336

J. Physiol. (I959) I45, 336-352

THE EFFECTS OF LESIONS IN THE HYPOTHALAMUSIN PARABIOTIC RATS

BY G. R. HERVEY*From the Medical Research Council Department of Experimental Medicine,

University of Cambridge

(Received 1 August 1958)

An adult animal's intake and expenditure of energy are normally almostequal over long periods. This equality is probably brought about mainly byadjustment of the amount of food eaten. Regulation of food intake is par-ticularly efficient in the young adult rat, which can adjust its intake to providea nearly constant supply of calories when the bulk it must eat is varied bydiluting the food with inert material (Adolph, 1947; Kennedy, 1950). In therat and other mammals which have been investigated, bilateral lesions in theregion of the ventromedial nuclei of the hypothalamus cause obesity if theanimal has free access to food which it finds palatable; the obesity is due,mainly if not entirely, to an increase in the amount of food eaten (Hethering-ton & Ranson, 1942; Brobeck, 1946; Kennedy, 1950). It has therefore beensuggested that the hypothalamus contains nervous centres which normallycontrol the intake of food, and that these are damaged by the lesions (Anand& Brobeck, 1951).

There has been much discussion of the way in which these centres work. Itis difficult to see how they can directly measure the number of caloriesexpended in a given period, and then regulate feeding to provide just thisamount of energy. Alternatively, the regulk ing centres may be sensitive tosome change in the body which follows the intake or expenditure ofenergy, and they may inhibit or encourage eating until the changed quantityhas been restored to normal. This would be a 'feedback' control system:the controlling centres would be acting on information, fed back to themfrom the periphery, about the behaviour of the quantity they stabilized.Mechanisms of this type are known to be important in the body, as wellas in artificial control mechanisms. Brobeck (1948), Mayer (1952) andKennedy (1953b) have proposed systems of the feedback type in relation tothe control of food intake. It has not, however, been directly demonstrated

* Present address: Department of Physiology, The University of Sheffield.

HYPOTHALAMIC LESIONS IN PARABIOTIC RATS 337

that the regulation of food intake involves a feedback system, and there isconflicting evidence as to the nature of the information used and the pathby which it reaches the centres. Food intake can be shown to be modified inresponse to many factors; the difficulty is to identify the mechanisms par-ticularly concerned in achieving an approximate balance of energy over longperiods.

In the present study a series of parabiotic rats have been subjected tohypothalamic lesions of the type which causes obesity. Parabiotic animalsare surgically united when young, and exchange blood throughout life; thepreparation has proved valuable in investigating a number of physiologicalinterrelationships involving long-term responses (Finerty, 1952). It hasalready been briefly reported that lesions made in one member of a para-biotic pair appear to influence the feeding of the other (Hervey, 1957). Thisresponse in the partner may provide some further evidence of the mechanismand pathways which in normal animals regulate the intake of food over longperiods, and so maintain energy balance.

METHODS

AnimalsThe rats were a hooded strain which originally came from the Lister Institute. They had been keptas a closed colony for some years. Initially animals for parabiosis were more closely inbred bymating litter-mates for three generations before using the offspring; later pairs were made fromordinary stock litters. In order to standardize the size of the animals as far as possible they werebred from females weighing between 200 and 250 g, and only second and third litters which con-tained 8-12 young were used. The young were separated from the mothers on the 21st day afterbirth. The diet was Mill Hill Diet 41 (Bruce & Parkes, 1949). Food and water were providedad libitum. When rats had been subjected to hypothalamic lesions, food was scattered around thecage as well as supplied in the usual hoppers.

ParabiosisThe pairs of rats united in parabiosis were litter-mates of the same sex whose weights did not

differ by more than 3% at the time of joining. Most pairs were united when 4 weeks old, but themost recent ones have been united when a week or two older. As many pairs as possible weremade from each litter, and any compargle animals left over were kept as single controls. Someof these single animals were subjected to sham operations, in which equally extensive surgerywas carried out without uniting animals. Both sexes were used. The anaesthetic was pentobarbi-tone sodium, given intraperitoneally in a dose of approximately 6 mg/100 g body weight. Thesurgical technique used for the earlier pairs was that of Bunster & Meyer (1933), in which theperitoneal cavities are opened and the four cut edges of muscle and peritoneum united in one

suture. In later pairs the upper and lower cut edges were sutured separately, making a 'coelio-anastomosis' (Sauerbruch & Heyde, 1908). This avoids the risk of small openings forming betweenthe peritoneal cavities and occasionally causing death through strangulation of loops of intestine.In the most recent pairs the abdominal cavities have not been opened and firm union of the pos-terior parts of the bodies has been secured by tying the femora together (J. M. Ledingham, per-sonal communication). Bunster & Meyer's technique of uniting the scapulae was followed, exceptthat the scapulae were scraped to expose raw bone before suturing. Aseptic technique was used as

