Region-specific projections from the subfornical organ to the paraventricular hypothalamic nucleus in the rat.

Neurons in the subfornical organ (SFO) project to the paraventricular hypothalamic nucleus (PVN) and there, in response to osmolar and blood pressure changes, regulate vasopressin neurons in the magnocellular part (mPVN) or neurons in the parvocellular part (pPVN) projecting to the cardiovascular center. The SFO is functionally classified in two parts, the dorsolateral peripheral (pSFO) and ventromedial core parts (cSFO). We investigated the possibility that neurons in each part of the SFO project region-specifically to each part of the PVN, using anterograde and retrograde tracing methods. Following injection of an anterograde tracer, biotinylated dextran amine (BDX) in the SFO, the respective numbers of BDX-uptake neurons in the pSFO and cSFO were counted and the ratio of the former to the latter was obtained. In addition, the respective areas occupied by BDX-labeled axons per unit area of the mPVN and pPVN were measured and the ratio of the former to the latter was obtained. Similarly, following injection of the retrograde tracer in the PVN, the respective areas occupied by tracer per unit area of the mPVN and pPVN were measured and the ratio of the former to the latter was obtained. The respective numbers of retrogradely labeled neurons in the pSFO and cSFO were also counted and the ratio of the former to the latter was obtained. It became clear by statistical analyses that there are strong positive correlations between the ratio of BDX-uptake neuron number in the SFO and the ratio of BDX-axon area in the PVN in anterograde experiment (correlation coefficient: 0.787) and between the ratio of retrograde neuron number in the SFO and the ratio of tracer area in the PVN in retrograde experiment (correlation coefficient: 0.929). The result suggests that the SFO projects region-specifically to the PVN, the pSFO to the mPVN and the cSFO to the pPVN.

Collateral projections from the subfornical organ to the median preoptic nucleus and paraventricular hypothalamic nucleus in the rat.

It is morphologically demonstrated that the subfornical organ (SFO) projects to the paraventricular hypothalamic nucleus (PVN) and also projects to the nucleus preopticus medianus (POMe), a relay nucleus of indirect projections from the SFO to PVN. However, it remains unknown, whether or not SFO neurons project collaterally to the POMe and PVN. To confirm this, a double retrograde labeling method was performed on rats using two fluorescent tracers. One tracer (red-colored FluoSpheres: FSR) was injected into the POMe and the other (Fast Blue: FB) was injected into the unilateral PVN at the same time. As a result, many retrogradely labeled neurons were found in the entire SFO. Of these, some neurons showed both FSR and FB fluorescence. Double-labeled neurons were found in about 8.7% of FSR-labeled neurons and 15.5% of FB-labeled neurons. The existence of double-labeled neurons indicates that single neurons in the SFO project simultaneously to the POMe and PVN via collateral axon branches. The data suggest that there are complicated neuronal pathways originating from the SFO in regulating cardiovascular and body fluid homeostasis.

The circumventricular organs: an atlas of comparative anatomy and vascularization.

The circumventricular organs are small sized structures lining the cavity of the third ventricle (neurohypophysis, vascular organ of the lamina terminalis, subfornical organ, pineal gland and subcommissural organ) and of the fourth ventricle (area postrema). Their particular location in relation to the ventricular cavities is to be noted: the subfornical organ, the subcommissural organ and the area postrema are situated at the confluence between ventricles while the neurohypophysis, the vascular organ of the lamina terminalis and the pineal gland line ventricular recesses. The main object of this work is to study the specific characteristics of the vascular architecture of these organs: their capillaries have a wall devoid of blood-brain barrier, as opposed to central capillaries. This particular arrangement allows direct exchange between the blood and the nervous tissue of these organs. This work is based on a unique set of histological preparations from 12 species of mammals and 5 species of birds, and is taking the form of an atlas.

Peptidergic and catecholaminergic synaptic contacts onto nucleus preopticus medianus neurons projecting to the subfornical organ in the rat.

