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

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Scienceselected on April 25, 1995.

Hypothalamic integration of body fluid regulationD. A. DENTON, M. J. MCKINLEY, AND R. S. WEISINGERHoward 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 thepaleozoic ocean to rivers and diverse econiches on the planet'ssurface, as well as the subsequent reinvasion of the ocean,involved many different stresses on ionic pattern, osmoticpressure, and volume of the extracellular fluid bathing bodycells. The relatively constant ionic pattern of vertebratesreflects a genetic "set" of many regulatory mechanisms-particularly renal regulation. Renal regulation of ionic pat-tern when loss of fluid from the body is disproportionaterelative to the extracellular fluid composition (e.g., gastricjuice with vomiting and pancreatic secretion with diarrhea)makes manifest that a mechanism to produce a biologicallyrelatively inactive extracellular anion HCO3 exists, whereasno comparable mechanism to produce a biologically inactivecation has evolved. Life in the ocean, which has three times thesodium concentration of extracellular fluid, involves quitedifferent osmoregulatory stress to that in freshwater. Terres-trial life involves risk of desiccation and, in large areas of theplanet, salt deficiency. Mechanisms integrated in the hypo-thalamus (the evolutionary ancient midbrain) control waterretention and facilitate excretion of sodium, and also controlthe secretion of renin by the kidney. Over and above themultifactorial processes of excretion, hypothalamic sensorsreacting to sodium concentration, as well as circumventricu-lar organs sensors reacting to osmotic pressure and angio-tensin II, subserve genesis of sodium hunger and thirst. Thesebehaviors spectacularly augment the adaptive capacities ofanimals. Instinct (genotypic memory) and learning (pheno-typic memory) are melded to give specific behavior apt to themetabolic status of the animal. The sensations, compellingemotions, and intentions generated by these vegetative sys-tems focus the issue of the phylogenetic emergence of con-sciousness and whether primal awareness initially came fromthe interoreceptors and vegetative systems rather than thedistance receptors.

In the higher mammals, the functions centered in the hypo-thalamus play a paramount role in integrating the manyphysiological systems controlling the milieu interieur. Thesehypothalamic processes range from genetically determinedpatterns of ingestive behavior that correct body deficits and, inturn, involve associated cognitive and memory functions of thecortex, to the other extreme of the control of excretoryprocesses, in a mode apt to the metabolic status of the animal.Some evolutionary aspects of body fluid control will bedescribed first as a general biological context of the mecha-nisms in mammals.Mountain building, like the Grand Canyon uplift in the

Cambrian and subsidence in the Ordivician periods, providedconditions of rivers flowing into the ocean, and, probablyduring this time, protovertebrates with spindle body andsegmentally arranged muscles adapted to rhythmic contrac-tions evolved (reviewed in refs. 1 and 2). Later, irradiation ofvertebrates from estuaries, rivers, and swamps involved pro-

gressive invasion of diverse regions of the planet-jungles, thedry interior of continents, deserts, savannah, temperate for-ests, and the Alps. Also, there was reinvasion of the oceans, allmigrations entailing diverse environmental influences withpotential to distort the stability of the fluid milieu of the tissuesof the organism.

Constancy of the Milieu

The constancy of the milieu interieur, the condition of free life,involves, inter alia, maintenance of a pattern of ionic compo-nents. John Fulton (3), in his book Selected Readings in theHistory of Physiology, pointed out that MacCallum (4) gavehistorical and phylogenetic meaning to the constancy of themilieu interieur when he demonstrated the striking resem-blance between the ionic components of the blood sera andthat of seawater, and, in particular, the ionic ratios. Hesuggested that tissue cells could only live within a relativelynarrow range of physicochemical conditions that representedthat of the ancient ocean, in which the cells of ancestralorganisms had originated. As Metazoan organisms evolvedand developed a closed circulatory system, they evolved organsthat regulated and kept the ionic pattern constant regardlessof changes the external milieu underwent with time. Thecomparative constancy of this pattern of the extracellularfluids from elasmobranchs to Australian marsupials and themammals (5, 6) reflects a basic genetic characteristic of animallife, reflecting the "set" of a number of regulatory systems-particularly renal regulation.With addition stress, as a result of an excess of a substance

entering the milieu, the rise in plasma concentration or load tothe renal tubules results in regulatory excretion.A broad evolutionary implication of pattern constancy

emerges clearly in the face of subtraction stress distorting theionic pattern, as can occur in higher mammals as a result ofdisease or, for that matter, human medical problems. Forexample, the stomach secretes fluid that has Cl- in large excessof Na (Cl/Na, -4-5), relative to the normal extracellularproportions (Cl/Na, 0.7), HI being the electrically equivalentcation. With large loss of acid gastric juice by vomiting, theplasma level of Cl- falls and the level of HCOj3, an anionconstantly produced in the body, may rise reciprocally 10-20meq/liter, the plasma pH being maintained near constant bya compensatory rise in pCO2 as a result of regulatory depres-sion of respiration. The replacement of plasma Cl- by arelatively biologically inactive anion, HCO-, obviates thephysiological need to excrete significant amounts of sodium inthe urine to control the pattern. In striking contrast, with lossof alkaline pancreatic secretion as a result of diarrhea orintestinal fistulae or loss of parotid salivary secretion in

Abbreviations: ANG, angiotensin; CSF, cerebrospinal fluid; OVLT,organum vasculosum of the lamina terminalis; AT1, ANG receptorsubtype 1; ICV, intracerebroventricular.

