Proc. Natl. Acad. Sci. USA Vol. 93, pp. 7397-7404, July 1996 Physiology

This contribution is part of the special series ofInaugural Articles by members of the National Academy of Sciences elected on April 25, 1995.

Hypothalamic integration of body fluid regulation D. A. DENTON, M. J. MCKINLEY, AND R. S. WEISINGER Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria 3052, Australia Contributed by D. A. Denton, March 26, 1996

ABSTRACT The progression of animal life from the gressive invasion of diverse regions of the planet-jungles, the paleozoic ocean to rivers and diverse econiches on the planet's dry interior of continents, deserts, savannah, temperate for- surface, as well as the subsequent reinvasion of the ocean, ests, and the Alps. Also, there was reinvasion of the oceans, all involved many different stresses on ionic pattern, osmotic migrations entailing diverse environmental influences with pressure, and volume of the extracellular fluid bathing body potential to distort the stability of the fluid milieu of the tissues cells. The relatively constant ionic pattern of vertebrates of the organism. reflects a genetic "set" of many regulatory mechanisms- particularly renal regulation. Renal regulation of ionic pat- Constancy of the Milieu tern when loss of fluid from the body is disproportionate relative to the extracellular fluid composition (e.g., gastric The constancy of the milieu interieur, the condition of free life, juice with vomiting and pancreatic secretion with diarrhea) involves, inter alia, maintenance of a pattern of ionic compo- makes manifest that a mechanism to produce a biologically nents. John Fulton (3), in his book Selected Readings in the relatively inactive extracellular anion HCO3 exists, whereas History of Physiology, pointed out that MacCallum (4) gave no comparable mechanism to produce a biologically inactive historical and phylogenetic meaning to the constancy of the cation has evolved. Life in the ocean, which has three times the milieu interieur when he demonstrated the striking resem- sodium concentration of extracellular fluid, involves quite blance between the ionic components of the blood sera and different osmoregulatory stress to that in freshwater. Terres- that of seawater, and, in particular, the ionic ratios. He trial life involves risk of desiccation and, in large areas of the suggested that tissue cells could only live within a relatively planet, salt deficiency. Mechanisms integrated in the hypo- narrow range of physicochemical conditions that represented thalamus (the evolutionary ancient midbrain) control water that of the ancient ocean, in which the cells of ancestral retention and facilitate excretion of sodium, and also control organisms had originated. As Metazoan organisms evolved the secretion of renin by the kidney. Over and above the and developed a closed circulatory system, they evolved organs multifactorial processes of excretion, hypothalamic sensors that regulated and kept the ionic pattern constant regardless reacting to sodium concentration, as well as circumventricu- of changes the external milieu underwent with time. The lar organs sensors reacting to osmotic pressure and angio- comparative constancy of this pattern of the extracellular tensin II, subserve genesis of sodium hunger and thirst. These fluids from elasmobranchs to Australian marsupials and the behaviors spectacularly augment the adaptive capacities of mammals (5, 6) reflects a basic genetic characteristic of animal animals. Instinct (genotypic memory) and learning (pheno- life, reflecting the "set" of a number of regulatory systems- typic memory) are melded to give specific behavior apt to the particularly renal regulation. metabolic status of the animal. The sensations, compelling With addition stress, as a result of an excess of a substance emotions, and intentions generated by these vegetative sys- entering the milieu, the rise in plasma concentration or load to tems focus the issue of the phylogenetic emergence of con- the renal tubules results in regulatory excretion. sciousness and whether primal awareness initially came from A broad evolutionary implication of pattern constancy the interoreceptors and vegetative systems rather than the emerges clearly in the face of subtraction stress distorting the distance receptors. ionic pattern, as can occur in higher mammals as a result of disease or, for that matter, human medical problems. For In the higher mammals, the functions centered in the hypo- example, the stomach secretes fluid that has Cl- in large excess thalamus play a paramount role in integrating the many of Na (Cl/Na, -4-5), relative to the normal extracellular physiological systems controlling the milieu interieur. These proportions (Cl/Na, 0.7), HI being the electrically equivalent hypothalamic processes range from genetically determined cation. With large loss of acid gastric juice by vomiting, the patterns of ingestive behavior that correct body deficits and, in plasma level of Cl- falls and the level of HCOj3, an anion turn, involve associated cognitive and memory functions of the constantly produced in the body, may rise reciprocally 10-20 cortex, to the other extreme of the control of excretory meq/liter, the plasma pH being maintained near constant by processes, in a mode apt to the metabolic status of the animal. a compensatory rise in pCO2 as a result of regulatory depres- Some evolutionary aspects of body fluid control will be sion of respiration. The replacement of plasma Cl- by a described first as a general biological context of the mecha- relatively biologically inactive anion, HCO-, obviates the nisms in mammals. physiological need to excrete significant amounts of sodium in Mountain building, like the Grand Canyon uplift in the the urine to control the pattern. In striking contrast, with loss Cambrian and subsidence in the Ordivician periods, provided of alkaline pancreatic secretion as a result of diarrhea or conditions of rivers flowing into the ocean, and, probably intestinal fistulae or loss of parotid salivary secretion in during this time, protovertebrates with spindle body and segmentally arranged muscles adapted to rhythmic contrac- Abbreviations: ANG, angiotensin; CSF, cerebrospinal fluid; OVLT, tions evolved (reviewed in refs. 1 and 2). Later, irradiation of organum vasculosum of the lamina terminalis; AT1, ANG receptor vertebrates from estuaries, rivers, and swamps involved pro- subtype 1; ICV, intracerebroventricular. 