The nucleus preopticus medianus (POMe) is known to be a key site in regulation of cardiovascular and body fluid homeostasis. To clarify the regulation mechanism to the POMe, the innervation pattern of synapses made by axon terminals immunoreactive to beta-endorphin, neuropeptide Y and tyrosine hydroxylase onto POMe neurons projecting to the subfornical organ (SFO) was investigated in the rat. After injection of a retrograde tracer, wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex, into the SFO, many neurons were retrogradely labeled in the POMe, more frequently in its dorsal part. Electron microscopy of the POMe revealed that beta-endorphin- and tyrosine hydroxylase-immunoreactive axon terminals formed predominantly axo-somatic synapses, and neuropeptide Y-immunoreactive axon terminals formed more axo-dendritic than axo-somatic synapses with retrogradely labeled neurons. The present localization patterns of POMe neurons retrogradely labeled from the SFO and the type of synapses of axon terminals immunoreactive to three neurochemical markers on these neurons were compared to those of POMe neurons retrogradely labeled from the paraventricular hypothalamic nucleus demonstrated in our previous report.

The neonatal treatment of rats with monosodium glutamate induces morphological changes in the subfornical organ.

The parenteral administration of monosodium glutamate (MSG) to neonatal rats induces specific lesions in the central nervous system that lead to a well characterized neuroendocrinological dysfunction. Additionally, it has been shown that MSG-treated rats present a blunted blood pressure response to the injection of nitric oxide synthase inhibitors. Recently, a similar cardiovascular alteration has been reported after the electrolytic lesion of the anteroventral region of the third ventricle affecting the connections of the subfornical organ (SFO). We hypothesized that the treatment of neonatal rats with MSG could affect the nitrergic cells of the SFO. In the present work, we have looked for alterations in the NADPH-diaphorase activity (a commonly used marker for nitrergic neurons) in the SFO of MSG-treated rats of either sex and at two different ages. Our results shown that the treatment of neonatal rats with MSG induced a substantial reduction in the volume of the SFO and in the number of its nitrergic cells with regard to control animals. These findings suggest that the SFO could be implicated in some of the cardiovascular alterations observed in MSG-treated rats.

The sensory circumventricular organs of the mammalian brain.

The brain's three sensory circumventricular organs, the subfornical organ, organum vasculosum of the lamina terminalis and the area postrema lack a blood brain barrier and are the only regions in the brain in which neurons are exposed to the chemical environment of the systemic circulation. Therefore they are ideally placed to monitor the changes in osmotic, ionic and hormonal composition of the blood. This book describes their. General structure and relationship to the cerebral ventricles Regional subdivisions Vasculature and barrier properties Neurons, glia and ependymal cells Receptors, neurotransmitters, neuropeptides and enzymes Neuroanatomical connections Functions.

Role of the subfornical organ in the chronic hypotensive response to losartan in normal rats.

Angiotensin II is known to act at a unique set of brain regions known as the circumventricular organs. These structures lack the normal blood-brain barrier and are therefore thought to participate in the central nervous system processing of neuroendocrine signals. We have reported that chronic treatment with the angiotensin type 1 (AT1) receptor antagonist, losartan, decreases arterial pressure in normotensive rats. Furthermore, this hypotension is attenuated in area postrema-lesioned rats, suggesting a role of endogenous angiotensin II at this circumventricular organ. Another circumventricular organ, the subfornical organ (SFO), has also been shown to mediate actions of angiotensin II. The present study tested the hypothesis that the SFO is a central site of action of endogenous angiotensin II at AT1 receptors. Adult male Sprague-Dawley rats were anesthetized and placed in a stereotaxic apparatus, and the SFO was sham or electrolytically lesioned. One week later, rats were instrumented with venous catheters and radiotelemetry pressure transducers for continuous infusion and monitoring of mean arterial pressure, respectively. After 3 days of control, losartan was administered intravenously (10 mg x kg(-1) x d(-1)) for 10 days in both SFO-lesioned and sham rats. By day 4 of losartan administration, mean arterial pressure had decreased to 75+/-2 mm Hg in sham rats (n=9) but had only fallen to 83+/-2 mm Hg in lesioned rats (n=10). This attenuated hypotensive response in SFO-lesioned rats continued through day 10 of losartan treatment. These results support the hypothesis that the SFO mediates part of the hypotensive effects of chronic AT1 receptor blockade in the normotensive rat.