far as practicable.22 PHYSIO. CXLV

338 G. R. HERVEY

Plasma exchangeThe dye Evans Blue (T-1824) was used to confirm the existence and amount of the exchange of

plasma between the members of pairs. A dose of 0.1 ml./100 g total weight of the pair, of an0-25% solution of the dye, was injected into a femoral vein of one rat under ether anaesthesia.A half, one, and sometimes two hours after the injection, about 0 3 ml. of blood was collected fromeach member of the pair by snipping the tip of the tail under light ether anaesthesia. Clottingwas prevented by a trace of solid heparin. After separating the plasma, 0 1 ml. from each specimenwas diluted with 3-4 ml. 0-2% sodium carbonate, and the optical densities were read at 620 m,uin a spectrophotometer. The rates of plasma exchange were calculated from the formula:

coth rt =C1/C2,where r is the rate of exchange as a fraction of one animal's plasma volume per minute, t is thetime in minutes after injection, and cl and c2 are the optical densities for the injected animal and

optic chiasma

Lesions

Pituiary stalk

Text-fig. 1. Under surface of rat's brain post-mortem, showing effective hypothalamic lesions(Traced from a colour photograph.)

its partner. This formula depends upon assumptions that the members of a pair have equal plasmavolumes, that disappearance of the dye from each circulation is exponential, and that the timeconstants of disappearance are the same in both animals. These assumptions were tested bygiving different doses of dye to single animals and following the rates of disappearance from theplasma. They appeared to be approximately true over the duration of the test. The calculationsfrom successive bleedings also usually agreed well. In view of existing evidence (Finerty, 1952)it was not thought necessary to verify exchange of blood cells as well as of plasma.

Hypothalamic lesionsThe lesions were made with a stereotaxic instrument based on Krieg's (1946) design, using a

direct current of 2 mA passed for 7j-10 sec. The position of the lesions was verified after deathby naked-eye examination of the under surface of the brain. The lesions are easily visible; theappearance when they are correctly placed is shown in Text-fig. 1. Variation in size of the animalswas allowed for by adjusting the antero-posterior co-ordinate (i.e. the distance forward fromthe line of the ear-bars of the instrument to the site of the electrode). The correct distances werefound for a number of animals by operating upon and killing them. The distances were thenplotted against a 'skull length index', obtained by measuring the distance between the ear-barsand jaw-bar of the stereotaxic instrument with the animal in place, using an engineering calliper.The graph was used to obtain the co-ordinate for any animal being operated upon. This methodwas found more reliable for parabiotic animals than working from the total weights of pairs. The

HYPOTHALAMIC LESIONS IN PARABIOTIC RATScorrect distances lay between 5 and 7 mm. The lateral position of the electrode was obtained bymoving it 05 mm to each side of the mid line (0.7 mm in the largest animals), and the verticalposition by lowering it until the tip touched bone and then withdrawing about 0-2 mm.

AnalysesThe animals were killed with ether. After the carcasses had been weighed, individual organs

were dissected out, blotted and weighed. Except in the earliest animals the contents were re-moved from the alimentary tract, and the loss of weight recorded. The bodies were placed onweighed trays in an oven at approximately 1050 C, chopped and spread out after 24 hr, and driedto constant weight. Fat was estimated in the dried material by extracting in successive changesofpetroleum ether until the supernatant appeared to be fat-free; this required three to five changes.The solids were separated from petroleum ether by filtering through cambric, and after the lastextraction were again dried in the oven and weighed. This method was checked by recovery ofthe fat from the solvent and by Soxhlet extraction of the residues; 98-99% of the fat was ex-tracted. Total nitrogen was estimated in some of the dry fat-free residues by a3 tandard micro-Kjeldahl method.

RESULTS

ParabiosisIn the course of the experiments 93 parabiotic pairs have been made. Out of39 pairs made from specially inbred stock, 32 (82%) survived to becomeyoung adults. All the animals lost from this group died during the operationor from infection or illness afterwards, and none from the condition known asparabiotic intoxication or disharmony, in which one member of a pair fails togrow, becomes thin and anaemic, and dies 2-3 weeks after parabiosis (Finerty& Panos, 1951). Two pairs killed later may have suffered from a mild form ofdisharmony. Of the 54 pairs made from ordinary stock 30 (55%) survivedto young adulthood. Eight of the pairs in this group which died appeared tobe typical cases of disharmony; one pair which died as adults were thought toshow disharmony; and two pairs apparently developed disharmony butrecovered later. The difference in the incidence of disharmony agrees withFinerty & Panos' suggestion that the incidence is inversely related to thecloseness of inbreeding. Close inbreeding, however, led to smaller litters andsmaller-sized young, and was given up for this reason.The pairs which survived grew well and seemed healthy and, as far as

could be judged, contented (Plate 1). Individuals in parabiosis, however,grew more slowly and weighed less at any age than comparable unpairedanimals. This was not simply due to the effect of a surgical operation in in-fancy, for the sham operations on single animals had no detectable effect.Twelve normal pairs could be compared with single litter-mates, of the samesex and similar weight at the time of parabiosis. At 5 months old the animalsin the parabiosis weighed 71-85% of the weights of their single litter-mates(average 78 %). For this comparison the parabiotic animals' weights weretaken to be half the total weights of the pairs. When parabiotic animals werekilled without further experimental treatment the weights of the individuals