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ruminants by drooling, there is a loss of Na+ in excess of C1-relative to the extracellular proportions. Whereas in the caseof Cl- in excess of sodium subtraction, the necessity ofelectrical neutrality is achieved by capacity to produce abiologically relatively inactive anion, HCO3, in the case ofexcess sodium subtraction, no analogous mechanism for theproduction of a biologically inactive cation has evolved. A riseof extracellular Cl/Na ratio begins and compensatory in-creased pulmonary ventilation with reduction of pCO2 obvi-ates, to an extent, a fall in plasma pH. However, a crucialmechanism in these circumstances is that the kidney excretesCl- with electrically equivalent amount of NH' or K+, and thisregulation occurs even though the plasma level of Cl- may bebelow the so-called "renal threshold" initially thought toregulate excretion of ions and even though there is a reductionof the load of Cl- to the renal tubules as a result of decline ofcirculation. Thus, ionic pattern and plasma pH are preserved,compatible with life, as a result of the renal mechanismoperating on electrolyte relativity, rather than absoluteamounts or concentrations delivered to the tubules (7, 8).

Osmoregulation. Notwithstanding the demands of this par-ticular facet of regulation involving pattern, the maintenanceof constancy of the volume of the tissue fluids and also theconcentration of the total solutes in the body are of primeimportance. The problems that land-dwelling organisms en-counter in keeping solute concentrations constant differ fromthose that organisms living either in the ocean or in freshwaterencounter. In the latter case, seawater has a [Na] of 450-480mmol/liter and an osmotic concentration of '1000 mosmol/liter, the present composition reflecting a large change sincethe paleozoic ocean in which metazoans evolved (4). To givea phylogenetic perspective, the contemporary marine teleostsfall into two clear groups according to whether they maintainosmotic concentration either near that of seawater or in thevicinity of one-third seawater-in parallel to the freshwaterteleosts and land animals (reviewed by Schmidt-Nielsen, ref.9). The former group, like the hagfish, have no large problemsof water balance, whereas the hypoosmotic ones are at con-stant risk of desiccation. The body surface and gills are notcompletely impermeable to water. But drinking seawater, andalso its intake with food, results in compensatory net watergain, because the gills can eliminate the sodium and chlorideat a concentration above seawater. The elasmobranches(sharks and rays) maintain ionic concentrations like mostvertebrates at about one-third that of the ocean but contriveosmotic pressure equivalent to ocean by retention in the bodyfluids of large amounts of urea. The blood urea of elasmo-branches is 100 times higher than that in mammals. However,this situation is not invariable for the elasmobranches, in thatthe freshwater stingray of the Amazon has blood urea similarto freshwater fish and correspondingly similar osmotic prob-lems (10).

Freshwater fish with osmotic concentration of blood near300 mosmol/liter have the problem of osmotically determinedinflow of water. Smith (1) proposed the evolution of thefiltering glomerular kidney with tubular reabsorption of sol-utes was the phylogenetic emergent, making feasible invasionof the freshwater rivers and eventually land. Again, the gills,and to a much lesser extent, the skin, are permeable to water,and excretion of excess water occurs as a dilute urine.As noted earlier, vertebrate life, as well as migrating to land,

reinvaded the ocean. Some species of air-breathing reptiles,birds, and mammals have a marine existence. This has involvedlarge intake of saltwater in the course of feeding, particularlyif marine invertebrates, isoosmotic with the ocean, are a majorfood source. With reptiles and birds, the kidneys of whichcannot produce a urine as concentrated as seawater, there aresalt-excreting glands that excrete fluid at 400-1100 meq,according to species. In reptiles, it may be in the orbit of theeye, or in birds, usually above the orbit with a duct entering into

the nasal cavity. The salt glands excrete intermittently inresponse to osmotic stress (9). Sensory elements in the cardiacarea transmit via the vagus nerve. The secretion of salt glandsis mediated by acetylcholine, with possibly some influence ofthe hormone prolactin during transition from freshwater toestuarine habitat, analogous to its role in osmoregulation ofmigratory fish (11).

In contrast, the marine mammals like whales and sealsproduce a urine much more concentrated than seawater andcan make a net water gain from intake of seawater and feedingon marine invertebrates and plankton.Comparative Anatomy of Hypothalamus. Given its major

function in body fluid control in mammals to be elaboratedbelow, it is salient to note comparatively that the hypothalamusis a major anatomical component of the brain in fishes,amphibians, and reptiles. The pars magnocellularis of thenucleus praeopticus is present in fishes and above, and inreptiles it is clearly split into the paraventricular nucleus andsupraoptic nucleus. There is progressive differentiation of thecytoarchitecture of the hypothalamus in the ascending series oflower vertebrates embodying cellular groups in the ventralregion proximate to the infundibular stalk, and the mammil-lary bodies have connections, such as the mammillothalamictract, as in higher mammals. The hypothalamus is large in theseprimitive forms relative to the brain as a whole, but this reflectsthe small size of the cerebral lobes (12).

Colonization of the Land Surface. Two of the major prob-lems inherent with the invasion of the land areas by animal lifewere need of adequate intake and conservation of water toavoid desiccation, and, likewise, in large areas of the planet,the need for salt. The need of sodium can be immediate andparamount, given its role as the major ionic component of thecirculating tissue fluids and its pivotal role in reproduction.Water economy of terrestrial animals is intimately interwovenwith the physiology of body temperature regulation. As well asthe hypothalamic mechanisms of thirst and water conservationwhich we will analyze, behavioral strategies for temperaturecontrol include burrowing in desert environments, particularlywith small animals with large body surface relative to weight,and anatomical adaptations, such as body fur and hair. Fur-thermore, in contrast to man, animals like the camel cansustain a higher body temperature (9). Thus they can, in effect,store heat, and so have a reduced environmental heat gain-they cool off at night. The classic studies of Schmidt-Nielsen(9) showed that the camel's temperature fluctuated from aslow as 30°C in the morning to nearly 41°C in late afternoon.The camel in the desert uses 0.28 liters/h of water comparedwith >1.0/h by a man weighing one-quarter as much and whokeeps body temperature constant at 37°C. Also, in the case ofmost reptiles and birds, nitrogen from protein metabolism andnucleic acid is excreted by way of uric acid thus resulting in asemisolid urine from the cloaca and large water saving.Large areas of the earth-the interior of continents, the