7397 Downloaded by guest on September 25, 2021 7398 Physiology: Denton et al. Proc. Natl. Acad. Sci. USA 93 (1996) ruminants by drooling, there is a loss of Na+ in excess of C1- the nasal cavity. The salt glands excrete intermittently in relative to the extracellular proportions. Whereas in the case response to osmotic stress (9). Sensory elements in the cardiac of Cl- in excess of sodium subtraction, the necessity of area transmit via the vagus nerve. The secretion of salt glands electrical neutrality is achieved by capacity to produce a is mediated by acetylcholine, with possibly some influence of biologically relatively inactive anion, HCO3, in the case of the hormone prolactin during transition from freshwater to excess sodium subtraction, no analogous mechanism for the estuarine habitat, analogous to its role in osmoregulation of production of a biologically inactive cation has evolved. A rise migratory fish (11). of extracellular Cl/Na ratio begins and compensatory in- In contrast, the marine mammals like whales and seals creased pulmonary ventilation with reduction of pCO2 obvi- produce a urine much more concentrated than seawater and ates, to an extent, a fall in plasma pH. However, a crucial can make a net water gain from intake of seawater and feeding mechanism in these circumstances is that the kidney excretes on marine invertebrates and plankton. Cl- with electrically equivalent amount of NH' or K+, and this Comparative Anatomy of Hypothalamus. Given its major regulation occurs even though the plasma level of Cl- may be function in body fluid control in mammals to be elaborated below the so-called "renal threshold" initially thought to below, it is salient to note comparatively that the hypothalamus regulate excretion of ions and even though there is a reduction is a major anatomical component of the brain in fishes, of the load of Cl- to the renal tubules as a result of decline of amphibians, and reptiles. The pars magnocellularis of the circulation. Thus, ionic pattern and plasma pH are preserved, nucleus praeopticus is present in fishes and above, and in compatible with life, as a result of the renal mechanism reptiles it is clearly split into the paraventricular nucleus and operating on electrolyte relativity, rather than absolute supraoptic nucleus. There is progressive differentiation of the amounts or concentrations delivered to the tubules (7, 8). cytoarchitecture of the hypothalamus in the ascending series of Osmoregulation. Notwithstanding the demands of this par- lower vertebrates embodying cellular groups in the ventral ticular facet of regulation involving pattern, the maintenance region proximate to the infundibular stalk, and the mammil- of constancy of the volume of the tissue fluids and also the lary bodies have connections, such as the mammillothalamic concentration of the total solutes in the body are of prime tract, as in higher mammals. The hypothalamus is large in these importance. The problems that land-dwelling organisms en- primitive forms relative to the brain as a whole, but this reflects counter in keeping solute concentrations constant differ from the small size of the cerebral lobes (12). those that organisms living either in the ocean or in freshwater Colonization of the Land Surface. Two of the major prob- encounter. In the latter case, seawater has a [Na] of 450-480 lems inherent with the invasion of the land areas by animal life mmol/liter and an osmotic concentration of '1000 mosmol/ were need of adequate intake and conservation of water to liter, the present composition reflecting a large change since avoid desiccation, and, likewise, in large areas of the planet, the paleozoic ocean in which metazoans evolved (4). To give the need for salt. The need of sodium can be immediate and a phylogenetic perspective, the contemporary marine teleosts paramount, given its role as the major ionic component of the fall into two clear groups according to whether they maintain circulating tissue fluids and its pivotal role in reproduction. osmotic concentration either near that of seawater or in the Water economy of terrestrial animals is intimately interwoven vicinity of one-third seawater-in parallel to the freshwater with the physiology of body temperature regulation. As well as teleosts and land animals (reviewed by Schmidt-Nielsen, ref. the hypothalamic mechanisms of thirst and water conservation 9). The former group, like the hagfish, have no large problems which we will analyze, behavioral strategies for temperature of water balance, whereas the hypoosmotic ones are at con- control include burrowing in desert environments, particularly stant risk of desiccation. The body surface and gills are not with small animals with large body surface relative to weight, completely impermeable to water. But drinking seawater, and and anatomical adaptations, such as body fur and hair. Fur- also its intake with food, results in compensatory net water thermore, in contrast to man, animals like the camel can gain, because the gills can eliminate the sodium and chloride sustain a higher body temperature (9). Thus they can, in effect, at a concentration above seawater. The elasmobranches store heat, and so have a reduced environmental heat gain- (sharks and rays) maintain ionic concentrations like most they cool off at night. The classic studies of Schmidt-Nielsen vertebrates at about one-third that of the ocean but contrive (9) showed that the camel's temperature fluctuated from as osmotic pressure equivalent to ocean by retention in the body low as 30°C in the morning to nearly 41°C in late afternoon. fluids of large amounts of urea. The blood urea of elasmo- The camel in the desert uses 0.28 liters/h of water compared branches is 100 times higher than that in mammals. However, with >1.0/h by a man weighing one-quarter as much and who this situation is not invariable for the elasmobranches, in that keeps body temperature constant at 37°C. Also, in the case of the freshwater stingray of the Amazon has blood urea similar most reptiles and birds, nitrogen from protein metabolism and to freshwater fish and correspondingly similar osmotic prob- nucleic acid is excreted by way of uric acid thus resulting in a lems (10). semisolid urine from the cloaca and large water saving. Freshwater fish with osmotic concentration of blood near Large areas of the earth-the interior of continents, the 300 mosmol/liter have the problem of osmotically determined Alps, and jungles-are sodium-impoverished. In