Journal of Chemical Ecology, Vol. 17, No. 8, 1991
EXPOSURE TO ODORS FROM STRESSED CONSPECIFICS INCREASES PREFERENCE FOR HIGHER AMBIENT TEMPERATURES IN C57BL/6J MICE
DEL
THIESSEN,*
CHANA
AKINS,
and CARLOS
ZALAQUETT
Department of Psychology University of Texas Austin, Texas 78712 (Received January 28, 1991; accepted March 8, 1991)
Abstract--Exposure to odors from stressed conspecifics increases preference for higher ambient temperatures in C57BL/6J mice. C57BL/6J male mice were individually allowed preferences on a thermal gradient ranging in temperature from 22~ to 42~ Group 1 (N = 10) was exposed to odors from triads of foot-shocked conspecifics during the first 2-hr temperature preference trial. Group 2 (N = 10) was exposed to odors from triads of nonstressed conspecifics during similar testing. Body temperature (TB) variations were measured in three animals of each group. Thermal preference was significantly higher for animals exposed to odors from stressed conspecifics than for animals exposed to odors from nonstressed animals (32.0~ vs. 29.0~ TB changes on the heated gradient were significantly higher for animals exposed to odors from stressed animals ( + 1 . 5 ~ than for animals exposed to odors from nonstressed animals ( - 0 . 3 3 ~ Additional animals on a nonheated thermogradient were tested for T8 when exposed to odors from stressed or from nonstressed animals (N = 3 per condition). There was no difference in T8 between these two groups. Increases in TB on the heated gradient are apparently due to the higher ambient temperature choices and not due to the odor per se.
Key Words--Stress, odors, body temperature, ambient temperature, C57BL/ 6J mice, Mus musculus.
INTRODUCTION
Odors from animals under stress often signal injury, distress, or the presence of predators. The conspecific response to these signals is avoidance, dispersal, *To whom correspondence should be addressed. 1611 0098-0331/91/0800-161 I $06.50/0 9 1991 Plenul"o Publishing Corporation
1612
THIESSEN ET AL.
or increased activity (Muller-Velten, 1966; Carr et al., 1970; Rottman and Snowdon, 1972; Zalaquett and Thiessen, 1991). Observations of the gerbil, Meriones unguiculatus, and the mouse, Mus musculus, suggest a relationship between conspecific odors, physiological changes, and behavior (Cocke and Thiessen, 1986; Thiessen and Cocke, 1990). When gerbils are exposed to odor from stressed conspecifics, body temperatures are elevated about 0.38 ~ within 20 rain. This effect is consistent with other findings that many forms of stress elevate body temperatures (Long et al., 1990). The gerbil becomes inactive when confined with the odor and will avoid the odor if given the opportunity. We have noted similar behavioral effects with BALB/cJ and C57BL/6J mice (Zalaquett and Thiessen, 1991; Zalaquett, unpublished observations). In addition, in mitogen assays of BALB/cJ mice, continuous exposure to odors from stressed mice for 24 hr results in immunological suppression of T-cell proliferation (Thiessen and Cocke, 1990). These observations led us to speculate that animals may benefit from increased body temperatures. This appears true for animals exposed to pathogens and may also be the case for exposure to stressful stimuli. When a pathogen invades the body, the initial response often observed among most mammals is an increase in body temperature (Jampal et al., 1983). The febrile response is adaptive in that the spread of the infection is retarded and longevity is increased (Covert and Reynolds, 1977; Hart, 1988; Kluger et al., 1975; Kluger and Vaughn, 1978). Benefits are gained by the reduction of blood levels of iron and zinc, which are necessary for the proliferation of pathogens (Hart, 1988, 1990; Kluger, 1989). Increased body temperature also facilitates T-cell activity and interferon synthesis, and thus brings the force of the immune system into play at an early stage of infection (Downing et al., 1988; Duff and Durum, 1982). Recent studies in this laboratory show that C57BL/6J mice seek out a higher ambient temperature when exposed to lipopolysaccharide (LPS) or to the odors from animals made ill with LPS (Akins et al., 1991; Akins and Thiessen, 1990). The similarity of reactions suggests that animals exposed to odors from stressed animals may also cause the recipients to seek higher ambient temperatures, a reaction that may facilitate immune responses. This study tests the possibility that C57BL/6J mice subjected to body odors from stressed conspecifics will increase their temperature preferences and hence their body temperatures. METHODS AND MATERIALS
Subjects. Fifty male C57BL/6J mice, approximately 60 days of age at the beginning of the study, were obtained from the Animal Resources Center at The University of Texas at Austin. Mice were housed in colony cages mea-
ODORS FROM STRESSED CONSPECIFICS IN MICE
1613