Salt appetite of adrenalectomized rats after a lesion of the SFO.

Circumventricular organs such as the subfornical organ (SFO) may mediate the effects of circulating angiotensin (ANG) II on salt appetite under conditions of sodium depletion in the rat. We studied the effects of an electrolytic lesion of SFO on salt appetite after adrenalectomy (ADX) in Long-Evans rats. The SFO lesion had no effect on saline intake, but it did abolish water intake after acute peripheral treatments with 2 mg/kg of captopril or a 10 mg/kg of furosemide. These findings contrast with other recent data from this laboratory demonstrating large reductions in salt appetite in adrenal-intact rats with lesions of either SFO or the organum vasculosum laminae terminalis during acute iv infusions of ANG II. Thus, the SFO may contribute to the salt appetite response to circulating ANG II, but it is not essential for the response to adrenalectomy.

Vasopressin acts in the subfornical organ to decrease blood pressure.

In addition to its traditional role as a circulating vasoactive peptide, vasopressin (VP) has been shown to play significant roles in central cardiovascular processing. The recent description of VP receptors within the subfornical organ (SFO) has suggested this circumventricular organ (CVO) as a potential locus for feedback actions of circulating VP on the brain. The well-established anatomical connections between SFO and hypothalamic autonomic control centers provide further arguments in support of such a view. This study was undertaken to determine the physiological consequences of activation of VP receptors within the SFO of urethane anesthetized rats. Microinjection (0.5 microliter) of 5 pmol VP into SFO resulted in significant decreases in blood pressure (BP, mean AUC -638.3 +/- 110.3 mm Hg.s, p < 0.01, n = 13) without a change in heart rate (HR, mean AUC 7.9 +/- 14.0 beats, p > 0.05, n = 12), effects which were repeatable. These depressor effects were specific to microinjection locations within this CVO as similar VP microinjections into non-SFO tissue were without effect on BP (mean AUC 245.4 +/- 111.5 mm Hg.s, p > 0.05, n = 10), or HR (mean AUC 1.8 +/- 3.1 beats, p > 0.05, n = 9). In contrast to the former depressor effects, VP microinjection (5 pmol in 0.5 microliter) into the third ventricle produced large increases in BP (mean AUC 1,461.8 +/- 368.97 mm Hg.s, p < 0.05, n = 6) again with no change in HR (mean AUC 1.4 +/- 5.96 beats, p > 0.05, n = 6). The hypotensive effects observed in response to VP microinjection into SFO were abolished by systemic treatment with a V1 receptor antagonist (mean AUC 89.5 +/- 67.7 mm Hg.s, p > 0.05) compared to BP response before V1 receptor blockade (mean AUC -605.9 +/- 119.8 mm Hg.s, n = 4). These results suggest that the SFO may be an essential structure in the feedback control loop through which circulating VP influences descending autonomic pathways involved in cardiovascular control.

Paraventricular nucleus neurons projecting to the spinal cord receive excitatory input from the subfornical organ.