the absence inflow of water. Smith (1) proposed the evolution of the of geological sources, marine aerosols embodied in rain are the filtering glomerular kidney with tubular reabsorption of sol- main source of sodium. The sodium content of rain diminishes utes was the phylogenetic emergent, making feasible invasion with distance from the sea coast, so that by 150-200 km, it is of the freshwater rivers and eventually land. Again, the gills, nearly absent. Soil and also plants may have extremely low and to a much lesser extent, the skin, are permeable to water, sodium content (1 mmol/kg wet weight), and, accordingly, and excretion of excess water occurs as a dilute urine. herbivorous animals may become sodium-deficient (reviewed As noted earlier, vertebrate life, as well as migrating to land, in ref. 13). Carnivores avoid the problem because of the reinvaded the ocean. Some species of air-breathing reptiles, obligatory sodium content of muscles and viscera of their prey birds, and mammals have a marine existence. This has involved (50 mmol of sodium per kg). Folklore over centuries from large intake of saltwater in the course of feeding, particularly various continents has recounted how animals will trek large if marine invertebrates, isoosmotic with the ocean, are a major distances to salt licks and springs. That severe sodium defi- food source. With reptiles and birds, the kidneys of which ciency caused this behavior was first demonstrated formally by cannot produce a urine as concentrated as seawater, there are studies of native and introduced animals in the Alps of salt-excreting glands that excrete fluid at 400-1100 meq, Australia in 1968 (14). Very low sodium content of urine, high according to species. In reptiles, it may be in the orbit of the blood levels of aldosterone and renin, and spectacular enlarge- eye, or in birds, usually above the orbit with a duct entering into ment of adrenal glands and electrolyte-reabsorbing ducts of Downloaded by guest on September 25, 2021 Physiology: Denton et al. Proc. Natl. Acad. Sci. USA 93 (1996) 7399 salivary glands was found. Sodium deficiency is a powerful Increased secretion of aldosterone determines a major selection pressure. Mechanisms to obviate the consequences mechanism in the evolutionary development of the capacity of have carried high survival value. Deficiency of sodium can the Ruminatia and Tylopoedea-the pastoral and game her- seriously compromise circulation and thus ranging efficiency bivores of the earth-to adapt successfully to the large areas of and flight from predators, and, also, the all-important biolog- continents that are sodium-deficient (13). The Na/K ratio of ical process of reproduction, where there is the need to the copious digestive salivary flow of ruminants is 170/5 sequestrate sodium in the tissues of the developing young in mmol/liter; with progressive sodium deficiency, the composi- utero and secrete it during lactation (13). Experiments with tion may change as far as to 10/160 mmol/liter and there will mice, for example, have shown that with a low-sodium diet of be corresponding change in the rumen fluid. Thus the animal 5 mmol/kg (-12-15 ,umol/day) whereas body weight is main- can, in effect, draw upon the large sodium reservoir of the tained and may increase, and the frequency of mating is rumen by means of changing over to K+, abundant in grass, as unchanged, the successful outcome of pregnancy is greatly the main cation of its digestive system (19). A striking instance compromised (Fig. 1). Sodium deficiency limits reproduction. of this remarkable adaptive process is the moose of the Isle The paramount extracerebral mechanism in control of so- Royale in Canada, where analysis of herbage of the land dium homeostasis is secretion of the salt-retaining hormone environment indicates that the sodium supply is inadequate to aldosterone by the glomerulosa of the adrenal gland. Inter alia, support the observed reproduction of the biomass. In spring/ it controls sodium excretion by the kidney. The control of early summer, the animals wade into the surrounding shallows secretion of this steroid is multifactorial (15, 16). A main and eat submerged aquatic plants, which are sodium accumu- stimulus is the peptide angiotensin (ANG II), determined by lators (500-fold greater sodium than land plants). The moose the secretion of renin by the kidney in response to sodium enter the autumn and winter, which will involve sodium deficiency. As well, reduced concentration of Na+ in the demands contingent on reproduction, with a large rumen pool adrenal arterial blood or increased concentration of K+ di- of high-sodium fluid that serves as a reservoir to be slowly rectly stimulates secretion, as does ACTH released by stress. depleted (reviewed in ref. 13). Inhibition of secretion of aldosterone in the sodium-deficient sheep is caused when sodium deficiency is corrected by the Hypothalamic Control of Vegetative Systems satiating act of rapid drinking sodium solution. In 3-5 min, sodium-deficient sheep will rapidly drink an amount of This litany of adaptive processes representative of some of the NaHCO3 solution commensurate with deficit. In sheep with diverse mechanisms controlling body fluid homeostasis pro- autotransplanted adrenal glands, it is seen that 5-25 min after vides a context for the hypothalamic vegetative elements of rapid drinking of sodium solution, the aldosterone secretion control. may fall by 30-90%. This change antecedes any change in any In the last 150 years, some seven hypothalamic-brain stem factors known to stimulate aldosterone secretion. The aldo- sensors, which monitor the milieu, have been discovered (see sterone secretion subsequently increases again over 90-120 ref. 6). Isaac Ott of Philadelphia discovered the temperature min. It falls to basal or near basal by 240 min with absorption sensor. Haldane and Priestley discovered the intracranial of the NaHCO3 from the gut. When the same amount of elements reacting to blood gases (mechanisms elucidated by sodium solution was rapidly administered to the forestomach Pappenheimer). Glucose sensors influencing hunger were of the sodium-depleted animal by tube, there was no "psychic" recognized by Anand and Brobeck, and Verney discovered the fall of aldosterone secretion. The data indicate the existence of osmoreceptors controlling antidiuretic hormone release. an unidentified hormone, which is released by cerebral events, Andersson and Olsson delineated the [Na] sensor