336

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

THE EFFECTS OF LESIONS IN THE HYPOTHALAMUS IN 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 almost equal over long periods. This equality is probably brought about mainly by adjustment of the amount of food eaten. Regulation of food intake is particularly efficient in the young adult rat, which can adjust its intake to provide a nearly constant supply of calories when the bulk it must eat is varied by diluting the food with inert material (Adolph, 1947; Kennedy, 1950). In the rat and other mammals which have been investigated, bilateral lesions in the region of the ventromedial nuclei of the hypothalamus cause obesity if the animal 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 (Hetherington & Ranson, 1942; Brobeck, 1946; Kennedy, 1950). It has therefore been suggested that the hypothalamus contains nervous centres which normally control 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. It is difficult to see how they can directly measure the number of calories expended in a given period, and then regulate feeding to provide just this amount of energy. Alternatively, the regulk ing centres may be sensitive to some change in the body which follows the intake or expenditure of energy, and they may inhibit or encourage eating until the changed quantity has been restored to normal. This would be a 'feedback' control system: the controlling centres would be acting on information, fed back to them from the periphery, about the behaviour of the quantity they stabilized. Mechanisms of this type are known to be important in the body, as well as in artificial control mechanisms. Brobeck (1948), Mayer (1952) and Kennedy (1953b) have proposed systems of the feedback type in relation to the control of food intake. It has not, however, been directly demonstrated
*

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

337 HYPOTHALAMIC LESIONS IN PARABIOTIC RATS that the regulation of food intake involves a feedback system, and there is conflicting evidence as to the nature of the information used and the path by which it reaches the centres. Food intake can be shown to be modified in response to many factors; the difficulty is to identify the mechanisms particularly concerned in achieving an approximate balance of energy over long periods. In the present study a series of parabiotic rats have been subjected to hypothalamic lesions of the type which causes obesity. Parabiotic animals are surgically united when young, and exchange blood throughout life; the preparation has proved valuable in investigating a number of physiological interrelationships involving long-term responses (Finerty, 1952). It has already been briefly reported that lesions made in one member of a parabiotic pair appear to influence the feeding of the other (Hervey, 1957). This response in the partner may provide some further evidence of the mechanism and pathways which in normal animals regulate the intake of food over long periods, and so maintain energy balance.
METHODS

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

Parabiosis The 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 the most recent ones have been united when a week or two older. As many pairs as possible were made from each litter, and any compargle animals left over were kept as single controls. Some of these single animals were subjected to sham operations, in which equally extensive surgery was carried out without uniting animals. Both sexes were used. The anaesthetic was pentobarbitone sodium, given intraperitoneally in a dose of approximately 6 mg/100 g body weight. The surgical technique used for the earlier pairs was that of Bunster & Meyer (1933), in which the peritoneal 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 'coelioanastomosis' (Sauerbruch & Heyde, 1908). This avoids the risk of small openings forming between the 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 posterior parts of the bodies has been secured by tying the femora together (J. M. Ledingham, personal communication). Bunster & Meyer's technique of uniting the scapulae was followed, except that the scapulae were scraped to expose raw bone before suturing. Aseptic technique was used as far as practicable. PHYSIO. CXLV 22

338

G. R. HERVEY
Plasma exchange

The 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 an 0-25% 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 from each member of the pair by snipping the tip of the tail under light ether anaesthesia. Clotting was prevented by a trace of solid heparin. After separating the plasma, 0 1 ml. from each specimen was diluted with 3-4 ml. 0-2 % sodium carbonate, and the optical densities were read at 620 m,u in 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 the time 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 plasma volumes, that disappearance of the dye from each circulation is exponential, and that the time constants of disappearance are the same in both animals. These assumptions were tested by giving different doses of dye to single animals and following the rates of disappearance from the plasma. They appeared to be approximately true over the duration of the test. The calculations from 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 lesions The 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 death by naked-eye examination of the under surface of the brain. The lesions are easily visible; the appearance when they are correctly placed is shown in Text-fig. 1. Variation in size of the animals was allowed for by adjusting the antero-posterior co-ordinate (i.e. the distance forward from the line of the ear-bars of the instrument to the site of the electrode). The correct distances were found for a number of animals by operating upon and killing them. The distances were then plotted against a 'skull length index', obtained by measuring the distance between the ear-bars and 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 method was found more reliable for parabiotic animals than working from the total weights of pairs. The

