Physiology and Behavior, Vol. 12, pp. 259-264. Brain Research Publications Inc., 19"/4. Printed in the U.S.A.

Hypothalamic Hyperphagia Without Plateau in Ground Squirrels

N. MROSOVSKY

Departments o f Zoology and Psychology, University o f Toronto, Ontario, Canada

(Received 25 January 1973)

MROSOVSKY, N. Hypothalamic hyperphagia without plateau in ground squirrels. PHYSIOL. BEHAV. 12(2) 259-264, 1974. - Hypothalamic lesions infringing on the ventromedial nuclei in thirteen-lined ground squirrels, Citellus tridecemlineatus, were followed by weight gains, despite the fact that the animals were in a weight-loss phase of their annual cycle. Lesioning through chronically implanted electrodes and sham operations showed that the results did not depend on non-specific effects of the surgery. There was no plateau after the lesion-induced weight gain: instead weight loss and continuation of cyclic changes in body weight resumed. The results suggest that a balance between medial and lateral hypothalamic systems is still functional during annual cycles of body weight in ground squirrels.

Body weight Food intake Ground squirrel Hibernation Hypothalamus Ventromedial nucleus

ANNUAL or near annual changes in the body weight of ground squirrels are associated with slowly changing levels in the set points for body weight [1]. It has been sug- gested that the mechanisms underlying such naturally occurring changes depend on functional adjustments in the balance between medial and lateral hypothalamic areas [1,6]. If this is the case, then it should be possible to alter the level of these cycles experimentally. For instance, animals with partial lesions in the ventromedial areas of the hypothalamus should continue to show seasonal changes in body weight, but about higher absolute levels than pre- viously. The experiments reported here were designed to test this prediction.

It is known that medial hypothalamic lesions in thirteen- lined ground squirrels can result in a rapid weight gain [9] but whether this is followed by a long lasting plateau or by a resumption in cyclical changes has not been studied. The present investigations concentrated on this point. Since hibernators without any experimental lesions can some- times suddenly gain weight [4], and since the environ- mental and internal conditions controlling and triggering such weight gains are not fully understood in thirteen-lined ground squirrels [7 ], several control procedures for possible non-specific effects of hypothalamic operations were employed, and the lesions were also made in the phase of the cycle when the animals were losing weight.

METHOD General

Thirteen-lined ground squirrels, Citellus tridecemline-

atus, were kept in three rooms at different temperatures but all on a 12/12 hr l ight-dark cycle. The squirrels lived in individual cages 37 x 22 x 19 cm, provided with nesting material, and were given Purina Laboratory chow pellets and water ad lib.

Body weights and food intakes were usually recorded once a week but were taken more frequently following lesions and less frequently when the animals were hiber- nating to avoid disturbing them. In the latter case animals were only weighed when they were already awake. Weigh- ing took place within the animal rooms. Hibernation was monitored by the sawdust technique with daily inspections [4,8]. Sex was checked at autopsy.

Criteria for Hibernation

Weekly periods were considered and, on the basis of the daily inspections, put into one of three classes for hiber- nation. Well developed hibernation was considered to have occurred in that week if an animal had remained in hiber- nation at least once from one day to the next without throwing the sawdust off its back (tall vertical black bars on the figures). This is relatively easy to judge. Slight hiber- nation was scored if the animal was found hibernating on at least two days in the week, but not hibernating long enough for the sawdust to be in place on the subsequent day (black bars of small vertical extent on the figures). Requiring two such occasions, to meet the criteria for slight hibernation, guards against errors in judgment. Anything less than being found in hibernation on two occasions during the daily inspections each week was not counted, and the animal was classed as active (blank spaces on figures),

The National Research Council of Canada supported these studies. The author thanks Kirsteen Lang, Hugh Craske, Virginia Jackson, Rosemary Crean and John Hallonquist for help and comments.

259

260 MROSOVSKY

Surgical Methods

The surgical techniques have been described elsewhere [3]. The same methods, with some minor modifications, were used in the present experiments. Lesions of 2 ma for 25 secs each side, through nichrome wire of approximately 0.25 mm dia., and bare of insulation 0 .3-0 .5 mm at the tip, were made in most of the animals. For the squirrels in one of the rooms similar lesions were made through previously implanted stainless steel electrodes (MS 333, Plastic Pro- ducts, Roanoke, Va.). The hypothalamic electrode was the anode and the cathode was attached to a saline soaked piece of cotton wrapped around one leg. The coordinates used, with the skull approximately level, were as follows: Anterior from the ear bars (A) 6 .4 -7 .0 ; Lateral from the central sinus (L) 0 .6 -0 .7 ; Down from the dura (D) 8 .5-9 .6 . The mouth bar was generally slightly below the interaural line (2.7 below to 1.5 above). Some unresolved difficulties in getting consistent lesion placements in this species, even when the same coordinates were used, were the fusion of the parts of the skull and lack of a visible bregma, the marked variation in skull shape, and the fact that there was more than one location in which the head could be firmly held by the ear bars.

