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Transient hypoxic episodes: a mechanism to support associative fetal learning
Anita. Behav., 1991,41, 477-480
Transient hypoxic episodes: a mechanism to support associative fetal learning P E T E R G. H E P P E R School o f Psychology, The Queen's University o f Belfast, Belfast BT71NN, U.K. (Received 16 June 1989; initial acceptance 20 December 1989; final acceptance 20 August 1990; MS. number: 3417)
Abstract. Fetal learning has been demonstrated to play an important role in neonatal rodents' response to chemical stimuli. However, the mechanism of such learning under natural conditions~ unknown. In this experiment, fetal rats, Rattus norvegicus, were subjected to transient hypoxic episodes before or after being exposed to orange essence through the uterine and amniotic membranes. At 12 days after birth, pups that experienced orange followed by hypoxia avoided orange in a two-choice preference test whereas those that experienced orange during recovery from hypoxia preferred it. These results suggest that transient hypoxic episodes present a naturally occurring mechanism that is capable of supporting fetal learning in utero. The importance of hypoxia-supported learning for the avoidance of intra-uterine neurological damage is discussed. Recent research has indicated that experiences gained during the prenatal period may have important consequences for later behaviour (e.g. Hepper 1987). Of particular interest is learning prior to birth or hatching, which is not restricted to mammals but is found in invertebrates (e.g. Isingrini et al. 1985), amphibians (e.g. Waldman 1981) and birds (e.g. Shindler 1984). A number of experiments have demonstrated that the mammalian fetus is capable of learning in the womb (e.g. Smotherman 1982a, b; Hepper 1989). However, many have involved experimental manipulation of the uterine environment (e.g. Smotherman & Robinson 1986; Hepper, in press) and this raises the question of how such learning can take place naturally (Hepper, in press). Recently, experiments have indicated how prenatal exposure learning could occur naturally and influence dietary and social preferences (Hepper 1987, 1988). Associative learning may be a more powerful means of learning, yet to date there has been no indication of how this could occur naturally within the womb. Growth of the fetus, reduction in the volume of amniotic fluid and other changes that occur within the uterine environment increasingly confine the fetus as pregnancy progresses and may result in compression of the umbilical cord during the latter stage of pregnancy (Smotherman & Robinson 1988a). In third trimester human pregnancies transient compression of the umbilical cord is often seen (personal observations) and cord compression is frequently observed in the rat, Rattus norvegicus, 0003-3472/91/030477 + 04 $03.00/0
fetus especially during the last few days of gestation (Smotherman & Robinson 1988a). Experimental occlusion of the umbilical cord in the rat fetus resuits in a stereotyped response consisting of behavioural suppression, activation and then suppression (Smotherman & Robinson 1987), the exact pattern of behavioural change being dependent upon the age of the fetus (Smotherman & Robinson 1988b). Acute hypoxia resulting from compression of the umbilical cord in utero is common during the latter stages of pregnancy. Hypoxia has been suggested as a significant source of brain damage and/or mortality among human fetuses (Mann 1986). Hypoxic events during pregnancy are thus frequent naturally occurring phenomena which may have important consequences for later development. In this paper I investigate whether hypoxic episodes can influence and possibly support learning. METHODS
Subjects I used 24 Sprague-Dawley female rats (supplied by B & K Animal Suppliers) in this experiment. They were 'time mated' with Sprague Dawley males (i.e. the day of conception was known, and this was denoted as day 1 of gestation). Females were housed in groups of three until day 18 of gestation and then housed individually in NKP plastic cages (41 • 18cm). Food and water were freely available throughout the study and subjects were
9 1991 The Association for the Study of Animal Behaviour 477
478
A n i m a l B e h a v i o u r , 41, 3
kept on a 12:12 h light:dark cycle, darkness starting at 1200 hours.
Preparation of the Pregnant Female Females on day 20 of gestation were placed under ether anaesthesia, and were injected with 70 ~tl of 2% lidocaine plus 1% adrenaline between T1 and T2 vertebrae, to produce a reversible chemical transection of the spinal cord (Smotherman et al. 1987). This eliminates afferent and efferent stimuli from the lower part of the body without the use of general anaesthesia and lasts for approximately 1 h. The females were supported in a holding apparatus and the uterus was externalized, onto sterile pads, by midline laparotomy. Endoscopic observations of the fetus in situ (e.g. Smotherman & Robinson 1986) indicate that externalization of the uterus exerts little effect on fetal behaviour (personal observations). The females were then left for 10 min to recover fully from the ether anaesthesia.
Preparation of the Fetus I used four fetuses from each female and randomly assigned them to one of four conditions. (1) Stimulus Hypoxia: 15 ~tl pure orange essence was injected into the mouth of the fetus through the uterine and amniotic membranes. After 30 s, the umbilical cord was clamped using a specially designed clamp, which consisted of a modified needle and hook. This was inserted through the uterus and amnion and placed around the umbilical cord. The hook was then pulled up against the body of the needle, trapping the umbilical cord and effectively stopping circulation of fetal blood. The clamp remained in place for 79 s and was then removed. The fetus was checked to ensure normal blood flow resumed. (2) Hypoxia Stimulus: the umbilical cord was clamped as described above and 79 s of hypoxia were induced. Within 5 s of removal of the clamp 15 ~1 pure orange essence was injected into the mouth of the fetus. (3) Hypoxia control: the clamp was fixed and 79 s of hypoxia followed. No chemical stimuli were introduced into the amniotic cavity. An orange only control was not used since the comparison was between stimuli associated with hypoxia onset and hypoxia offset. Also the effects of orange only exposure have previously been reported (Hepper, in press).