22-2

339

in a pair were normally found to be approximately equal. Ten normal pairswere killed at various ages; the weights of the separated individuals differedfrom the mean weights of the pairs to which they belonged by 1-8% of themean weights (average 4%; see Tables 3 and 4).The rate of exchange of plasma was measured in eight pairs. The results

ranged from 0-3 to 2-1 % of one animal's plasma volume exchanged per minute(mean 1-0 %, S.E. + 0-2 %). The mean value is a usual one for parabiotic rats(Finerty, 1952; J. M. Ledingham, personal communication). The presence orabsence of a coelio-anastomosis made no apparent difference to the exchangeof plasma. The two pairs with the lowest exchange rates were early pairs,made by the unmodified Bunster & Meyer technique, in which the scapulaehad become widely separated.

Hypothalamic lesionsAn attempt was made to produce hypothalamic lesions of the type which

lead to obesity in the right-hand animals of 32 parabiotic pairs. The lesionswere normally made when the animals had just passed the stage of rapidgrowth. Whenever possible a second pair of the same sex and litter was keptunoperated on for comparison, and so also were the single litter-mates. Thusthree groups of animals were obtained, normal single animals, normal pairs,and pairs with lesions in the right-hand animal, all from similar stock andincluding litter-mate comparisons where possible. Hypothalamic lesions werealso made in a number of single animals; the animals in this group were notspecially selected to be comparable with the others. In a first series of11 operations on members of pairs, 4 were successful. In a later series of21 pairs, one pair died during the operation, and in all others the lesions wereeffective.Five pairs with effective lesions, however, were lost soon after the operation.

Three of the operated animals died from aspirating food into the trachea; onedied from intestinal obstruction caused by eating sawdust; and one died fromexcessive drinking. Brobeck, Tepperman & Long (1943a) described chokingfrom voracious eating soon after hypothalamic lesions. Parabiotic animalsseemed to be particularly liable to death in this way, and latterly care wastaken to withdraw food and water and anything the animals could swallowfor 24 hr after making the lesions. On the other hand, no single animals withlesions were lost as a result of post-operative voracity, although no specialprecautions were taken with them.

In single animals successful lesions were followed by the hyperphagia andobesity which are now well known. In parabiotic pairs, the effects in theanimals which received the lesions (i.e. the right-hand animals of operatedpairs) were in general similar. On recovering from the anaesthetic most ofthese animals were overactive, and they would immediately eat voraciously

340 G. R. HERVEY

HYPOTHALAMIC LESIONS IN PARABIOTIC RATS 341

and unselectively. The overactivity disappeared within 24 hr, though theoperated animals often remained somewhat irritable and more difficult tohandle than before. The grossly voracious eating also disappeared, but pairs ofwhich one member had received lesions continued to show a greater food

* R of pair/ with lesions

I

I

II

_(Mean of air/ with lesions]

.'I

ISingle rat

R NormalL pair

L of pairwith lesions

(in right-hand ratof one pair) All killed

MonthsText-fig. 2. Growth curves and post-mortem body weights of a group of two parabiotic pairs and

one single rat. All were males and litter-mates. At approximately 5 months old, lesions weremade in the hypothalamus of the right-hand member of one pair. The left-hand member ofthis pair appeared to be moribund approximately 21 months later; all the animals were thenkilled. , i x total weight of each pair during life. - --, weight of single control rat.- presumed weights of individuals of pair in which lesions were made, after placing of

the lesions. 0, individual body weights after death and separation of the parabiotic pairs.

intake than before the operation, and their total weights rose rapidly. Text-fig. 2 shows the weight curves for a group of five male litter-mate animals,which provided a normal single animal, a normal parabiotic pair, and aparabiotic pair in which hypothalamic lesions were made in the right-handanimal. The weight curves for some pairs which were kept long enough appearedto approach a plateau 2-3 months after lesions had been made.

342 G. R. HERVEYThe live weights and food intakes of parabiotic animals have so far only

been satisfactorily measured as the totals for pairs. Tables 1 and 2 show theincreases in weight, and the intakes of food where known, for single rats and

TABLE 1. Weight gains and food intakes of single rate in 28 days after hypothalamic lesions

Serial no.