Alps, and jungles-are sodium-impoverished. In the absenceof geological sources, marine aerosols embodied in rain are themain source of sodium. The sodium content of rain diminisheswith distance from the sea coast, so that by 150-200 km, it isnearly absent. Soil and also plants may have extremely lowsodium content (1 mmol/kg wet weight), and, accordingly,herbivorous animals may become sodium-deficient (reviewedin ref. 13). Carnivores avoid the problem because of theobligatory sodium content of muscles and viscera of their prey(50 mmol of sodium per kg). Folklore over centuries fromvarious continents has recounted how animals will trek largedistances to salt licks and springs. That severe sodium defi-ciency caused this behavior was first demonstrated formally bystudies of native and introduced animals in the Alps ofAustralia in 1968 (14). Very low sodium content of urine, highblood levels of aldosterone and renin, and spectacular enlarge-ment of adrenal glands and electrolyte-reabsorbing ducts of

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salivary glands was found. Sodium deficiency is a powerfulselection pressure. Mechanisms to obviate the consequenceshave carried high survival value. Deficiency of sodium canseriously compromise circulation and thus ranging efficiencyand flight from predators, and, also, the all-important biolog-ical process of reproduction, where there is the need tosequestrate sodium in the tissues of the developing young inutero and secrete it during lactation (13). Experiments withmice, for example, have shown that with a low-sodium diet of5 mmol/kg (-12-15 ,umol/day) whereas body weight is main-tained and may increase, and the frequency of mating isunchanged, the successful outcome of pregnancy is greatlycompromised (Fig. 1). Sodium deficiency limits reproduction.The paramount extracerebral mechanism in control of so-

dium homeostasis is secretion of the salt-retaining hormonealdosterone by the glomerulosa of the adrenal gland. Inter alia,it controls sodium excretion by the kidney. The control ofsecretion of this steroid is multifactorial (15, 16). A mainstimulus is the peptide angiotensin (ANG II), determined bythe secretion of renin by the kidney in response to sodiumdeficiency. As well, reduced concentration of Na+ in theadrenal arterial blood or increased concentration of K+ di-rectly stimulates secretion, as does ACTH released by stress.Inhibition of secretion of aldosterone in the sodium-deficientsheep is caused when sodium deficiency is corrected by thesatiating act of rapid drinking sodium solution. In 3-5 min,sodium-deficient sheep will rapidly drink an amount ofNaHCO3 solution commensurate with deficit. In sheep withautotransplanted adrenal glands, it is seen that 5-25 min afterrapid drinking of sodium solution, the aldosterone secretionmay fall by 30-90%. This change antecedes any change in anyfactors known to stimulate aldosterone secretion. The aldo-sterone secretion subsequently increases again over 90-120min. It falls to basal or near basal by 240 min with absorptionof the NaHCO3 from the gut. When the same amount ofsodium solution was rapidly administered to the forestomachof the sodium-depleted animal by tube, there was no "psychic"fall of aldosterone secretion. The data indicate the existence ofan unidentified hormone, which is released by cerebral events,inhibiting aldosterone secretion (13). Similarly, a large imme-diate reduction of antidiuretic hormone secretion by theposterior pituitary has been observed when water-deprivedanimals rapidly satiate thirst. This again is indicative of be-havioral events involving hypothalamic processes giving rise tosystemic changes anticipatory of subsequent metabolicchanges (17, 18).

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FIG. 1. The effect of sodium intake on reproduction in Balb/cmice. The high-sodium food contained 120 mmol/kg. The low-sodiumfood contained 5 mmol/kg. With low-sodium food and access to 30mM NaCl, the mice drank a mean of 2.3 ml (69 gmol of sodium perday) during control conditions, and intake increased to a mean of 10.9ml on day 18 of gestation (327 ,tmol of sodium per day; McBurnie, M.,Blair-West, J. R., D.A.D. & R.S.W., unpublished data). *,P < 0.05; * *,P = 0.005; and ***, P = 0.0005, low-sodium diet versus high-sodiumfood and low-sodium food with access to 30 mM NaCl.

Increased secretion of aldosterone determines a majormechanism in the evolutionary development of the capacity ofthe Ruminatia and Tylopoedea-the pastoral and game her-bivores of the earth-to adapt successfully to the large areas ofcontinents that are sodium-deficient (13). The Na/K ratio ofthe copious digestive salivary flow of ruminants is 170/5mmol/liter; with progressive sodium deficiency, the composi-tion may change as far as to 10/160 mmol/liter and there willbe corresponding change in the rumen fluid. Thus the animalcan, in effect, draw upon the large sodium reservoir of therumen by means of changing over to K+, abundant in grass, asthe main cation of its digestive system (19). A striking instanceof this remarkable adaptive process is the moose of the IsleRoyale in Canada, where analysis of herbage of the landenvironment indicates that the sodium supply is inadequate tosupport the observed reproduction of the biomass. In spring/early summer, the animals wade into the surrounding shallowsand eat submerged aquatic plants, which are sodium accumu-lators (500-fold greater sodium than land plants). The mooseenter the autumn and winter, which will involve sodiumdemands contingent on reproduction, with a large rumen poolof high-sodium fluid that serves as a reservoir to be slowlydepleted (reviewed in ref. 13).

Hypothalamic Control of Vegetative Systems

This litany of adaptive processes representative of some of thediverse mechanisms controlling body fluid homeostasis pro-vides a context for the hypothalamic vegetative elements ofcontrol.

