Physiology & Behavior, Vol. 49, pp. 289--294. © Pergamon Press plc, 1991. Printed in the U.S.A.
0031-9384/91 $3.00 + .00
Effects of Cycloheximide on the Retention of Olfactory Learning and Maternal Experience Effects in Postpartum Rats S Y L V I E A. M A L E N F A N T , M A U R E E N B A R R Y A N D A L I S O N S. F L E M I N G 1
Department of Psychology, Erindale College, University o f Toronto, Mississauga, Ontario R e c e i v e d 4 April 1990
MALENFANT, S. A., M. BARRY AND A. S. FLEMING. Effects of cycloheximide on the retention of olfactory learning and maternal experience effects in postpartum rats. PHYSIOL BEHAV 49(2) 289-294, 1991. --We have previously shown that cycloheximide, a protein synthesis inhibitor, blocks retention of experientially based maternal responding in postpartum rats. In the following study, we investigated further the effect of this drug on maternal experience effects and, in particular, on the retention of olfactory learning. Dams were injected SC with cycloheximide or saline following a one-hour maternal experience with pups scented with one of two artificial odorants. When tested eight days later, saline but not cycloheximide animals demonstrated a preference for the odor which they had experienced on pups. Animals were then tested for maternal responsiveness with pups that were scented with an odor that was either the same or different from the one used in the maternal experience. All animals receiving cycloheximide, regardless of the pup odor at testing, exhibited long latencies to become maternal, replicating our earlier finding that the retention of learning in maternal behavior is susceptible to disruption by protein synthesis inhibitors. A similar disruption of the experience effect was found when the pup odor at testing was different from the exposure odor. This suggests that olfactory learning may normally play a role in the mediation of postpartum experience effects. Protein synthesis inhibitor
Cycloheximide
Maternal behavior
PRIMIPAROUS female rats show immediate maternal behavior at the time of parturition when hormones are exerting an effect (12,28). If separated from their young at birth or following caesarian delivery, their maternal responsiveness declines such that by day 10 postpartum they are no longer maternally responsive. However, if a postpartum female receives as little as one-half hour of maternal experience soon after c-section, her responsiveness remains elevated for at least 10 days. (25). Though postpartum experience has long-lasting effects on maternal responsiveness, it seems that this learning differs in some respects from other forms of learning. For instance, the maternal experience effect is strengthened by the parturitional hormones. Cohen and Bridges (5) found that animals obtaining one day of experience postpartum were more maternally responsive 25 days later than were nulliparous animals receiving equivalent experience following maternal induction procedures. We have replicated these findings and observed, in addition, that similar effects can be obtained if nuUiparous animals receive maternal experience after priming with progesterone and estradiol (11). Although hormones such as ACTH, epinephrine and opioid peptides can modulate learning and memory for more traditional forms of learning (18), similar effects on these types of tasks have not yet been demonstrated for the parturitional hormones. However, the maternal experience effect bears some similari-
Olfactory learning
Postpartum rats
ties to other forms of learning in that it can be blocked by protein synthesis inhibitors. In a recent series of studies we have found that if the antibiotic cycloheximide (CXM) is infused intraventricularly into c-sectioned females immediately before or after a two-hour maternal experience, the experience effect is blocked and 10 days later animals respond to test pups like inexperienced animals. If cycloheximide is infused 24 hours after the experience, once "consolidation" has occurred, the maternal experience is retained (9). Cycloheximide, therefore, has similar effects on maternal experience as it has on conditioned avoidance or appetitive learning (8); long-term retention is blocked but "acquisition" or on-going expression of the behavior is spared. It is not clear what precisely is learned during that first brief interaction with pups or what aspect of the learning is essential for the long-term retention. Orpen and Fleming (25) found that heightened responsiveness at 10 days post c-section required at least 30 minutes of interactive contact between dam and pups; retrieval alone or retrieval plus 15 minutes of licking and some crouching was not adequate. These data suggest that a minimal amount of proximal stimulation from the pups, including ventral stimulation, is required for the maternal experience effect; smelling, seeing and hearing the pups from a distance, with no direct interaction, produces only a weak experience effect in more highly responsive animals (15, 25, 31). Furthermore, the maternal expe-
~Requests for reprints should be addressed to A. S. Fleming.
