Pharmacologic independence of subfornical organ receptors mediating drinking

In rats with chronically implanted cannulae in the subfornical organ (SFO), the relationship between cholinergic- and angiotensin (AII)-induced drinking was investigated pharmacologically. All substances were injected via SFO cannulae which did not rupture ventricular ependyma. Pretreatment with low doses of the muscarinic antagonist atropine abolished carbachol-induced drinking, while nicotinic antagonists had no effect. Nonetheless, pretreatment with much larger doses of atropine had no effect on AII-induced drinking. Similarly, relatively small doses of the AII antagonist, saralasin, blocked AII-induced drinking, yet a much larger dose of saralasin had no effect on carbachol-induced drinking. The receptors mediating cholinergic- and AII-induced drinking therefore cannot be in series and must be in parallel. A hypothesis is proposed to account for this independence and for the significance of the SFO cholinergic innervation.     

Transection of subfornical organ neural connections diminishes the pressor response to intravenously infused angiotensin II

Knife-cut lesions were used to assess the participation of the subfornical organ (SFO) in the central pressor action of intravenously administered angiotensin. Knife-cuts of the ventral stalk of the SFO significantly attenuated pressor responses during infusion of 3 doses of angiotensin, although responses to bolus injections were unaffected. These results are consistent with previous work in implicating the SFO as an important mediator of the central pressor action of circulating angiotensin.     

Drinking behavior following electrical stimulation of the subfornical organ in the rat

Electrical stimulation of the subfornical organ (SFO) through implanted stainless-steel electrodes produced drinking in water-sated rats. Drinking was elicited primarily during the interstimulation (OFF) periods. Water intake in rats with hippocampal electrode placements occurred during both ON and OFF periods at current intensities (24–100 μA) similar to rats with SFO placements. These findings support the hypothesis that the SFO is involved in the central control of fluid balance.     

The site of the dipsogenic action of angiotensin II in the North American opossum

Adult North American opossums (Didelphis virginiana), predominantly nocturnal water drinkers, were found to respond by drinking to water deprivation, cellular dehydration produced by intravenous infusion of hypertonic saline, and to both intravenous and intracerebroventricular administration of angiotensin II (AII). by taking advantage of the absence of the corpus callosum, an anatomical characteristic of the marsupial, we removed the subfornical organ and implanted a cannula into the anterior third ventricle under direct visual guidance. In these animals: (1) spontaneous drinking, and responses to water deprivation and cellular dehydration were unaffected; (2) after a postoperative period of unresponsiveness, attenuated and unreliable responses only to high doses of intracerebroventricular AII could be elicited; and (3) the dipsogenic effect of intravenous AII was abolished. We conclude that the subfornical organ is necessary for the physiologically appropriate effect of AII on drinking but that, in the absence of the subfornical organ, the brain can mediate the dipsogenic effect of intracranially administered AII, although the physiological significance of the latter phenomenon is unclear.     

Reduced dipsogenic response induced by angiotensin II activation of subfornical organ projections to the median preoptic nucleus in estrogen-treated rats

The present study was carried out to investigate whether estrogen modulates the drinking response induced by activation of angiotensinergic neural pathways from the subfornical organ (SFO) to the median preoptic nucleus (MnPO). Microinjection of angiotensin II (ANG II, 10(-10) M, 0.2 microl) into the SFO elicited drinking in ovariectomized (OVX) female rats that were treated with either propylene glycol (PG) vehicle or estrogen benzoate (EB). The amount of water intake induced by the ANG II injection was significantly greater in the PG-treated than in the EB-treated animals. In both groups of female rats, previous injections of saralasin (Sar, 10(-10) M, 0.2 microl), a specific ANG II antagonist, into the MnPO resulted in the significant attenuation of the drinking response to ANG II, showing that the ANG II-induced drinking response may be mediated in part by the angiotensinergic SFO projections to the MnPO. Injections of ANG II (10(-10) M, 0.2 microl) into the MnPO caused drinking in both groups, while no significant difference was found between the groups in the amount of water intake. These results suggest that increases in the circulating level of estrogen may attenuate the drinking response induced by ANG II activation of the SFO projections to the MnPO.     

The effects of disconnecting the subfornical organ on behavioral and physiological responses to alterations of body sodium

Rats with knife cuts transecting the subfornical organ efferent projections were examined for behavioral and renal responses to experimental alterations in body sodium. Lack of subfornical organ efferent projections did not impair behavioral and renal compensatory responses either in the latency to respond or in magnitude of the response.     