suring 16 x 16 x 13.5 cm at 26~ ambient temperature and 40% relative humidity, with ad libitum food and water under a 12:12 hr light-dark cycle. Twenty-four of these mice were assigned randomly to form four triads of stress odor (SO) donors and four triads of control odor (CO) donors. The remaining 26 animals were divided randomly into four groups housed individually and assigned one of the following conditions: Group 1 (N = 10): stress odor recipients placed on the preheated temperature gradient. Body temperature was measured in three of these recipients (see Procedure below). Group 2 (N = 10): control odor recipients placed on the preheated temperature gradient. Body temperature was measured in three of these recipients. Group 3 (N = 3): stress odor recipients placed on the nonheated temperature gradient, measured for body temperature. Group 4 (N = 3): control odor recipients placed on the nonheated temperature gradient, measured for body temperature. Thus, animals were tested for temperature reactions to the stress and control odors and for changes in body temperature due to odor exposure or to temperature gradient exposure. Test Apparatus. A schematic of the temperature gradient apparatus as described by Thiessen et al. (1970) is shown in Figure 1. The gradient was constructed of metal, measuring 147.3 cm in length and 11.4 cm in width. A tube attached to the bottom of the gradient carried water at room temperature one fourth the length of the gradient. Metal heating tapes were placed along the bottom adjacent to the water conduit and were spaced so that the temperature of the gradient ranged from 22 ~ to 42~ when heated. The heating tapes were regulated with a rheostat, and the gradient runway was enclosed with a removable Plexiglas hood. The odor donor chambers (enclosed in a cabinet that precluded any visual or auditory contact between donors or experimental subjects) measured 50.6 • 30.8 x 30.8 cm and were sealed with weatherstripping. Colony cages, provided with wood shavings and Purina Lab Chow, were placed inside the donor chambers. The donor chambers were connected to the temperature gradient with Tygon tubing. Airflow from the donor chambers entered at the runway and was vacuumed out at a rate of 7-8 liters/min. The apparatus was housed in the test room adjacent to the colony room. The test room was maintained at room temperature (26~ An RCA video camera attached to an RCA video recorder monitored the behavior of animals on the temperature gradient. A Bailey type T probe needle (model BAT-12) was used to measure preferred ambient temperature along the length of the gradient. Body Temperature Measurements. Biotelemetry devices (Mini-mitter thermistors model M) were used to measure mouse body temperature (TB) (Thiessen
1614
THIESSENET AL. 147.3 crn I
I
I
22~
42"C
/ I\ /
F
\
FiG. 1. Schematic diagram of the temperature gradient apparatus.
and Kittrell, 1980). Mini-mitters were reduced in size to accommodate mice by removing the plastic capping and coating the transmitter and battery with candle wax. These thermistors then were calibrated individually over a range of 11 ~ to 41 ~ in three steps. The interval between audible beats, which is temperature sensitive, was counted in milliseconds using a receiver and standard AM radio during testing and converted to body temperature using calibration data. As the termistor increases in temperature, the interval between beats decreases. Mini-mitters were surgically implanted under anesthesia five days prior to testing. A calibrated thermistor was inserted into the interperitoneal cavity through a small incision on the fight abdominal surface. The wound was sutured and mice were allowed to recover. Shock Apparatus. The foot-shock apparatus was constructed of stainless steel with Plexiglas walls. The apparatus measured 24 • 24 x 35 cm with 0.2cm stainless-steel grid bars spaced 0.4 cm apart forming the floor. Scrambled foot shock was supplied to the floor of the chamber by the Lafayette foot-shock apparatus (model 82404/5 SS). Shock parameters for the individual animals were three successive, 7-sec scrambled electric shocks, separated by a 60-sec rest interval (0.5 mA, 600 V). Procedure. On the day of testing, mice were transferred from the colony
1615
ODORS FROM STRESSED CONSPECIFICS IN MICE
room to the test room. The temperature gradient apparatus was either preheated for 2 hr prior to testing or left at room temperature (nonheated) during the test period. Each triad of the control odor donors was habituated in the donor chamber with the active airstream 1 hr prior to testing. Each recipient mouse was placed individually on the gradient for 2 hr and received either odor from stressed donors or odor from control donors. Odors were introduced into either end of the gradient (warm or cool) in a random order to eliminate any possible position bias. Temperature preference was defined as that temperature where the mouse first rested for at least 5 rain. Body temperature was recorded preceding each trial and immediately after each trial. The two groups were equated for original body temperature. RESULTS A t test for independent samples was used to test for differences in preferred ambient temperature between SO and CO recipients. These data are seen in Figure 2. Animals that received the odor from stressed conspecifics preferred
33.0 O o UJ
0 Z LU s 111
32.0
LL