289
290
MALENFANT. BARRY AND FLEMING
rience effect is intact if the experience is obtained while the animal is anosmic (19), hyposmic (Fleming, Garvath and Sarker, submitted), anaptic (Morgan, Fleming and Stern, in preparation), or thelectomized (15). Clearly, no single sensory system is essential if proximal interaction occurs. Whether it is the ventral stimulation itself, other forms of proximal stimulation occurring while the animal crouches, or the act of crouching over pups that is necessary for the maternal experience is not known. Although these studies indicate that chemosensory input is not necessary for the maternal experience effect, such input may still play a role in the intact mother and altering odor characteristics of the young may well influence the strength of the maternal experience. Mother rats develop an attraction to lactating nest odors (2), an effect that can be produced in the virgin by administration of maternal hormones (10). They can also distinguish the sex of their offspring based on chemosensory signals (20) and learn to discriminate their own from alien offspring, probably based on their odors (3,4). In this study we explore further the role of pup odors in the maternal experience effect and focus on three main issues: Will mother rats develop a preference for a familiar artificial odorant used to scent pups during a previous maternal experience over another novel, artificial odorant? Will the maternal experience effect be disrupted if the odor of the pups at the time of the experience is different from the odor of pups at retention testing? And, will cycloheximide block memory for the " p u p " odor in the context of both olfactory preference testing and maternal induction tests'? METHOD
Subjects and Housing Subjects were 120-day-old Sprague-Dawley virgin female rats obtained from Charles River, Canada. Animals were housed in opaque plastic cages (22 x 44 x 15 cm) and provided with wood chips. For maternal behavior testing, females were moved into large transparent cages (37 x 47 x 21 cm). Purina rat chow and water were available ad lib for the duration of the experiment. Room temperature was maintained at 20--33°C with humidity at 23%. A 12:12 illumination cycle was in effect with lights on at 0800 h.
Experimental Design Rats were assigned to one of 8 groups and all received a lhour maternal experience 36 hours after caesarian delivery. Females were presented with four foster pups scented with one of two artificial odors (vanilla or rum) and, following the exposure, were injected with either cycloheximide (CXM) or saline (SAL). Maternal induction tests were conducted 10 days after c-section; females were presented with pups that were scented with either the same (SAME) or different (DIFF) odor than that to which they had already been exposed. Thus the groups were: SAMECXM (rum-rum, n = 5 ; van-van, n = 5 ) ; SAME-SAL (rum-rum, n = 5; van-van, n = 5); DIFF-CXM (rum-van, n = 5; van-rum, n = 5); DIFF-SAL (rum-van, n = 5; van-rum, n = 5). In addition to these groups, a condition was run to control for the possibility that olfactory preferences might derive simply from differential familiarity with the two odors. Females were exposed to pups scented with either rum (n = 8) or van (n = 6), just as before, and injected with saline after the exposure. However, 3-4 hours later, these animals also received exposure, for an equal period of time, to wood chips scented with the odor that wasn't used to scent pups. In this way, each female was exposed to both odors, one odor being on the pups (PUP odor) and the other on
the bedding material (NON-PUP odor). At induction testing, females were presented with either the SAME- (rum-rum, n = 5: van-van, n =4) or DIFF- (rum-van, n = 3: van-rum, n = 2) scented pups.