HYPOTHALAMIC LESIONS IN PARABIOTIC RATS

339

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

Analyses The 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 removed from the alimentary tract, and the loss of weight recorded. The bodies were placed on weighed trays in an oven at approximately 1050 C, chopped and spread out after 24 hr, and dried to constant weight. Fat was estimated in the dried material by extracting in successive changes of petroleum 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 last extraction were again dried in the oven and weighed. This method was checked by recovery of the fat from the solvent and by Soxhlet extraction of the residues; 98-99% of the fat was extracted. Total nitrogen was estimated in some of the dry fat-free residues by a3 tandard microKjeldahl method.
RESULTS

Parabiosis In the course of the experiments 93 parabiotic pairs have been made. Out of 39 pairs made from specially inbred stock, 32 (82%) survived to become young adults. All the animals lost from this group died during the operation or from infection or illness afterwards, and none from the condition known as parabiotic intoxication or disharmony, in which one member of a pair fails to grow, 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 of disharmony. Of the 54 pairs made from ordinary stock 30 (55%) survived to young adulthood. Eight of the pairs in this group which died appeared to be typical cases of disharmony; one pair which died as adults were thought to show disharmony; and two pairs apparently developed disharmony but recovered later. The difference in the incidence of disharmony agrees with Finerty & Panos' suggestion that the incidence is inversely related to the closeness of inbreeding. Close inbreeding, however, led to smaller litters and smaller-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 unpaired animals. This was not simply due to the effect of a surgical operation in infancy, for the sham operations on single animals had no detectable effect. Twelve normal pairs could be compared with single litter-mates, of the same sex and similar weight at the time of parabiosis. At 5 months old the animals in the parabiosis weighed 71-85% of the weights of their single litter-mates (average 78 %). For this comparison the parabiotic animals' weights were taken to be half the total weights of the pairs. When parabiotic animals were killed without further experimental treatment the weights of the individuals
22-2

340 G. R. HER VEY in a pair were normally found to be approximately equal. Ten normal pairs were killed at various ages; the weights of the separated individuals differed from the mean weights of the pairs to which they belonged by 1-8 % of the mean 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 or absence of a coelio-anastomosis made no apparent difference to the exchange of plasma. The two pairs with the lowest exchange rates were early pairs, made by the unmodified Bunster & Meyer technique, in which the scapulae had become widely separated.

Hypothalamic lesions An attempt was made to produce hypothalamic lesions of the type which lead to obesity in the right-hand animals of 32 parabiotic pairs. The lesions were normally made when the animals had just passed the stage of rapid growth. Whenever possible a second pair of the same sex and litter was kept unoperated on for comparison, and so also were the single litter-mates. Thus three groups of animals were obtained, normal single animals, normal pairs, and pairs with lesions in the right-hand animal, all from similar stock and including litter-mate comparisons where possible. Hypothalamic lesions were also made in a number of single animals; the animals in this group were not specially selected to be comparable with the others. In a first series of 11 operations on members of pairs, 4 were successful. In a later series of 21 pairs, one pair died during the operation, and in all others the lesions were effective. Five pairs with effective lesions, however, were lost soon after the operation. Three of the operated animals died from aspirating food into the trachea; one died from intestinal obstruction caused by eating sawdust; and one died from excessive drinking. Brobeck, Tepperman & Long (1943a) described choking from voracious eating soon after hypothalamic lesions. Parabiotic animals seemed to be particularly liable to death in this way, and latterly care was taken to withdraw food and water and anything the animals could swallow for 24 hr after making the lesions. On the other hand, no single animals with lesions were lost as a result of post-operative voracity, although no special precautions were taken with them. In single animals successful lesions were followed by the hyperphagia and obesity which are now well known. In parabiotic pairs, the effects in the animals which received the lesions (i.e. the right-hand animals of operated pairs) were in general similar. On recovering from the anaesthetic most of these animals were overactive, and they would immediately eat voraciously

341 HYPOTHALAMIC LESIONS IN PARABIOTIC RATS and unselectively. The overactivity disappeared within 24 hr, though the operated animals often remained somewhat irritable and more difficult to handle than before. The grossly voracious eating also disappeared, but pairs of which one member had received lesions continued to show a greater food
* R of pair / with lesions

I
I
I .'I

_(Mean of air with lesions]

ISingle rat
R Normal L pair L of pair with lesions

(in right-hand rat

of one pair)

All killed

Months curves and Growth body weights of a group of two parabiotic pairs and 2. post-mortem Text-fig. one single rat. All were males and litter-mates. At approximately 5 months old, lesions were made in the hypothalamus of the right-hand member of one pair. The left-hand member of this pair appeared to be moribund approximately 21 months later; all the animals were then killed. , 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. Textfig. 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 a parabiotic pair in which hypothalamic lesions were made in the right-hand animal. The weight curves for some pairs which were kept long enough appeared to approach a plateau 2-3 months after lesions had been made.