12345678910111213141516171819202122232425

Average

SexMFFFMMMFFMMMMMMMMM

MMMM

MM

Initialweight

(g)236169178135260212232171172250313245285280217173178152207234266184188167126209

Weight gainedin 28 days

(g)7679

11213011648149163150131192252183113771361711349314215215712610944129

Food intakein 28 days

(g)

700

730

1360

1350980

1020

Ratio:exces food (g)exces gain (g)

5*0

3-9

3-8

4-15'2

4.4Mean age at operation: 4*1 months.

TABLE 2. Weight

Serial no.8121418424953545658606671798185

Average

gains and food intakes of parabiotic pairs in 28 days after hypothalamiclesions in the right-hand animal

Initial Weight gained Food intake Ratio:weight in 28 days in 28 days excess food (I

Sex (g) (g) (g) excess gain (^F 210 109 -M 247 128M 349 126 1320 4*8M 285 55 990 4*0F 367 183M 505 119 1530 3*5F 329 83 -F 318 128 -F 354 131 -M 492 164M 443 103 1450 5*9M 454 52 1190 3-6M 481 225 -M 445 155 1540 4B5F 294 184M 568 100 -

384 128 1340 4-4Mean age at operation : 5-1 months.

g)g)

HYPOTHALAMIC LESIONS IN PARABIOTIC RATS

parabiotic pairs during the 28 days after the placing of lesions in the hypo-thalamus (of the right-hand animal in the case of the pairs). Both tablesinclude all animals in which the lesions led to a definite increase in weight andwhich survived for 28 days or longer. Within each group the gains of weightdid not seem to be influenced by the animal's age, sex or initial weight, so thelack of comparability in these respects may not invalidate comparison of theeffects of the lesions.The average increases of weight in the 28 days were the same in both

groups. The average rate of weight gain over the period was 4-6 g/day in bothsingle animals and parabiotic pairs. The food intakes of single and parabioticanimals are not directly comparable, and satisfactory measurements were onlymade on a minority of animals in each group. It also so happens that five ofthe six pairs with lesions, for which food intake measurements were available,showed less than average gains of weight (perhaps because choking tended toremove the pairs with the most effective lesions, if food was continuouslyavailable, as it was if intake measurements were being made). If, however,the increases in food intake per unit of extra weight gained are calculated,(by comparison with pre-operative data), the ratios so obtained are in thesame range in both groups (final columns in Tables 1 and 2). As the averageweight gains in the complete groups were the same, this suggests that theaverage increases in food intake might also have been the same if measure-ments had been available for the complete groups. The ratios also show that,in both groups, an average of 023 g body weight was gained for every gramof extra food eaten, or about 0O08 g for each excess kilocalorie. The efficiency ofconversion of food into body weight was apparently of the same order in singleand parabiotic animals.

It was clear from observation that the hyperphagia and weight gain ofpairs with lesions in one member were entirely due to the animals which re-ceived the lesions (i.e. the right-hand animals). These animals showed theinitial overactivity and voracity, their stomachs were distended within a dayof the operation, and they rapidly became obviously obese. The partners withintact hypothalami (the left-hand animals) were certainly not eating raven-ously, and appeared to be eating less than before. They became obviously thin.

After the first 24 hr after operation the animals with lesions did not inter-fere with their partners' feeding in any way that could be seen. They were notseen to attack their partners, drag them around the cage, or impede theiraccess to food, which was freely available. If a pair was held and offered food,the obese, right-hand member of the pair investigated and usually ate thefood; the left-hand animal, although in the later stages it appeared emaciated,took no interest in food held under its nose.Many of the left-hand animals became listless and inactive. Two of them

died, both about 8 weeks after lesions had been made in the right-hand

343

animal. In one instance the obese animal was still alive when the death wasdiscovered. In the other, both animals were dead, but the prior death of theleft-hand animal was shown by the accumulation of blood in its body. Aone-way movement of blood takes place when one member of a parabioticpair dies before the other; this effect appears to be a very reliable indicationof which animal has died first when a pair is found dead. Two other pairs werekilled because the left-hand partners appeared to be moribund, 8 and 10 weeksafter the operation.

Three pairs with effective lesions in the right-hand animal were subjectedto further hypothalamic lesions in the left-hand animal (see below). Fourpairs were lost in the course of other experimental surgery or from infection.The remaining eight pairs with lesions in one member were killed arbitrarily1-3 months after the operation.