Procedures Mating and caesarian sections. Daily vaginal smears were performed and a male was placed into a female's cage if she was found to be in proestrus or estrus. The male was removed on the following day and the female was smeared to check for the presence of sperm. The day of sperm detection was called day 1 of pregnancy. On day 21 of pregnancy (1000-1400 h), foeti were removed by caesarian section. Sections were performed under ether anaesthesia using a single 2-3-cm mid-ventral incision. Foeti were removed through two incisions made along each uterine horn. The uterus was left in place and the abdominal muscle wall was sutured whereas the skin was closed using Mikron 9-ram wound clips. Odorants and pup scenting. Pups were scented with either artificial rum or vanilla extract (Loretta Foods Manufacturing Co.) by covering them in scented wood chips for 10 minutes prior to the 1-hour experience. (These two odors were found to be the most attractive to females from among four different artificial odors that were used for preference testing in a pilot study.) Chips were scented by adding 5 ml of vanilla or 2.5 ml of rum diluted with an equal volume of tap water to 200 ml of clean bedding chips. An additional 25 ml of wood chips scented with 2 ml of odor were placed over the pups at the midpoint of the maternal experience. Maternal experience and injections. On the morning following caesarian section females were transferred to clear plastic cages (22 x 44 x 15 cm) and placed into one of the two observation rooms, according to their assigned odor. Pup exposure was given 36 hours after caesarian section, Four foster pups (1-3 days old) obtained from donor mothers in the breeding colony were scented with one of the two odors and placed, along with the scented chips, into the corner of the cage opposite the female's nest. The female was observed for 16 minutes, beginning with the retrieval of the first pup to the nest site. On some occasions the female did not retrieve the pups but instead changed her nest site. In these cases, the observation was initiated once the female had stayed with and licked the pups for at least 1 minute. Behaviors that were observed included retrieval, crouching, pup licking (general body and genital) and nest building. Observations were conducted using a one-zero 5-second interval sampling procedure in which each behavior is given a score of either 1 (observed) or 0 (not observed) every 5 seconds. The pups were then left with the female for an additional 44 minutes, for a total of one hour, during which the female's behavior was observed for a brief period of time every 15 minutes. Females that were not maternal during the initial 15 minutes of the exposure were observed briefly every 15 minutes thereafter; observation of maternal experience was initiated if pups were found retrieved to the nest. Females who cannibalized or did not respond were excluded from the study. At the end of the maternal experience the pups were removed and the female was given a subcutaneous injection of either saline (0.9%, 1.25 ml/kg) or cycloheximide (1.25 mg/kg, 1.25 mg/ ml) at the back of the neck. (This dose was chosen, based on a pilot study, as the highest dose that did not lead to any apparent motor side-effects.) The female was then transferred to a clean cage and isolated in another room until olfactory and induction testing. Exposure to scented bedding material. For those animals in
CYCLOHEXIMIDE AND MATERNAL BEHAVIOR
the control condition, wood chips were scented as described for the olfactory preference test and placed into the female's cage for a duration of one hour, 3--4 hours after the pups had been removed. Olfactory and maternal induction testing. Females were moved into large observation cages (37 × 47 x 21 cm) and placed into the olfactory testing room on day 9 post c-section (c-section = day 0). Olfactory preference tests. On the following morning, two circular metal cups (5.5 × 4 cm) each containing 25 ml of wood chips mixed with 2 ml of one of the two odors were placed into opposite comers of the female's cage (location of the odors was counterbalanced across animals). The female was then placed into the center of the cage and observed for five minutes using a onezero 5-second interval sampling procedure of the following behaviors: sniffing around the cup, sniffing with nose in the cup and tipping the cup. The cups were then removed and the female transferred to the appropriate test room. Maternal induction tests. Females were first tested for maternal behavior on day 10 post c-section, following the olfactory preference test, and then tested daily until designated maternal or cannibalistic, or for a maximum of 11 days. Females were provided with 2 shredded paper towels for nesting material. Each morning foster pups from the previous day were removed and four recently fed 1-3-day-old foster pups were scented with one of two odors as previously described and placed opposite the nest site. The female was observed, using a one-zero 5-second interval sampling procedure, for a period of 8 minutes. Brief observations of the female's behavior and position were made 2 hours after the test, at the end of the day, and on the following morning before removal of the pups. Additional scented wood chips were added at the end of the day. The following behaviors were recorded: pup retrieval, crouch posture, licking (general body), genital licking, and nest building. A female was considered maternal when she retrieved all four pups to a common nest site on two consecutive days and adopted a crouch posture over the pups on at least one of these days. The latency to maternal behavior was the number of days to the first of these two consecutive days. RESULTS
Behavior During the Initial Pup Exposure Seventy-six percent of the females tested became maternal during the exposure phase; that is, they either retrieved the pups or changed their nest site to the pups' location and in addition licked the pups and adopted a crouch posture over them. Frequencies for the various maternal behaviors observed during the first 16 minutes of the exposure phase were compared across drug conditions and no significant differences were found. There were also no differences in the frequencies of maternal behaviors between females exposed to rum or to vanilla pups. In addition, there appeared to be no qualitative differences in the behavior of these dams and dams from other studies carded out in our laboratory in which nonscented pups were used.