342 G. R. HER VEY The 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 the increases 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 Ratio: Initial Weight gained Food intake in 28 days exces food (g) in 28 days weight exces gain (g) Serial no. Sex (g) (g) (g) M 1 236 76 5*0 700 F 2 169 79 F 178 112 3 F 3-9 730 4 130 135 M 116 5 260 M 6 212 48 M 7 232 149 F 163 8 171 F 172 150 9 M 10 131 250 M 3-8 1360 11 192 313 M 12 252 245 4-1 M 1350 285 183 13 5'2 M 980 14 280 113 M 77 15 217 16 173 136 M 17 178 171 M 18 152 134 M 207 93 19 M 234 142 20 M 266 21 152 M 22 157 184 M 188 126 23 M 109 24 167 126 44 25 4.4 1020 129 Average 209 Mean age at operation: 4*1 months.

TABLE 2. Weight gains and food intakes of parabiotic pairs in 28 days after hypothalamic lesions in the right-hand animal Ratio: Initial Weight gained Food intake in 28 days in 28 days excess food (Ig) weight excess gain (^g) (g) (g) (g) Sex Serial no. 210 109 F 8 M 128 247 12 4*8 M 126 1320 349 14 M 4*0 55 990 285 18 367 183 F 42 3*5 1530 M 119 505 49 83 329 F 53 128 318 F 54 131 F 354 56 164 M 492 58 1450 5*9 M 443 103 60 3-6 M 1190 52 454 66 M 225 481 71 155 1540 M 445 4B5 79 184 294 F 81 100 M 568 85 4-4 128 1340 384 Average Mean age at operation : 5-1 months.

343 HYPOTHALAMIC LESIONS IN PARABIOTIC RATS parabiotic pairs during the 28 days after the placing of lesions in the hypothalamus (of the right-hand animal in the case of the pairs). Both tables include all animals in which the lesions led to a definite increase in weight and which survived for 28 days or longer. Within each group the gains of weight did not seem to be influenced by the animal's age, sex or initial weight, so the lack of comparability in these respects may not invalidate comparison of the effects 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 both single animals and parabiotic pairs. The food intakes of single and parabiotic animals are not directly comparable, and satisfactory measurements were only made on a minority of animals in each group. It also so happens that five of the six pairs with lesions, for which food intake measurements were available, showed less than average gains of weight (perhaps because choking tended to remove the pairs with the most effective lesions, if food was continuously available, 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 the same range in both groups (final columns in Tables 1 and 2). As the average weight gains in the complete groups were the same, this suggests that the average increases in food intake might also have been the same if measurements 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 gram of extra food eaten, or about 0O08 g for each excess kilocalorie. The efficiency of conversion of food into body weight was apparently of the same order in single and parabiotic animals. It was clear from observation that the hyperphagia and weight gain of pairs with lesions in one member were entirely due to the animals which received the lesions (i.e. the right-hand animals). These animals showed the initial overactivity and voracity, their stomachs were distended within a day of the operation, and they rapidly became obviously obese. The partners with intact hypothalami (the left-hand animals) were certainly not eating ravenously, 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 interfere with their partners' feeding in any way that could be seen. They were not seen to attack their partners, drag them around the cage, or impede their access 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 the food; 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