Polydipsia and abolition of angiotensin-induced drinking after transections of subfornical organ efferent projections in the rat

Rats with transections of subfornical organ (SFO) efferent projections failed to drink to intravenous angiotensin-II (AII) but responded to intracellular dehydration and water deprivation and suppressed drinking when food deprived. However, the transected rats were polydipsic and polyuric. Thus SFO efferent projections mediate AII-induced drinking and may be involved in the regulation of body fluid balance.     

Vasopressin release to central and peripheral angiotensin II in rats with lesions of the subfornical organ

Ovine corticotropin releasing factor (CRF) given intracerebroventricularly to rhesus monkeys at doses of 0.1-1 microgram/kg activates the pituitary-adrenal axis. The increases of plasma cortisol concentrations after intraventricular injection of CRF were similar to those observed after intravenous administration of this releasing factor and occurred without elevation of plasma concentrations of CRF, measured by radioimmunoassay. Thus, the cerebrospinal fluid (CSF) can serve as a conduit for delivery of CRF to the pituitary gland.     

Neural projections subserving the initiation of a specific motivated behavior in the rat: new projections from the subfornical organ

The activation of neurons in the subfornical organ (SFO) by angiotensin II in the blood is known to stimulate thirst. The projections of these neurons were examined with anterograde and retrograde axonal transport methods, and were found to end in specific parts of the prefrontal cortex, septal region, substantia innominata, medial preoptic area, zona incerta and lateral hypothalamic area. These regions in turn are thought to be involved in arousal, somatomotor control and cognitive functions, essential components of the initiation and procurement phases of motivated behavior.     

Electrophysiological identification of forebrain connections of the subfornical organ

Experiments were performed in 17 urethane-anesthetized rats to investigate electrophysiologically neurons in the subfornical organ (SFO), which send efferent axons directly to the region of the paraventricular nucleus of the hypothalamus (PVH), the supraoptic nucleus (SON) and the nucleus medianus (NM). Extracellular single unit recordings were made from spontaneously active and silent neurons in the region of SFO (n = 130) and the nucleus triangularis (NT; n = 20). Sixty-five units in SFO were antidromically activated by stimulation of either PVH, SON or NM with latencies corresponding to conduction velocities of 0.54 +/- 0.07 (n = 24), 0.44 +/- 0.05 (n = 17) and 0.23 +/- 0.02 (n = 24) m/s, respectively. Axons of SFO units projecting to NM conducted at significantly slower velocities than those to PVH and SON. An additional 11 units were antidromically activated in NT by stimulation of these forebrain structures. Sixty-seven units were found to respond orthodromically to stimulation of PVH, SON and NM: 58 in SFO and 9 in NT. Orthodromic responses were primarily excitation or inhibition. These data have demonstrated bidirectional pathways between SFO and forebrain structures which are likely involved in the dipsogenic and arterial pressure responses to activation of SFO by blood-borne angiotensin II.     

The subfornical organ: A novel site for prolactin action

Prolactin (PRL ) is a peptide hormone that performs over 300 biological functions, including those that require binding to prolactin receptor (PRL ‐R) in neurones within the central nervous system (CNS ). To enter the CNS , circulating PRL must overcome the blood‐brain barrier. Accordingly, areas of the brain that do not possess a blood‐brain barrier, such as the subfornical organ (SFO ), are optimally positioned to interact with systemic PRL . The SFO has been classically implicated in energy and fluid homeostasis but has the potential to influence oestrous cyclicity and gonadotrophin release, which are also functions of PRL . We aimed to confirm and characterise the expression of PRL ‐R in the SFO , as well as identify the effects of PRL application on membrane excitability of dissociated SFO neurones. Using a quantitative real‐time polymerase chain reaction, we found that PRL ‐R mRNA in the SFO of male and female Sprague Dawley rats did not significantly differ between juvenile and sexually mature rats (P  = .34), male and female rats (P  = .97) or across the oestrous cycle (P  = .54). Patch‐clamp recordings were obtained in juvenile male rats to further investigate the actions of PRL at the SFO . Dissociated SFO neurones perfused with 1 μmol L^‐1 PRL resulted in 2 responsive subpopulations of neurones; 40% depolarised (n = 15/43, 11.3 ± 1.7 mV ) and 14% hyperpolarised (n = 6/43, ?6.7 ± 1.4 mV ) to PRL application. Within the range of 10 pmol L^‐1 to 1 μmol L^‐1, the concentrations of PRL were not significantly different in either the magnitude (P  = .53) or proportion (P  = .19) of response. Furthermore, PRL application significantly reduced the transient K⁺ current in 67% of SFO neurones in voltage‐clamp configuration (n = 6/9, P  = .02). The stability in response to PRL and expression of PRL ‐R in the SFO suggests that PRL function is conserved across physiological states and circulating PRL concentrations, prompting further investigations aiming to clarify the nature of PRL function in the SFO.     