Post-mortem findingsAt post-mortem examination the individuals with effective lesions in the

hypothalamus were obviously obese, as would be expected. The thin andstarved appearance of their parabiotic partners was almost equally striking(Plates 2, 3). In the animals with lesions much fat was visible in the sub-cutaneous tissues and omentum; the alimentary tracts, particularly thestomachs, were hypertrophied and full; the livers were large, pale and mottled.The appearances were in no way different from those seen in single rats witheffective hypothalamic lesions. The bodies of the partners contained no visiblefat; the viscera appeared atrophic and empty; the livers were small and dark.Where a coelio-anastomosis had been made, the mass of viscera and omentumbelonging to the animal with lesions almost filled the combined peritonealcavity; but the partners showed the same changes whether or not a coelio-anastomosis was present. The partners which died spontaneously, or appearedto be moribund, showed no abnormalities beyond those described. Normalparabiotic animals contained some visible fat, although less than singleanimals; their viscera, although smaller and less well-filled than in singleanimals, looked essentially normal and did not show the appearance ofatrophy and emptiness seen in the partners of animals with lesions.

Numerical data obtained at post-mortem and in subsequent analyses aregiven in Tables 3-6. Tables 3 and 4 give the body weights and fat contentsof normal single animals, normal parabiotic pairs, parabiotic pairs with lesionsin the hypothalamus of the right-hand member, and single animals withlesions (males only). All animals are included in which the techniques werecarried out successfully and which were not lost from intercurrent causes. Theindividual data have been given for the parabiotic animals, and mean valuesonly for the single animals. Males and females are grouped separately. Tables5 and 6 give other measurements as mean values for the groups.

G. R. HERVEY344

HYPOTHALAMIC LESIONS IN PARABIOTIC RATS 345

Body weight. The normal findings in parabiotic animals have already beenmentioned. When hypothalamic lesions were made in one member of a pairthey led, as in single animals, to a great increase in the weight of that animal.The partners of animals with lesions weighed less, on average, than members ofnormal parabiotic pairs. Where pairs with lesions in one member could becompared individually with litter-mate pairs which had weighed approxi-mately the same at the time of the operation, the partners of the animals withlesions always weighed less at death than the members of the normal pairs.Also, assuming that the members of the operated pair were of equal weight at

TABLE 3. Post-mortem body weights and fat contents of male animals

Normal single rats(mean of 14)

Normal parabioticpairs

mean of 5Parabiotic pairs withlesions in right-handrat (average age atoperation 5-0 months)

mean of 8Single rats with lesions(average age at opera-tion 4-7 months)(mean of 8)

Age atSerial deathno. (months)

7-2

1959616980

6-87-65-18-04-46-44-98-28-67-57-58-76-98-67-67-6

1214495860667985

Body weight(g)

L R327

234276182275221238161185220236182236217242210

206282203319197241347412525600488394475507469

Fat(% body weight)

L R12-4

8-36-16-18-05-16-72-12-11-72-11-21-61-00-61-6

501

6-56-15-68-55-46-4

52-250-845-249-849-441-247-951-348-5

49-4

TABLE 4. Post-mortem

Serialno.

Normal single rats(mean of 4)Normal parabioticpairs

mean of 5Parabiotic pairs withlesions in right-handrat (average age atoperation 5-3 months)

mean of 5

2643445582

842545681

body weights and fat contentsBody weight

Age at (g)death A

(months) L R6-9

6-48-17-48-66-17-35-08-18-66-96-16-9

197

151179162190176172107174143166147147

154174191184157172307381362315395352

of female animalsFat

(% body weight)L R

16-5

9-710-16-7

14-48-59-93-45-11-12-84-23-3

7-69-16-9

14-57-09-0

65-841-248-635-057-149-5

G. R. HERVEY

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HYPOTHALAMIC LESIONS IN PARABIOTIC RATS

the time of the operation, in most instances the partners of the animals inwhich lesions were made evidently lost weight after the operation (Text-fig. 2).Fat content. In unoperated parabiotic pairs the two members contained

approximately equal percentages of fat. The average percentage of fat foundin normal parabiotic animals was of the order of half that found in singleanimals of the same sex. Where parabiotic animals could be compared in-dividually with single litter-mates, an approximately 1: 2 relation was found,within a scatter of 1-2% of fat. The parabiotic animals in which effectivelesions had been made showed greatly increased fat percentages, similar to thelevels found in single animals with lesions. The partners of animals with lesionswere found to contain very low percentages of fat, in keeping with theirnaked-eye appearance. This effect appears to have been a little more marked inmale than in female animals. In both sexes the range of fat percentages foundin the partners of animals with lesions did not overlap the range for normalparabiotic animals.Body lengqth. Members of normal parabiotic pairs were evidently smaller in

skeletal measurements as well as lighter and thinner than single animals.Hypothalamic lesions had no clear effect on body length. This might beexpected since the animals were usually nearly full-grown when the lesionswere made.

Contents of the alimentary tract. It is reasonable to suppose that the amount ofthe contents of the alimentary tract would bear some relation to the turnoverof food. The contents of the alimentary tracts of normal parabiotic pairs werea little less than the amounts found in single animals. The animals with hypo-thalamic lesions showed much increased contents; their partners gave figuresslightly lower than those for members of normal pairs. Measuring the contentsof the alimentary tract as a whole may have minimized these differences. Atpost-mortem examination the emptiness of the stomachs of the partners ofanimals with lesions was very striking. The small intestines also appeared to becomparatively empty, but the caeca and colons of these animals containedquite large amounts of rather dry material. If the contents of the stomachsalone had been measured the differences between the groups might havebeen more marked.

