Physiology & Behavior, Vol. 16, pp. 543--546. Pergamon
Press and Brain Research Pubi., 1976. Printed in the U.S.A.
Sex Difference in the Effect of Dexamethasone on Open-Field Behavior in Rats: Gonadal Hormones ' JUSTIN M. J O F F E , JAMES A. MULICK, JOHN M. PETERSON
Department o f Psychology, University o f Vermont, Burlington, VT 05401 AND J A SEN K A PAUNOVIC
Department o f Biology, Medical Faculty, University o f Zagreb, Zagreb, Yugoslavia (Received 28 April 1975) JOFFE, J. M., J. A. MULICK, J. M. PETERSON AND J. PAUNOVIC. Sex difference in the effect ofdexamethasone on open-field behavior in rats: gonadal hormones. PHYSIOL. BEHAV. 16(5) 543-546, 1976. - Previous experiments indicated that dexamethasone phosphate (DEX) increases open-field defecation in females but not in males. To examine the question of whether this sex difference in response to DEX is due to sex differences in gonadal hormones, female rats were (a) ovariectomized; (b) ovariectomized and injected with testosterone propionate prior to and during open-field testing; or (c) sham operated. Half the females in each group received DEX in their drinking water during the 4 day period of testing. DEX produced the expected reductions in body weight, adrenal weight, and plasma corticosterone levels. Despite the differences between the three groups on a variety of physiological measures, DEX produced comparable increases in open-field defecation in all cases. This indicates that the sex difference in the response of open-field defecation to DEX is not dependent on sex differences in circulating gonadal hormones. Activity Body weight Corticosterone Defecation Dexamethasone phosphate Ovarian hormones Ovariectomy Pituitary-adrenocortical system Sex differences
EXPERIMENTAL manipulations of pituitary-adrenal status affect open-field behavior. Moyer found that adrenalectomy significantly increases open-field defecation in female rats [ 12] but that low doses of ACTH do not affect activity or defecation [13] and adrenal demedullation does not affect defecation or urination [14]. Joffe et al. [5] confirmed Moyer's [12] findings concerning adrenalectomy, and showed that the effect occurs in both male and female rats. In an attempt to determine which of the possible hormonal changes might be involved in the effects on open-field behavior, Mulick et al. [ 15 ] treated animals with dexamethasone phosphate (DEX), a synthetic glucocorticoid known to produce strong inhibition of corticotrophic stimulation of the adrenal cortex [ 1, 16, 19] and a decrease in adrenocorticosteroid production [2,17]. Since DEX is itself a potent glucocorticoid the pattern of pituitary-adrenocortical hormonal changes resulting from DEX administration is the reverse of that produced by
Open-field behavior Testosterone propionate
adrenalectomy - the DEX treated animal has high circulating levels of steroids and low levels of ACTH. Nevertheless DEX treatment had similar effects to adrenalectomy in female rats but not in males; that is, females showed a significant increase in open-field defecation [15]. That this f'mding was specific to the open field, and not simply a general effect on overall defecation, was indicated by the substantial and significant decrease in home-cage defecation observed in both male and female rats. Both males and females lost weight, and showed suppressed levels of plasma corticosterone; in addition males tested in an activity wheel showed an increase in activity similar to that reported by others [8]. The present experiment was designed to investigate whether or not the sex difference in the effect of DEX on open-field defecation was due to the difference in circulating gonadal hormones in normal male and female rats. This was done by comparing the effects of DEX treatment on the open-field behavior of ovariectomized females
1Supported by grant USPHS R01-05571 from the National Institute of Child Health and Human Development. The authors wish to thank Dr. Clement A. Stone of Merck, Sharp, and Dohme Research Laboratories for supplying the dexamethasone phosphate and Joel Najman for help with preparation of figures. A preliminary version of this paper was presented at the Fifteenth Annual Meeting of the Psychonomic Society, Boston, in November, 1974. Reprints may be obtained from Dr. J. M. Joffe, Department of Psychology, University of Vermont, Burlington, VT 05401. 543
J O F F E ET AL.