involved in inhibiting aldosterone secretion (13). Similarly, a large imme- thirst, and we elucidated interactive control by both osmore- diate reduction of antidiuretic hormone secretion by the ceptors and [Na] sensors (20). Weisinger and colleagues posterior pituitary has been observed when water-deprived discovered the specific [Na] sensor regulating the avid sodium animals rapidly satiate thirst. This again is indicative of be- appetite in ruminants. It is highly probably that a seventh havioral events involving hypothalamic processes giving rise to sensor reactive to plasma P04 exists and determines bone systemic changes anticipatory of subsequent metabolic appetite of phosphate deficient ruminants, but the locale, changes (17, 18). (possibly in a circumventricular organ), is yet to be established (21). The phylogenetic emergence of specific appetites for water, salt, and other minerals-calcium and phosphorus (i.e., bone chewing in herbivores)-effectively takes regulatory capacities to active seeking and ingestion of substances of which the body is depleted. It was a spectacular advance over and above regulation based upon multifactorial processes of conservation in the face of deficit. Hypothalamus-sensed deviations from the normal osmotic pressure and ionic content of the milieu interieur, as well as signals from extracranial volume receptors through the hind brain, entrain diverse integrative physiolog- ical mechanisms involving powerful motivations of thirst, qV~~~~~~~~~~~~~~~~~~~~~~~~~~~~~,l specific hungers, and cravings. In effect, the behavior of animals in the wild represents the meld of instinct (genotypic memory) with learning (phenotypic memory) to give specific behavior apt to the metabolic state of the creature. FIG. 1. The effect of sodium intake on reproduction in Balb/c Some general biological aspects of thirst to note include that mice. The high-sodium food contained 120 mmol/kg. The low-sodium ANG II, the peptide which appears to be implicated in the food contained 5 mmol/kg. With low-sodium food and access to 30 genesis of thirst, or else involved as a neurotransmitter in the mM NaCl, the mice drank a mean of 2.3 ml (69 gmol of sodium per neural or day) during control conditions, and intake increased to a mean of 10.9 circuitry subserving thirst, both, evokes water drink- ml on day 18 of gestation (327 ,tmol of sodium per day; McBurnie, M., ing upon intracerebral injection in the iguana, substantiating Blair-West, J. R., D.A.D. & R.S.W., unpublished data). *, P < 0.05; * *, the thirst mechanism is developed in reptiles. ANG is an P = 0.005; and ***, P = 0.0005, low-sodium diet versus high-sodium effective dipsogen in those animals that drink water in nature, food and low-sodium food with access to 30 mM NaCl. but it is ineffective in creatures that do not. In higher mammals Downloaded by guest on September 25, 2021 7400 Physiology: Denton et al. Proc. Natl. Acad. Sci. USA 93 (1996) it has been shown that increase of systemic osmotic pressure (iii) Surprisingly, infusion of 700 mM hypertonic mannitol >2% causes drinking behavior contingent on thirst. Clearly it artificial CSF, which raised CSF osmotic pressure equivalently, is important that there be a threshold before the specific doubled sodium intake. It was found that due to the osmoti- behavior is evoked. Otherwise the animal's attention would be cally driven movement of water, CSF [Na] was reduced by aroused by any minor deviation from normal, and other 10-15 mM (25). biologically relevant, behaviors compromised by constant pre- (iv) Infusion of equiosmotic 340 mM mannitol in artificial occupation with drinking. Further, ruminants (who, as a group, CSF with [Na] = 330 mM, which increased osmolality and, due are a major component of the animal biomass) are exposed to to combined effect of the elevated [Na] of infusate and carnivores at water holes, and the ability to correct large body osmotically driven movement of water, caused no change of deficits by rapidly drinking large volumes in 2-5 min and [Na] of CSF, was without influence on sodium appetite. Thus getting out of the locale probably carries high survival advan- sodium intake was not affected by osmolality change but by tage. A camel, for example, can drink 100 liters in 10 min, and change of [Na] of CSF. sheep and cattle can correct large deficits in 2-5 min, an ability These effects on sodium appetite were replicated in sodium- subserved by the capacious rumen. deplete cows (26) and also in rats by R. Sakai and colleagues The primary impact of a sodium-deficient ecosystem falls (personal communication), but not in rabbits or mice. Increas- directly on the ruminant and herbivorous animals, and they ing the [Na] of cerebral arterial blood by infusion of hypertonic have been ideal creatures in which to study the mechanism of NaCl into the carotid arteries also reduced sodium appetite. sodium appetite. Sodium-deficient rabbits and kangaroos in Changes within the brain were operative (infusion of 1 ml/h of the Snowy Mountains of Australia will avidly select sodium hypertonic NaCl CSF having negligible effect on sodium salts from a cafeteria of NaCl, KCl, CaCl2, or MgCl2 sources balance), and this was ratified by the fact that if the rise of CSF (14). Sutcliffe and Redman have observed that elephants will [Na] caused by the intracarotid infusion was prevented by enter the Kitum and other caves of Mount Elgon in Kenya and infusion into the CSF of 700 mM hypertonic mannitol, no will proceed 100 m in pitch darkness to gouge Na2SO4 from the decrease of sodium appetite occurred (27). walls and ingest it (reviewed in refs. 13 and 22). In the Gabon, (v) Further, it was determined that infusion into the ventricle elephants in the jungle will excavate large volumes of earth of 700 mM mannitol, L-glucose, or sucrose increased sodium from under large trees and discard it and then ingest the soil appetite, whereas infusion of D-glucose, D-mannose, 2-deoxy- adjacent to the root system. Gorillas follow them at the sites D-glucose, 3-0-methyl glucose, or distilled water did not (28). and also eat the soil. Analysis has shown this root soil to be high All infusions decreased CSF [Na], but because the latter group in sodium. Questions arise as to the cognitive processes of saccharides penetrated cells and capillaries and thus the determinant of the intentions of elephants proceeding at risk blood-brain barrier, whereas the former did not, it strongly over suggested that the