Evidence for intraventricular secretion of angiotensinogen and angiotensin by the subfornical organ using transgenic mice.

Direct intracerebroventricular injection of angiotensin II (ANG II) causes increases in blood pressure and salt and water intake, presumably mimicking an effect mediated by an endogenous mechanism. The subfornical organ (SFO) is a potential source of cerebrospinal fluid (CSF), ANG I, and ANG II, and thus we hypothesized that the SFO has a secretory function. Endogenous levels of angiotensinogen (AGT) and renin are very low in the brain. We therefore examined the immunohistochemical localization of angiotensin peptides and AGT in the SFO, and AGT in the CSF in two transgenic models that overexpress either human AGT (A+ mice), or both human AGT (hAGT) and human renin (SRA mice) in the brain. Measurements were made at baseline and following volumetric depletion of CSF. Ultrastructural analysis with immunoelectron microscopy revealed that superficially located ANG I/ANG II and AGT immunoreactive cells in the SFO were vacuolated and opened directly into the ventricle. Withdrawal of CSF produced an increase in AGT in the CSF that was accompanied by a large decline in AGT immunoreactivity within SFO cells. Our data provide support for the hypothesis that the SFO is a secretory organ that releases AGT and possibly ANG I/ANG II into the ventricle at least under conditions when genes that control the renin-angiotensin system are overexpressed in mice.

COX-1-derived PGE2 and PGE2 type 1 receptors are vital for angiotensin II-induced formation of reactive oxygen species and Ca(2+) influx in the subfornical organ.

Regulation of blood pressure by angiotensin II (ANG II) is a process that involves the reactive oxygen species (ROS) and calcium. We have shown that ANG-II type 1 receptor (AT1R) and prostaglandin E2 (PGE2) type 1 receptors (EP1R) are required in the subfornical organ (SFO) for ROS-mediated hypertension induced by slow-pressor ANG-II infusion. However, the signaling pathway associated with this process remains unclear. We sought to determine mechanisms underlying the ANG II-induced ROS and calcium influx in mouse SFO cells. Ultrastructural studies showed that cyclooxygenase 1 (COX-1) codistributes with AT1R in the SFO, indicating spatial proximity. Functional studies using SFO cells revealed that ANG II potentiated PGE2 release, an effect dependent on AT1R, phospholipase A2 (PLA2) and COX-1. Furthermore, both ANG II and PGE2 increased ROS formation. While the increase in ROS initiated by ANG II, but not PGE2, required the activation of the AT1R/PLA2/COX-1 pathway, both ANG II and PGE2 were dependent on EP1R and Nox2 as downstream effectors. Finally, ANG II potentiated voltage-gated L-type Ca(2+) currents in SFO neurons via the same signaling pathway required for PGE2 production. Blockade of EP1R and Nox2-derived ROS inhibited ANG II and PGE2-mediated Ca(2+) currents. We propose a mechanism whereby ANG II increases COX-1-derived PGE2 through the AT1R/PLA2 pathway, which promotes ROS production by EP1R/Nox2 signaling in the SFO. ANG II-induced ROS are coupled with Ca(2+) influx in SFO neurons, which may influence SFO-mediated sympathoexcitation. Our findings provide the first evidence of a spatial and functional framework that underlies ANG-II signaling in the SFO and reveal novel targets for antihypertensive therapies.

Ultrastructural changes in the circumventricular organs after experimental subarachnoid hemorrhage.

OBJECTIVES: Circumventricular organs (CVOs) are fine, periventricular, neurotransmitter-rich structures that are devoid of a blood-brain barrier and are known for their secretory role controlling fluid and electrolyte balance, thirst and even reproduction. Common pathologies of the brain such as trauma or bleeding affect CVOs, and hence their function. However, at what stage of these disease processes are CVOs affected and the time sequence of their recovery is still not clear. The aim of this study was to detect the morphological changes in CVOs using electron microscopy after experimental subarachnoid hemorrhage (SAH). METHODS: Experimental SAH was induced by transclival puncture of the basilar artery. Both scanning and transmission electron microscopic examination of the representive sections from each CVO was undertaken. RESULTS: Electron microscopy has shown that after SAH, the cells that form the CVOs exhibit signs of cellular necrosis with margination of the nucleus as well as cytoplasmic, mitochondrial and axonal edema. The subfornicial organ and organum vasculosum lamina terminalis appear to be more vulnerable to the effects of SAH than the median eminence or area postrema. DISCUSSION: Considering the fact that the experimental SAH model we have used is very similar to the momentary rupture of an aneurysm with secondary reflex spasm to seal the hole, it will not be unrealistic to consider that similar effects may also take place in the clinical setting.

Structure of the subfornical organ: a review.