The present study utilized electrophysiological techniques to determine the effects of subfornical organ (SFO) stimulation on the activity of neurons in the paraventricular nucleus (PVN) projecting to the spinal cord. Single-unit recordings were obtained from 79 PVN neurons antidromically identified as projecting to the intermediolateral cell column (IML). Antidromically evoked action potentials showed a mean latency of 94.6 +/- 5.3 ms and a mean threshold for activation of 1.58 +/- 0.11 mA. Electrical stimulation of SFO (100 microA-1.5 mA, 0.1 ms) resulted in excitatory responses in 18 of the 27 neurons tested (67%). Peristimulus histogram analysis of such effects demonstrated a duration of < 50 ms in 14 of the 18 cells so influenced (78%), whereas the remaining 4 cells (22%) showed excitatory responses with a longer duration. Systemic administration of the nonpeptidergic angiotensin II (ANG) type 1 (AT1) receptor antagonist losartan (3 mg/kg) blocked the long-duration excitatory responses in 100% (3 of 3) of the cells tested but was without effect on the short-duration excitations (0 of 5). Twenty-two identified PVN neurons were also tested for their responses to systemic ANG (20-500 ng), which had no observable effect on the activity of any of these cells. These data demonstrate that neurons in SFO provide excitatory input to PVN cells that project to the IML. One of the neurotransmitters responsible for communication in this pathway is ANG.

The parabrachio-subfornical organ projection in the rat.

Previous physiological studies have shown that both the parabrachial nucleus and the subfornical organ are involved in drinking behavior and cardiovascular controls. The purpose of the present work was to study the direct connections between these two structures by using anterograde and retrograde transport methods. A mixture of wheat germ agglutinin conjugated with horseradish peroxidase and free horseradish peroxidase or Fluorogold was injected into either the parabrachial nucleius (PBN) or the subfornical organ (SFO). The results indicated that the parabrachial nucleus sends a substantial projection to the entirety of the subfornical organ, and this input appears to be distributed to both the central and peripheral regions of this structure. Neurons that give origin to this projection are mainly located in the outer layer of the lateral division of the parabrachial nucleus, including the superior, internal, dorsal, and external lateral subnuclei. These findings suggest that, besides the already known connections, there is an additional parabrachio-subfornical pathway that may be involved in the central integration of cardiovascular function and drinking behavior.

Regional differences in the blood-brain-barrier of the subfornical organs of rats and ducks (Anas platyrhynchos).

Recently published electrophysiological data investigated the effect of blood borne and brain intrinsic substances on the activity of neurons in the duck subfornical organ (SFO). This study defines histologically the region in the duck SFO, where blood borne substances can possibly influence neuronal activity. Intravenous injection of Evans blue, a dye which labels brain structures devoid of a blood brain barrier (BBB), resulted in diffuse labelling of the duck SFO from the anterior commissure to the end of the organ in rostrocaudal extension. In addition, specifically labelled neurons could be observed just rostral to the diffuse Evans blue labelling and in an area dorsomedial to the large central blood vessel. The majority of the somata of these heavily stained neurons were located inside the BBB, whereas in the areas with diffuse Evans blue labelling, thus being outside the BBB, labelled cells were rarely observed. Intravenous injection of Evans blue in rats resulted similarly in diffuse labelling of the parenchyma of the medial and caudal part of the SFO, with only a few, but heavily stained cells with fusiform somata. The rostral region of the rat SFO, which is known to have a functional BBB, shows hardly any diffuse labelling, but there the majority of neurons show strong Evans blue fluorescence. It is concluded that the heavily labelled somata inside the BBB have axonal or dendritic projections to BBB-free areas, where they can take up the dye. This study gives a functional description of the extension of the SFO areas without a BBB of rats and ducks. It is concluded that blood borne agents can affect those SFO neurons which have their somata located outside the BBB as well as those located inside the BBB which have terminals projecting to BBB free regions.

Accumulation of blood-borne horseradish peroxidase in medial portions of the mouse hippocampus.

The intracerebral distribution of intravenously injected horseradish peroxidase (HRP) in young adult DDD mice was examined. HRP-tetramethylbenzidine reaction products were observed in the medial portions of the hippocampus, particularly the medial CA1 region and medial dentate gyrus. Reaction products were observed in the subfornical organ in mice decapitated 5 min after HRP injection, and then also progressively more caudally in the medial portions of the hippocampus as postinjection survival time increased. These findings suggest that blood-borne macromolecules have ready access to the medial portions of the hippocampus, particularly the medial CA1 region and medial dentate gyrus.