sensors subserving sodium appetite were boulders in pitch darkness and, also, the excavation distant from the ventricular wall, and it required a front of processes around the root system of trees before any actual soil fluid of changed composition to travel through the intercel- ingestion (C. Tutin and D.A.D., unpublished data). lular channels to deep within the neuropil. In clear contrast, all For laboratory examination of sodium appetite, an ideal of these intraventricular infusions that lower CSF [Na] inhib- method is the surgical preparation of a permanent unilateral ited the thirst induced by infusion of hypertonic NaCl into the parotid fistula in sheep or cattle. Phosphate in saliva is replaced carotid artery, suggesting the elements subserving thirst are by its addition to the diet, and thus the fistula acts to all intents very close to the ventricular wall. The sodium appetite and as a NaHCO3 tap on the blood stream, continuously producing thirst sensors are anatomically quite separate, a conclusion large sodium deficit (19). It is found that the naive animal, from functional data which is ratified by lesion studies reported which has never experienced sodium deficiency hitherto, rap- below. idly develops an appetite for sodium. The appetite is specific (vi) That infusions changing brain extracellular fluid [Na] act for sodium in a cafeteria situation of various salts. They prefer by change of intracellular [Na] is supported by the fact that NaHCO3 solution to NaCl, an apt choice in face of NaHCO3 intraventricular infusion of 2.3 mM phlorizin, which reduces loss. When presented with a solution of NaHCO3 after 1, 2, or sodium-coupled transport of glucose into cells, and thus 3 days of depletion, a sheep will rapidly (over 2-5 min) drink intracellular [Na], enhances sodium appetite, and, also, pre- an amount of sodium commensurate with deficit (P < 0.001) vents the reduction of sodium appetite caused by increasing (13). The major questions that arise, parallel to those with CSF [Na] (28). Further, push-pull microirrigation of the other aspects of ingestive behavior, are: how is an appetite anterior third ventricle (Fig. 2) with 10-6 ouabain, which would generated in the brain that is commensurate with audited body inhibit sodium and potassium ATPase and increase intracel- deficit?; and, how is it determined that the animal stops lular sodium, reduced sodium appetite, just as did raising the drinking after rapid intake of the apt amount long before the local CSF [Na] by push-pull -of 200 mM NaCl (29). material drunk could be absorbed from gut and correct any Role ofANG. Parallel to the ionic changes in the brain which blood or brain chemical changes consequent on sodium defi- would follow systemic changes in electrolytes caused by sodi- ciency, and putatively generative of the sodium appetite? (13) um-impoverished food, temperature regulation, or loss ofbody fluids, a major issue presenting in analysis of sodium appetite The Genesis of Sodium Appetite is the role of ANG. Systemically, ANG II present in plasma is formed by action of the enzyme renin, released from the Cerebral Sodium Sensors. The hypothesis that the sodium kidney, on its substrate ANG, which is formed in the liver. appetite of sodium-deplete sheep is caused by changes in the ANG I, a decapeptide, is formed first and it is converted to intracellular sodium concentration of cells subserving sodium ANG II, an octapeptide, by ANG-converting enzyme. Regard- appetite was advanced first over 30 years ago (23, 24). Evi- ing mode of formation of ANG II in the brain, the precursor dence consistent with this hypothesis has included the follow- angiotensinogen is present in neuroglia. Experiments suggest ing. that ANG may act as a neuropeptide transmitter in neurones (i) Slow intraventricular infusion of isotonic artificial cere- but whether or how it is formed from glial angiotensinogen is brospinal fluid (CSF; [Na] = 150 mM) had no influence on the unresolved (30). sodium appetite of a sodium-deficient sheep. The activities of ANG II are mediated by at least two (ii) Infusion of hypertonic 500 mM NaCl CSF, which raised different ANG II receptor subtypes-AT1 and type 2-and CSF [Na] by 10-15 mM, reduced sodium intake by 50-75% selective, nonpeptide receptor antagonists are available. ANG (25). II-induced water and sodium intakes of rats and sheep appear Downloaded by guest on September 25, 2021 Physiology: Denton et al. Proc. Natl. Acad. Sci. USA 93 (1996) 7401 The anteroventral third ventricle region AV3V includes the OVLT and the ventral median preoptic nucleus, a midline brain structure with a blood-brain barrier, located on the front wall of the third ventricle (Fig. 2). This brain area is clearly involved in body fluid homeostasis. Rats with lesion of the AV3V have impaired water drinking responses to peripheral and central administration of ANG II and to water deprivation (39), but sodium appetite induced by formalin, which causes both fluid sequestration and stress, appears to be intact (40). The complexity of the rat data may reflect the different physiological effects of different stimuli used to produce sodium appetite. Sheep with lesion of the AV3V are normal in regard to sodium intake induced by sodium depletion or by ICV infusion of hypertonic mannitol-CSF, which decreases brain extracel- lular fluid [Na]. They have an impaired water drinking re- sponse to intracarotid infusion of ANG II, water deprivation, or hypertonic stimuli and may become temporarily or perma- nently adipsic. Furthermore, in these sheep with lesion of the FIG. 2. A diagram of major sites of ANG II receptors (stippled) in AV3V, even though systemic infusion of hypertonic NaCl the mammalian brain projected onto a midsagittal diagram of the no sheep brain. The area shown in red is the lamina terminalis in the longer causes increased water intake, it continued to cause a midline anterior wall of the third ventricle, which contains osmore- decrease in sodium intake. The results clearly ratify that sponsive neurons as well as ANG II receptors. The third ventricle is different brain areas are involved in the control of thirst and shown in blue. AP, area postrema; BNST, bed nucleus of the stria sodium appetite in sheep (41). terminalis; CVLM, caudal ventrolateral medulla; DMV dorsal motor Not only do the loci of neural aggregates subserving sodium nucleus of vagus; LC, locus ceruleus; LPBN, lateral parabrachial appetite contingent on sodium deficiency (possibly