Odor Preference Test To determine whether animals exhibited a preference for the odor to which they had been preexposed, a preference ratio was computed for each animal by summing the number of 5-second intervals during which the female sniffed at or nosed the cup containing the PUP odor and dividing it by the summed interval value for both PUP and NON-PUP odor (PUP/PUP + NON-PUP). Rum- and van-exposed animals did not differ statistically on this measure and were combined within drug condition for analysis of
291
A4)E100] u)+
80]
)4
W
60
0
Zi,i n40 l,l u. W n(I, 20
SALINE
CXM
DRUG CONDITION FIG. 1. Preference ratio for PUP odor at olfactory testing for groups injected SC with SAL or CXM.
preference test results. In addition, animals were not separated into SAME and DIFF groups for these analyses since preference testing preceded induction testing, and thus SAME and DIFF animals had received identical treatment to this point. Finally, animals in the control condition, which had been exposed to both odors (on pup and on wood chips), did not differ in preference from SAL animals exposed to only one of the odors (on pups), and thus the two groups were combined for statistical analysis. Comparisons, then, were made between all SAL animals and all CXM animals. As indicated by the preference ratios, SAL animals demonstrated a significantly greater preference for the PUP odor they had experienced 8 days earlier than did the CXM animals (U = 209, n I = 32, n 2 = 20, p50). In other words, there was a significantly greater number of animals exhibiting a preference for the PUP than for the NON-PUP odor within the SAL group, ×2(1)=6.3, p
0031-9384/91 $3.00 + .00
Effects of Cycloheximide on the Retention of Olfactory Learning and Maternal Experience Effects in Postpartum Rats S Y L V I E A. M A L E N F A N T , M A U R E E N B A R R Y A N D A L I S O N S. F L E M I N G 1
Department of Psychology, Erindale College, University o f Toronto, Mississauga, Ontario R e c e i v e d 4 April 1990
MALENFANT, S. A., M. BARRY AND A. S. FLEMING. Effects of cycloheximide on the retention of olfactory learning and maternal experience effects in postpartum rats. PHYSIOL BEHAV 49(2) 289-294, 1991. --We have previously shown that cycloheximide, a protein synthesis inhibitor, blocks retention of experientially based maternal responding in postpartum rats. In the following study, we investigated further the effect of this drug on maternal experience effects and, in particular, on the retention of olfactory learning. Dams were injected SC with cycloheximide or saline following a one-hour maternal experience with pups scented with one of two artificial odorants. When tested eight days later, saline but not cycloheximide animals demonstrated a preference for the odor which they had experienced on pups. Animals were then tested for maternal responsiveness with pups that were scented with an odor that was either the same or different from the one used in the maternal experience. All animals receiving cycloheximide, regardless of the pup odor at testing, exhibited long latencies to become maternal, replicating our earlier finding that the retention of learning in maternal behavior is susceptible to disruption by protein synthesis inhibitors. A similar disruption of the experience effect was found when the pup odor at testing was different from the exposure odor. This suggests that olfactory learning may normally play a role in the mediation of postpartum experience effects. Protein synthesis inhibitor
Cycloheximide
Maternal behavior
PRIMIPAROUS female rats show immediate maternal behavior at the time of parturition when hormones are exerting an effect (12,28). If separated from their young at birth or following caesarian delivery, their maternal responsiveness declines such that by day 10 postpartum they are no longer maternally responsive. However, if a postpartum female receives as little as one-half hour of maternal experience soon after c-section, her responsiveness remains elevated for at least 10 days. (25). Though postpartum experience has long-lasting effects on maternal responsiveness, it seems that this learning differs in some respects from other forms of learning. For instance, the maternal experience effect is strengthened by the parturitional hormones. Cohen and Bridges (5) found that animals obtaining one day of experience postpartum were more maternally responsive 25 days later than were nulliparous animals receiving equivalent experience following maternal induction procedures. We have replicated these findings and observed, in addition, that similar effects can be obtained if nuUiparous animals receive maternal experience after priming with progesterone and estradiol (11). Although hormones such as ACTH, epinephrine and opioid peptides can modulate learning and memory for more traditional forms of learning (18), similar effects on these types of tasks have not yet been demonstrated for the parturitional hormones. However, the maternal experience effect bears some similari-
