Low Non-NMDA Receptor Current Density as Possible Protection Mechanism from Neurotoxicity of Circulating Glutamate on Subfornical Organ Neurons in Rats

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, p0.05). 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.

Angiotensin-II-induced reactive oxygen species along the SFO-PVN-RVLM pathway: implications in neurogenic hypertension

Neurogenic hypertension has been the subject of extensive research worldwide. This review is based on the premise that some forms of neurogenic hypertension are caused in part by the formation of angiotensin-II (Ang-II)-induced reactive oxygen species along the subfornical organ-paraventricular nucleus of the hypothalamus-rostral ventrolateral medulla pathway (SFO-PVN-RVLM pathway). We will discuss the recent contribution of our laboratory and others regarding the mechanisms by which neurons in the SFO (an important circumventricular organ) are activated by Ang-II, how the SFO communicates with two other important areas involved in sympathetic activity regulation (PVN and RVLM) and how Ang-II-induced reactive oxygen species participate along the SFO-PVN-RVLM pathway in the pathogenesis of neurogenic hypertension.

Differential brain angiotensin-II type I receptor expression in hypertensive rats

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.

Administration of adrenomedullin into subfornical organ inhibits Na(+),K(+)-ATPase activity in single proximal renal tubule of rats / 生理学报

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.

Effect of micro-injection angiotensin II into subfornical organ in rats on Na+, K(+)-ATPase activity in proximal tubules / 中国应用生理学杂志

<p><b>AIM</b>To 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.</p><p><b>METHODS</b>SFO 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).</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>Inhibition 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.</p>

The dynamic changes of heme oxygenase-1 mRNA and protein express at subfornical organ in rats with experimental allergic encephalomyelitis / 中国应用生理学杂志

<p><b>AIM</b>To 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.</p><p><b>METHODS</b>EAE 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>SFO 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.</p>

Effects of agmatine on the electrical activity of subfornical organ neurons / 生理学报

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.

Dynamic changes of heme oxygenase-1 protein and mRNA in the brains of rats with experimental allergic encephalomyelitis / 生理学报

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.

Drinking behavior and c-fos expression induced by chemical or electrical stimulation of SFO in rat brain / 中国应用生理学杂志

<p><b>AIM</b>To compare the drinking behavior and c-fos expression induced by chemical or electrical stimulation of subfornical organ (SFO) in rat brain.</p><p><b>METHODS</b>L-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.</p><p><b>RESULTS</b>A 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).</p><p><b>CONCLUSION</b>The drinking behavior and Fos expression in brain induced by SFO stimulation are the results of activation of the neuronal bodies in SFO.</p>

Los fosfoinositidos como señalización de las endotelinas en la eminencia media y el órgano subfornical de la rata / The fosfoinositidos as endotheling signaling in the half prominence and the subfornical organ of the rat

La endotelina-1 es un péptido derivado del endotelio con potentes efectos vasoconstrictores. Existen tres isoformas de endotelinas (ETs):ET-1, ET-2 y ET-3 que actúan en receptores designados como ETA y ETB. Se ha demostrado que la administración central de los isopéptidos de ETs produce efectos autonómicos y cerebrovasculares, lo que sugiere la existencia de un papel clave para las ETs en el SNC. Las endotelinas son capaces de activar diferentes sistemas de segundos mensajeros. Se ha descrito que los receptores de ETs en el SNC están acoplados a la activación de la fosfolipasa C y el subsecuente incremento de los niveles de trifosfato de inositol (IP3). Consistente con la presencia de una alta densidad de receptores específicos para las ETs en el órgano subfornical (OSF) y la eminencia media (EM) del cerebro de la rata, nuestros resultados muestran que el incremento en la hidrólisis de fosfoinosítidos (PI) de membrana inducido por las ET-1 y ET-3 en el OSF y la EM, es dependiente de la dosis y muestra valores de DE50 similares, lo que sugiere que este efecto es mediado por el subtipo de receptor ETB. Se caracterizaron los receptores que median dicho efecto, mediante el uso de agonistas y antagonistas selectivos de ambos subtipos de receptores. El BQ 123 y BQ 610, antagonistas selectivos del recptor ETA, no afectaron significativamente el incremento en el recambio de inositoles de membrana inducidos por las ETs en las estructuras cerebrales. Mientras que el IRL 1620, un agonista selectivo del subtipo de receptor ETB, incrementó la acumulación de InsP1 en el OSF y la EM de modo comparable al producido por las endotelinas, siendo este efecto bloqueado por el BQ 788, un antagonista selectivo de este subtipo de receptor. Nuestros hallazgos demuestran que el receptor ETB media el incremento de recambio de fosfoinosítidos inducido por la ET-1 y la ET-3 en el OSF y la EM de cerebro de la rata

Effect of injection of L-NAME on drinking response

The drinking behavior responses to centrally administered NG-nitro-L-arginine methyl ester (L-NAME; 10, 20 or 40 µg/µl), an inhibitor of nitric oxide synthase, were studied in satiated rats, with cannulae stereotaxically implanted into the lateral ventricle (LV) and subfornical organ (SFO). Water intake increased in all animals after angiotensin II (ANG II) injection into the LV, with values of 14.2 + or - 1.4 ml/h. After injection of L-NAME at doses of 10, 20 or 40 µg/µl into the SFO before injection of ANG II (12 ng/µl) into the LV, water intake decreased progressively and reached basal levels after treatment with 0.15 M NaCl and with the highest dose of L-NAME (i.e., 40 µg). The water intake obtained after 40 µg/µl L-NAME was 0.8 + or - 0.01 ml/h. Also, the injection of L-NAME, 10, 20 or 40 µg/µl, into the LV progressively reduced the water intake induced by hypertonic saline, with values of 5.3 + or - 0.8, 3.2 + or - 0.8 and 0.7 + or - 0.01 ml/h, respectively. These results indicate that nitric oxide is involved in the regulation of drinking behavior induced by centrally administered ANG II and cellular dehydration and that the nitric oxide of the SFO plays an important role in this regulation.