Composition of the fat-free bodies. The mean weights of the fat-free portionsof the bodies showed differences between the groups generally in the samedirection as the differences in whole body weight but less marked. The animalswith hypothalamic lesions evidently only gained small amounts of fat-freetissue compared with their large gains of total weight. The partners of animalswith lesions showed small losses of fat-free weight, in comparison with controlpairs. The percentages of water in the fat-free tissue, together with the smallnumber of nitrogen analyses, show no evidence of any differences between thegroups in the composition of the fat-free parts of the body.

347

Weights of individual organs. The livers of normal parabiotic animals onaverage weighed less than the livers of normal single animals, but there was agood deal of variation. The livers of animals subjected to hypothalamic lesionsshowed a substantial increase in weight; their partners' livers on averageweighed a little less than the livers of normal parabiotic animals. The weightsof the hearts and kidneys showed differences in the same direction as theweights of the livers, but less marked, and with the exception that the heartsof the partners of animals with lesions were not reduced in weight.

Second hypothalamic lesionsIn three parabiotic pairs hypothalamic lesions were made in the left-hand

animals when these had become obviously thin, after earlier lesions in theright-hand animals. All three pairs were males, and the intervals betweenthe two hypothalamic operations were 21-31 months. In the first pair,the newly operated animal ate avidly as soon as food was offered; before theoperation it had shown no interest in offered food. Unfortunately, however,it then choked, and, despite attempts at treatment, died a day later with,apparently, inhalation pneumonia. Food and water were withheld from thesecond pair during the remainder of the day and the night following thesecond operation. The newly operated animal died during the night. It hadpreviously shown marked overactivity, alternating periods of running roundthe cage with lying immobile, and it may have died from exhaustion. Thethird pair survived the post-operative period, and were killed some 2 monthslater. After the operation the weight of this pair increased rapidly. At deaththe fat contents of the carcasses were: left-hand animal, 224%; right-handanimal, 48-5%. Both animals had fairly heavy livers and alimentary tractcontents. The lesion on one side of the left-hand animal's brain was displacedabout 05 mm from the optimum position. An unoperated single litter-matecontained 8-1% of fat.

DISCUSSION

It is known that appropriately placed lesions in the hypothalamus will causehyperphagia and obesity in single animals. Lesions made by the same techniqueproduced essentially similar results in rats which were members of parabioticpairs. The partners of the animals with lesions became underweight and thin.The immediate cause of this was, almost certainly, that they ate less. Observa-tion during life suggested that they were eating little, and the small livers andamounts found in the alimentary tracts after death are evidence in the samedirection. Also, larger amounts of nutrients than normal were presumablycirculating in the animals which were overeating and laying down fat, ofwhich some portion would have been carried by the cross-circulation to thepartners; the partners would therefore have been expected to gain weight if

348 G. R. HBRVEY

HYPOTHALAMIC LESIONS IN PARABIOTIC RATS 349

they had not eaten less. The only alternative explanations to reduction in foodintake would appear to be (a) increased metabolism, or (b) some sort of suckingin of nutrients by the tissues of the obese animals. The thin animals did notshow any visible increase in activity. The finding of similar weight gains perunit of extra food eaten in single animals and pairs also does not support asuggestion that there was an appreciable increase in metabolism in the un-operated members of the pairs. On either of these explanations, moreover,the left-hand animals might have been expected to show a visible increase infood intake in response to the loss of nutrients.

Ifthe unoperated partners dideat less, itmaybe suggested that this inturnwasdue to the action of an internal, hypothalamic control of feeding. The possibilitythat the animals with lesions interfered with their partners' feeding in some non-specific way is difficult to exclude entirely, but observation of the animals didnot support it. Overactivity only lasted for 1 day, and thereafter no behaviourlikely to interfere with the partners' feeding was seen. Furthermore, if thepartners were being prevented from eating by external interference, it wouldhave been expected that when held in the hand and offered food they wouldhave shown signs of hunger.The results of placing lesions in the hypothalami of the partners after they

had become thin provide further evidence on the same point. Two of theanimals in which this was done began to eat when their hypothalami had beendamaged (the third had no opportunity to eat). The fact that the right-hand,previously operated, animal in the pair which survived did not apparentlylose fat may also be significant. This result would be expected if loss of fatafter a partner's operation depended on the hypothalamus; but not if itdepended on external interference with feeding, for rats with chronic hypo-thalamic lesions are sensitive to any deterrents to feeding (Kennedy, 1953 a).