Sensory circumventricular organs and brain homeostatic pathways.

Circumventricular organs (CVOs), small structures bordering the ventricular spaces in the midline of the brain, have common morphological and endocrine-like characteristics that distinguish them from the rest of the nervous system. Among their unique features are cellular contacts with two fluid phases--blood and cerebrospinal fluid--and neural connections with strategic nuclei establishing circuitry for communications throughout the neuraxis. A variety of additional morphological and functional characteristics of the CVOs implicates this group of structures in a wide array of homeostatic processes. For three of the circumventricular organs--the subfornical organ (SFO), the organum vasculosum of the lamina terminalis (OVLT), and the area postrema (AP)--recent findings demonstrate these structures as targets for blood-borne information reaching the brain. We propose that these three sensory CVOs interact with other nuclei in the maintenance of several homeostatic processes by way of neural and humoral links. We emphasize the collective role of brain CVOs in the maintenance of body fluid homeostasis as a model for the functional integration of these fascinating "windows of the brain" within central neurohumoral systems.

ANP and naloxone reduce postdeprivation drinking after subfornical organ lesions.

We tested a report that atrial natriuretic peptide (ANP) injected into, or near, the subfornical organ (SFO) will reduce the water consumption of previously water deprived rats and that suggested ANP acts on neurons in the SFO to bring about this action. We tested this suggestion and the hypothesis that the SFO is involved in the facilitation of drinking produced by opioids. ANP (5 nmol in 4 microliters, IVT) or naloxone (2 mg/ml/kg, SC, or 200 micrograms in 2 microliters, IVT) when given to rats deprived of water for 16 h (SC treatment) or 23 h (IVT treatment) significantly depressed postdeprivation drinking measured at 15 and 60 min. Rats with complete, partial, or control lesions of the SFO, after the same treatment, also showed a significant depression of postdeprivation drinking and, after 23-h deprivation, a significant hyperdipsia. There was no interaction between drug effects and lesion effects (two-factor analysis of variance, Tukey's post-hoc tests). The hyperdipsia declined exponentially and was lost 45-50 days after lesioning. Our results do not support the hypothesis that the SFO is involved in the actions of ANP or of opioids on postdeprivation drinking.

Morphology and physiology of capillary systems in subregions of the subfornical organ and area postrema.

From recent morphological and physiological studies of capillaries, I shall review four new or revised concepts about blood-tissue communication in the subfornical organ (SFO) and area postrema (AP). First, the capillary systems of SFO and AP exhibit subregional differentiation correlated topographically with cytoarchitecture, densities of immunoreactivity for several peptides and amines, cellular sensitivity to neuroactive substances, afferent neural terminations, and tissue metabolic activity. Thus, contrary to frequent citations, the angioarchitecture and microcirculatory physiology of these small sensory nuclei are not homogeneous. Second, electron microscopic, morphometric, and topographical studies reveal that SFO contains three different types of capillary and AP has two. The differentiated capillary morphology appears to be well organized for specialized functions particularly in SFO subregions. No other body organ or small tissue region is known to have such capillary diversity, further highlighting the complex functions served by SFO. Third, pools of interstitial fluid (Virchow-Robin spaces) surrounding type I and III capillaries in SFO and AP may participate in the receptive properties of these organs as low-resistance pathways for rapid dispersion of blood-borne hormones inside their organ boundaries. The parenchymal walls of Virchow-Robin spaces appear to harbour metabolic mechanisms for hormones such as angiotensin II, and thus could vastly extend the effective blood-brain surface area of permeable capillaries in SFO and AP. Fourth, SFO and AP bear similar physiological characteristics of high blood volume, yet relatively low rates of blood flow. Accordingly, intracapillary blood velocity must be quite slow in these organs, and the duration of transit by blood and circulating messengers rather protracted. This feature of slow blood transit time likely compounds the sensory capability of SFO and AP, rendering increased contact time for blood-borne hormones to penetrate the permeable capillaries of these structures and interact with their known dense populations of receptors for several homeostatic substances involved in regulation of blood pressure and body fluids.