lateral nucleus; LS, lateral septum; ME, median eminence; MnPO, median hypothalamic), but also their functional interaction with other preoptic nucleus; NTS, nucleus tractus solitarii; OB, olfactory bulb, centers involved in sodium appetite generated by different OVLT, organum vasculosum of the lamina terminalis; PVN, hypo- physiological circumstances, need to be thalamic paraventricular nucleus; RVLM, rostral ventrolateral me- determined. The latter dulla, SFO, subfornical organ; and SON, supraoptic nucleus. include the hedonic (need-free) appetite for salt, the salt appetite of stress caused by adrenal hormones (13), which to be mediated by predominately AT1 receptors, because appears to involve neural groups of the amygdala, and the bed intakes are blocked by losartan (30), a AT1 antagonist. nucleus of the stria terminalis (22). The locale of action of the Peripheral administration of ANG-converting enzyme in- quintet of steroid and peptide hormones determinant of the hibitors such as captopril or enalapril decreased sodium intake powerful appetite of pregnancy and lactation (13) has yet to be of sodium-deplete rats, sheep, cows, rabbits, and mice. The determined. decrease of sodium appetite of the captopril-treated sodium- With regard to satiation, ruminant animals, for example, deplete animal is obviated by peripheral administration of have the remarkable capacity to drink an amount of sodium solution in 3-5 min, which commensurately corrects body ANG II, sufficient only to give basal plasma levels. Peripheral deficit. A precipitate decline of motivation follows. Various ANG II has an important role in the genesis of sodium experiments on animals with an esophageal fistula, which appetite, which may be permissive of other severally necessary contrives that fluid drunk is lost from the neck, indicate that factors, or it may be a major factor causal of sodium appetite the satiation process involved a "gestalt" of sensory inflow (31). arising from tongue taste receptors responding to salt concen- The role of cerebral ANG II in sodium appetite is equivocal. tration, pharyngoesophageal impulses metering volume swal- In sheep and cows, intracerebroventricular (ICV) administra- lowed, and nerve afferents from the forestomach responding tion of ANG II failed to effect the decrease in sodium appetite to volume of fluid drunk and distension (13). How this cascade resulting from systemic treatment with captopril (31). In rats, of inflow caused by the consummatory act of drinking rapidly the findings are not consistent. Thunhorst and colleagues (32) and completely inhibits the neural systems hitherto exciting were unable to demonstrate a role of brain ANG II in sodium sodium appetite is unknown. It is established that intraven- appetite, whereas Sakai and coworkers found brain-generated tricular infusion of several neuropeptides-somatostatin (42), ANG II was causal (33). Epstein (34) proposes that in the rat, atrial natriuretic peptide (43), and basic fibroblast growth sodium appetite is generated in the brain by a synergistic action factor (44)-will inhibit sodium appetite, but whether they of cerebrally derived ANG II and aldosterone. The controversy have a physiological role is not known. regarding the contribution of peripheral and central ANG II to sodium depletion-induced sodium appetite has yet to be Thirst and Sodium Excretion resolved. Cerebral Regulation ofWater Intake and Sodium Sodium intake of sodium-deplete sheep or cows is not Excretion. Sodium homeostasis is inextricably linked with body water decreased by ICV infusion of captopril or saralasin or losartan balance. When an animal becomes dehydrated, the [Na] and suggesting II not that brain ANG is involved in sodium appetite osmolality of extracellular fluid increase. If water is available, either directly or indirectly in these species (31, 35). However, it can be ingested, but if it is not accessible, sodium may be captopril and losartan may cross the blood-brain barrier, and, excreted by the kidneys. In the first case, both extracellular therefore, it is possible that the neural elements subserving volume and [Na] will be restored to normal, whereas in the sodium appetite, which are some distance from the ventricle, second case, body fluid volumes remain depressed, but the rise are not accessed by ICV infusion. in [Na] is ameliorated. Not surprisingly, brain regions involved Where are the Sensors? Sodium appetite caused by the in the regulation of thirst also have a role in the regulation of natriuretic agent furosemide was decreased or abolished in rats renal sodium excretion. with lesion of the subfornical organ (36, 37) or the organum Early this century, Mayer (45) concluded that thirst was vasculosum of the lamina terminalis (OVLT; ref. 38). related to the osmotic pressure of blood. Systemic infusions of Downloaded by guest on September 25, 2021 7402 Physiology: Denton et aL Proc. Natl. Acad. Sci. USA 93 (1996) concentrated solutions of NaCl or various saccharides (but not location of the sodium sensors remains to be determined, D-glucose) caused drinking behavior. However, similarly con- though permanent adipsia in animals and humans results from centrated solutions of urea or glycerol were considerably less lesions, which encompass completely the lamina terminalis effective dipsogens (46, 47). NaCl or sucrose molecules do not and surrounding preoptic/anterior hypothalamic tissue (59). penetrate cell membranes rapidly and cause an osmotic gra- ANG-Induced Water Intake. As well as thirst of cellular dient across cell membranes and thus cellular dehydration. dehydration, depletion of the extracellular fluid leads to However, urea or glycerol molecules traverse cell membranes increased water intake and dilution of the extracellular fluid relatively quickly, so there is no osmotic gradient and little [Na]. While neural input carried by vagal afferents from cellular dehydration. Hyperosmolar solutions that induce cel- thoracic volume sensors plays a role in the drinking, extracel- lular dehydration cause thirst, and the concept of sensors lular fluid depletion causes renin secretion, and the resulting (osmoreceptors) responding to cellular dehydration was pro- octapeptide ANG II delivered to the brain in the bloodstream posed (48, 49). As to the bodily location, although there is is also dipsogenic (60). Fitzsimons and colleagues showed that evidence of hepatic osmoreceptors, the major site of detection ANG II was dipsogenic when administered