In this review, the light microscopic and fine structural characteristics of neurons, axons, dendrites, glial cells, and capillaries and their topography within the subfornical organ are summarized, with an emphasis on recent findings. Structure-function relationships are discussed whenever possible and put into perspective in a concluding section.

GABAergic inhibitory inputs to subfornical organ neurons in rat slice preparations.

To investigate GABAergic inhibitory inputs to neurons of the subfornical organ (SFO), intracellular recordings were made in rat brain slice preparations. Inhibitory postsynaptic potentials, which occurred spontaneously or were evoked by focal electric stimulation, had reversal potentials of approximately -60 mV, and were almost totally abolished by the GABAA antagonists bicuculline at 3-100 microM or picrotoxin at 50 microM. Following the application of bicuculline or picrotoxin, the resting membrane potentials were decreased by 4-8 mV. GABA at 10-100 microM and the GABAA agonist muscimol at 1-100 microM decreased the membrane resistance and the firing rate in all neurons tested. The reversal potential of the response to muscimol was similar to that for inhibitory postsynaptic potentials. The actions of muscimol persisted in the presence of 1 microM tetrodotoxin, implying that muscimol must act directly on the recorded neurons. These results suggest that there is a tonic inhibitory GABAergic input to SFO neurons which are mainly mediated through GABAA receptors.

Expression of a novel angiotensin II receptor subtype in gerbil brain.

Angiotensin II receptors are highly localized in adult gerbil brain. Apparent receptor number is high in subfornical organ, vascular organ of the lamina terminalis, nucleus of the solitary tract, hippocampus, and in the anterior pituitary gland. In the hippocampus, binding is localized to the stratum oriens, radiatum, the lacunar molecular layers of the CA1 subfield, and the molecular layer of the gyrus dentatus, with a medial to lateral and anterior to posterior gradient in receptor expression. Binding is absent from the pyramidal layer of the CA1 subfield and from the granular cell layer of the gyrus dentatus, areas rich in angiotensin IV binding. Characterization in the hippocampus revealed the presence of a high affinity receptor, sensitive to incubation with the guanine nucleotide GTP gamma S, and displaced by angiotensin II = angiotensin III < Sar1-Ile8-angiotensin II, but not by angiotensin IV or other angiotensin fragments, the AT1 receptor antagonist losartan, or the AT2 ligands CGP 42112 or PD 123177. In other brain areas, binding was equally insensitive to displacement by AT1 or AT2 ligands, with the exception of binding in the olfactory bulb, which was totally displaced by CGP 42112 and PD 123177, but not by losartan. In the gerbil, most of the brain and pituitary angiotensin II receptors are different from the AT1, AT2 and AT4 subtypes, and should be considered 'atypical' until further characterization.

Met5-enkephalin is localized within axon terminals in the subfornical organ: vascular contacts and interactions with neurons containing gamma-aminobutyric acid.

Met5-enkephalin inhibits sodium and water excretion and antagonizes the central actions of angiotensin II in subfornical organ of rat brain. We examined the ultrastructural basis for enkephalin modulation in this circumventricular region. Additionally, we examined the possibility that there might be cellular sites for functional interactions involving Met5-enkephalin and gamma-aminobutyric acid (GABA), a known inhibitory transmitter throughout the central nervous system. Met5-enkephalin and GABA were identified in single coronal sections through the subfornical organ using immunoperoxidase and silver-enhanced immunogold labeling methods, respectively. Enkephalin-like immunoreactivity was most prominently localized within axon terminals. These were distributed primarily in the central, highly vascular, regions of the subfornical organ. Enkephalin-labeled terminals were apposed to the basement membranes of fenestrated capillaries and also formed symmetric, inhibitory type synapses with neurons. In terminals associated with either blood vessels or neurons, the enkephalin immunoreactivity was enriched in large (80-150 nm) dense core vesicles. The immunoreactive vesicles were usually located within portions of the axon in close proximity to astrocytic processes. In contrast, smaller vesicles in the same terminals were more often aggregated near the basement membrane of the capillaries and the active zone of the synapse. The targets of enkephalin-immunoreactive terminals were either unlabeled or GABA-labeled dendrites of local neurons. Enkephalin was also co-localized with GABA in perikarya and in axon terminals. Terminals containing only GABA were far more abundant than those containing enkephalin or enkephalin and GABA. GABA-immunoreactive terminals formed symmetric synapses on unlabeled dendrites some of which also received convergent input from terminals containing enkephalin. Additionally, the enkephalin-immunoreactive terminals were closely apposed to GABA-labeled and unlabeled terminals. These results suggest sites for nonsynaptic release of Met5-enkephalin from dense core vesicles in contact with astrocytes near blood vessels and synaptic complexes in the rat subfornical organ. Moreover, the observed dual localization and pre- and postsynaptic associations between neurons containing Met5-enkephalin and GABA indicate that inhibitory effects of opioids in the subfornical organ may be mediated or potentiated by GABA.