In the last 150 years, some seven hypothalamic-brain stemsensors, which monitor the milieu, have been discovered (seeref. 6). Isaac Ott of Philadelphia discovered the temperaturesensor. Haldane and Priestley discovered the intracranialelements reacting to blood gases (mechanisms elucidated byPappenheimer). Glucose sensors influencing hunger wererecognized by Anand and Brobeck, and Verney discovered theosmoreceptors controlling antidiuretic hormone release.Andersson and Olsson delineated the [Na] sensor involved inthirst, and we elucidated interactive control by both osmore-ceptors and [Na] sensors (20). Weisinger and colleaguesdiscovered the specific [Na] sensor regulating the avid sodiumappetite in ruminants. It is highly probably that a seventhsensor reactive to plasma P04 exists and determines boneappetite of phosphate deficient ruminants, but the locale,(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., bonechewing in herbivores)-effectively takes regulatory capacitiesto active seeking and ingestion of substances of which the bodyis depleted. It was a spectacular advance over and aboveregulation based upon multifactorial processes of conservationin the face of deficit. Hypothalamus-sensed deviations fromthe normal osmotic pressure and ionic content of the milieuinterieur, as well as signals from extracranial volume receptorsthrough the hind brain, entrain diverse integrative physiolog-ical mechanisms involving powerful motivations of thirst,specific hungers, and cravings. In effect, the behavior ofanimals in the wild represents the meld of instinct (genotypicmemory) with learning (phenotypic memory) to give specificbehavior apt to the metabolic state of the creature.Some general biological aspects of thirst to note include that

ANG II, the peptide which appears to be implicated in thegenesis of thirst, or else involved as a neurotransmitter in theneural circuitry subserving thirst, or both, evokes water drink-ing upon intracerebral injection in the iguana, substantiatingthe thirst mechanism is developed in reptiles. ANG is aneffective dipsogen in those animals that drink water in nature,but it is ineffective in creatures that do not. In higher mammals

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it has been shown that increase of systemic osmotic pressure>2% causes drinking behavior contingent on thirst. Clearly itis important that there be a threshold before the specificbehavior is evoked. Otherwise the animal's attention would bearoused by any minor deviation from normal, and otherbiologically relevant, behaviors compromised by constant pre-occupation with drinking. Further, ruminants (who, as a group,are a major component of the animal biomass) are exposed tocarnivores at water holes, and the ability to correct large bodydeficits by rapidly drinking large volumes in 2-5 min andgetting out of the locale probably carries high survival advan-tage. A camel, for example, can drink 100 liters in 10 min, andsheep and cattle can correct large deficits in 2-5 min, an abilitysubserved by the capacious rumen.The primary impact of a sodium-deficient ecosystem falls

directly on the ruminant and herbivorous animals, and theyhave been ideal creatures in which to study the mechanism ofsodium appetite. Sodium-deficient rabbits and kangaroos inthe Snowy Mountains of Australia will avidly select sodiumsalts from a cafeteria of NaCl, KCl, CaCl2, or MgCl2 sources(14). Sutcliffe and Redman have observed that elephants willenter the Kitum and other caves of Mount Elgon in Kenya andwill proceed 100 m in pitch darkness to gouge Na2SO4 from thewalls and ingest it (reviewed in refs. 13 and 22). In the Gabon,elephants in the jungle will excavate large volumes of earthfrom under large trees and discard it and then ingest the soiladjacent to the root system. Gorillas follow them at the sitesand also eat the soil. Analysis has shown this root soil to be highin sodium. Questions arise as to the cognitive processesdeterminant of the intentions of elephants proceeding at riskover boulders in pitch darkness and, also, the excavationprocesses around the root system of trees before any actual soilingestion (C. Tutin and D.A.D., unpublished data).For laboratory examination of sodium appetite, an ideal

method is the surgical preparation of a permanent unilateralparotid fistula in sheep or cattle. Phosphate in saliva is replacedby its addition to the diet, and thus the fistula acts to all intentsas a NaHCO3 tap on the blood stream, continuously producinglarge sodium deficit (19). It is found that the naive animal,which has never experienced sodium deficiency hitherto, rap-idly develops an appetite for sodium. The appetite is specificfor sodium in a cafeteria situation of various salts. They preferNaHCO3 solution to NaCl, an apt choice in face of NaHCO3loss. When presented with a solution of NaHCO3 after 1, 2, or3 days of depletion, a sheep will rapidly (over 2-5 min) drinkan amount of sodium commensurate with deficit (P < 0.001)(13). The major questions that arise, parallel to those withother aspects of ingestive behavior, are: how is an appetitegenerated in the brain that is commensurate with audited bodydeficit?; and, how is it determined that the animal stopsdrinking after rapid intake of the apt amount long before thematerial drunk could be absorbed from gut and correct anyblood or brain chemical changes consequent on sodium defi-ciency, and putatively generative of the sodium appetite? (13)

The Genesis of Sodium Appetite

Cerebral Sodium Sensors. The hypothesis that the sodiumappetite of sodium-deplete sheep is caused by changes in theintracellular sodium concentration of cells subserving sodiumappetite was advanced first over 30 years ago (23, 24). Evi-dence consistent with this hypothesis has included the follow-ing.

(i) Slow intraventricular infusion of isotonic artificial cere-brospinal fluid (CSF; [Na] = 150 mM) had no influence on thesodium appetite of a sodium-deficient sheep.

(ii) Infusion of hypertonic 500 mM NaCl CSF, which raisedCSF [Na] by 10-15 mM, reduced sodium intake by 50-75%(25).