systemically and of changes in the tonicity of body fluids is the preoptic/ especially intracerebrally-a pivotal discovery (reviewed in hypothalamic region of the brain (48), as Verney determined ref. 60). Circulating ANG II acts mainly on the subfornical in elucidating the control of antidiuretic hormone, the water- organ for the stimulation of drinking behavior, thus circum- conserving peptide secreted from the neurohypophysis. venting the blood-brain barrier, which excludes peptides from In regard to thirst, Andersson showed that injection of brain interstitium (61). Autoradiographic binding studies in a microliter quantities of hypertonic saline into the hypothala- number of species, hybridization histochemistry, and immu- mus of the goat induced them to drink large amounts of water nohistochemistry have shown that ANG II receptors (of the (50). When hypertonic solutions of NaCl or saccharides were AT1 subtype) are distributed throughout the lamina terminalis administered into the CSF of the third ventricle, only hyper- (OVLT and median preoptic nucleus) and many other sites in tonic sodium salts elicited water drinking and vasopressin the central nervous system associated with body fluid ho- secretion (47). They proposed that the osmoreceptors were meostasis and cardiovascular control (e.g., hypothalamic para- really [Na] sensors in the anterior wall of the third ventricle. ventricular nucleus, parabrachial nucleus, rostral and caudal We investigated this hypothesis in conscious sheep and found ventrolateral medulla, nucleus of the solitary tract, and inter- that, if prepared in artificial CSF with normal sodium con- mediolateral cell column of the thoraco-lumbar spinal cord) centration, injection of hypertonic sucrose into the third (62). The endogenous ligand for these receptors is probably an ventricle did elicit drinking, but the response was considerably ANG of cerebral origin (62). Angiotensinergic circuits ema- less than that obtained with injection of equiosmolar hyper- nating from the subfornical organ to preoptic and hypothala- tonic NaCl (51). We proposed that both osmoreceptors and mus regions have been shown by electrophysiological and sodium sensors existed in the brain. In a further investigation immunohistochemical studies (63). Intravenous ANG II in- in sheep, we observed that infusions of 1 M NaCl, 2 M sucrose duces c-fos expression in the OVLT, just as in the subfornical or fructose, and 2-4.6 M urea in the carotid artery of sheep all organ, although an OVLT role in ANG-induced drinking increased the [Na] of ventricular CSF because the blood-brain behavior remains to be demonstrated (64). Further, different barrier excludes all these molecules from the brain. Thus, populations of neurons seem to be excited by hypertonicity hyperosmolar urea is similar to hypertonic NaCl or sucrose in than are by ANG II, in both the subfornical organ and OVLT that when it is infused systemically it dehydrates cells in the (Fig. 3). brain. Yet hypertonic urea is relatively a poor dipsogen. To Recently, we found in five species that centrally adminis- explain this result, we proposed that the relevant osmorecep- tered AT1 receptor antagonist losartan blocks drinking initi- tors were situated in a region(s) lacking a blood-brain barrier, ated by intraventricular infusion of hypertonic saline (65), and and suggested the OVLT and/or subfornical organ as likely in sheep natriuresis and vasopressin secretion, induced by sites (20). central hypertonic saline, is also blocked by losartan (66). The Consistent with this idea, ablation of brain tissue in the data point to the involvement of angiotensinergic pathways in region of the ventral lamina terminalis, which included the the central organization of osmoregulatory responses, but the OVLT, did disrupt osmoregulatory water drinking (39, 52, 53). locus of action of losartan is unknown. Ablation of the subfornical organ may also reduce osmotically Osmoregulatory Sodium Excretion. Despite a reduction of stimulated drinking in sheep (54), as does ablation of the extracellular volume, dehydration results in a natriuretic re- adjacent median preoptic nucleus (55), which receives rich sponse in many mammals (54). This dehydration-induced neural input from both circumventricular organs. Neurons in natriuresis is under central control because it can be abolished both the OVLT and subfornical organ, have been shown by if the lamina terminalis is ablated, or if the [Na] within the electrophysiological techniques to increase activity in response periventricular brain tissue is prevented from increasing by to a local increase in tonicity (56, 57). Single unit recordings means of slow ICV infusion of isotonic or hypertonic mannitol from neurons in the median preoptic nucleus show that they solutions in dehydrated sheep (54, 67). Gross systemic hyper- are responsive to systemic hypertonicity, although not neces- natraemia results with these experiments showing the impor- sarily directly stimulated by it (57). Recently, mapping of tant hemostatic role (54). In sheep, ablation of brain tissue in expression of the immediate early gene c-fos shows that the region of the lamina terminalis also inhibits the excretion populations of neurons in the periphery of the subfornical of i.v. hypertonic NaCl loads but does not affect the excretion organ, in the dorsal part of the OVLT, and throughout the of intravenous volume loads of isotonic saline (68). Conversely, median preoptic nucleus are activated in response to i.v. it has been shown that injection of hypertonic saline into the infusion of hypertonic solutions or dehydration (57). third cerebral ventricle increases sodium excretion greatly, and In addition to osmoreceptors in the subfornical organ and push-pull perfusion of hypertonic saline in the anterodorsal OVLT, periventricular sodium sensors may also play a role in part of the ventricle adjacent to the median preoptic nucleus detecting changes in systemic tonicity. Thus reduction of CSF is the most effective site for inducing such a natriuretic [Na] by 10-15 mM by means of ICV infusion of isoosmotic or response (69). Reduction of CSF [Na] also inhibits the excre- hyperosmotic sugars or mannitol can severely inhibit drinking tion of hypertonic NaCl loads, as well as abolishing the responses to water deprivation or systemically infused hyper- "escape" from mineralocorticoid-induced sodium retention, tonic saline (47, 58). This suggests that the ambient [Na] in the