u..I r 13. ILl n,"
31.0
I-,
EXPOSURE TO ODORS FROM STRESSED CONSPECIFICS INCREASES PREFERENCE FOR HIGHER AMBIENT TEMPERATURES IN C57BL/6J MICE
DEL
THIESSEN,*
CHANA
AKINS,
and CARLOS
ZALAQUETT
Department of Psychology University of Texas Austin, Texas 78712 (Received January 28, 1991; accepted March 8, 1991)
Abstract--Exposure to odors from stressed conspecifics increases preference for higher ambient temperatures in C57BL/6J mice. C57BL/6J male mice were individually allowed preferences on a thermal gradient ranging in temperature from 22~ to 42~ Group 1 (N = 10) was exposed to odors from triads of foot-shocked conspecifics during the first 2-hr temperature preference trial. Group 2 (N = 10) was exposed to odors from triads of nonstressed conspecifics during similar testing. Body temperature (TB) variations were measured in three animals of each group. Thermal preference was significantly higher for animals exposed to odors from stressed conspecifics than for animals exposed to odors from nonstressed animals (32.0~ vs. 29.0~ TB changes on the heated gradient were significantly higher for animals exposed to odors from stressed animals ( + 1 . 5 ~ than for animals exposed to odors from nonstressed animals ( - 0 . 3 3 ~ Additional animals on a nonheated thermogradient were tested for T8 when exposed to odors from stressed or from nonstressed animals (N = 3 per condition). There was no difference in T8 between these two groups. Increases in TB on the heated gradient are apparently due to the higher ambient temperature choices and not due to the odor per se.
Key Words--Stress, odors, body temperature, ambient temperature, C57BL/ 6J mice, Mus musculus.
INTRODUCTION
Odors from animals under stress often signal injury, distress, or the presence of predators. The conspecific response to these signals is avoidance, dispersal, *To whom correspondence should be addressed. 1611 0098-0331/91/0800-161 I $06.50/0 9 1991 Plenul"o Publishing Corporation
1612
THIESSEN ET AL.
or increased activity (Muller-Velten, 1966; Carr et al., 1970; Rottman and Snowdon, 1972; Zalaquett and Thiessen, 1991). Observations of the gerbil, Meriones unguiculatus, and the mouse, Mus musculus, suggest a relationship between conspecific odors, physiological changes, and behavior (Cocke and Thiessen, 1986; Thiessen and Cocke, 1990). When gerbils are exposed to odor from stressed conspecifics, body temperatures are elevated about 0.38 ~ within 20 rain. This effect is consistent with other findings that many forms of stress elevate body temperatures (Long et al., 1990). The gerbil becomes inactive when confined with the odor and will avoid the odor if given the opportunity. We have noted similar behavioral effects with BALB/cJ and C57BL/6J mice (Zalaquett and Thiessen, 1991; Zalaquett, unpublished observations). In addition, in mitogen assays of BALB/cJ mice, continuous exposure to odors from stressed mice for 24 hr results in immunological suppression of T-cell proliferation (Thiessen and Cocke, 1990). These observations led us to speculate that animals may benefit from increased body temperatures. This appears true for animals exposed to pathogens and may also be the case for exposure to stressful stimuli. When a pathogen invades the body, the initial response often observed among most mammals is an increase in body temperature (Jampal et al., 1983). The febrile response is adaptive in that the spread of the infection is retarded and longevity is increased (Covert and Reynolds, 1977; Hart, 1988; Kluger et al., 1975; Kluger and Vaughn, 1978). Benefits are gained by the reduction of blood levels of iron and zinc, which are necessary for the proliferation of pathogens (Hart, 1988, 1990; Kluger, 1989). Increased body temperature also facilitates T-cell activity and interferon synthesis, and thus brings the force of the immune system into play at an early stage of infection (Downing et al., 1988; Duff and Durum, 1982). Recent studies in this laboratory show that C57BL/6J mice seek out a higher ambient temperature when exposed to lipopolysaccharide (LPS) or to the odors from animals made ill with LPS (Akins et al., 1991; Akins and Thiessen, 1990). The similarity of reactions suggests that animals exposed to odors from stressed animals may also cause the recipients to seek higher ambient temperatures, a reaction that may facilitate immune responses. This study tests the possibility that C57BL/6J mice subjected to body odors from stressed conspecifics will increase their temperature preferences and hence their body temperatures. METHODS AND MATERIALS
Subjects. Fifty male C57BL/6J mice, approximately 60 days of age at the beginning of the study, were obtained from the Animal Resources Center at The University of Texas at Austin. Mice were housed in colony cages mea-
ODORS FROM STRESSED CONSPECIFICS IN MICE
1613