Histology

Animals were perfused after an overdose of Nembutal anesthesia with saline solution followed by neutral formol saline. After fixing in neutral formol saline the brains were embedded in paraffin, some double-embedded in paraffin and celloidin. Sections 10-25 ~ were cut and stained with cresyl violet or thionin.

Warm R o o m : Procedure

Three animals that had been born in the laboratory on May 18-20 , 1968 were given lesions between Nov. 13 and Dec. 12, 1968 at which time they were all losing weight. These animals were kept at 22±3.5°C. They were operated on in an adjacent room at similar temperatures and re- turned to their home room shortly after, so that there were no major thermal changes associated with the operation. The animals were kept thereafter for periods up to just over a year.

Cool R o o m : Procedure

The six animals used in this part of the experiment had been in captivity for over a year at the time of lesioning, and came from a larger group of animals that had been used in previous descriptive investigations of hibernation cycles, not involving any physiological interventions [6,7]. They were selected on the basis of showing an unmistakable phase of fattening during their second autumn in captivity (1968), followed by a weight-loss phase when the lesions were made. They were kept in a cool room, 11.5 ± 1.75°C, over the period of the experiment, except for three occa- sions when the temperature temporarily rose to 14-17°C and one occasion when it temporarily fell to 7.25°C.

For lesioning three animals were removed from the cool room. The animals were returned to the cool room about 4 - 1 0 hr after being taken out, when they had recovered from the anesthetics. In addition, three animals were given sham operations, and treated in the same way except that electrodes were lowered varying distances into the brain and withdrawn without passing current.

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FIG. 1. Body weights of ground squirrels kept in a warm room. Figures on the left are the body weights at the start of the graphs. Underlined figures are numbers assigned to each animal. Arrows show dates of lesions.

HYPOTHALAMIC HYPERPHAGIA IN HIBERNATORS 261

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FIG. 2. Body weights of ground squirrels kept in a cool room. Figures on the left are the body weights at the start of the graphs. Underlined figures are the numbers assigned to each animal. Arrows show the dates of lesions or sham operations (S). Tall solid bars represent well developed hiberation and solid bars of smaller vertical

extent represent slight hibernation as defined in the text.

Cold Room: Procedure

Sixteen ground squirrels from near Lawrence, Kansas, were obtained from a dealer and arrived in the laboratory on September 3, 1968. From that time until July 2, 1969 they were kept at approximately 8.5°C, with fluctuations between 2 ° and 14°C. All but two of these animals showed some hibernation during their first winter in captivity. From July 2, 1969 to October 29, 1969 the cooling in the room was turned off because there is some evidence that prehibernation weight gains do not occur as readily in

thirteen-lined ground squirrels kept in constant cold tem- peratures [7]. In the present case there were rapid weight gains during the time the cooling was off (temperature approximately 26°C with fluctuations between 22 ° and 33°C). On October 29, 1969 the cooling was turned on again and for the rest of the experiment the animals were kept at 10.0 ± 3.5°C except for some temporary tempera- ture drops below this range in June 1970 when the main part of the experiment was already over.

Between January 27, 1969 and June 12, 1969 the

262 MROSOVSKY

animals were temporarily removed from the cold room and electrodes were bilaterally implanted. Some of the animals developed infections around their implants which were treated with neohydracaine opthalmic ointment. Between December 15, 1969 and January 12, 1970, at times when all animals were in a weight-loss phase, lesions were made wh i l e t he animals were in a state of hibernation (11.0-13.75°C rectal temperatures, 4 cm immersion of temperature probe) without removing from their environ- ment. In two animals larger lesions (3 ma, 30 sec) were made later through the same electrodes.

Of the 16 animals originally intended for this experi- ment, three died under anesthetic or shortly afterwards, four pulled out their implants and one was discarded on account of a broken tooth. Data are presented on the remaining eight animals.