Olfactory learning
Postpartum rats
ties to other forms of learning in that it can be blocked by protein synthesis inhibitors. In a recent series of studies we have found that if the antibiotic cycloheximide (CXM) is infused intraventricularly into c-sectioned females immediately before or after a two-hour maternal experience, the experience effect is blocked and 10 days later animals respond to test pups like inexperienced animals. If cycloheximide is infused 24 hours after the experience, once "consolidation" has occurred, the maternal experience is retained (9). Cycloheximide, therefore, has similar effects on maternal experience as it has on conditioned avoidance or appetitive learning (8); long-term retention is blocked but "acquisition" or on-going expression of the behavior is spared. It is not clear what precisely is learned during that first brief interaction with pups or what aspect of the learning is essential for the long-term retention. Orpen and Fleming (25) found that heightened responsiveness at 10 days post c-section required at least 30 minutes of interactive contact between dam and pups; retrieval alone or retrieval plus 15 minutes of licking and some crouching was not adequate. These data suggest that a minimal amount of proximal stimulation from the pups, including ventral stimulation, is required for the maternal experience effect; smelling, seeing and hearing the pups from a distance, with no direct interaction, produces only a weak experience effect in more highly responsive animals (15, 25, 31). Furthermore, the maternal expe-
~Requests for reprints should be addressed to A. S. Fleming.
289
290
MALENFANT. BARRY AND FLEMING
rience effect is intact if the experience is obtained while the animal is anosmic (19), hyposmic (Fleming, Garvath and Sarker, submitted), anaptic (Morgan, Fleming and Stern, in preparation), or thelectomized (15). Clearly, no single sensory system is essential if proximal interaction occurs. Whether it is the ventral stimulation itself, other forms of proximal stimulation occurring while the animal crouches, or the act of crouching over pups that is necessary for the maternal experience is not known. Although these studies indicate that chemosensory input is not necessary for the maternal experience effect, such input may still play a role in the intact mother and altering odor characteristics of the young may well influence the strength of the maternal experience. Mother rats develop an attraction to lactating nest odors (2), an effect that can be produced in the virgin by administration of maternal hormones (10). They can also distinguish the sex of their offspring based on chemosensory signals (20) and learn to discriminate their own from alien offspring, probably based on their odors (3,4). In this study we explore further the role of pup odors in the maternal experience effect and focus on three main issues: Will mother rats develop a preference for a familiar artificial odorant used to scent pups during a previous maternal experience over another novel, artificial odorant? Will the maternal experience effect be disrupted if the odor of the pups at the time of the experience is different from the odor of pups at retention testing? And, will cycloheximide block memory for the " p u p " odor in the context of both olfactory preference testing and maternal induction tests'? METHOD
Subjects and Housing Subjects were 120-day-old Sprague-Dawley virgin female rats obtained from Charles River, Canada. Animals were housed in opaque plastic cages (22 x 44 x 15 cm) and provided with wood chips. For maternal behavior testing, females were moved into large transparent cages (37 x 47 x 21 cm). Purina rat chow and water were available ad lib for the duration of the experiment. Room temperature was maintained at 20--33°C with humidity at 23%. A 12:12 illumination cycle was in effect with lights on at 0800 h.