(iii) Surprisingly, infusion of 700 mM hypertonic mannitolartificial CSF, which raised CSF osmotic pressure equivalently,doubled sodium intake. It was found that due to the osmoti-cally driven movement of water, CSF [Na] was reduced by10-15 mM (25).

(iv) Infusion of equiosmotic 340 mM mannitol in artificialCSF with [Na] = 330 mM, which increased osmolality and, dueto combined effect of the elevated [Na] of infusate andosmotically driven movement of water, caused no change of[Na] of CSF, was without influence on sodium appetite. Thussodium intake was not affected by osmolality change but bychange of [Na] of CSF.

These effects on sodium appetite were replicated in sodium-deplete cows (26) and also in rats by R. Sakai and colleagues(personal communication), but not in rabbits or mice. Increas-ing the [Na] of cerebral arterial blood by infusion of hypertonicNaCl into the carotid arteries also reduced sodium appetite.Changes within the brain were operative (infusion of 1 ml/h ofhypertonic NaCl CSF having negligible effect on sodiumbalance), and this was ratified by the fact that if the rise of CSF[Na] caused by the intracarotid infusion was prevented byinfusion into the CSF of 700 mM hypertonic mannitol, nodecrease of sodium appetite occurred (27).

(v) Further, it was determined that infusion into the ventricleof 700 mM mannitol, L-glucose, or sucrose increased sodiumappetite, whereas infusion of D-glucose, D-mannose, 2-deoxy-D-glucose, 3-0-methyl glucose, or distilled water did not (28).All infusions decreased CSF [Na], but because the latter groupof saccharides penetrated cells and capillaries and thus theblood-brain barrier, whereas the former did not, it stronglysuggested that the sensors subserving sodium appetite weredistant from the ventricular wall, and it required a front offluid of changed composition to travel through the intercel-lular channels to deep within the neuropil. In clear contrast, allof these intraventricular infusions that lower CSF [Na] inhib-ited the thirst induced by infusion of hypertonic NaCl into thecarotid artery, suggesting the elements subserving thirst arevery close to the ventricular wall. The sodium appetite andthirst sensors are anatomically quite separate, a conclusionfrom functional data which is ratified by lesion studies reportedbelow.

(vi) That infusions changing brain extracellular fluid [Na] actby change of intracellular [Na] is supported by the fact thatintraventricular infusion of 2.3 mM phlorizin, which reducessodium-coupled transport of glucose into cells, and thusintracellular [Na], enhances sodium appetite, and, also, pre-vents the reduction of sodium appetite caused by increasingCSF [Na] (28). Further, push-pull microirrigation of theanterior third ventricle (Fig. 2) with 10-6 ouabain, which wouldinhibit sodium and potassium ATPase and increase intracel-lular sodium, reduced sodium appetite, just as did raising thelocal CSF [Na] by push-pull -of 200 mM NaCl (29).

Role ofANG. Parallel to the ionic changes in the brain whichwould follow systemic changes in electrolytes caused by sodi-um-impoverished food, temperature regulation, or loss ofbodyfluids, a major issue presenting in analysis of sodium appetiteis the role of ANG. Systemically, ANG II present in plasma isformed by action of the enzyme renin, released from thekidney, on its substrate ANG, which is formed in the liver.ANG I, a decapeptide, is formed first and it is converted toANG II, an octapeptide, by ANG-converting enzyme. Regard-ing mode of formation of ANG II in the brain, the precursorangiotensinogen is present in neuroglia. Experiments suggestthat ANG may act as a neuropeptide transmitter in neuronesbut whether or how it is formed from glial angiotensinogen isunresolved (30).The activities of ANG II are mediated by at least two

different ANG II receptor subtypes-AT1 and type 2-andselective, nonpeptide receptor antagonists are available. ANGII-induced water and sodium intakes of rats and sheep appear

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FIG. 2. A diagram of major sites ofANG II receptors (stippled) inthe mammalian brain projected onto a midsagittal diagram of thesheep brain. The area shown in red is the lamina terminalis in themidline anterior wall of the third ventricle, which contains osmore-sponsive neurons as well as ANG II receptors. The third ventricle isshown in blue. AP, area postrema; BNST, bed nucleus of the striaterminalis; CVLM, caudal ventrolateral medulla; DMV dorsal motornucleus of vagus; LC, locus ceruleus; LPBN, lateral parabrachialnucleus; LS, lateral septum; ME, median eminence; MnPO, medianpreoptic nucleus; NTS, nucleus tractus solitarii; OB, olfactory bulb,OVLT, organum vasculosum of the lamina terminalis; PVN, hypo-thalamic paraventricular nucleus; RVLM, rostral ventrolateral me-dulla, SFO, subfornical organ; and SON, supraoptic nucleus.

to be mediated by predominately AT1 receptors, becauseintakes are blocked by losartan (30), a AT1 antagonist.

Peripheral administration of ANG-converting enzyme in-hibitors such as captopril or enalapril decreased sodium intakeof sodium-deplete rats, sheep, cows, rabbits, and mice. Thedecrease of sodium appetite of the captopril-treated sodium-deplete animal is obviated by peripheral administration ofANG II, sufficient only to give basal plasma levels. PeripheralANG II has an important role in the genesis of sodiumappetite, which may be permissive of other severally necessaryfactors, or it may be a major factor causal of sodium appetite(31).The role of cerebral ANG II in sodium appetite is equivocal.