544 (OVX), ovariectomized females given testosterone propionate (OVX-TP), and sham operated females. The OVX group provides information on whether or not circulating female gonadal hormones are needed to obtain the increase in open-field defecation usually seen in DEX treated females, However, if the sex difference is due to the presence of male gonadal hormones, OVX-TP females should respond to DEX like normal males, which show either no change in open-field defecation or a nonsignificant decrease when their score8 are corrected for the concomitant decrease in home cage d~fecation [ 15 ]. METHOD
Animals Sixty experimentally naive adult female Long-Evans hooded rats weighing 2 0 0 - 2 5 0 g at the start of the experiment were used. The animals were housed in pairs since this is our usual procedure in open-field experiments [5,15] and had free access to food and water (or DEX solution for experimental animals) in the home cage.
Apparatus The open field is a walled circular arena 83.2 cm in dia.; wails and floor are painted white, and black concentric circles and sections of radii divide the floor into segments used to score activity. Testing is carried out under standard lighting conditions and a speaker 1.2 m above the center of the field provides white noise to mask extraneous sound (79 dB at floor level).
Procedure Surgery and hormone treatment. On arrival the animals were housed in pairs and randomly assigned to either the ovariectomized (n = 40) or the sham operated (n = 20) group. Three days later animals were weighed and appropriate surgery performed. The OVX animals were bilaterally ovariectomized via dorsal approach under ether anesthesia using standard techniques. Shams underwent the same operation without ligation or removal of the ovaries. Following a 3 day recovery period the animals were randomly assigned to numerically equal subgroups (n = 10). The OVX animals were assigned to the following groups: (a) Dexamethasone in drinking water (OVX-DEX); (b) No dexamethasone (OVX-NO DEX); (c) Daffy injections of testosterone propionate (TP) and dexamethasone in drinking water (OVX-TP-DEX); (d) Daily injections of TP and no dexamethasone (OVX-TP-NO DEX). Similarly, Shams were randomly assigned to subgroups (n = 10) and either given DEX in their drinking water (Sham-DEX) or not (Sham-NO DEX). These groups form a 3 (gonadal hormones: Sham, OVX, OVX-TP) × 2 (corticosteroids: DEX or NO DEX) factorial design. Animals in the OVX-TP-DEX and OVX-TP-NO DEX groups received subcutaneous injections of TP (660 ug in 0.1 ml peanut oil) once each day in the late afternoon. A similar dosage (250 t~g TP per 100 g body weight) has been reported to reinstate sexual behavior following castration in adult male rats [ 10]. All remaining animals in both OVX and Sham subgroups received injections of an equal volume of vehicle at the same time of day. The injections were administered for 10 days prior to the start of testing as well as throughout testing.
Animals were again weighed 24 hr prior to the start of open-field testing and the DEX treatment was begun. Animals in DEX groups received 1.0 ug DEX per ml distilled water in their water bottles, to obtain an approximate daily dosage of 4 0 - 5 0 ug DEX per rat per day. The bottles were refilled with freshly mixed DEX solution every 48 hr, and the amount consumed was recorded. Body weight changes and posttesting plasma corticosterone levels provided additional indications of DEX consumption. Animals in NO DEX groups had nothing added to their drinking water. Open field (OF). All animals, in random order each day, received a 2 min trial daily for 4 consecutive days. Testing was always completed between 8:30 and noon each day. Home cage defecation for each pair of animals housed together was recorded daily over the 4 days of testing. Blood sampling. Following the last test trial a blood sample ( 0 . 4 - 0 . 5 ml) was taken from each animal by jugular venipuncture under ether anesthesia. Blood was taken 15 rain after a rat was removed from its home cage for testing. Each animal was retained in a holding cage after leaving the open field for the remainder of the interval prior to sampling. Plasma was removed by centrifugation and frozen for later assay of corticosterone concentrations using the method of Glick et al. [3]. Organ weights. Following blood sampling the animals were weighed and sacrificed by an overdose of ether. Adrenals and uteri were removed, cleaned and weighed. In addition the ovaries of Shams were removed and weighed. RESULTS Since many of the effects of ovariectomy and testosterone administration on body, uterine, and adrenal weights and plasma corticosterone levels were in accord with previous findings (e.g. [7, 11, 17, 18]), data on the effects of these treatments are not reported here in detail except where these treatments interacted with DEX administration.
Body Weight The mean daffy DEX consumption of all experimental animals was approximately 45.3 ~g DEX per rat per day. This dosage resulted in large weight reductions compared to weights prior to DEX treatment. Analysis of weights, which were recorded prior to surgery, 9 days postoperatively, and on the last day of open-field testing indicated a significant main effect for DEX treatment, F(1,54) = 24.07; p
Press and Brain Research Pubi., 1976. Printed in the U.S.A.
