the brains reached the peak on day 14. The immunoreactive cells of HO-1 were mainly located at the choroid plexuses and subfornical organ (SFO), as well as in regions around the "sleeve-like" lesion foci, all of which were coincident with the locations of lesions of EAE. The levels of HO-1 mRNA and its protein expression were lowered gradually on day 21, which were in parallel with the severities of symptoms and signs of EAE. After a specific inhibitor of HO-1, Snpp-9, was applied, both of the symptoms and pathological lesions of EAE in the rat brains were mitigated markedly. Therefore, these results may suggest that the dynamic changes of HO-1 mRNA and its protein expression are in parallel with the changes of symptoms and pathological lesions of EAE in the brain. In conclusion, the levels of HO-1 mRNA and its protein expression in brains may play an important role in the pathogenesis of EAE, and application of inhibitors of HO-1 may be one of the potential therapeutic ways for the prevention and treatment of EAE.
Animals ; Brain ; enzymology ; metabolism ; Encephalomyelitis, Autoimmune, Experimental ; enzymology ; genetics ; physiopathology ; Female ; Heme Oxygenase (Decyclizing) ; biosynthesis ; genetics ; Heme Oxygenase-1 ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Subfornical Organ ; metabolism ; pathology

Water transport is mediated by two distinct pathways, diffusional and channel-mediated water transport. The first molecular water channel was identified from human erythrocytes in 1992. Genetically-related proteins from other mammalian tissues have subsequently been identified to transport water, and the group is referred to as the "Aquaporins". Aquaporin-4 (AQP4) is most abundant in the brain, which may be involved in CSF reabsorption and osmoregulation. However, ontogeny and regulatory mechanisms of AQP4 channels have not been reported. Northern blot analysis showed that AQP4 mRNA began to be expressed in the brain just before birth and that its expression gradually increased by PN7 and then decreased at adult level. AQP4 was expressed predominantly in the ependymal cells of ventricles in newborn rats. And then its expression decreased in ependymal cells and increased gradually in other regions including supraoptic and paraventricular nuclei. AQP4 is also expressed in the subfornical organ, in which the expression level is not changed after birth. Cryogenic brain injury did not affect expression of AQP4 mRNA, while ischemic brain injury decreased it. Osmotic water permeability of AQP4 channel expressed in Xenopus oocytes was inhibited by the pretreatment of BAPTA/AM and calmidazolium, a Ca2+/ Calmodulin kinase inhibitor, in a dose-dependent manner. These results indicate that the expression and the function of AQP4 channel are regulated by developmental processes and various pathophysiological conditions. These results will contribute to the understanding of fluid balance in the central nervous system and the osrmoregulatory mechanisms of the body.
Adult ; Animals ; Blotting, Northern ; Brain Injuries ; Brain* ; Calcium-Calmodulin-Dependent Protein Kinases ; Central Nervous System ; Diffusion ; Erythrocytes ; Humans ; Infant, Newborn ; Ischemia* ; Oocytes ; Osmoregulation ; Parturition ; Permeability ; Rats ; RNA, Messenger ; Subfornical Organ ; Water* ; Water-Electrolyte Balance ; Xenopus

The subfornical organ (SFO) is one of circumventricular organs characterized by the lack of a normal blood brain barrier. The SFO neurons are exposed to circulating glutamate (60~100 microM), which may cause excitotoxicity in the central nervous system. However, it remains unclear how SFO neurons are protected from excitotoxicity caused by circulating glutamate. In this study, we compared the glutamate-induced whole cell currents in SFO neurons to those in hippocampal CA1 neurons using the patch clamp technique in brain slice. Glutamate (100 microM) induced an inward current in both SFO and hippocampal CA1 neurons. The density of glutamate-induced current in SFO neurons was significantly smaller than that in hippocampal CA1 neurons (0.55 vs. 2.07 pA/pF, p<0.05). To further identify the subtype of the glutamate receptors involved, the whole cell currents induced by selective agonists were then compared. The current densities induced by AMPA (0.45 pA/pF) and kainate (0.83 pA/pF), non-NMDA glutamate receptor agonists in SFO neurons were also smaller than those in hippocampal CA1 neurons (2.44 pA/pF for AMPA, p<0.05; 2.34 pA/pF for kainate, p< 0.05). However, the current density by NMDA in SFO neurons was not significantly different from that of hippocampal CA1 neurons (1.58 vs. 1.47 pA/pF, p>0.05). These results demonstrate that glutamate-mediated action through non-NMDA glutamate receptors in SFO neurons is smaller than that of hippocampal CA1 neurons, suggesting a possible protection mechanism from excitotoxicity by circulating glutamate in SFO neurons.
alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid ; Animals ; Blood-Brain Barrier ; Brain ; Central Nervous System ; Glutamic Acid* ; Hippocampus ; Kainic Acid ; N-Methylaspartate ; Neurons* ; Rats* ; Receptors, Glutamate ; Subfornical Organ*