RESULTS AND DISCUSSION

A few of the animals showed marked weight increases after the lesion (No. 73, Fig. 1; No. 37, Fig. 2; No. 102, Fig. 3). Several considerations suggest that specific lesion effects rather than non-specific effects associated with the surgery are responsible. First, weight gains could be pro- duced with minimal interference in animals bearing chron- ically implanted electrodes. Second, for animals in the warm room, no great thermal changes were associated with the operation. Third, weight gains were correlated with damage in the ventromedial hypothalamus (Table 1, Fig. 4). Fourth, weight gains following sham operations were slight, 4-7 g, compared to those after successful lesions, 55-188 g.

The most interesting feature of the results was that both in animals that showed marked weight gains and those that showed slighter gains, there was no permanent plateau, but changes in body weight continued as long as the experiment lasted, in some cases over a year.

A second general point was that hibernation was inter- rupted during the lesion-induced weight gain. This is not surprising since food intakes, although often taking a few days to reach their maximum values, were much elevated compared to prelesion levels and ground squirrels do not eat while in deep torpor. However, the relationship between hibernation and eating is probably more complicated than direct response competition [5]. Hibernation generally reappeared when the animals began to lose weight again (Figs. 2 and 3), an apparent discrepancy with previous reports [10].

Although in a few animals the weight changes were rather erratic (No. 28, Fig. 2) the principal effect of lesions infringing on the ventromedial nucleus was quite consis- tent: a weight gain, followed by a weight-loss phase. It is not possible, however, to determine from the present data whether the annual variation continues around an elevated mean level or whether there is a greater peak to trough difference.

Since the lesions occurred when the animals were losing weight, the results show that medial hypothalamic systems involved in weight control are still operating in a hibernator even when it is in a weight-loss phase of the cycle. With such systems still operational, it is plausible that the natur- ally occurring cycle of set point changes in hibernators depends in part on a shifting balance between medial and lateral hypothalamic systems [1 ]. Suggesting this does not necessarily imply that the primary factor in producing rhythmicity is hypothalamic; other regions in the brain

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FIG. 3. Body weights of ground squirrels kept in a cold room. Figures on the left are body weights at the start of the graphs. Underlined figures are the numbers assigned to each animal. Arrows show dates of lesions through chronically implanted electrodes. R, lesion in the right side only; L, lesion in the left side only; b, relesioning at higher current (see text). Tall solid bars represent well developed hibernation and solid bars of smaller vertical extent repre-

sent slight hibernation as defined in the text.

HYPOTHALAMIC H Y P E R P H A G I A IN H I B E R N A T O R S 263

TABLE 1

LOCATION OF LESIONS IN THIRTEEN-LINED GROUND SQUIRRELS

Coordinates* at Maximum Extent

Limits of Lesion Animal of Lesion Number (left and right side) Ant. Post.

D L A A A

Main Damaged Areas

70 1.7 0.7 9.5 9.8 9.5

1.7 0.7 9.5 9.8 9.5

83 2.7 0.7 >9.8 >9.8 9.5

2.7 0.7 >9.8 >9.8 9.8

73 1.7 0.7 7.4 7.7 7.1

1.7 0.7 7.4 8.0 7.1

37 2.5 1.0 9.2 9.5 7.7

2.5 0.5 9.2 9.5 7.7

28 2.2 1.2 8.6 9.5 8.0

2.0 0.5 8.6 9.5 8.3

25 1.5 1.2 8.6 9.2 8.0

2.0 0.5 8.6 8.9 8.0

105 1.5 0.8 8.9 9.2 8.6

1.5 0.8 8.9 9.2 8.6

104 2.0 0.7 9.5 9.8 9.2

2.0 0.7 9.5 9.8 9.5

102 1.7 0.7 8.0 8.9 7.7

2.0 0.7 8.3 9.2 7.7

100 t 3.2 0.7 9.5 >9.8 8.9

3.5 0.5 9.8 >9.8 9.5

96y 2.0 0.5 8.3 9.2 7.7

2.0 1.0 8.6 9.5 8.3

93 t 1.5 0.5 8.3 8.9 7.7

1.5 0.7 8.3 8.6 8.0

92 2.2 0.5 9.8 >9.8 9.5

2.2 0.7 9.8 >9.8 9.5

91 2.0 1.0 8.9 9.2 8.6

2.0 0.5 8.9 9.5 8.6

WARM ROOM (FIG. 1)

