Effects of the rearing temperature on the temporal feeding pattern of the staggerer mutant mouse

Physiology& Behavior, Vol. 49, pp. 405--409. ©Pergamon Press plc, 1991. Printed in the U.S.A.

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Effects of the Rearing Temperature on the Temporal Feeding Pattern of the Staggerer Mutant Mouse J E A N - M A R I E G U A S T A V I N O , * R. B E R T I N t A N D R. P O R T E T t

*Laboratoire d'Ethologie et Sociobiologie, URA C.N.R.S. 667 Universit~ Paris Nord, 93430 Villetaneuse, France "?Laboratoire d'Adaptation Energ~tique ?t l'Environment, E.P.H.E. CollOge de France, 75231 Paris, France R e c e i v e d 10 M a y 1989

GUASTAVINO, J.-M., R. BERTIN AND R. PORTET. Effects of the rearing temperature on the temporalfeeding pattern of the staggerer mutant mouse. PHYSIOL BEHAV 49(2) 405--409, 1991.--The temporal feeding pattern of the staggerer mutant mouse was tested under two thermal rearing conditions, 28°C and 22°C, and compared to that of the normal mouse. At a temperature of 28°C, the mutants were observed to eat more than the normal mice did although they showed a drastic body weight deficit. At 22°C, this peculiarity was still observed, but with an altered feeding pattern, wherein the mutants ate as much during both the dark and light phases. Mutant mouse

Staggerer

Temporal pattern

Food intake

DURING the last decade, mutant mice have been studied from many different points of view. Among mutants, the staggerer mouse has been used extensively as a tool for investigating mechanisms involved in neurophysiology (14), embryology (5,10), biochemistry (13), and behavior (4, 6, 7, 9, 11, 12). For an ethologist trying to identify the influence of a single gene on behavior, it is advantageous to tap a wide data base from other sciences. This may allow him to relate the observed behavioral abnormalities to the alterations caused by the mutation in the central nervous system of the animals studied. No obvious neurological alterations, other than those related to the cerebellum, have been described in the staggerer mutant. The staggerer mutation reduces the size of the cerebellum to onethird of the normal size and is responsible for the lack of lamination: Purkinje cells are scattered in the base of the cerebellum, granula cells are absent, and connections between the elements of the cerebellum are erratic and severely altered (22). Previous studies of the behavior of staggerer mice have demonstrated clear relationships between the alterations in the CNS and behavioral abnormalities. These studies have confirmed the role of the altered nervous structure, i.e., the cerebellum, in a wide range of behaviors. Most clearly exemplified was the role of the cerebellum in motor coordination and body balance. However, a number of studies, most of them carried out in our laboratory (6-9, 11, 12), have demonstrated the existence of other behavioral abnormalities not obviously linked to the cerebellar alterations, e.g., alterations in sexual, maternal, and exploratory behaviors (7, 8, 11, 12). These findings suggest two possibilities:

Rearing temperature

Behavior

1) that structures other than those described in the cerebellum could be altered by this mutation, or 2) that the cerebellum is involved in functions other than those most frequently reported (15,23). In the present study, we demonstrated the existence of alterations in food intake and in the temporal pattern of the feeding activity of the staggerer. We observed that these mice engaged in feeding activity much more frequently than did normal mice. In fact, undernourishment seemed to be one of the causes of early death of young staggerer mice in standard rearing conditions (6). We showed that young staggerers continued to suckle their mothers up to the age of 60 days vs. 18-20 days for normal pups (8). However, in spite of such prolonged care by the mother and access to food ad lib, the body weight of the mutant pups remained about half of that of normal pups, indicating possible metabolic abnormality, depending on the staggerer gene (8). This observation of frequent feeding activity helped us to detect young mutants in a litter, as they stayed either in the bottom of the nest or close to the containers in which mashed food was delivered (6). However, we had not carried out a quantitative study of this behavior in adult staggerers until now. We investigated, further, the circadian pattern of feeding activity and the quantity of food intake in staggerer and control mice, under two different temperature conditions (22°C and 28°C). METHOD