The effects of chronic administration of captopril on the mouse subfornical organ and area postrema.

We have studied by morphometric procedures the chronic effect of captopril on the subfornical organ (SFO) and area postrema (AP) of the adult mouse. Oral administration of captopril does not produce any change in the size of individual nuclei of the ependymocytes and neurons in both centers. However, there are other quantitative effects of captopril on the global volume of the SFO and on the neuropil and vascular elements of both the SFO and AP which present a significant increase. It is suggested that this increase is due to metabolic processes at the level of both circumventricular organs.

Terminations of LHRH-immunoreactive fibers in the subfornical organ of the opossum: an ultrastructural study.

Electron microscopic immunocytochemical approaches were used to analyze LHRH-containing elements in the subfornical organ of the opossum, a species in which this input to the subfornical organ is prominent. Not only were LHRH synaptic specializations easily demonstrated in the subfornical organ, forming axo-dendritic and axo-axonal contacts, but also LHRH-immunoreactive fibers contacted astrocytic end-feet on fenestrated capillaries and were found in the subependymal layer. LHRH-carrying elements in the subfornical organ may be important for relating reproductive functions to body fluid balance.

Differential rates of glucose metabolism across subregions of the subfornical organ in Brattleboro rats.

We applied [14C]deoxyglucose autoradiography and imaging techniques to determine rates of glucose metabolism in distinct subdivisions of the subfornical organ (SFO) of conscious Brattleboro rats. Seven anatomically-defineD SFO subregions were discerned having metabolic activities that differed from one another by as much as 29% in water-sated Brattleboro rats. The highest metabolic activity was found in the ventromedial zone of central and caudal subregions where previous studies identified the greatest densities of neurons, capillaries, putative angiotensin receptors, and angiotensin-immunoreactive fibers. Homozygous Brattleboro rats had rates of glucose metabolism that were 39-68% greater than those in corresponding SFO subregions of Long-Evans rats; these differences were accentuated by about 50% following 18 h of water deprivation. Exogenous treatment of Brattleboro rats with vasopressin uniformly normalized subregional glucose metabolism in the SFO. In Sprague-Dawley rats, water deprivation over 120 h provoked greater increases in metabolism of ventromedial than of dorsolateral SFO zones in amounts similar to the differences between Long-Evans and Brattleboro rats. The findings identify focal areas of high metabolic activity within subregions of the SFO where central responses are likely initiated to defend against homeostatic disturbances. The data represent further evidence for the probability that angiotensin II, as both hormone and neurotransmitter, is a metabolic stimulant of its target cells in the nervous system.

[The subfornical organ today: morphological aspects and functional role]. / L'organo subfornicale oggi: aspetti morfologici e ruolo funzionale.

The recognition of the role played by the subfornical organ (SFO) in the central regulation of body water balance has recently aroused new interest in this anatomical formation which remained ignored for a long time. The SFO is included in the group of the circumventricular organs. In higher vertebrates it is adherent to the ventral surface of the fornix and protrudes into the third ventricle at the level of the interventricular foramina, partially covered by the choroid plexus. The SFO appears as a small nodule, rounded or ovoidal in shape, consisting of highly vascularized nervous tissue and lined by ependyma at the ventricular surface. Its structural organization is fundamentally constant and presents only minor differences in the various species. The SFO neuronal perikarya show different aspects which have been classified in four types. However, it is not yet clearly defined if such aspects refer to distinct cell types or to different transitional features. Nerve and glial cell processes form a dense plexus through the SFO and the subependymal area, as well as in the connective tissue perivascular spaces. These may be narrow or wide and surround fenestrated and non-fenestrated capillaries, assuming sometimes a labyrinthine aspect. The ependymal lining of the SFO ventricular surface shows large variations and regional differences concerning the cell height, the number and development of microvilli, the cilia distribution. The structural properties of SFO, which is characterized by a rich and highly permeable capillary bed, by a wide surface area of contact and exchange with the cerebrospinal fluid, by direct and indirect neural connections with a number of regulatory structures, have been considered as the basis for the role of neurohumoral integration that SFO plays in regulating physiological and behavioral responses to water-mineral changes. Much experimental evidence substantiates this function. However, the studies on SFO are increasingly enriching the literature with new experimental, especially physiological and cytochemical, data which may suggest for this organ connections even more extensive and functions even more complex than those until now ascertained.

Are close contacts between astrocytes and endothelial cells a prerequisite condition of a blood-brain barrier? The rat subfornical organ as an example.