The present study has been performed to investigate the neural axis of rat digastric muscle using viral tracer, pseudorabies virus. The upper nuclei to innervate digastric muscle were in accumbens nucleus, agran-ular insular cortex, central nucleus of amygaloid, lateral septal nucleus, frontal cortex, and subfornical organ etc, in telencephalon ; arcuate hypothalamic nucleus, lateral hypot-halamic area, medial preoptic nucleus, bed nucleus of stria terminalis, dorsomedial hypot-halamic nucleus, suprachiasmatic nucleus, paraventricular nucleus, and retrochiasmatic area etc, in diencephalon ; nucleus Darkschewitsch, interstitial nucleus of the medial logitudinal fasciculus, parabrachial nucleus, locus ceruleus, Kolliker-Fuse nucleus, trigeminal mesencephalic nucleus, red nucleus, substantia nigra, nucleus of posterior commissure, Edinger-Westphal nucleus, and dorsal raphe nucleus etc, in mesencephalon ; giganto-cellular reticular nucleus, raphe magnus nucleus, raphe pallidus nucleus, raphe obscuous nucleus, nucleus of solitary tracts, lateral reticular nucleus, parvocellular reticular nucleus, area postrema, facial nucleus, pontine reticular nucleus, pontine nucleus of trigeminal nerve and spinal nucleus of trigeminal nerve etc, in rhombencephalon. There are significant difference of numbers of PRV-Ba immunoreactive cells between right and left sides of brain in almost nuclei[P< 0.05]. But PRV-Ba immunoreactive cells were observed only ipsilaterally in accessory trigeminal motor nucleus, accessory facial nucleus and agranular insular cortex. Frontal cortex was the only area which were shown contralateral immunoreactivity. The results of this study provide anatomical support that both the cranial and caudal bellies are innervated by the same upper nuclei. The results also support the suggestion that the lower nuclei of digastric muscle, accessory trigeminal motor nucleus and accessory facial nucleus consist of somatotopic motor complex.
Animals ; Area Postrema ; Axis, Cervical Vertebra* ; Brain ; Diencephalon ; Herpesvirus 1, Suid ; Hypothalamic Area, Lateral ; Immunohistochemistry ; Locus Coeruleus ; Mesencephalon ; Paraventricular Hypothalamic Nucleus ; Raphe Nuclei ; Rats* ; Red Nucleus ; Rhombencephalon ; Septal Nuclei ; Subfornical Organ ; Substantia Nigra ; Suprachiasmatic Nucleus ; Telencephalon ; Trigeminal Nerve ; Trigeminal Nuclei

The aim of this study was to investigate the effects of agmatine (Agm) on the electrical activity of neurons in subfornical organ (SFO) slices using extracellular recording technique. The results are as follows. (1) In response to the application of Agm (1.0 micromol/L) into the superfusate for 2 min, the discharge rate of 24/28 (85.7%) subfornical neurons was decreased significantly, while the discharge rate of 4/28 (14.3%) neurons were not affected. (2) Pretreatment with L-glutamate (0.3 mmol/L) led to a marked increase in the discharge rate of 19/24 (79.2%) subfornical neurons in an epileptiform pattern and the activity of the remaining 5/24 (20.8%) neurons was unaffected. By application of Agm (1.0 micromol/L) into the superfusate for 2 min, the epileptiform dicharge of 15/19 (78.9%) neurons was suppressed significantly, while that of the other 4 (21.1%) neurons was not inhibited. (3) In 12 neurons, perfusion of the selective L-type calcium channel agonist, Bay K-8644 (0.1 micromol/L), induced a significant increase in the discharge rate of 10/12 (83.3%) neurons, while the other 2 (16.7%) neurons showed no change. The increased discharge of 8/10 (80%) neurons was reduced by application of Agm (1.0 micromol/L) into the superfusate and that of 2/10 (20%) neurons was not affected. (4) Application of nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 50 micromol/L) into the superfusate also significantly increased the discharge rate of 6/9 (66.7%) neurons, and that of 3/9 (33.3%) neurons had no response. Agm (1.0 micromol/L) applied into the superfusate reduced the increased discharge of all 6/6 (100%) neurons. These results suggest that Agm can inhibit the spontaneous discharge, and L-glutamate, Bay K-8644- or L-NAME-induced discharge of neurons in SFO. These inhibitory effects of Agm may be related to the blockade of NMDA receptors and reduction in calcium influx in SFO neurons.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester ; pharmacology ; Action Potentials ; drug effects ; Agmatine ; pharmacology ; Animals ; Calcium Channel Agonists ; pharmacology ; Female ; Glutamic Acid ; pharmacology ; Hippocampus ; physiology ; Male ; Neurons ; physiology ; Rats ; Rats, Sprague-Dawley ; Receptors, Drug ; agonists ; Receptors, N-Methyl-D-Aspartate ; antagonists & inhibitors ; Subfornical Organ ; drug effects ; physiology