Sex Difference in the Effect of Dexamethasone on Open-Field Behavior in Rats: Gonadal Hormones ' JUSTIN M. J O F F E , JAMES A. MULICK, JOHN M. PETERSON
Department o f Psychology, University o f Vermont, Burlington, VT 05401 AND J A SEN K A PAUNOVIC
Department o f Biology, Medical Faculty, University o f Zagreb, Zagreb, Yugoslavia (Received 28 April 1975) JOFFE, J. M., J. A. MULICK, J. M. PETERSON AND J. PAUNOVIC. Sex difference in the effect ofdexamethasone on open-field behavior in rats: gonadal hormones. PHYSIOL. BEHAV. 16(5) 543-546, 1976. - Previous experiments indicated that dexamethasone phosphate (DEX) increases open-field defecation in females but not in males. To examine the question of whether this sex difference in response to DEX is due to sex differences in gonadal hormones, female rats were (a) ovariectomized; (b) ovariectomized and injected with testosterone propionate prior to and during open-field testing; or (c) sham operated. Half the females in each group received DEX in their drinking water during the 4 day period of testing. DEX produced the expected reductions in body weight, adrenal weight, and plasma corticosterone levels. Despite the differences between the three groups on a variety of physiological measures, DEX produced comparable increases in open-field defecation in all cases. This indicates that the sex difference in the response of open-field defecation to DEX is not dependent on sex differences in circulating gonadal hormones. Activity Body weight Corticosterone Defecation Dexamethasone phosphate Ovarian hormones Ovariectomy Pituitary-adrenocortical system Sex differences
EXPERIMENTAL manipulations of pituitary-adrenal status affect open-field behavior. Moyer found that adrenalectomy significantly increases open-field defecation in female rats [ 12] but that low doses of ACTH do not affect activity or defecation [13] and adrenal demedullation does not affect defecation or urination [14]. Joffe et al. [5] confirmed Moyer's [12] findings concerning adrenalectomy, and showed that the effect occurs in both male and female rats. In an attempt to determine which of the possible hormonal changes might be involved in the effects on open-field behavior, Mulick et al. [ 15 ] treated animals with dexamethasone phosphate (DEX), a synthetic glucocorticoid known to produce strong inhibition of corticotrophic stimulation of the adrenal cortex [ 1, 16, 19] and a decrease in adrenocorticosteroid production [2,17]. Since DEX is itself a potent glucocorticoid the pattern of pituitary-adrenocortical hormonal changes resulting from DEX administration is the reverse of that produced by
Open-field behavior Testosterone propionate
adrenalectomy - the DEX treated animal has high circulating levels of steroids and low levels of ACTH. Nevertheless DEX treatment had similar effects to adrenalectomy in female rats but not in males; that is, females showed a significant increase in open-field defecation [15]. That this f'mding was specific to the open field, and not simply a general effect on overall defecation, was indicated by the substantial and significant decrease in home-cage defecation observed in both male and female rats. Both males and females lost weight, and showed suppressed levels of plasma corticosterone; in addition males tested in an activity wheel showed an increase in activity similar to that reported by others [8]. The present experiment was designed to investigate whether or not the sex difference in the effect of DEX on open-field defecation was due to the difference in circulating gonadal hormones in normal male and female rats. This was done by comparing the effects of DEX treatment on the open-field behavior of ovariectomized females
1Supported by grant USPHS R01-05571 from the National Institute of Child Health and Human Development. The authors wish to thank Dr. Clement A. Stone of Merck, Sharp, and Dohme Research Laboratories for supplying the dexamethasone phosphate and Joel Najman for help with preparation of figures. A preliminary version of this paper was presented at the Fifteenth Annual Meeting of the Psychonomic Society, Boston, in November, 1974. Reprints may be obtained from Dr. J. M. Joffe, Department of Psychology, University of Vermont, Burlington, VT 05401. 543
J O F F E ET AL.