The Studies on Central Neural Axis to Innervate Rat Digastric Muscle / 대한해부학회지

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.

Immunohistochemical Studies on Calcitonin Gene-related Peptide Cell in Rat Brain / 대한해부학회지

These studies were performed to identify the localization, and neuronal function of calcitonin gene-related peptide[CGRP] in the neural axis of rat stomach by retrograde tracing and immunohistochemical techniques. After injection of pseudorabies virus Bartha strain[PRV] as tracer between serosa and muscle layer of stomach, the rats were perfused and the brains were removed. PRV-immunoreactive cells were observed in central nucleus of amygdaloid, insular cortex, subfornical organ, bed nucleus of stria terminalis, paraventricular nucleus, organum vasculosum of terminalis, suprachiasmatic nucleus, lateral hypothalamic area, K lliker-Fuse nucleus, parabrachial nucleus, locus ceruleus, A1 noradrenaline area, A5 noradrenaline area, area postrema, dorsal motor nucleus of vagus nerve, nucleus tractus solitarius and raphe nuclei. CGRP-immunoreactive cells are observed in insular cortex, bed nucleus of stria terminalis, paraventricular nucleus, lateral hypothalamic area, parabrachial nucleus, area postrema, nucleus tractus solitarisu, neucleus ambiguus, facial nucleus, hypoglossal nucleus and raphe nuclei. The dobule immunofluorescent study was carried out to examine the coexistence of CGRP and PRV in several nuclei : insular cortex, bed nucleus of stria terminalis, paraventricular nucleus, later hypithalamic area, parabrachial nucleus, area postrema, nucleus tractus solitarius and raphe nuclei. At the results of double immunofluorescent study, we could not observe the double immunoreactive neurons CGRP and PRV in those nuclei but raphe nuclei. These results suggest that CGRP should not have a neural functions in the neurons in nuclei projecting to rat stomach except raphe nuclei.

Central Neural Pathway for the Rat Tongue / 대한해부학회지

Bartha strain of pseudorabies virus[PRV-Ba] was utilized as a tracer to identify the neuronal axis of rat tongue muscles ; intrinsic muscles and extrinsic muscles, styloglossus, genioglossus, and hyoglossus muscle. After injection of 10 microliter of PRV-Ba into tongue muscles and 48-96 hours survivals, rats were perfused with 4% paraformaldehyde lysine periodate and brains were removed. PRV-Ba were localized in neural circuits by immunohistochemistry employing rabbit anti PRV-Ba as a primary antibody and ABC method. Injection of PRV-Ba into the tongue muscles resulted in uptake and retrograde transport of PRV-Ba in the rat brain. The result showed a circuit specific connection of many nerve cell groups along the time sequence : PRV-Ba immunoreactive cells appeared in hypoglossal nucleus and motor trigeminal nucleus ipsilaterally as seen with conventional tracers. Raphe nucleus, prepositus hypoglossal nucleus, spinal trigeminal nucleus, Al, A5 and facial nucleus of rhombencephalon showed immunoreactivity bilaterally. There were positive neurons in parabrachial nucleus, locus ceruleus, mesencephalic trigeminal nucleus, periaqueductal gray and A7 of mesencephalon and paraventricular nucleus, suprachiasmatic nucleus, organum vasculosum of lamina terminalis of diencephalon. Also positive reactions were showed in amygdala, insular cortex, frontal cortex and subfornical organ in telencephalon. Early immunoreactivity was appeared in hypoglossal nucleus and motor trigeminal nucleus, and there were positive neurons in the nuclei of the medulla oblongate, midbrain, pons, hypothalamus, cerebellum and medial preoptic area at middle stage. Subsequently the viral antigens were found in forebrain cell groups, paraventricular nuclei, suprachiasmatic nucleus, lateral hypothalamic area and primary motor cortex in frontal lobe bilaterally at 80-90hrs postinjection. These data demonstrate that the PRV-Ba can across synapses in the central nervous system with projection specific pattern, and this virus defines many elements of the neural network governing tongue. Therefore PRV-Ba are proved as a excellent neurotracer in the tract-tracing researches.

Regulation of AQP-4 water channel expression in the brain during development and by ischemia

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.

Role of opiates in the regulation of water and salt metabolism: effects of the FK33-824 injection into the subfornical organ of both normal and hypophysectomized rats

The present study was performed to evaluate the participation of the subfornical organ (SFO) in the opioid modulation of urinary volume (Uv), and of sodium and potassium excretion. Intact and hypophysectomized (HYPOX) adult male rats were implanted with a cannula into the SFO, and injected with the opiate agonist FK 33-824 (FK). FK induced a significant decrease in Uv and in Na+ and K+ excretion in both intact and HYPOX rats. The data show that opioids play an important role in the regulation of hydromineral metabolism by the SFO