It is therefore suggested that the partners of animals with lesions mostprobably became thin because their own hypothalamic controlling centresreduced their intakes of food. If this is correct, these centres appear to haveresponded to some change which took place in animals attached in parabioticunion, when these had been made hyperphagic and obese. This implies thatsome influence or 'information' from an overfed body can affect an intacthypothalamus in such a way that feeding is reduced. On this interpretation,therefore, the experiments demonstrate what in a normal single animal wouldconstitute a feedback system. The results also imply that the informationreaches the hypothalamus in some form which is capable of crossing throughthe parabiotic union.At least three suggestions have been made as to the possible nature of the

information on which the centres governing long-term regulation offood intakeact. Kennedy (1953b) has suggested that adjustment of feeding is made inrelation to the amount of stored fat in the body, and that the controlling

G. R. HERVEYcentres in the hypothalamus may be sensitive to the concentration of somemetabolite in equilibrium with stored fat. Mayer (1952) has suggested thatthe difference between the arterial and venous blood levels of glucose is theimportant factor, though he has also (1955) given Kennedy's suggestion aplace in his scheme. Brobeck (1948; 1957) has suggested that a rise in tempera-ture after eating is the signal for cessation of eating, and that the regulationof feeding is therefore part of the control of body temperature.The present results seem to fit in most easily with Kennedy's suggestion.

The partners were found to be thin when a great excess of fat was present inthe bodies of the animals subjected to hypothalamic lesions; and this excessof fat is the most obvious and permanent abnormality which has been found inanimals with lesions. An indicating metabolite could cross through the para-biotic union, provided that it was a substance slowly metabolized or excretedin the recipient animals. Substances with a rapid metabolic turnover, how-ever, cannot produce effective concentrations in the recipient members ofparabiotic pairs (Huff, Trautmann & Van Dyke, 1950). With regard to theglucose theory, even large disturbances of blood glucose in one animal of aparabiotic pair do not affect the other (Fleming & Nugent, 1957), and thealterations in glucose levels found after hypothalamic lesions are small (Bro-beck, Tepperman & Long, 1943b; Mayer, Bates & Van Itallie, 1952; unpub-lished personal measurements). There appears to be no evidence as to how farchanges in temperature in one animal of a parabiotic pair affect the other.Both 'glucose' and 'temperature' hypotheses appear to face two general

difficulties, where they are concerned with the adjusting of the intake of foodto the expenditure of energy over long periods of time. First, in so far as aquantity is stabilized by regulating mechanisms of its own, it provides lessinformation for other purposes (Ashby, 1956). Both blood glucose and bodytemperature have their own regulating mechanisms. Secondly, since succes-sive transient deviations in sugar or temperature levels from the normal donot, as far as is known, produce a cumulative change in the body, the regu-lating centres must be supposed to have a perfect 'memory' for such changes(i.e. they would have to integrate fluctuations with mathematical accuracythroughout life), or long-term drift in energy balance would occur (Kennedy,1953a). In the present experiments there was no indication that the thinanimals began to recover their fat when the animals with lesions reached thestatic phase of hypothalamic obesity. Since in this phase the intake of foodfalls to near-normal levels (Kennedy, 1950), there is a suggestion here that theloss of fat in the partners depended on some maintained change in the operatedanimals' bodies, and not on transient changes immediately related to foodintake.

Finally, the suggestion that the amount of body fat in one parabioticanimal may have influenced the feeding of the other may possibly be relevant

350

HYPOTHALAMIC LESIONS IN PARABIOTIC RATSto the findings in normal parabiotic pairs, in which both hypothalami wereintact. It was observed that the members of normal pairs each contained apercentage of fat of the order of half that found in comparable single animals.Although there are difficulties in visualizing an exact mechanism, it seemslogical to suggest that each animal's hypothalamus may have been influencedby the fat in both bodies, and that feeding was being adjusted in each animalto hold constant the total fat in the two. It would follow from this thatthere would be a greater degree of hypothalamic inhibition of feeding innormal parabiotic animals than in normal single animals. This would be con-sistent with three observations made. (1) The rate of growth was reduced afterparabiosis. (2) After hypothalamic lesions had been made, the total gains ofweight and increases of food intake were as great in pairs as in single animals,although both quantities were initially at lower levels in pairs, and althoughboth were apparently diminished in the member of the pair not subjected tolesions. (3) The incidence of post-operative choking suggests that voracitywhen the hypothalamic inhibition was removed may have been greater inparabiotic than in single animals. (Smaller capacity of the stomachs may,however, be sufficient to explain this.)

SUMMARY

1. Lesions have been made in the hypothalami of rats which were membersof pairs joined in parabiosis.

2. The animals in which the lesions were made showed hyperphagia andobesity.

3. Their parabiotic partners with normal hypothalami became thin. Someof them died, or were killed when moribund.

4. Lesions subsequently made in the hypothalami of two of the partnerswhen thin caused hyperphagia.

5. It is suggested that the partners with normal hypothalami became thinbecause they ate less; and that this in turn was due to a response on the partof their hypothalamic controlling centres to the operated animals' overfeeding.The results may be evidence for a feedback control of food intake, and maythrow some light on the information used in such a system.