In sheep and cows, intracerebroventricular (ICV) administra-tion ofANG II failed to effect the decrease in sodium appetiteresulting from systemic treatment with captopril (31). In rats,the findings are not consistent. Thunhorst and colleagues (32)were unable to demonstrate a role of brain ANG II in sodiumappetite, whereas Sakai and coworkers found brain-generatedANG II was causal (33). Epstein (34) proposes that in the rat,sodium appetite is generated in the brain by a synergistic actionof cerebrally derived ANG II and aldosterone. The controversyregarding the contribution of peripheral and central ANG II

to sodium depletion-induced sodium appetite has yet to beresolved.Sodium intake of sodium-deplete sheep or cows is not

decreased by ICV infusion of captopril or saralasin or losartansuggesting that brain ANG II is not involved in sodium appetiteeither directly or indirectly in these species (31, 35). However,captopril and losartan may cross the blood-brain barrier, and,therefore, it is possible that the neural elements subservingsodium appetite, which are some distance from the ventricle,are not accessed by ICV infusion.Where are the Sensors? Sodium appetite caused by the

natriuretic agent furosemide was decreased or abolished in ratswith lesion of the subfornical organ (36, 37) or the organumvasculosum of the lamina terminalis (OVLT; ref. 38).

The anteroventral third ventricle region AV3V includes theOVLT and the ventral median preoptic nucleus, a midlinebrain structure with a blood-brain barrier, located on the frontwall of the third ventricle (Fig. 2). This brain area is clearlyinvolved in body fluid homeostasis. Rats with lesion of theAV3V have impaired water drinking responses to peripheraland central administration ofANG II and to water deprivation(39), but sodium appetite induced by formalin, which causesboth fluid sequestration and stress, appears to be intact (40).The complexity of the rat data may reflect the differentphysiological effects of different stimuli used to producesodium appetite.Sheep with lesion of the AV3V are normal in regard to

sodium intake induced by sodium depletion or by ICV infusionof 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 theAV3V, even though systemic infusion of hypertonic NaCl nolonger causes increased water intake, it continued to cause adecrease in sodium intake. The results clearly ratify thatdifferent brain areas are involved in the control of thirst andsodium appetite in sheep (41).Not only do the loci of neural aggregates subserving sodium

appetite contingent on sodium deficiency (possibly lateralhypothalamic), but also their functional interaction with othercenters involved in sodium appetite generated by differentphysiological circumstances, need to be determined. The latterinclude the hedonic (need-free) appetite for salt, the saltappetite of stress caused by adrenal hormones (13), whichappears to involve neural groups of the amygdala, and the bednucleus of the stria terminalis (22). The locale of action of thequintet of steroid and peptide hormones determinant of thepowerful appetite of pregnancy and lactation (13) has yet to bedetermined.With regard to satiation, ruminant animals, for example,

have the remarkable capacity to drink an amount of sodiumsolution in 3-5 min, which commensurately corrects bodydeficit. A precipitate decline of motivation follows. Variousexperiments on animals with an esophageal fistula, whichcontrives that fluid drunk is lost from the neck, indicate thatthe satiation process involved a "gestalt" of sensory inflowarising from tongue taste receptors responding to salt concen-tration, pharyngoesophageal impulses metering volume swal-lowed, and nerve afferents from the forestomach respondingto volume of fluid drunk and distension (13). How this cascadeof inflow caused by the consummatory act of drinking rapidlyand completely inhibits the neural systems hitherto excitingsodium appetite is unknown. It is established that intraven-tricular infusion of several neuropeptides-somatostatin (42),atrial natriuretic peptide (43), and basic fibroblast growthfactor (44)-will inhibit sodium appetite, but whether theyhave a physiological role is not known.

Thirst and Sodium Excretion

Cerebral Regulation ofWater Intake and Sodium Excretion.Sodium homeostasis is inextricably linked with body waterbalance. When an animal becomes dehydrated, the [Na] andosmolality of extracellular fluid increase. If water is available,it can be ingested, but if it is not accessible, sodium may beexcreted by the kidneys. In the first case, both extracellularvolume and [Na] will be restored to normal, whereas in thesecond case, body fluid volumes remain depressed, but the risein [Na] is ameliorated. Not surprisingly, brain regions involvedin the regulation of thirst also have a role in the regulation ofrenal sodium excretion.

Early this century, Mayer (45) concluded that thirst wasrelated to the osmotic pressure of blood. Systemic infusions of

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concentrated solutions of NaCl or various saccharides (but notD-glucose) caused drinking behavior. However, similarly con-centrated solutions of urea or glycerol were considerably lesseffective dipsogens (46, 47). NaCl or sucrose molecules do notpenetrate cell membranes rapidly and cause an osmotic gra-dient across cell membranes and thus cellular dehydration.However, urea or glycerol molecules traverse cell membranesrelatively quickly, so there is no osmotic gradient and littlecellular dehydration. Hyperosmolar solutions that induce cel-lular dehydration cause thirst, and the concept of sensors(osmoreceptors) responding to cellular dehydration was pro-posed (48, 49). As to the bodily location, although there isevidence of hepatic osmoreceptors, the major site of detectionof changes in the tonicity of body fluids is the preoptic/hypothalamic region of the brain (48), as Verney determinedin elucidating the control of antidiuretic hormone, the water-conserving peptide secreted from the neurohypophysis.

In regard to thirst, Andersson showed that injection ofmicroliter quantities of hypertonic saline into the hypothala-mus of the goat induced them to drink large amounts of water(50). When hypertonic solutions of NaCl or saccharides wereadministered into the CSF of the third ventricle, only hyper-tonic sodium salts elicited water drinking and vasopressinsecretion (47). They proposed that the osmoreceptors werereally [Na] sensors in the anterior wall of the third ventricle.We investigated this hypothesis in conscious sheep and foundthat, if prepared in artificial CSF with normal sodium con-centration, injection of hypertonic sucrose into the thirdventricle did elicit drinking, but the response was considerablyless than that obtained with injection of equiosmolar hyper-tonic NaCl (51). We proposed that both osmoreceptors andsodium sensors existed in the brain. In a further investigationin sheep, we observed that infusions of 1 M NaCl, 2 M sucroseor fructose, and 2-4.6 M urea in the carotid artery of sheep allincreased the [Na] of ventricular CSF because the blood-brainbarrier excludes all these molecules from the brain. Thus,hyperosmolar urea is similar to hypertonic NaCl or sucrose inthat when it is infused systemically it dehydrates cells in thebrain. Yet hypertonic urea is relatively a poor dipsogen. Toexplain this result, we proposed that the relevant osmorecep-tors were situated in a region(s) lacking a blood-brain barrier,and suggested the OVLT and/or subfornical organ as likelysites (20).