Experimental Design Rats were assigned to one of 8 groups and all received a lhour maternal experience 36 hours after caesarian delivery. Females were presented with four foster pups scented with one of two artificial odors (vanilla or rum) and, following the exposure, were injected with either cycloheximide (CXM) or saline (SAL). Maternal induction tests were conducted 10 days after c-section; females were presented with pups that were scented with either the same (SAME) or different (DIFF) odor than that to which they had already been exposed. Thus the groups were: SAMECXM (rum-rum, n = 5 ; van-van, n = 5 ) ; SAME-SAL (rum-rum, n = 5; van-van, n = 5); DIFF-CXM (rum-van, n = 5; van-rum, n = 5); DIFF-SAL (rum-van, n = 5; van-rum, n = 5). In addition to these groups, a condition was run to control for the possibility that olfactory preferences might derive simply from differential familiarity with the two odors. Females were exposed to pups scented with either rum (n = 8) or van (n = 6), just as before, and injected with saline after the exposure. However, 3-4 hours later, these animals also received exposure, for an equal period of time, to wood chips scented with the odor that wasn't used to scent pups. In this way, each female was exposed to both odors, one odor being on the pups (PUP odor) and the other on
the bedding material (NON-PUP odor). At induction testing, females were presented with either the SAME- (rum-rum, n = 5: van-van, n =4) or DIFF- (rum-van, n = 3: van-rum, n = 2) scented pups.
Procedures Mating and caesarian sections. Daily vaginal smears were performed and a male was placed into a female's cage if she was found to be in proestrus or estrus. The male was removed on the following day and the female was smeared to check for the presence of sperm. The day of sperm detection was called day 1 of pregnancy. On day 21 of pregnancy (1000-1400 h), foeti were removed by caesarian section. Sections were performed under ether anaesthesia using a single 2-3-cm mid-ventral incision. Foeti were removed through two incisions made along each uterine horn. The uterus was left in place and the abdominal muscle wall was sutured whereas the skin was closed using Mikron 9-ram wound clips. Odorants and pup scenting. Pups were scented with either artificial rum or vanilla extract (Loretta Foods Manufacturing Co.) by covering them in scented wood chips for 10 minutes prior to the 1-hour experience. (These two odors were found to be the most attractive to females from among four different artificial odors that were used for preference testing in a pilot study.) Chips were scented by adding 5 ml of vanilla or 2.5 ml of rum diluted with an equal volume of tap water to 200 ml of clean bedding chips. An additional 25 ml of wood chips scented with 2 ml of odor were placed over the pups at the midpoint of the maternal experience. Maternal experience and injections. On the morning following caesarian section females were transferred to clear plastic cages (22 x 44 x 15 cm) and placed into one of the two observation rooms, according to their assigned odor. Pup exposure was given 36 hours after caesarian section, Four foster pups (1-3 days old) obtained from donor mothers in the breeding colony were scented with one of the two odors and placed, along with the scented chips, into the corner of the cage opposite the female's nest. The female was observed for 16 minutes, beginning with the retrieval of the first pup to the nest site. On some occasions the female did not retrieve the pups but instead changed her nest site. In these cases, the observation was initiated once the female had stayed with and licked the pups for at least 1 minute. Behaviors that were observed included retrieval, crouching, pup licking (general body and genital) and nest building. Observations were conducted using a one-zero 5-second interval sampling procedure in which each behavior is given a score of either 1 (observed) or 0 (not observed) every 5 seconds. The pups were then left with the female for an additional 44 minutes, for a total of one hour, during which the female's behavior was observed for a brief period of time every 15 minutes. Females that were not maternal during the initial 15 minutes of the exposure were observed briefly every 15 minutes thereafter; observation of maternal experience was initiated if pups were found retrieved to the nest. Females who cannibalized or did not respond were excluded from the study. At the end of the maternal experience the pups were removed and the female was given a subcutaneous injection of either saline (0.9%, 1.25 ml/kg) or cycloheximide (1.25 mg/kg, 1.25 mg/ ml) at the back of the neck. (This dose was chosen, based on a pilot study, as the highest dose that did not lead to any apparent motor side-effects.) The female was then transferred to a clean cage and isolated in another room until olfactory and induction testing. Exposure to scented bedding material. For those animals in