It is a pleasure to thank Professor R. A. McCance for inspiring this work and for continuedadvice and encouragement; Dr E. M. Widdowson, Dr G. C. Kennedy and other colleagues formuch valuable discussion; and the Misses P. and S. Pledger for their help in looking after theanimals.

REFERENCESADOLPH, E. F. (1947). Urges to eat and drink in rats. Amer. J. Physiol. 151, 110-125.AND, B. K. & BROBECK, J. R. (1951). Hypothalamic control of food intake in rats and cats.

Yale J. Biol. Med. 24, 123-140.ASHBY, W. R. (1956). An Introduction to Cybernetics. London: Chapman and Hall.BROBEaK, J. R. (1946). Mechanism of the development of obesity in animals with hypothalamic

lesions. Physiol. Rev. 26, 541-559.

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352 G. R. HERVEYBROBECK, J. R. (1948). Food intake as a mechanism of temperature regulation. Yale J. Biol. Med.

20, 545-552.BROBECK, J. R. (1957). Neural control of hunger, appetite and satiety. Yale J. Biol. Med. 29,

565-574.BROBEC1K, J. R., TEPPERMAN, J. & LONG, C. N. H. (1943a). Experimental hypothalamic hyper-

phagia in the albino rat. Yale J. Biol. Med. 15, 831-853.BROBECK, J. R., TEPPmAN, J. & LONG, C. N. H. (1943b). The effect of experimental obesity

upon carbohydrate metabolism. Yale J. Biol. Med. 15, 893-904.BRuCE, H. M. & PARKEs, A. S. (1949). A complete cubed diet for mice and rats. J. Hyg., Camb.,

47, 202-208.BUNSTER, E. & MEYER, R. K. (1933). An improved method of parabiosis. Anat. Rec. 57, 339-343.FiENrTy, J. C. (1952). Parabiosis in physiological studies. Physiol. Rev. 32, 277-302.FmiTy, J. C. & PANos, T. C. (1951). Parabiosis intoxication. Proc. Soc. exp. Biol., N. Y., 76,

833-835.FLEmING, D. G. & NuGENT, M. A. (1957). Glucose tolerance curves in parabiotic rats. Fed. Proc.

16, 38-39.HERVEy, G. R. (1957). Hypothalamic lesions in parabiotic rats. J. Phy8iol. 138, 15-16P.HETHERINGTON, A. W. & RANSON, S. W. (1942). The relation of various hypothalamic lesions to

adiposity in the rat. J. coomp. Neurol. 76, 475-499.HuFF, R. L., TAuTmANN, R. & VAN DYKE, D. C. (1950). Nature of exchange in parabiotic rats.

Amer. J. Physiol. 161, 56-74.KENNEDY, G. C. (1950). The hypothalamic control of food intake in rats. Proc. Roy. Soc. B, 187,

535-549.KENNEDY, G. C. (1953a). The effect of lesions in the hypothalamus on appetite. Proc. Nutr. Soc.

12, 160-165.KENNEDY, G. C. (1953b). The role of depot fat in the hypothalamic control of food intake in the

rat. Proc. Roy. Soc. B, 140, 578-592.KRIEG, W. J. S. (1946). Accurate placement of minute lesions in the brain of the albino rat.

Quart. Bull. Northw. Univ. med. Sch. 20, 199-208.MAY=E, J. (1952). The glucostatic theory of regulation of food intake and the problem of obesity.

Bull. New Engl. med. Cent. 14, 43-49.MAYER, J. (1955). Regulation of energy intake and the body weight: the glucostatic theory and

the lipostatic hypothesis. Ann. N. Y. Acad. Sci. 63, 15-43.MAYER, J., BATES, M. W. & VAN ITALLIE, T. B. (1952). Blood sugar and food intake in rats with

lesions of the anterior hypothalamus. Metaboli8m, 1, 340-348.SAUERBRUCH, F. & HEYDE, M. (1908). Ueber Parabiose kilnstlich vereinigter Warmbluter.

Mlnch. med. W8chr. 55, 153-156.

EXPLANATION OF PLATES(All the plates are reproduced from colour transparencies)

PLATE 1Normal pair of young adult parabiotic rats.

PLATE 2Parabiotic pair with hypothalamic lesions in one member (the left as seen in the photograph),

after spontaneous death of the unoperated partner, approximately 2 months after the lesionshad been made.

PLATE 31, 2, Parabiotic pair, in which hypothalamic lesions were made in one member (1), approximately

1j months before killing. 3, 4, Normal parabiotic pair, litter-mates to 1, 2; killed at thesame time.

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