Consistent with this idea, ablation of brain tissue in theregion of the ventral lamina terminalis, which included theOVLT, did disrupt osmoregulatory water drinking (39, 52, 53).Ablation of the subfornical organ may also reduce osmoticallystimulated drinking in sheep (54), as does ablation of theadjacent median preoptic nucleus (55), which receives richneural input from both circumventricular organs. Neurons inboth the OVLT and subfornical organ, have been shown byelectrophysiological techniques to increase activity in responseto a local increase in tonicity (56, 57). Single unit recordingsfrom neurons in the median preoptic nucleus show that theyare responsive to systemic hypertonicity, although not neces-sarily directly stimulated by it (57). Recently, mapping ofexpression of the immediate early gene c-fos shows thatpopulations of neurons in the periphery of the subfornicalorgan, in the dorsal part of the OVLT, and throughout themedian preoptic nucleus are activated in response to i.v.infusion of hypertonic solutions or dehydration (57).

In addition to osmoreceptors in the subfornical organ andOVLT, periventricular sodium sensors may also play a role indetecting changes in systemic tonicity. Thus reduction of CSF[Na] by 10-15 mM by means of ICV infusion of isoosmotic orhyperosmotic sugars or mannitol can severely inhibit drinkingresponses to water deprivation or systemically infused hyper-tonic saline (47, 58). This suggests that the ambient [Na] in thebrain is sensed and interacts with information from osmore-ceptors in the circumventricular organ. However, the precise

location of the sodium sensors remains to be determined,though permanent adipsia in animals and humans results fromlesions, which encompass completely the lamina terminalisand surrounding preoptic/anterior hypothalamic tissue (59).ANG-Induced Water Intake. As well as thirst of cellular

dehydration, depletion of the extracellular fluid leads toincreased water intake and dilution of the extracellular fluid[Na]. While neural input carried by vagal afferents fromthoracic volume sensors plays a role in the drinking, extracel-lular fluid depletion causes renin secretion, and the resultingoctapeptide ANG II delivered to the brain in the bloodstreamis also dipsogenic (60). Fitzsimons and colleagues showed thatANG II was dipsogenic when administered systemically andespecially intracerebrally-a pivotal discovery (reviewed inref. 60). Circulating ANG II acts mainly on the subfornicalorgan for the stimulation of drinking behavior, thus circum-venting the blood-brain barrier, which excludes peptides frombrain interstitium (61). Autoradiographic binding studies in anumber of species, hybridization histochemistry, and immu-nohistochemistry have shown that ANG II receptors (of theAT1 subtype) are distributed throughout the lamina terminalis(OVLT and median preoptic nucleus) and many other sites inthe central nervous system associated with body fluid ho-meostasis and cardiovascular control (e.g., hypothalamic para-ventricular nucleus, parabrachial nucleus, rostral and caudalventrolateral medulla, nucleus of the solitary tract, and inter-mediolateral cell column of the thoraco-lumbar spinal cord)(62). The endogenous ligand for these receptors is probably anANG of cerebral origin (62). Angiotensinergic circuits ema-nating from the subfornical organ to preoptic and hypothala-mus regions have been shown by electrophysiological andimmunohistochemical studies (63). Intravenous ANG II in-duces c-fos expression in the OVLT, just as in the subfornicalorgan, although an OVLT role in ANG-induced drinkingbehavior remains to be demonstrated (64). Further, differentpopulations of neurons seem to be excited by hypertonicitythan are by ANG II, in both the subfornical organ and OVLT(Fig. 3).

Recently, we found in five species that centrally adminis-tered AT1 receptor antagonist losartan blocks drinking initi-ated by intraventricular infusion of hypertonic saline (65), andin sheep natriuresis and vasopressin secretion, induced bycentral hypertonic saline, is also blocked by losartan (66). Thedata point to the involvement of angiotensinergic pathways inthe central organization of osmoregulatory responses, but thelocus of action of losartan is unknown.Osmoregulatory Sodium Excretion. Despite a reduction of

extracellular volume, dehydration results in a natriuretic re-sponse in many mammals (54). This dehydration-inducednatriuresis is under central control because it can be abolishedif the lamina terminalis is ablated, or if the [Na] within theperiventricular brain tissue is prevented from increasing bymeans of slow ICV infusion of isotonic or hypertonic mannitolsolutions in dehydrated sheep (54, 67). Gross systemic hyper-natraemia results with these experiments showing the impor-tant hemostatic role (54). In sheep, ablation of brain tissue inthe region of the lamina terminalis also inhibits the excretionof i.v. hypertonic NaCl loads but does not affect the excretionof intravenous volume loads of isotonic saline (68). Conversely,it has been shown that injection of hypertonic saline into thethird cerebral ventricle increases sodium excretion greatly, andpush-pull perfusion of hypertonic saline in the anterodorsalpart of the ventricle adjacent to the median preoptic nucleusis the most effective site for inducing such a natriureticresponse (69). Reduction of CSF [Na] also inhibits the excre-tion of hypertonic NaCl loads, as well as abolishing the"escape" from mineralocorticoid-induced sodium retention,which causes increased CSF [Na]. Thus, when the [Na] andosmolality of the extracellular fluid increase, the ambient [Na]within the brain also increases and this is detected by tissue in