Cardiovascular actions of orexin-A in the rat subfornical organ

Orexin-A is a neuropeptide, primarily produced in the lateral hypothalamic/perifornical hypothalamus. Orexin receptors and immunoreactive neuronal fibres are widely distributed throughout the brain, suggesting integrative neurotransmitter roles in a variety of physiological systems. Intracerebroventricular injections of orexin-A increase blood pressure and stimulate drinking, and the subfornical organ (SFO), a circumventricular structure implicated in autonomic control, is a potential site at which orexin may act to exert these effects. We have therefore used microinjection techniques to examine the effects of orexin-A administered directly into the SFO on blood pressure and heart rate in urethane anaesthetised male Sprague-Dawley rats. Orexin-A microinjection (50 fmol) into the SFO caused site-specific decreases in blood pressure (SFO: mean area under curve (AUC) = -681.7 +/- 46.8 mmHg*s, n = 22 versus non-SFO: 63.68 +/- 54.69 mmHg*s, n = 15, P < 0.001), and heart rate (SFO: mean AUC = -26.7 +/- 2.8 beats, n = 22, versus non-SFO: mean AUC = 1.62 +/- 2.1 beats, n = 15, P < 0.001). Vagotomy did not alter the hypotensive or bradycardic responses elicited by orexin-A microinjection. Prior alpha-adrenoceptor blockade with phenoxybenzamine (1 mg/kg, i.v.) masked the orexin-A induced blood pressure (mean AUC = -122.6 +/- 17.6 mmHg*s, n = 4, P < 0.01 paired t-test) and heart rate (mean AUC = -6.7 +/- 1.7 beats, n = 4, P < 0.05, paired test) response. The orexin-A induced heart rate response was attenuated when beta-adrenoceptors were blocked with propranolol (1 mg/kg, i.v.; mean AUC = 0.6 +/- 2.8 beats, n = 5, P < 0.01 paired t-test). These studies demonstrate that microinjection of orexin-A into the SFO causes site specific decreases in blood pressure and heart rate which is mediated by a reduction in sympathetic tone.     

Cardiovascular actions of leptin in the subfornical organ are abolished by diet-induced obesity

The subfornical organ (SFO), a sensory circumventricular organ lacking the normal blood-brain barrier with well documented roles in cardiovascular regulation, has recently been identified as a potential site at which the adipokine, leptin, may act to influence central autonomic pathways. Systemic and central leptin administration has been shown to increase blood pressure and it has been suggested that selective leptin resistance contributes to obesity-related hypertension. Given the relationship between obesity and hypertension, the present study aimed to investigate the cardiovascular consequences of the direct administration of leptin into the SFO of young lean rats and in the diet-induced obesity (DIO) rat model, which has been shown to be leptin-resistant. Leptin administration (500 fmol) directly into the SFO of young rats resulted in rapid decreases in blood pressure (BP) [mean area under the curve (AUC) = -677.8 ± 167.1 mmHg*s; n = 9], without an effect on heart rate (mean AUC = -21.2 ± 13.4 beats; n = 9), and these effects were found to be dose-related as microinjection of 5 pmol of leptin into the SFO had a larger effect on BP (mean AUC = -972.3 ± 280.1 mmHg*s; n = 4). These BP effects were also shown to be site-specific as microinjection of leptin into non-SFO regions or into the ventricle was without effect on BP (non-SFO: mean AUC = -22.4 ± 55.3 mmHg*s; n = 4; ventricle: mean AUC = 194.0 ± 173.0 mmHg*s; n = 6). By contrast, microinjection of leptin into leptin-resistant DIO rats was without effect on BP (mean AUC = 205.2 ± 75.1 mmHg*s; n = 4). These observations suggest that the SFO may be an important relay centre through which leptin, in normal weight, leptin responsive rats, acts to maintain BP within normal physiological limits through descending autonomic pathways involved in cardiovascular control and that, in obese, leptin-resistant, rats leptin no longer influences SFO neurones, resulting in an elevated BP, thus contributing to obesity-related hypertension.     

Effect of transection of subfornical organ efferent projections on vasopressin release induced by angiotensin or isoprenaline in the rat

Transection of subfornical organ efferents in the rat prevented the vasopressin release in response to intravenous angiotensin II infusion or following a small dose of the beta-sympathomimetic amine isoprenaline (30 micrograms/kg i.m.). In contrast, this lesion had no effect on vasopressin release after hypertonic saline injection or a high dose of isoprenaline (480 micrograms/kg i.m.). We conclude that blood-borne angiotensin II induces vasopressin release by acting on the subfornical organ; depending on the dose of isoprenaline, activation of the endogenous renin-angiotensin system may mediate isoprenaline-induced vasopressin release.     