Animals All the mice used in this study belonged to the C57BL/6 strain 405

406

and were seven weeks old. The mutants had the sg/sg genotype, and the control animals were either + / + or +/sg. At this age, it is very difficult to distinguish between + / s g and + / + , as the staggerer mutation is recessive. The staggerer gene can have a deleterious effect even if only one allele is altered. However, these effects are clearly observed only in aged animals (21). Thus, they would not be of major importance for the present study. The mice in this study were divided into four groups: Group 1:10 staggerer mutants born, reared, and tested at an ambient temperature of 28°C. Mean weight 5.49 ---0.16 g SEM. Group 2:10 control mice born, reared, and tested at an ambient temperature of 28°C. Mean weight 11.17---0.24 g SEM. Group 3 : 1 0 staggerer mutants born, reared, and tested at an ambient temperature of 22°C. Mean weight 5.6-+ 0.20 g SEM. Group 4 : 1 0 control mice born, reared, and tested at an ambient temperature of 22°C. Mean weight 9.61-+0.47 g SEM. These temperatures were chosen because 28°C is a temperature included in the thermoneutral zone of mice, and because 22°C is a moderately cold temperature, found in most animal rooms. All the animals, mutants and controls, were weaned at 35 days of age. [We have previously shown that delayed weaning may be one of the factors increasing the life span of staggerer mutants (6).] The mice were isolated just after weaning and housed in individual cages (24 × 11 × 7.5 cm). They were fed ad lib with a standard laboratory diet (UAR A04, hydrated diet prepared daily by mixing 40% chow with 60% water). All the mice had free access to food and water and were subjected to a photoperiod of 12 h darkness and a 12 h period of lightness (lights going on at 6:00 a.m.).

Procedure Twenty grams of the hydrated diet described above were delivered in a small plastic container. Every four hours, day and night, the plastic container was removed and weighed in order to determine the quantity of food ingested. A new container with 20 g of fresh food was then given to the mouse, and fresh food was substituted every four hours, to avoid a possible artifact due to the different evaporation rates of the two experimental temperatures. About one week before the experiment began, the animals were placed in the experimental conditions, i.e., isolated in individual cages, subjected to their group temperature (28°C or 22°C), and fed ad lib with the hydrated diet. However, the food was delivered only twice a day, 8:00 a.m. and 8:00 p.m. The experiment lasted for a five-day period, beginning at 10:00 a.m. on the first day. The quantity of food intake was recorded for each individual animal six times (periods) daily: 3 for the dark phase and 3 for the light phase. Results (by group of animals and by period of time) were expressed in means and compared by multifactorial ANOVA, first with 4 factors: strain, temperature, periods, and days; and then with 3 factors: strain, temperature, and periods. In addition, in order to compare quantities of food eaten during the dark phase and the light phase, and to check the trigger effect of cutting off the light, paired differences t-test was used. Two aspects were considered in the temporal pattern of food intake: 1) the amount of food ingested during the light and dark phases (mean for 10 mice), and 2) the amount of food ingested during the last period of the dark phase and the first period of the light phase. This was designed in order to check for the existence of a trigger effect due to cutting off the light in the animal room for the onset of feeding activity. The amount of food ingested by staggerers and controls during the light and the dark phases, and during the whole day, was

GUASTAVINO, BERTIN AND PORTET

TABLE 1 FOOD INTAKEPER DAY (MEANIN mg PER g OF BODY WEIGHT) General Means for the 5-Day Period

Highest Means 780.5 951.6 1268.7 1243.7

mg mg mg mg

>Mean >Mean >Mean >Mean

Controls Controls Staggerers Staggerers

28°C = 22°C = 28°C = 22°C =

770.7 938.6 1246.3 1234.7

Lowest Means rag> 760.3 rag> 902.6 rng> 1213.3 mg>1226.0

mg mg mg nag

Strain: "Controls." Source "day": F(4,72) = 1.320, p =0.26. Interaction "day" × "periods": F(20,360) = 1.27, p = 0.19. Strain: "Staggerers." Source "day": F(4,72)= 1.25, p=0.29. Interaction "day" × "periods": F(20,360)=0.76, p=0.38.

expressed in mg of hydrated diet per g of body weight and compared. RESULTS

Influence of the Day The experiment lasted for a 5-day period. The day factor, according to the statistical treatment, was not significant and no significant interaction between " d a y " and " p e r i o d " was observed. We then decided to pool the data for each animal per period and per day and to consider the mean.