Functional identification of central pressor pathways originating in the subfornical organ.

The functional projections from pressor sites in the subfornical organ (SFO) were identified using the 2-deoxyglucose (2-DG) autoradiographic method in urethane-anesthetized, sinoaortic-denervated rats. Autoradiographs of brain and spinal cord sections taken from rats whose SFO was continuously stimulated electrically for 45 min with stereotaxically placed monopolar electrodes (150 microA, 1.5-ms pulse duration, 15 Hz) following injection of tritiated 2-DG were compared with control rats that received intravenous infusions of pressor doses of phenylephrine to mimic the increase in arterial pressure observed during SFO stimulation. Comparisons were also made to autoradiographs from rats in which the ventral fornical commissure (CFV), just dorsal to the SFO, was electrically stimulated. The pressor responses during either electrical stimulation of the SFO or intravenous infusion of phenylephrine were similar in magnitude. On the other hand, stimulation of the CFV did not elicit a significant pressor response. Electrical stimulation of the SFO increased 2-DG uptake, in comparison to the phenylephrine-infused rats, in the nucleus triangularis, septofimbrial nucleus, lateral septal nucleus, nucleus accumbens, bed nucleus of the stria terminalis, dorsal and ventral nucleus medianus (median preoptic nucleus), paraventricular nucleus of the thalamus, hippocampus, supraoptic nucleus, suprachiasmatic nucleus, paraventricular nucleus of the hypothalamus, and the intermediolateral nucleus of and central autonomic area of the thoracic spinal cord. In contrast, in rats whose CFV was stimulated, these nuclei did not demonstrate changes in 2-DG uptake compared with control animals that received pressor doses of phenylephrine. These data have demonstrated some of the components of the neural circuitry likely involved in mediating the pressor responses to stimulation of the SFO and the corrective responses to activation of the SFO by disturbances to circulatory and fluid balance homeostasis.

Focal metabolic effects of angiotensin and captopril on subregions of the rat subfornical organ.

Angiotensin infusion increased glucose metabolism in 4 of 7 subdivisions of the rat subfornical organ, the effect being stronger in ventromedial compared to dorsolateral zones across the rostrocaudal axis. [Sar1-Leu8]Angiotensin II attenuated metabolic responses to intravenous angiotensin in all subfornical organ subregions. Brattleboro rats, having high circulating levels of angiotensin, displayed greater rates of glucose metabolism than Long-Evans rats in all subregions, differences that were eliminated by captopril, an inhibitor of angiotensin converting enzyme. The studies reveal focal subfornical organ zones where in vivo metabolic activity corresponds to cytoarchitectonic evidence for topographical processing within this angiotensin-sensitive structure.

Endotoxin inhibition of drinking behaviour in the rat.

Intravenous (i.v. 320 and 640 micrograms/kg) and intracerebroventricular (i.c.v.; 1 microgram/rat) injection of Escherichia coli lipopolysaccharide (LPS) powerfully inhibited drinking induced by 24 h water deprivation. Pretreatment with acetylsalicylic acid (ASA) into the preoptic area (POA) completely abolished the effect induced by i.v. LPS, but did not modify that elicited by i.c.v. LPS. Intraperitoneal ASA injections significantly reduced the antidipsogenic effect of i.c.v. LPS. Electrolytic ablation of the subfornical organ (SFO) did not modify the effect induced by either i.v. or i.c.v. LPS. Present findings indicate that: (1) the antidipsogenic effect of i.v. LPS is mediated by prostaglandin synthesis into the POA, (2) the SFO is not involved in this effect, and (3) prostaglandins in other brain areas, besides POA, modulate the effect of i.c.v. LPS. It is suggested that at least two different brain sites, inside the blood-brain barrier, might be involved in the antidipsogenic effect of LPS.