544 (OVX), ovariectomized females given testosterone propionate (OVX-TP), and sham operated females. The OVX group provides information on whether or not circulating female gonadal hormones are needed to obtain the increase in open-field defecation usually seen in DEX treated females, However, if the sex difference is due to the presence of male gonadal hormones, OVX-TP females should respond to DEX like normal males, which show either no change in open-field defecation or a nonsignificant decrease when their score8 are corrected for the concomitant decrease in home cage d~fecation [ 15 ]. METHOD
Animals Sixty experimentally naive adult female Long-Evans hooded rats weighing 2 0 0 - 2 5 0 g at the start of the experiment were used. The animals were housed in pairs since this is our usual procedure in open-field experiments [5,15] and had free access to food and water (or DEX solution for experimental animals) in the home cage.
Apparatus The open field is a walled circular arena 83.2 cm in dia.; wails and floor are painted white, and black concentric circles and sections of radii divide the floor into segments used to score activity. Testing is carried out under standard lighting conditions and a speaker 1.2 m above the center of the field provides white noise to mask extraneous sound (79 dB at floor level).
Procedure Surgery and hormone treatment. On arrival the animals were housed in pairs and randomly assigned to either the ovariectomized (n = 40) or the sham operated (n = 20) group. Three days later animals were weighed and appropriate surgery performed. The OVX animals were bilaterally ovariectomized via dorsal approach under ether anesthesia using standard techniques. Shams underwent the same operation without ligation or removal of the ovaries. Following a 3 day recovery period the animals were randomly assigned to numerically equal subgroups (n = 10). The OVX animals were assigned to the following groups: (a) Dexamethasone in drinking water (OVX-DEX); (b) No dexamethasone (OVX-NO DEX); (c) Daffy injections of testosterone propionate (TP) and dexamethasone in drinking water (OVX-TP-DEX); (d) Daily injections of TP and no dexamethasone (OVX-TP-NO DEX). Similarly, Shams were randomly assigned to subgroups (n = 10) and either given DEX in their drinking water (Sham-DEX) or not (Sham-NO DEX). These groups form a 3 (gonadal hormones: Sham, OVX, OVX-TP) × 2 (corticosteroids: DEX or NO DEX) factorial design. Animals in the OVX-TP-DEX and OVX-TP-NO DEX groups received subcutaneous injections of TP (660 ug in 0.1 ml peanut oil) once each day in the late afternoon. A similar dosage (250 t~g TP per 100 g body weight) has been reported to reinstate sexual behavior following castration in adult male rats [ 10]. All remaining animals in both OVX and Sham subgroups received injections of an equal volume of vehicle at the same time of day. The injections were administered for 10 days prior to the start of testing as well as throughout testing.
Animals were again weighed 24 hr prior to the start of open-field testing and the DEX treatment was begun. Animals in DEX groups received 1.0 ug DEX per ml distilled water in their water bottles, to obtain an approximate daily dosage of 4 0 - 5 0 ug DEX per rat per day. The bottles were refilled with freshly mixed DEX solution every 48 hr, and the amount consumed was recorded. Body weight changes and posttesting plasma corticosterone levels provided additional indications of DEX consumption. Animals in NO DEX groups had nothing added to their drinking water. Open field (OF). All animals, in random order each day, received a 2 min trial daily for 4 consecutive days. Testing was always completed between 8:30 and noon each day. Home cage defecation for each pair of animals housed together was recorded daily over the 4 days of testing. Blood sampling. Following the last test trial a blood sample ( 0 . 4 - 0 . 5 ml) was taken from each animal by jugular venipuncture under ether anesthesia. Blood was taken 15 rain after a rat was removed from its home cage for testing. Each animal was retained in a holding cage after leaving the open field for the remainder of the interval prior to sampling. Plasma was removed by centrifugation and frozen for later assay of corticosterone concentrations using the method of Glick et al. [3]. Organ weights. Following blood sampling the animals were weighed and sacrificed by an overdose of ether. Adrenals and uteri were removed, cleaned and weighed. In addition the ovaries of Shams were removed and weighed. RESULTS Since many of the effects of ovariectomy and testosterone administration on body, uterine, and adrenal weights and plasma corticosterone levels were in accord with previous findings (e.g. [7, 11, 17, 18]), data on the effects of these treatments are not reported here in detail except where these treatments interacted with DEX administration.
Body Weight The mean daffy DEX consumption of all experimental animals was approximately 45.3 ~g DEX per rat per day. This dosage resulted in large weight reductions compared to weights prior to DEX treatment. Analysis of weights, which were recorded prior to surgery, 9 days postoperatively, and on the last day of open-field testing indicated a significant main effect for DEX treatment, F(1,54) = 24.07; p