Temporal Pattern of Food Intake Ambient temperature. At an ambient temperature of 28°C, controls ingested about twice as much food during the dark phase (520 mg vs. 254 mg) than during the light phase, t(9)= 70.8, p<0.001. Moreover, the trigger effect of turning off the light, resulting in an increase of food intake, was also clearly noticed: the difference between the food intake during the last 4 hours of the light phase (112 mg) and the first period of the dark phase (180 mg) was significant at the level p<0.001, t(9)=30.8. The mutants also displayed a certain rhythmicity in food intake activity: 744 mg during the dark phase vs. 498 mg during the light phase, t(9)= 29.9, p<0.001. The ratio was about 1.5. This rhythmicity was expressed also by the presence of the trigger effect of turning off the light: during the first 4 hours of the dark phase, the food intake was about twice as high [215 mg vs. 109 mg, t(9)= 17.1] as during the last period of the light phase. When animals of both strains were pooled together for all periods of the day, those exposed to a temperature of 22°C ate more than those exposed to 28°C [1086 mg vs. 1008 rag, F(1,36)= 57.62, p<0.001]. However, a strong "Strain-Temperature" interaction was seen: F(1,36)=82.3, p<0.0001, indicating a differential strain sensitivity to temperature changes. Raising the temperature from 22°C to 28°C resulted in a decreased food intake by the control animal (469 mg vs. 385 mg) but did not decrease the staggerer's food intake (1234 mg vs. 1246 mg). This indicates that the general result observed is attributable solely to the controls' reaction following the lowering of the ambient temperature. Amount of Food A significant difference between nocturnal and diurnal food

TEMPORAL FEEDING PATFERN OF THE STAGGERER MOUSE

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about 1.5 times more than the controls: 1240 mg of food per g of body weight vs. 854 mg, F(1,36)= 1417.668, p<0.0001. At the ambient temperature of 28°C, a clear difference was observed between the animals of both strains. The staggerers ate 1.62 times more mashed food than the controls (1246 per g of body weight vs. 770 mg for the controls). The difference is seen during both phases: dark (744 mg vs. 520 mg) and light (498 mg vs. 254 mg), t(18) = 34.04, p<0.001. For the temperature of 22°C we notice also that mutants are seen to eat more than controls. The ratio was 1.31 (1234 mg vs. 938 mg). The difference is particularly noticeable for the light phase during which staggerers are seen to eat 610 mg vs. 267 mg for the controls, t(18)=49.45, p<0.001.

intake appeared, t(9)=59.9; the controls ate 2.5 times more mashed food during the nocturnal phase than during the light phase. Moreover, after light turn-off, a significant increase in the food intake was seen. During the first 4 hours of the dark phase, the controls ate twice as much as during the last 4 hours of the light phase (214 mg vs. 98 mg). Thus, the lowering of the ambient temperature from 28°C to 22°(2 did not significantly change the behavioral patterns of feeding activity of mice in the control group, as far as the rhythmicity "light phase-dark phase" and the trigger effect of cutting off the light are concerned. In the mutant group, the temporal pattern of feeding activity was very different from that of the animals in the control group. The mutants ate the same amount of food during the dark phase as they did during the light phase [623 mg vs. 610 mg, t(9)= 1.57, p = 0 . 3 4 ] . Furthermore, the trigger effect of turning off the light (264 mg vs. 251 mg) was observed, but was only significant at p<0.042, t(9) = 2.37. The strong "Periods-Strain" interaction, F(1,36)=547.7, p<0.0001, indicates a differential sensitivity of the animals of the two strains to the light and dark phase alternation.

DISCUSSION The staggerer mutation affects the feeding pattern of the mouse. Its effects are clearly visible in relation to the total food intake, but it also affects the temporal pattern of feeding activity. When subjected to temperatures of either 28°C or 22°C, the staggerer is observed to eat more than the control animal (in g per g of body weight). The ratio is about 1.5. It could be argued that an artifact, linked to the method of weighing the residual food in the container to evaluate the intake, may be responsible for the differences observed. This method

Total Amount of Food Ingested A clear difference between staggerers and controls is observed: for both temperatures and all periods of the day the staggerers eat