Angiotensin II immunoreactivity in the neural afferents and efferents of the subfornical organ of the rat

Angiotensin II (AII) immunoreactive cells and fibers were identified in the subfornical organ (SFO) of the rat. Cells were distributed in an annulus around the periphery of the SFO and were most visible in the Brattleboro rat treated with colchicine. Fibers were observed in a plexus, located centrally within the ring of cells, and knife-cuts suggested that they arise from parent cell bodies lying outside of the SFO. Studies combining immunohistochemistry with retrograde transport identified the perifornical zone of the lateral hypothalamus, the rostral zona incerta, and the nucleus reuniens of the thalamus as the source of AII-stained inputs to the SFO, and the region of the median preoptic nucleus as a recipient of AII-immunoreactive SFO efferents. It is suggested that these biochemically defined connections of the SFO participate in the central neural control of fluid balance.     

Role of the subfornical organ (SFO) in the control of gonadotropin secretion

Radiofrequency lesions have been placed in the subfornical organ (SFO) or normally cycling and of long-term castrated female rats. The data have shown that, in normal female rats, SFO lesions bring about a disruption of estrous cyclicity in a large number of animals, the majority of which enter a state of prolonged diestrus. In these animals, serum gonadotropin levels are similar to those of regularly cycling animals in the second day of diestrus. In the animals in which estrous cyclicity was still present, an LH surge occurred which was regular in timing and magnitude. On the contrary the proestrous FSH surge was absent, but the usual FSH elevation of the day of estrus was present. SFO lesions were not able to change the high gonadotropin levels typical of the post-castration situation. These results suggest that the SFO may play a role in the control of the "cyclic" but not of the "tonic" release of the two gonadotropins. In particular, it appears from the results that the SFO might be involved in the regulation of the hypersecretion of FSH which occurs during the day of proestrus.     

Estrogen decreases the responsiveness of subfornical organ neurons to angiotensinergic neural inputs from the lateral hypothalamic area in the female rat

Twenty-eight subfornical organ (SFO) neurons in ovariectomized (OVX) female rats that were treated with propylene glycol (PG) vehicle and 26 SFO neurons in OVX female rats that were treated with estrogen benzoate (EB) were antidromically activated by electrical stimulation of the hypothalamic paraventricular nucleus (PVN) under urethane anesthesia. No significant differences were observed between the PG-treated and EB-treated OVX animals in the latency, conduction velocity, or threshold of antidromic activation. The mean spontaneous discharge rate was significantly lower in the EB-treated than in the PG-treated OVX animals. In both groups, the activity of the majority (86% in the PG-treated animals and 88% in the EB-treated animals) of identified SFO neurons were activated by microiontophoretic application of angiotensin II (ANG II). Electrical stimulation of the lateral hypothalamic area (LHA) increased the excitability of these ANG II-sensitive SFO neurons (58% in the PG-treated animals and 52% in the EB-treated animals). The excitatory response to either ANG II or LHA stimulation was blocked by microiontophoretic application of the ANG II antagonist saralasin (Sar), suggesting that the excitatory response to LHA stimulation may be mediated by angiotensinergic LHA projections to the SFO. The magnitude of excitatory response to either ANG II or the LHA stimulation was much greater in the PG-treated than in the EB-treated animals. These results suggest that estrogen decreases the responsiveness of SFO neurons projecting to the PVN to angiotensinergic inputs from the LHA.     

Terminal degeneration in supraoptic nucleus following subfornical organ lesions: ultrastructural observations in the rat

A projection from the subfornical organ (SFO) to the supraoptic nucleus, recently identified in light microscopic studies, was examined at the ultrastructural level following lesions in SFO. After 18–36 h, axon terminal degeneration was identified in axosomatic contacts with supraoptic neurosecretory neurons, and in axodendritic contacts within and around the supraoptic nucleus. These observations confirm a monosynaptic pathway from SFO to supraoptic neurosecretory neurons that may participate in the release of vasopressin following activation of angiotensin II receptors in SFO.     

Effects of electrical and chemical stimulation of the paraventricular nucleus on neurons in the subfornical organ of cats

Single unit recordings were made from neurons of the subfornical organ (SFO) in hemispherectomized cats following electrical stimulation in the paraventricular nucleus (PVN) of the hypothalamus. Of 132 neurons tested, 48 (36%) were inhibited, 3 (2.3%) excited and two (1.5%) antidromically activated. In separate experiments the wall of the third ventricle near the PVN was locally perfused with glutamate solution using a concentric push-pull cannula. Of 35 SFO neurons tested, 6 were inhibited, 5 were facilitated and 24 unaffected. The results indicate that one-third of SFO neurons receive synaptic inputs from cells in or near the PVN.