The microvessels of the rat subfornical organ (SFO) are heterogeneous: those of the caudal part lack a blood-brain barrier (BBB) unlike those of the rostral part. The astroglial environment of these microvessels has been studied by combining an immunocytochemical technique employing an anti-GFAP (glial fibrillary acidic protein) antiserum with the morphological detection of a barrier to the protein-silver complex. All the SFO microvessels are surrounded by astrocytes characterized by a tumescent aspect; however, the relative proximity between the astrocytic feet and the endothelial cells varies considerably. The capillaries provided with a barrier (rostral SFO) are contiguous with the astrocytes from which they are only separated by a basement membrane. The capillaries devoid of BBB (caudal SFO) are surrounded by a pericapillary space that keeps the astrocytes at a short distance (capillaries with a very rich vesicular endothelium) or at a long distance (capillaries with a fenestrated endothelium). The astrocytes are absent in the choroid plexus where all microvessels are fenestrated and lack a barrier. These data suggest that the astrocytes release one or more signals which in their vicinity inhibit the expression of endothelial morphological characteristics (fenestrations, vesicles) responsible for the leakage of plasmatic proteins from the blood to the cerebral parenchyma of the circumventricular organs.

Fine structural organization of the subfornical organ. A concise review.

This review of the subfornical organ, with special emphasis on the rat, summarizes the fine structural characteristics of the capillaries, the access route for blood-borne substances, the ependyma through which cerebrospinal fluid-borne substances penetrate the organ, neuronal perikarya, and types of synapses and axons, together with a brief discussion of the principal as yet unresolved problems.

The human subfornical organ: an anatomic and ultrastructural study.

The subfornical organ (SFO), one of the circumventricular organs (CVO) and a thirst-regulating structure, was examined in humans. In 21 autopsy specimens, the SFO was identified as one mm grey nodule on the ventral surface of the fornix at the foramina of Monro. The SFO is a neuronal-vascular organ on a loose glial background, lined by ependyma. Ultrastructural examination reveals deep, narrow invaginations in most neuronal nuclei, characteristically seen in the SFO of other species, but unlike neuronal nuclei in the rest of the human brain. Ovoid, clear vesicles in synaptic complexes and dense-core granules in non-synaptic neuronal process are seen. Ependymal channels are observed. Capillaries have luminal tongue-like projections and pinocytotic vesicles in the endothelial wall, as well as both tight and non-tight junctions between endothelial cells; endothelial fenestrations are not found. These specializations may permit access of macromolecules to receptor sites in the SFO, facilitating its functions as a chemoreceptor organ in drinking behavior. The anatomy of the human SFO is consistent with that of other CVO's and with that of the SFO in other species.

Fine structural cytology of the rat subfornical organ during ontogenesis.

The main developmental events in the subfornical organ take place between 17 fetal days (fd) and 5 days post natum (dpn) at which time it possesses most of its mature fine structural characteristics. The surface regional characteristics of ependymal cells differentiate primarily during this time as well, while the ependymal cellular fine structure, shape and relationship with neurons and the vascularity are well established prior to birth. Undifferentiated neurons contain glycogen prior to 19 fd and then differentiate by developing processes and organelles characteristic of neurons. By 5 dpn, the various types of neurons found in the mature subfornical organ are all present, except for giant vacuolated cells. Synapses containing only electron-lucent vesicles are first present at 20 fd, those containing additional electron-dense vesicles at 3 dpn. Microglial cells are first identifiable at 17 fd, and the first protoplasmic astrocytes are recognizable at 21 fd, while fibrous astrocytes are not detectable prior to 7 dpn. By 5 dpn, the cytological elements of the subfornical organ are all in place, and further developmental changes leading to adult fine structural characteristics by 30 dpn are essentially quantitative in nature.

Effects of propranolol on induced water intake and on the subfornical organ surface.

The chronic effect of propranolol on induced water intake and on the subfornical organ was studied. Propranolol reduced water intake postdehydration. It did not inhibit the increase in plasma renin activity due to dehydration or the dipsogenic response to angiotensin II. Propranolol decreased the subfornical organ large protrusion cells. This result suggests that propranolol impairs the thirst not related to angiotensin II. The subfornical organ changes may indicate that propranolol blocks a beta-adrenergic system originating in ependymal cells.

Luliberin and somatostatin fiber-terminals in the subfornical organ of the rat.

With the aid of light- and electron- microscopic immunocytochemistry, somatostatin- and luliberin (LRF)-positive fibers can be demonstrated in the rat subfornical organ (SFO). Each of the neurohormones has a specific location: LRF in the lateral parts of the organ, and somatostatin in the center of the posterior zone. Common to both neurohormone-containing fibers is the pattern in which they reach the organ as well as the fact that their terminals are located in the perivascular spaces of fenestrated vessels, i.e., within the limited neurohemal regions of the organ. Since injection of India ink of different colors demonstrates that the capillary bed of the SFO is connected with the central capillaries of the choroid plexus, the question arises as to whether the neurohormones released in the area of the SFO influence the choroid plexus.