suring 16 x 16 x 13.5 cm at 26~ ambient temperature and 40% relative humidity, with ad libitum food and water under a 12:12 hr light-dark cycle. Twenty-four of these mice were assigned randomly to form four triads of stress odor (SO) donors and four triads of control odor (CO) donors. The remaining 26 animals were divided randomly into four groups housed individually and assigned one of the following conditions: Group 1 (N = 10): stress odor recipients placed on the preheated temperature gradient. Body temperature was measured in three of these recipients (see Procedure below). Group 2 (N = 10): control odor recipients placed on the preheated temperature gradient. Body temperature was measured in three of these recipients. Group 3 (N = 3): stress odor recipients placed on the nonheated temperature gradient, measured for body temperature. Group 4 (N = 3): control odor recipients placed on the nonheated temperature gradient, measured for body temperature. Thus, animals were tested for temperature reactions to the stress and control odors and for changes in body temperature due to odor exposure or to temperature gradient exposure. Test Apparatus. A schematic of the temperature gradient apparatus as described by Thiessen et al. (1970) is shown in Figure 1. The gradient was constructed of metal, measuring 147.3 cm in length and 11.4 cm in width. A tube attached to the bottom of the gradient carried water at room temperature one fourth the length of the gradient. Metal heating tapes were placed along the bottom adjacent to the water conduit and were spaced so that the temperature of the gradient ranged from 22 ~ to 42~ when heated. The heating tapes were regulated with a rheostat, and the gradient runway was enclosed with a removable Plexiglas hood. The odor donor chambers (enclosed in a cabinet that precluded any visual or auditory contact between donors or experimental subjects) measured 50.6 • 30.8 x 30.8 cm and were sealed with weatherstripping. Colony cages, provided with wood shavings and Purina Lab Chow, were placed inside the donor chambers. The donor chambers were connected to the temperature gradient with Tygon tubing. Airflow from the donor chambers entered at the runway and was vacuumed out at a rate of 7-8 liters/min. The apparatus was housed in the test room adjacent to the colony room. The test room was maintained at room temperature (26~ An RCA video camera attached to an RCA video recorder monitored the behavior of animals on the temperature gradient. A Bailey type T probe needle (model BAT-12) was used to measure preferred ambient temperature along the length of the gradient. Body Temperature Measurements. Biotelemetry devices (Mini-mitter thermistors model M) were used to measure mouse body temperature (TB) (Thiessen
1614
THIESSENET AL. 147.3 crn I
I
I
22~
42"C
/ I\ /
F
\
FiG. 1. Schematic diagram of the temperature gradient apparatus.
and Kittrell, 1980). Mini-mitters were reduced in size to accommodate mice by removing the plastic capping and coating the transmitter and battery with candle wax. These thermistors then were calibrated individually over a range of 11 ~ to 41 ~ in three steps. The interval between audible beats, which is temperature sensitive, was counted in milliseconds using a receiver and standard AM radio during testing and converted to body temperature using calibration data. As the termistor increases in temperature, the interval between beats decreases. Mini-mitters were surgically implanted under anesthesia five days prior to testing. A calibrated thermistor was inserted into the interperitoneal cavity through a small incision on the fight abdominal surface. The wound was sutured and mice were allowed to recover. Shock Apparatus. The foot-shock apparatus was constructed of stainless steel with Plexiglas walls. The apparatus measured 24 • 24 x 35 cm with 0.2cm stainless-steel grid bars spaced 0.4 cm apart forming the floor. Scrambled foot shock was supplied to the floor of the chamber by the Lafayette foot-shock apparatus (model 82404/5 SS). Shock parameters for the individual animals were three successive, 7-sec scrambled electric shocks, separated by a 60-sec rest interval (0.5 mA, 600 V). Procedure. On the day of testing, mice were transferred from the colony
1615
ODORS FROM STRESSED CONSPECIFICS IN MICE
room to the test room. The temperature gradient apparatus was either preheated for 2 hr prior to testing or left at room temperature (nonheated) during the test period. Each triad of the control odor donors was habituated in the donor chamber with the active airstream 1 hr prior to testing. Each recipient mouse was placed individually on the gradient for 2 hr and received either odor from stressed donors or odor from control donors. Odors were introduced into either end of the gradient (warm or cool) in a random order to eliminate any possible position bias. Temperature preference was defined as that temperature where the mouse first rested for at least 5 rain. Body temperature was recorded preceding each trial and immediately after each trial. The two groups were equated for original body temperature. RESULTS A t test for independent samples was used to test for differences in preferred ambient temperature between SO and CO recipients. These data are seen in Figure 2. Animals that received the odor from stressed conspecifics preferred
33.0 O o UJ
0 Z LU s 111
32.0
LL
u..I r 13. ILl n,"
31.0
I-,
