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Water intakes in response to hypertonic, hypovolemic, and dehydrational stimuli were investigated in mice lacking angiotensin II as a result of deletion of the angiotensinogen gene (Agt-/- mice), and in C57BL6 wild-type (WT) mice. Baseline daily water intake in Agt-/- mice was approximately threefold that of WT mice because of a renal developmental disorder of the urinary concentrating mechanisms in Agt-/- mice. Intraperitoneal injection of hypertonic saline (0.4 and 0.8 mol/l NaCl) caused a similar dose-dependent increase in water intake in both Agt-/- and WT mice during the hour following injection. As well, Agt-/- mice drank appropriate volumes of water following water deprivation for 7 h. However, Agt-/- mice did not increase water or 0.3 mol/l NaCl intake in the 8 h following administration of a hypovolemic stimulus (30% polyethylene glycol sc), whereas WT mice increased intakes of both solutions during this time. Osmoregulatory regions of the brain [hypothalamic paraventricular and supraoptic nuclei, median preoptic nucleus, organum vasculosum of the lamina terminalis (OVLT), and subfornical organ] showed an increased number of neurons exhibiting Fos-immunoreactivity in response to intraperitoneal hypertonic NaCl in both Agt-/- mice and WT mice. Polyethylene glycol treatment increased Fos-immunoreactivity in the subfornical organ, OVLT, and supraoptic nuclei in WT mice but only increased Fos-immunoreactivity in the supraoptic nucleus in Agt-/- mice. These data show that brain angiotensin is not essential for the adequate functioning of neural pathways mediating osmoregulatory thirst. However, angiotensin II of either peripheral or central origin is probably necessary for thirst and salt appetite that results from hypovolemia.

The aim of this study was to determine, in conscious rats, whether elevated concentrations of circulating angiotensin II activate neurones in both the subfornical organ and organum vasculosum of the lamina terminalis (OVLT) that project to the bed nucleus of the stria terminalis (BNST). The strategy employed was to colocalize retrogradely transported cholera toxin B subunit (CTB) from the BNST, with elevated levels of Fos protein in response to angiotensin II. Circulating angiotensin II concentrations were increased by either intravenous infusion of angiotensin II or subcutaneous injection of isoproterenol. Neurones exhibiting Fos in response to angiotensin II were present in the subfornical organ, predominantly in its central core but with some also seen in its peripheral aspect, the dorsal and lateral margins of the OVLT, the supraoptic nucleus and the parvo- and magnocellular divisions of the paraventricular nucleus. Fos-labelling was not apparent in control rats infused with isotonic saline intravenously or injected with either CTB or CTB conjugated to gold particles (CTB-gold) only. Of the neurones in the subfornical organ that were shown by retrograde labelling to project to BNST, approximately 50% expressed Fos in response to isoproterenol. This stimulus also increased Fos in 33% of neurones in the OVLT that project to BNST. Double-labelled neurones were concentrated in the central core of the subfornical organ and lateral margins of the OVLT in response to increased circulating angiotensin II resulting from isoproterenol treatment. These data support a role for circulating angiotensin II acting either directly or indirectly on neurones in subfornical organ and OVLT that project to the BNST and provide further evidence of functional regionalization within the subfornical organ and the OVLT. The function of these pathways is yet to be determined; however, a role in body fluid homeostasis is possible.

Angiotensinogen, the precursor molecule of the peptides angiotensin I, II, and III, is synthesized in the brain and the liver. Evidence is reviewed that angiotensin II, and possibly angiotensin III, that are generated within the brain act within neural circuits of the central nervous system to regulate body fluid balance. Immunohistochemical studies in the rat brain have provided evidence of angiotensin-containing neurons, especially in the hypothalamic paraventricular nucleus, subfornical organ, periventricular region, and nucleus of the solitary tract, as well as in extensive angiotensin-containing fiber pathways. Angiotensin immunoreactivity is observed by electron microscope in synaptic vesicles in several brain regions, the most prominent of these being the central nucleus of the amygdala. Neurons in many parts of the brain (lamina terminalis, paraventricular and parabrachial nuclei, ventrolateral medulla, and nucleus of the solitary tract) known to be involved in the regulation of body fluid homeostasis exhibit angiotensin receptors of the AT(1) subtype. Pharmacological studies in several species show that intracerebroventricular administration of AT(1) receptor antagonist drugs inhibit homeostatic responses to the central administration of hypertonic saline, intravenous infusion of the hormone relaxin, or thermal dehydration. Responses affected by centrally administered AT(1) antagonists are water drinking, vasopressin secretion, natriuresis, increased arterial pressure, reduced renal renin release, salt hunger, and thermoregulatory adjustments. We conclude that angiotensinergic neural pathways in the brain probably have an important homeostatic function, especially in regard to osmoregulation and thermoregulation, and the maintenance of arterial pressure.