G. R. HER VEY animal. In one instance the obese animal was still alive when the death was discovered. In the other, both animals were dead, but the prior death of the left-hand animal was shown by the accumulation of blood in its body. A one-way movement of blood takes place when one member of a parabiotic pair dies before the other; this effect appears to be a very reliable indication of which animal has died first when a pair is found dead. Two other pairs were killed because the left-hand partners appeared to be moribund, 8 and 10 weeks after the operation. Three pairs with effective lesions in the right-hand animal were subjected to further hypothalamic lesions in the left-hand animal (see below). Four pairs were lost in the course of other experimental surgery or from infection. The remaining eight pairs with lesions in one member were killed arbitrarily 1-3 months after the operation.
Post-mortem findings At post-mortem examination the individuals with effective lesions in the hypothalamus were obviously obese, as would be expected. The thin and starved appearance of their parabiotic partners was almost equally striking (Plates 2, 3). In the animals with lesions much fat was visible in the subcutaneous tissues and omentum; the alimentary tracts, particularly the stomachs, were hypertrophied and full; the livers were large, pale and mottled. The appearances were in no way different from those seen in single rats with effective hypothalamic lesions. The bodies of the partners contained no visible fat; the viscera appeared atrophic and empty; the livers were small and dark. Where a coelio-anastomosis had been made, the mass of viscera and omentum belonging to the animal with lesions almost filled the combined peritoneal cavity; but the partners showed the same changes whether or not a coelioanastomosis was present. The partners which died spontaneously, or appeared to be moribund, showed no abnormalities beyond those described. Normal parabiotic animals contained some visible fat, although less than single animals; their viscera, although smaller and less well-filled than in single animals, looked essentially normal and did not show the appearance of atrophy and emptiness seen in the partners of animals with lesions. Numerical data obtained at post-mortem and in subsequent analyses are given in Tables 3-6. Tables 3 and 4 give the body weights and fat contents of normal single animals, normal parabiotic pairs, parabiotic pairs with lesions in the hypothalamus of the right-hand member, and single animals with lesions (males only). All animals are included in which the techniques were carried out successfully and which were not lost from intercurrent causes. The individual data have been given for the parabiotic animals, and mean values only for the single animals. Males and females are grouped separately. Tables 5 and 6 give other measurements as mean values for the groups.

344

345 HYPOTHALAMIC LESIONS IN PARABIOTIC RATS Body weight. The normal findings in parabiotic animals have already been mentioned. When hypothalamic lesions were made in one member of a pair they 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 of normal parabiotic pairs. Where pairs with lesions in one member could be compared individually with litter-mate pairs which had weighed approximately the same at the time of the operation, the partners of the animals with lesions 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 Fat Body weight (% body weight) Age at (g) Serial death L R L R (months) no. 12-4 327 Normal single rats 7-2 (mean of 14) 6-5 206 8-3 Normal parabiotic 6-8 234 19 6-1 6-1 276 282 7-6 59 pairs 5-6 6-1 203 5-1 182 61 8-5 8-0 319 275 8-0 69 5-1 5-4 197 4-4 221 80 6-7 6-4 241 mean of 5 6-4 238 2-1 52-2 161 347 Parabiotic pairs with 4-9 12 2-1 50-8 412 lesions in right-hand 185 8-2 14 1-7 45-2 rat (average age at 220 525 8-6 49 2-1 49-8 236 600 7-5 operation 5-0 months) 58 1-2 49-4 488 7-5 182 60 41-2 1-6 236 394 8-7 66 1-0 47-9 475 217 6-9 79 51-3 0-6 507 242 8-6 85 1-6 48-5 469 210 mean of 8 7-6 49-4 501 7-6 Single rats with lesions (average age at operation 4-7 months) (mean of 8) TABLE 4. Post-mortem body weights and fat contents of female animals Fat Body weight (g) Age at body weight) (% A death Serial R L R L no. (months) 16-5 6-9 197 Normal single rats (mean of 4) 7-6 9-7 154 151 6-4 Normal parabiotic 26 10-1 9-1 174 179 43 8-1 pairs 6-7 6-9 191 162 7-4 44 14-4 14-5 184 190 8-6 55 7-0 8-5 157 176 6-1 82 172 9-0 9-9 172 mean of 5 7-3 65-8 3-4 307 107 8 5-0 Parabiotic pairs with 41-2 5-1 381 174 42 8-1 lesions in right-hand 48-6 1-1 362 143 54 8-6 rat (average age at 35-0 2-8 315 56 166 6-9 operation 5-3 months) 57-1 4-2 395 81 147 6-1 49-5 3-3 352 147 6-9 mean of 5