chiasma opticum

chiasma opticum

chiasma opticum

chiasma opticum

n. ventromedialis, n. arcuatus

n. ventromedialis, n. arcuatus

COOL ROOM (FIG. 2)

n. ventromedialis, chiasma opticum, gyrus diagonalis

n. ventromedialis, n. arcuatus, n. suprachiasmaticus, chiasma opticum

chiasma opticum, gyrus diagonalis, n. suprachiasmaticus, n. supraopticus, n. preopticus lateralis

chiasma opticum, gyrus diagonalis, n. suprachiasmaticus, n. preopticus medialis

chiasma opticum, gyrus diagonalis

n. suprachiasmaticus, n. preopticus medialis, chiasma opticum

COLD ROOM (FIG. 3)

chiasma opticum

chiasma opticum

chiasma opticum, gyrus diagonalis

chiasma opticum

n. ventromedialis, n. preopticus medialis, chiasma opticum

n. ventromedialis, n. preopticus medialis, chiasma opticum, gyrus diagonalis

n. preopticus medialis, n. septi lateralis, n. accubens, gyrus diagonalis

gyrus diagonalis

chiasma opticum, n. preopticus medialis, n. anterior hypothalami, n. ventromedialis, n. suprachiasmaticus

n. preopticus medialis, n. supraopticus, chiasma opticum

chiasma opticum, n. preopticus medialis, n. arcuatus, n. ventromedialis, n. suprachiasmaticus

n. supraopticus, chiasma opticum

gyrus diagonalis, chiasma opticurn

gyrus diagonalis, chiasma opticum

n. preopticus medialis, n. supraopticus, chiasma opticum

n. preopticus medialis, n. suprachiasmaticus, gyrus diagonalis, chiasma opticum

*From the atlas of Joseph et aL [2] tSome infection around implant

264 M R O S O V S K Y

FIG. 4. Coronal section of ground squirrel No. 73 (Fig. 1) showing damage in ventromedial areas (cresyl violet).

might m o d i f y h y p o t h a l a m i c areas in h i b e r n a t o r s on a sea- sonal basis [ 4 ] . Whe the r the ba lance b e t w e e n media l and lateral sys tems changes s p o n t a n e o u s l y or is con t ro l l ed b y s t imul i f rom elsewhere , a par t ia l lesion in the media l hypo-

t ha l amus upsets this ba lance bu t leaves e n o u g h t issue avail- able for r h y t h m i c changes to c o n t i n u e to lower set po in t s and this leads to the p h e n o m e n o n of h y p o t h a l a m i c hyper - phagia w i t h o u t a p la teau phase.

REFERENCES

1. Barnes, D. S. and N. Mrosovsky. Body weight regulation in ground squirrels and hypothalamically lesioned rats: slow and sudden set point changes. Physiol. Behav. 12: 251-258, 1974.

2. Joseph, S. A., K. A. Knigge, L. M. Kalejs, R. A. Hoffman and P. Reid. A Stereotaxic Atlas o f the Brain of the 13-line Ground Squirrel (Citellus tridecemlineatus). Maryland: U. S. Army, Edgewood Arsenal, 1966.

3. Mrosovsky, N. Self-stimulation in hypothermic hibernators. Cryobiology, 2: 229-239, 1966.

4. Mrosovsky, N. Hibernation and the Hypothalamus. New York: Appleton-Century-Crofts, 1971.

5. Mrosovsky, N. and D. S. Barnes. Anorexia, food deprivation and hibernation. Physiol. Behav. 12: 265-270, 1974.

6. Mrosovsky, N. and K. C. Fisher. Sliding set points for body weight in ground squirrels during the hibernation season. Can. J. Zool. 48: 241-247, 1970.

7. Mrosovsky, N. and K. Lang. Disturbances in the annual weight and hibernation cycles of thirteen-lined ground squirrels kept in constant conditions and the effects of temperature changes. J. interdiscipl. Cycle Res. 2: 79-90 , 1971.

8. Pengelley, E. T. and K. C. Fisher. Rhythmical arousal from hibernation in the golden-mantled ground squirrel, Citellus lateralis tescorum. Can. J. Zool. 39: 105-120, 1961.

9. Satinoff, E. Aberrations of regulation in ground squirrels following hypothalamic lesions. Am. J. Physiol. 212: 1215-1220, 1967.

10. Satinoff, E. Disruption of hibernation caused by hypothalamic lesions. Science 155: 1031-1033, 1967.