1. Autoradiographic binding studies have shown that the AT(1) receptor is the predominant angiotensin II (AngII) receptor subtype in the central nervous system (CNS). Major sites of AT(1) receptors are the lamina terminalis, hypothalamic paraventricular nucleus, the lateral parabrachial nucleus, rostral and caudal ventrolateral medulla, nucleus of the solitary tract and the intermediolateral cell column of the thoraco-lumbar spinal cord. 2. While there are differences between species, AT(2) receptors are found mainly in the cerebellum, inferior olive and locus coeruleus of the rat. 3. Circulating AngII acts on AT(1) receptors in the subfornical organ and organum vasculosum of the lamina terminalis (OVLT) to stimulate neurons that may have a role in initiating water drinking. 4. Centrally administered AngII may act on AT(1) receptors in the median preoptic nucleus and elsewhere to induce drinking, sodium appetite, a sympathetic vasoconstrictor response and vasopressin secretion. 5. Recent evidence shows that centrally administered AT(1) antagonists inhibit dipsogenic, natriuretic, pressor and vasopressin secretory responses to intracerebroventricular infusion of hypertonic saline. This suggests that an angiotensinergic neural pathway has a role in osmoregulatory responses. 6. Central angiotensinergic pathways which include neural inputs to the rostral ventrolateral medulla may use AT(1) receptors and play a role in the function of sympathetic pathways maintaining arterial pressure., (1996 Blackwell Publishing Asia Pty Ltd.)

In the sheep spinal cord, a high density of [125I]angiotensin IV binding sites was localized to the perikaryon and processes of all somatic motor neurons, the autonomic motor neurons in the lateral horns of thoracic and lumbar segments and all dorsal root ganglia, but was low in lamina II of all dorsal horns. At supraspinal levels, [125I]angiotensin IV binding was abundant in numerous motor associated regions, with weaker binding observed in the sensory regions. This wide distribution pattern suggests an important role for the binding site in the central nervous system.

Fos immunoreactivity in the rat brain after intracerebroventricular (ICV) angiotensin II (ANG II) was compared with that induced by intravenous ANG II. ANG II was infused into the lateral ventricle (at 1 ng/min) or femoral vein (at 5 micrograms/h) of conscious rats. After 90 min, rats were killed and Fos was detected by immunohistochemistry. Both infusions caused Fos immunoreactivity to be present in the lamina terminalis, hypothalamic supraoptic, and paraventricular nuclei, bed nucleus of the stria terminalis, and central amygdaloid nucleus. However, distributions of Fos immunoreactivity within the lamina terminalis differed with the different routes of infusion. Intravenous ANG II caused intense Fos immunoreactivity mainly in the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT). By contrast, ICV ANG II caused intense Fos immunoreactivity predominantly in the median preoptic nucleus and juxtaventricular neurons of the SFO and OVLT. These results suggest that IV ANG II induces behavioural and endocrine responses by direct actions on the SFO and OVLT, whereas ICV ANG II directly stimulates neurons in the median preoptic nucleus as well neurons in the SFO and OVLT.

The beta adrenergic agonist isoproterenol has been used in these studies to elevate circulating levels of angiotensin II. Neurons in the brain responsive to the subcutaneous infusion of isoproterenol were identified using an antibody to Fos, the protein product of c-fos which is now used extensively as a marker of activated neurons. Fos-positive neurons were present in a range of specific forebrain and hind brain regions. Infusion of losartan (an angiotensin II type receptor antagonist) showed that neurons in the lamina terminalis were activated directly or indirectly by angiotensin II, whereas other neurons in the hypothalamus and brain stem were responsive as a consequence of the peripheral vasodilation caused by isoproterenol. The distribution of activated neurons in the lamina terminalis was consistent with that of neurons thought to be involved in water drinking.