which causes increased CSF [Na]. Thus, when the [Na] and brain is sensed and interacts with information from osmore- osmolality of the extracellular fluid increase, the ambient [Na] ceptors in the circumventricular organ. However, the precise within the brain also increases and this is detected by tissue in Downloaded by guest on September 25, 2021 Physiology: Denton et aL Proc. Natl. Acad. Sci. USA 93 (1996) 7403 orientated to a physiologically apt behavior. The cognitive A C -''''- process involves past experience relevant to satiation of thirst, salt hunger, or for that matter, whatever appetite (e.g., hunger or sex), the midbrain-limbic systems may be generating. Con- sideration of these mesencephalic structures in the context of the phylogenetic emergence of consciousness raises basic questions. Edelman (75) has argued cogently in relation to the phylogenetic emergence of consciousness: "It is the evolution- ary development of the ability to create a scene that led to the ovft . .... emergence of primary consciousness-for this to have sur- vived, it must have resulted in increased fitness." He emphasized also that categorization by the thalamocor- tical system so as to "organize different perceptions is deeply rV influenced by limbic-brain stem systems that confer values of or biological relevance." But basically his proposal suggests that primary consciousness comes from processing of distance ---r * W.: ; -. .; .: receptor input ("helps to abstract and organize complex t c 0A -' '. E. :. .:.- changes in an environment involving multiple parallel signals S::: with reentry of value category memory"), and by a scene he FIG. 3. Photomicrographs of coronal sections through the orga- proposes a spatiotemporal ordered set of categorizations of num vasculosum of the lamina terminalis (ovlt) in A and B and familiar and unfamiliar events, some with and some without subfornical organ (sfo) in C and D of rats showing the distribution of necessary physical or causal connections to others in the same Fos detected by immunohistochemistry in the nucleus of cells and seen scene. The issue that the limbic-brain stem systems may dictate as the brown dots. Fos, the .:protein .. .. : ...... encoded . : ... by the protooncogene saliency or biological relevance, seemingly a key component c-fos, is a marker of increased neuronal activity. InA and C, the rat had within the primal conscious process, merits the question of an been infused i.v. with ANG II (1 ,ug/h for 2 h), and neurons at the periphery of the ovlt (arrowheads) and throughout the subfornical alternate viewpoint on the first emergence of dim awareness. organ have been activated. In B and D, the rat had been infused i.v. It is feasible its genesis may have been in sensations and primal with hypertonic saline (5.5 ml/kg of 1.5 M NaCl) and increased activity compelling emotions determined by internal sensors and re- was observed in neurons in the dorsal cap of the ovlt and the periphery ceptors of the vegetative systems signaling threat to exis- of the subfornical organ. Data from experiments described in refs. 57 tence-e.g., hunger for air, thirst in the face of desiccation of and 64. hc, hippocampal commissure; oc, optic chiasm; or, optic recess the body, hunger (including specific hungers), extreme tem- of the third ventricle; and v, third ventricle. Scale bar = 200 ,um. perature change, and pain, all to a considerable extent involv- ing the phylogenetically ancient areas of the brain, including the region of the lamina terminalis which signals the kidney to hypothalamus and reticular activating system. initiate a natriuretic response. The signaling mechanism to the Many biologists entertain the possibility of conscious pro- kidney is still to be fully understood, but it is almost certainly cesses in animals lower in the phylogenetic scale than mammals hormonal, because renal denervation does not prevent such (1, 75, 76, 77). Pertinent to this, the spinoreticular system centrally mediated natriuresis (67, 68). Adrenalectomy or carrying pain fibers is conspicuous in vertebrates at the level hypophysectomy do not prevent dehydration-induced natri- of fish, amphibians, and reptiles. The reticular system is uresis, and neither can the response be attributed to increases present in all vertebrates (78, 79), and certain components of in atrial natriuretic peptide secretion or reduced activity of the the limbic system such as septum and habenula are recogniz- renin-ANG system (70). able in all vertebrates (80). The thesis that these vegetative The lamina terminalis may also play a role in the regulation systems, which antecede much cortical development were of renin secretion from the kidney. Reduction of CSF [Na] indeed involved in genesis of primal awareness, gives close increases plasma renin levels in dogs (71) and sheep (72), conjunction to the fact of these phylogenetically ancient areas whereas an increase in the tonicity of carotid arterial blood of the brain having a plenipotentiary role in higher mammals reduces it in conscious sheep, an effect abolished by ablation in the orchestration of arousal, focus of attention, intention of the lamina terminalis (73). Consequently, we have proposed and also sleep, and complex neuroanatomical pathways be- that there is an inhibitory cerebral osmoregulatory influence tween these structures and the thalamus and cortex subserve on renin secretion by the kidney (73). Consistent with this these functions. It is, of course, possible that rather than either proposal was the observation that dehydrated sheep with interceptor-generated sensation of threat to existence or mul- lesions encompassing the anterior wall of the third ventricle tiple parallel signals from distance receptors having temporal have inappropriately high plasma renin concentrations despite primacy in phylogenesis of consciousness, there was contem- their grossly hypernatremic state (70). ICV infusion of ANG poraneity of influence in this physiological emergent of very II strongly depresses plasma renin levels, an effect abolished by high survival value. ablation of the lamina terminalis (70), while centrally admin- istered AT1 antagonist losartan increases plasma renin con- This work has been supported by the National Health and Medical centration (66) and blocks the inhibitory effect of increasing Research Council of Australia, The Howard Florey Biomedical Foun- the ambient [Na] in the brain (74). 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