CYCLOHEXIMIDE AND MATERNAL BEHAVIOR
the control condition, wood chips were scented as described for the olfactory preference test and placed into the female's cage for a duration of one hour, 3--4 hours after the pups had been removed. Olfactory and maternal induction testing. Females were moved into large observation cages (37 × 47 x 21 cm) and placed into the olfactory testing room on day 9 post c-section (c-section = day 0). Olfactory preference tests. On the following morning, two circular metal cups (5.5 × 4 cm) each containing 25 ml of wood chips mixed with 2 ml of one of the two odors were placed into opposite comers of the female's cage (location of the odors was counterbalanced across animals). The female was then placed into the center of the cage and observed for five minutes using a onezero 5-second interval sampling procedure of the following behaviors: sniffing around the cup, sniffing with nose in the cup and tipping the cup. The cups were then removed and the female transferred to the appropriate test room. Maternal induction tests. Females were first tested for maternal behavior on day 10 post c-section, following the olfactory preference test, and then tested daily until designated maternal or cannibalistic, or for a maximum of 11 days. Females were provided with 2 shredded paper towels for nesting material. Each morning foster pups from the previous day were removed and four recently fed 1-3-day-old foster pups were scented with one of two odors as previously described and placed opposite the nest site. The female was observed, using a one-zero 5-second interval sampling procedure, for a period of 8 minutes. Brief observations of the female's behavior and position were made 2 hours after the test, at the end of the day, and on the following morning before removal of the pups. Additional scented wood chips were added at the end of the day. The following behaviors were recorded: pup retrieval, crouch posture, licking (general body), genital licking, and nest building. A female was considered maternal when she retrieved all four pups to a common nest site on two consecutive days and adopted a crouch posture over the pups on at least one of these days. The latency to maternal behavior was the number of days to the first of these two consecutive days. RESULTS
Behavior During the Initial Pup Exposure Seventy-six percent of the females tested became maternal during the exposure phase; that is, they either retrieved the pups or changed their nest site to the pups' location and in addition licked the pups and adopted a crouch posture over them. Frequencies for the various maternal behaviors observed during the first 16 minutes of the exposure phase were compared across drug conditions and no significant differences were found. There were also no differences in the frequencies of maternal behaviors between females exposed to rum or to vanilla pups. In addition, there appeared to be no qualitative differences in the behavior of these dams and dams from other studies carded out in our laboratory in which nonscented pups were used.
Odor Preference Test To determine whether animals exhibited a preference for the odor to which they had been preexposed, a preference ratio was computed for each animal by summing the number of 5-second intervals during which the female sniffed at or nosed the cup containing the PUP odor and dividing it by the summed interval value for both PUP and NON-PUP odor (PUP/PUP + NON-PUP). Rum- and van-exposed animals did not differ statistically on this measure and were combined within drug condition for analysis of
291
A4)E100] u)+
80]
)4
W
60
0
Zi,i n40 l,l u. W n(I, 20
SALINE
CXM
DRUG CONDITION FIG. 1. Preference ratio for PUP odor at olfactory testing for groups injected SC with SAL or CXM.
preference test results. In addition, animals were not separated into SAME and DIFF groups for these analyses since preference testing preceded induction testing, and thus SAME and DIFF animals had received identical treatment to this point. Finally, animals in the control condition, which had been exposed to both odors (on pup and on wood chips), did not differ in preference from SAL animals exposed to only one of the odors (on pups), and thus the two groups were combined for statistical analysis. Comparisons, then, were made between all SAL animals and all CXM animals. As indicated by the preference ratios, SAL animals demonstrated a significantly greater preference for the PUP odor they had experienced 8 days earlier than did the CXM animals (U = 209, n I = 32, n 2 = 20, p50). In other words, there was a significantly greater number of animals exhibiting a preference for the PUP than for the NON-PUP odor within the SAL group, ×2(1)=6.3, p