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408

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does not take into account possible spillage by the animals. On the contrary, the differences are underestimated since the staggerers have never been observed to scatter food, whereas this is a normal occurrence in the controls. The hyper-food intake is in discrepancy with the low body weight--a drastic deficit--of the staggerer, which weighs only about half as much as the control animal. This ponderal deficit has been reported by several authors (6, 8, 20, 22). This hyperfood intake was first suspected in 1978 (6), and evidenced in 1982 (8), when young staggerers were found to suckle the mother twice as long as controls did during the day and until a more advanced age (60 days vs. 18-20 days for controls). Surprisingly, however, this over-suckling never resulted in a compensation for the body weight deficit. We have previously reported abnormalities in the young staggerer's weight development (8). When the staggerer is nursed by a mutant mother which cares for her pups less than a normal mother does, the staggerer pup does not develop normally, and its body weight remains at a low level for a period of several months. Normal pups, under the same conditions, show a transient body weight deficit but soon recover after weaning. The abnormal body weight of the staggerer pup, however, is unaltered even when nursed by a normal mother. This body weight deficit, which appears to be a peculiarity linked to the staggerer gene, remains constant regardless of different feeding conditions. Hence, it is obvious that some, as yet unknown, internal factors--metabolic or anatomic--prevent normal growth in the staggerer. Investigations are presently underway to analyze alterations of metabolism. As mentioned previously, the mutation affects not only the total food intake, but also the feeding pattern according to the temperature to which the animals are subjected to from birth. When experiments are undertaken at an ambient temperature of 28°C, which is close to the thermal neutrality of the mouse, both the mutant and nonmutant show a rhythmicity related to the alternation of dark/light phases. Laboratory rodents demonstrate the same pattern of behavior--eating more during the dark phase (1). Thus, the behavior of the staggerers at 28°C is similar to that of controls and of other laboratory rodents (19), as far as rhythmicity is concerned. However, when mice were reared at a low ambient temperature (22aC), which is the standard temperature in most animal rooms, a clear difference was observed between staggerer mutants and controls. In the control animals, the differences in the quantities of food ingested during the dark and light phases were consistent, regardless of changes in the ambient temperature. These differences disappeared in the mutants when the temperature was lowered to 22°C, while the total amount of food ingested did not differ between the two phases (dark and light) of the cycle. This phenomenon was also observed in laboratory rats

kept at a cold temperature (5°C) (17,18), and genetically obese rats (2,3). For example, the obese Zucker rat's hyperphagia is essentially due to an abnormal diurnal food intake, while its nocturnal intake is comparable to a control animal's intake (2,3). Similarly, when a rat is exposed to low ambient temperatures, it compensates for the loss of calories by increasing its diurnal food intake (3,18). Present findings extend this deficiency to adult staggerers, who show insensitivity to changes in environment. The normal mouse adapts its food intake to the ambient temperature; when the temperature decreases from 28°C to 22°C, the food intake increases. By contrast, the staggerer is seen to eat the same amount of food regardless of the thermal conditions. The insensitivity to this particular environmental change is in contrast to the usual behavioral plasticity of the staggerer, which is known to react very strongly to modifications of environmental conditions (6-8). Similarly, the obese Zucker rat, with a comparable hyperphagia, does not increase its food intake when subjected to a higher temperature (2,3). This shows that the behavior of staggerer mutant mice, at a temperature of 22°C, is similar to that of rats showing a genetic hyperphagia or subjected to a low temperature of 5°C. Lack of understanding of the mechanisms by which the temperature affects the day-night pattern prevents us from drawing any conclusion at this point. However, they suggest that a temperature of 22°C can be perceived as comparatively colder by staggerers than by controls. It would be interesting to study the staggerer in higher temperature conditions, such as 30°C or 32°C. We could then observe if the staggerer might adjust its food intake to the high thermal conditions, as do normal mice, or if the quantity of food ingested would remain constant, independent of higher temperatures. It is important to note that experimental conditions can reveal aspects of behavior which may be hidden in standard environmental conditions. Since we conducted experiments only at the usual animal room temperature, we may have erroneously concluded that the staggerer failed to show rhythmicity in the feeding pattern. One question that has arisen from this study is whether the cerebellar abnormalities may contribute to the pecularities of the staggerer metabolism. Until now, this nervous structure has not been known to contribute directly to the control of the temporal pattern of feeding behavior, nor to the control of food intake. Thus, this mutation may affect other nervous structures and/ or metabolic processes which could be responsible, directly or indirectly, for the observed alterations. Current studies are devoted to investigating alterations in other parts of the body in order to explain the observed alterations of the behavioral feeding pattern.

T E M P O R A L F E E D I N G P A I I ' E R N OF THE S T A G G E R E R M O U S E

409

ACKNOWLEDGEMENTS The authors wish to thank Albert Ly for outstanding technical assistance, Gilles Gheusi for the statistics, Laura K. Killian and Esther Ronn for the translation, and Referee #2 for her excellent advice.

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