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A

ovft . ....

or

t c

---r * W.: ;

-. .; .:

0A -' '.E. :. .:.-

S:::.: .. .. : . ..... . : ... C -''''-

rV

FIG. 3. Photomicrographs of coronal sections through the orga-num vasculosum of the lamina terminalis (ovlt) in A and B andsubfornical organ (sfo) in C and D of rats showing the distribution ofFos detected by immunohistochemistry in the nucleus of cells and seenas the brown dots. Fos, the protein encoded by the protooncogenec-fos, is a marker of increased neuronal activity. InA and C, the rat hadbeen infused i.v. with ANG II (1 ,ug/h for 2 h), and neurons at theperiphery of the ovlt (arrowheads) and throughout the subfornicalorgan have been activated. In B and D, the rat had been infused i.v.with hypertonic saline (5.5 ml/kg of 1.5 M NaCl) and increased activitywas observed in neurons in the dorsal cap of the ovlt and the peripheryof the subfornical organ. Data from experiments described in refs. 57and 64. hc, hippocampal commissure; oc, optic chiasm; or, optic recessof the third ventricle; and v, third ventricle. Scale bar = 200 ,um.

the region of the lamina terminalis which signals the kidney toinitiate a natriuretic response. The signaling mechanism to thekidney is still to be fully understood, but it is almost certainlyhormonal, because renal denervation does not prevent suchcentrally mediated natriuresis (67, 68). Adrenalectomy orhypophysectomy do not prevent dehydration-induced natri-uresis, and neither can the response be attributed to increasesin atrial natriuretic peptide secretion or reduced activity of therenin-ANG system (70).The lamina terminalis may also play a role in the regulation

of renin secretion from the kidney. Reduction of CSF [Na]increases plasma renin levels in dogs (71) and sheep (72),whereas an increase in the tonicity of carotid arterial bloodreduces it in conscious sheep, an effect abolished by ablationof the lamina terminalis (73). Consequently, we have proposedthat there is an inhibitory cerebral osmoregulatory influenceon renin secretion by the kidney (73). Consistent with thisproposal was the observation that dehydrated sheep withlesions encompassing the anterior wall of the third ventriclehave inappropriately high plasma renin concentrations despitetheir grossly hypernatremic state (70). ICV infusion of ANGII strongly depresses plasma renin levels, an effect abolished byablation of the lamina terminalis (70), while centrally admin-istered AT1 antagonist losartan increases plasma renin con-centration (66) and blocks the inhibitory effect of increasingthe ambient [Na] in the brain (74). In homeostatic terms, acentral osmoregulatory inhibitory influence on renin secretionshould secondarily result in reduced aldosterone levels, andfacilitate natriuresis.

Physiological Integration

In terms of physiological integration and a biological overview,it is evident that the hypothalamic and midbrain sensors, intheir interoreceptor role, generate powerful emotions andbehavioral drives. The stream of consciousness may be dom-inated by such appetites and hungers, and the intentions are

orientated to a physiologically apt behavior. The cognitiveprocess involves past experience relevant to satiation of thirst,salt hunger, or for that matter, whatever appetite (e.g., hungeror sex), the midbrain-limbic systems may be generating. Con-sideration of these mesencephalic structures in the context ofthe phylogenetic emergence of consciousness raises basicquestions. Edelman (75) has argued cogently in relation to thephylogenetic emergence of consciousness: "It is the evolution-ary development of the ability to create a scene that led to theemergence 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 deeplyinfluenced by limbic-brain stem systems that confer values ofbiological relevance." But basically his proposal suggests thatprimary consciousness comes from processing of distancereceptor input ("helps to abstract and organize complexchanges in an environment involving multiple parallel signalswith reentry of value category memory"), and by a scene heproposes a spatiotemporal ordered set of categorizations offamiliar and unfamiliar events, some with and some withoutnecessary physical or causal connections to others in the samescene. The issue that the limbic-brain stem systems may dictatesaliency or biological relevance, seemingly a key componentwithin the primal conscious process, merits the question of analternate viewpoint on the first emergence of dim awareness.It is feasible its genesis may have been in sensations and primalcompelling emotions determined by internal sensors and re-ceptors of the vegetative systems signaling threat to exis-tence-e.g., hunger for air, thirst in the face of desiccation ofthe body, hunger (including specific hungers), extreme tem-perature change, and pain, all to a considerable extent involv-ing the phylogenetically ancient areas of the brain, includinghypothalamus and reticular activating system.Many biologists entertain the possibility of conscious pro-

cesses in animals lower in the phylogenetic scale than mammals(1, 75, 76, 77). Pertinent to this, the spinoreticular systemcarrying pain fibers is conspicuous in vertebrates at the levelof fish, amphibians, and reptiles. The reticular system ispresent in all vertebrates (78, 79), and certain components ofthe limbic system such as septum and habenula are recogniz-able in all vertebrates (80). The thesis that these vegetativesystems, which antecede much cortical development wereindeed involved in genesis of primal awareness, gives closeconjunction to the fact of these phylogenetically ancient areasof the brain having a plenipotentiary role in higher mammalsin the orchestration of arousal, focus of attention, intentionand also sleep, and complex neuroanatomical pathways be-tween these structures and the thalamus and cortex subservethese functions. It is, of course, possible that rather than eitherinterceptor-generated sensation of threat to existence or mul-tiple parallel signals from distance receptors having temporalprimacy in phylogenesis of consciousness, there was contem-poraneity of influence in this physiological emergent of veryhigh survival value.

This work has been supported by the National Health and MedicalResearch Council of Australia, The Howard Florey Biomedical Foun-dation of the U.S., and The Robert J., Jr., and Helen C. KlebergFoundation.

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