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347 HYPOTHALAMIC LESIONS IN PARABIOTIC RATS the time of the operation, in most instances the partners of the animals in which 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 found in normal parabiotic animals was of the order of half that found in single animals of the same sex. Where parabiotic animals could be compared individually with single litter-mates, an approximately 1: 2 relation was found, within a scatter of 1-2% of fat. The parabiotic animals in which effective lesions had been made showed greatly increased fat percentages, similar to the levels found in single animals with lesions. The partners of animals with lesions were found to contain very low percentages of fat, in keeping with their naked-eye appearance. This effect appears to have been a little more marked in male than in female animals. In both sexes the range of fat percentages found in the partners of animals with lesions did not overlap the range for normal parabiotic 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 be expected since the animals were usually nearly full-grown when the lesions were made. Contents of the alimentary tract. It is reasonable to suppose that the amount of the contents of the alimentary tract would bear some relation to the turnover of food. The contents of the alimentary tracts of normal parabiotic pairs were a little less than the amounts found in single animals. The animals with hypothalamic lesions showed much increased contents; their partners gave figures slightly lower than those for members of normal pairs. Measuring the contents of the alimentary tract as a whole may have minimized these differences. At post-mortem examination the emptiness of the stomachs of the partners of animals with lesions was very striking. The small intestines also appeared to be comparatively empty, but the caeca and colons of these animals contained quite large amounts of rather dry material. If the contents of the stomachs alone had been measured the differences between the groups might have been more marked. Composition of the fat-free bodies. The mean weights of the fat-free portions of the bodies showed differences between the groups generally in the same direction as the differences in whole body weight but less marked. The animals with hypothalamic lesions evidently only gained small amounts of fat-free tissue compared with their large gains of total weight. The partners of animals with lesions showed small losses of fat-free weight, in comparison with control pairs. The percentages of water in the fat-free tissue, together with the small number of nitrogen analyses, show no evidence of any differences between the groups in the composition of the fat-free parts of the body.

348 G. R. HBR VEY Weights of individual organs. The livers of normal parabiotic animals on average weighed less than the livers of normal single animals, but there was a good deal of variation. The livers of animals subjected to hypothalamic lesions showed a substantial increase in weight; their partners' livers on average weighed a little less than the livers of normal parabiotic animals. The weights of the hearts and kidneys showed differences in the same direction as the weights of the livers, but less marked, and with the exception that the hearts of the partners of animals with lesions were not reduced in weight.

Second hypothalamic lesions In three parabiotic pairs hypothalamic lesions were made in the left-hand animals when these had become obviously thin, after earlier lesions in the right-hand animals. All three pairs were males, and the intervals between the two hypothalamic operations were 21-31 months. In the first pair, the newly operated animal ate avidly as soon as food was offered; before the operation 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 the second pair during the remainder of the day and the night following the second operation. The newly operated animal died during the night. It had previously shown marked overactivity, alternating periods of running round the cage with lying immobile, and it may have died from exhaustion. The third pair survived the post-operative period, and were killed some 2 months later. After the operation the weight of this pair increased rapidly. At death the fat contents of the carcasses were: left-hand animal, 224%; right-hand animal, 48-5%. Both animals had fairly heavy livers and alimentary tract contents. The lesion on one side of the left-hand animal's brain was displaced about 05 mm from the optimum position. An unoperated single litter-mate contained 8-1 % of fat.
DISCUSSION

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

349 HYPOTHALAMIC LESIONS IN PARABIOTIC RATS they had not eaten less. The only alternative explanations to reduction in food intake would appear to be (a) increased metabolism, or (b) some sort of sucking in of nutrients by the tissues of the obese animals. The thin animals did not show any visible increase in activity. The finding of similar weight gains per unit of extra food eaten in single animals and pairs also does not support a suggestion that there was an appreciable increase in metabolism in the unoperated members of the pairs. On either of these explanations, moreover, the left-hand animals might have been expected to show a visible increase in food intake in response to the loss of nutrients. If the unoperated partners did eat less, it may be suggested that this in turn was due to the action of an internal, hypothalamic control of feeding. The possibility that the animals with lesions interfered with their partners' feeding in some nonspecific way is difficult to exclude entirely, but observation of the animals did not support it. Overactivity only lasted for 1 day, and thereafter no behaviour likely to interfere with the partners' feeding was seen. Furthermore, if the partners were being prevented from eating by external interference, it would have been expected that when held in the hand and offered food they would have 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 the animals in which this was done began to eat when their hypothalami had been damaged (the third had no opportunity to eat). The fact that the right-hand, previously operated, animal in the pair which survived did not apparently lose fat may also be significant. This result would be expected if loss of fat after a partner's operation depended on the hypothalamus; but not if it depended on external interference with feeding, for rats with chronic hypothalamic lesions are sensitive to any deterrents to feeding (Kennedy, 1953 a). It is therefore suggested that the partners of animals with lesions most probably became thin because their own hypothalamic controlling centres reduced their intakes of food. If this is correct, these centres appear to have responded to some change which took place in animals attached in parabiotic union, when these had been made hyperphagic and obese. This implies that some influence or 'information' from an overfed body can affect an intact hypothalamus in such a way that feeding is reduced. On this interpretation, therefore, the experiments demonstrate what in a normal single animal would constitute a feedback system. The results also imply that the information reaches the hypothalamus in some form which is capable of crossing through the 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 of food intake act. Kennedy (1953b) has suggested that adjustment of feeding is made in relation to the amount of stored fat in the body, and that the controlling