The lamina terminalis consists of neurons which are activated by both osmotic and angiotensinergic stimuli and which project axons to many sites including regions of the hypothalamus responsible for vasopressin production. Combination of retrograde neuronal tracing procedures with the identification of Fos protein following discrete stimuli shows populations of neurons, projecting to the supraoptic nuclei, which are preferentially activated by intravenous infusion of either hypertonic saline or angiotensin II. Following infusion of hypertonic saline, the greatest percentage of neurons both labelled with cholera toxin-gold and having elevated levels of Fos protein occurred in that part of the lamina terminalis called the organum vasculosum lamina terminalis. Conversely, angiotensin infusion resulted in greatest numbers of Fos and cholera toxin-gold-labelled neurons in the subfornical organ with fewer double-labelled cells represented in the other components of the lamina terminalis, the median preoptic nucleus and the organum vasculosum lamina terminalis. While these data do not support more than a general separation of the functions examined among neurons of the lamina terminalis, they do highlight a discrete group of osmoresponsive neurons in the dorsal cap of the organum vasculosum lamina terminalis. These cells, by virtue of their response to infusions of hypertonic saline and their axonal connections to regions of the hypothalamus responsible for vasopressin production, are likely candidates for cerebral osmoreceptors.

Hypotensive haemorrhage increased the production of Fos, a marker of cell activation, in cells of the subfornical organ (SFO) and the organum vasculosum of the lamina terminalis (OVLT) as well as in the supraoptic (SON) and paraventricular (PVN) nuclei in conscious rats. Pretreatment with captopril (100 mg/kg i.p.) inhibited the production of Fos in cells of the SFO and OVLT but did not reduce the concentration of Fos-positive cell nuclei in the PVN and SON. The results suggest that the production of Fos in the SFO and OVLT induced by the haemorrhage is probably due to elevated levels of circulating angiotensin II.

Conscious rats were infused intravenously with either angiotensin II (30-55 pmol/kg/min), isotonic saline or phenylephrine for 2 h, then killed. Fos was identified by immunohistochemistry in the brains. Fos expression occurred in many neurons of the subfornical organ and organum vasculosum of the lamina terminalis (OVLT) with angiotensin infusion but not with isotonic NaCl or phenylephrine. Fos immunoreactivity was induced in cells in several medullary, hypothalamic and limbic structures with infusions of angiotensin II or phenylephrine at pressor doses. The results suggest that blood-borne angiotensin II at physiological levels causes angiotensin receptive neurons in the subfornical organ and OVLT to express Fos. Activation of baroreceptor pathways may also induce Fos expression at several other sites.

Angiotensin II (Ang II) acts centrally to modulate autonomic activity and cardiovascular function. Using in vitro autoradiography we have determined the distribution of putative receptors for ANG II in brain regions associated with the baroreflex pathway. Ang II receptor binding sites were observed in the nucleus of the solitary tract (NTS), the dorsal motor nucleus of vagus, the rostral and caudal ventrolateral medulla and intermediolateral cell column of the spinal cord. Receptor binding sites were also observed in the nodose ganglion and in association with nerve fibres in the heart. Nodose ganglionectomy and vagal ligation studies revealed that Ang II receptor binding sites are produced in the nodose ganglion and transported in the vagus nerve to terminals of vagal afferent neurones. In the NTS these presynaptic receptors could mediate the known baroreflex inhibitory action of Ang II by inhibition of neurotransmitter release. In the cat, Ang II receptors were sharply localized in the rostral ventrolateral medulla to the subretrofacial nucleus. Microinjection of Ang II (10-50 pmoles) into this region induced a sympathetically-mediated pressor response. Together these results demonstrate several regions within the baroreflex arc which contain Ang II receptors, at which Ang II may modulate cardiovascular control and autonomic function.

1. Most circulating peptide hormones are excluded from much of the brain by the blood-brain barrier. However, they do have access to the circumventricular organs (CVO), which lack the blood-brain barrier. Three of the CVO, the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT) and area postrema, contain neurons responsive to peptides such as angiotensin II (Angll), atrial natriuretic peptide and relaxin. 2. We have studied the patterns of neuronal activation, as shown by Fos expression, in the SFO and OVLT in response to systemically infused AngII, relaxin or hypertonic saline and have found subgroups of neurons activated by the different stimuli. 3. Systemic infusion of relaxin or hypertonic saline activated neurons almost exclusively in the outer regions of the SFO and in the dorsal cap of the OVLT. Many of these neurons send axonal projections to regions of the brain subserving vasopressin secretion and thirst, such as the median preoptic, supraoptic and hypothalamic paraventricular nuclei. 4. At moderate blood concentrations, AngII only stimulates neurons in the inner core of the SFO and lateral regions of the OVLT. Higher levels of AngII in the bloodstream activate additional neurons in the outer parts of the SFO that connect to the supraoptic, paraventricular and median preoptic nuclei and these probably mediate water drinking and vasopressin secretion induced by blood-borne AngII. The efferent connections and the functions mediated by angiotensin-sensitive neurons in the inner core of the SFO and lateral part of the OVLT are unknown. [ABSTRACT FROM AUTHOR]