350 G. R. HERVEY centres in the hypothalamus may be sensitive to the concentration of some metabolite in equilibrium with stored fat. Mayer (1952) has suggested that the difference between the arterial and venous blood levels of glucose is the important factor, though he has also (1955) given Kennedy's suggestion a place in his scheme. Brobeck (1948; 1957) has suggested that a rise in temperature after eating is the signal for cessation of eating, and that the regulation of 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 in the bodies of the animals subjected to hypothalamic lesions; and this excess of fat is the most obvious and permanent abnormality which has been found in animals with lesions. An indicating metabolite could cross through the parabiotic union, provided that it was a substance slowly metabolized or excreted in the recipient animals. Substances with a rapid metabolic turnover, however, cannot produce effective concentrations in the recipient members of parabiotic pairs (Huff, Trautmann & Van Dyke, 1950). With regard to the glucose theory, even large disturbances of blood glucose in one animal of a parabiotic pair do not affect the other (Fleming & Nugent, 1957), and the alterations in glucose levels found after hypothalamic lesions are small (Brobeck, Tepperman & Long, 1943b; Mayer, Bates & Van Itallie, 1952; unpublished personal measurements). There appears to be no evidence as to how far changes 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 food to the expenditure of energy over long periods of time. First, in so far as a quantity is stabilized by regulating mechanisms of its own, it provides less information for other purposes (Ashby, 1956). Both blood glucose and body temperature have their own regulating mechanisms. Secondly, since successive transient deviations in sugar or temperature levels from the normal do not, as far as is known, produce a cumulative change in the body, the regulating centres must be supposed to have a perfect 'memory' for such changes (i.e. they would have to integrate fluctuations with mathematical accuracy throughout life), or long-term drift in energy balance would occur (Kennedy, 1953a). In the present experiments there was no indication that the thin animals began to recover their fat when the animals with lesions reached the static phase of hypothalamic obesity. Since in this phase the intake of food falls to near-normal levels (Kennedy, 1950), there is a suggestion here that the loss of fat in the partners depended on some maintained change in the operated animals' bodies, and not on transient changes immediately related to food intake. Finally, the suggestion that the amount of body fat in one parabiotic animal may have influenced the feeding of the other may possibly be relevant

351 HYPOTHALAMIC LESIONS IN PARABIOTIC RATS to the findings in normal parabiotic pairs, in which both hypothalami were intact. It was observed that the members of normal pairs each contained a percentage of fat of the order of half that found in comparable single animals. Although there are difficulties in visualizing an exact mechanism, it seems logical to suggest that each animal's hypothalamus may have been influenced by the fat in both bodies, and that feeding was being adjusted in each animal to hold constant the total fat in the two. It would follow from this that there would be a greater degree of hypothalamic inhibition of feeding in normal parabiotic animals than in normal single animals. This would be consistent with three observations made. (1) The rate of growth was reduced after parabiosis. (2) After hypothalamic lesions had been made, the total gains of weight 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 although both were apparently diminished in the member of the pair not subjected to lesions. (3) The incidence of post-operative choking suggests that voracity when the hypothalamic inhibition was removed may have been greater in parabiotic 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 members of pairs joined in parabiosis. 2. The animals in which the lesions were made showed hyperphagia and obesity. 3. Their parabiotic partners with normal hypothalami became thin. Some of them died, or were killed when moribund. 4. Lesions subsequently made in the hypothalami of two of the partners when thin caused hyperphagia. 5. It is suggested that the partners with normal hypothalami became thin because they ate less; and that this in turn was due to a response on the part of their hypothalamic controlling centres to the operated animals' overfeeding. The results may be evidence for a feedback control of food intake, and may throw 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 continued advice and encouragement; Dr E. M. Widdowson, Dr G. C. Kennedy and other colleagues for much valuable discussion; and the Misses P. and S. Pledger for their help in looking after the animals. REFERENCES ADOLPH, 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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BROBECK, 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 hyperphagia 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 1 Normal pair of young adult parabiotic rats. PLATE 2 Parabiotic 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 lesions had been made.

PLATE 3 1, 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 the same time.

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