(4) Control: the clamp was inserted into the uterine cavity and left without clamping the umbilical cord for 79 s. The above conditions were undertaken in a random order across females. After this procedure I marked the fetuses by subcutaneous injection of ink for identification after birth. The uterus was replaced in the abdominal cavity and the midline laparotomy sutured.
Care of Mothers The whole procedure from ether anaesthesia to replacing the mother in her cage took approximately 18 min. During the time in the holding apparatus the mothers did not appear to be stressed and made no attempts to 'escape' from the situation. Furthermore recording of maternal heartbeat during this procedure indicates that heart rate does not deviate significantly from unoperated controls. The mothers were administered analgesics (Tengesic; Reckitt & Coleman; 0.035 mg/kg by subcutaneous injection every 12 h for 48 h after the operation) and antibiotics (one injection of Pen. & Strep.; Norbrook; 0-25ml/kg by intramuscular injection immediately after surgery followed by Terramycin; Pfizer; 50mg/100ml added to the drinking water for 5 days following surgery). I paid close attention to the mothers after surgery and all gave birth naturally within 30-36 h after the operation. I observed two mothers giving birth and none exhibited any signs of distress or differences in behaviour from the normally parturient rat. Furthermore no sutures were broken before or during the birth of the pups. After birth all mothers successfully reared their litters with no pup deaths.
Testing The pups and mothers were left undisturbed until day 12 when testing took place. The pups were given a choice between orange essence and water in a two-choice preference test. They were tested singly in an aluminium box (30 • 15 • 15 cm) with mesh floor, and one side of clear Perspex. The apparatus stood 2 cm above the ground and orange and water stimuli were placed under the floor at either end of the box in glass petri dishes. The pups were left for 2 min in the box and the time spent over each side recorded. I noted the side over which the pups spent the most time and number of pups preferring the orange and water for each condition.
Hepper: Hypoxia andfetal learning 24
E R ~6 12
=
itimulus
Hypoxio
Hypoxio Stimulus
f
Hypoxia Control
Control
Figure 1. Number of pups in the four groups showing a preference for orange odour. Stimulus Hypoxia: the pups experienced orange followed by hypoxia; Hypoxia Stimulus: the pups experienced hypoxia followed by orange; Hypoxia Control: the pups experienced hypoxia only; Control: the pups experienced neither hypoxia nor orange. RESULTS Exposure to a hypoxic event before or after experiencing orange essence significantly affected the pups' later response to the stimulus (Fisher's exact test: P < 0.01; see Fig. 1). Pups experiencing orange followed by hypoxia avoided the orange (,~=7, N=24, P=0.032 one-tailed, binomial) whereas pups experiencing orange during recovery from hypoxia preferred the orange (.Y= 6, N = 24, P=0'011, one-tailed, binomial). In both control conditions, no preference for orange or water was found (Hypoxia Control: number that preferred orange 11, water 13; Control: number that preferred orange 12, water 12). DISCUSSION The results of this study indicate that prenatal hypoxia significantly affects the pup's response to the stimuli associated with this hypoxia. Stimuli associated with the onset of hypoxia become aversive while stimuli associated with the offset of hypoxia become attractive. The study demonstrates the existence of a naturally occurring circumstance capable of supporting associative learning by the fetus. Chemical stimuli assume importance for rodents in the neonatal period for the initiation of' suckling and attachment to the nipple (see Blass & Teicher 1980). Furthermore neonatal rats are responsive to odours produced by their conspecifics (Hepper
479
1983, 1987) and this is important for social interactions, present and future (Hepper, in press). Evidence suggests that the prenatal period is important for learning about these stimuli (Pedersen & Blass 1982; Hepper 1987, in press) although the exact mechanism has yet to be determined. This study demonstrated that a single hypoxic event is capable of supporting learning by the rat fetus. Since such transient hypoxic events are known to occur naturally during pregnancy (Smotherman & Robinson 1988a), experience of stimuli in conjunction with hypoxia may be one means through which the fetus can learn prenatally. Chemical stimuli used for both the initiation of suckling and in social interactions are present in the amniotic fluid throughout pregnancy (Hepper 1987) and thus could be associated with both the onset and offset ofhypoxia. This presents an apparent problem for learning by the fetus in that the same stimulus would be associated with both the onset and offset of hypoxia. The results from this experiment, however, suggest that a stronger effect is established by associating the stimulus with the offset of hypoxia than with the onset. Thus stimuli associated with both the onset and offset ofhypoxia may become preferred rather than avoided due to the greater 'reinforcing' association with hypoxia offset. It should be noted that prenatal exposure to chemical stimuli with no hypoxia also results in a postnatal preference for the familiar chemical (Hepper, in press). Thus stimuli presented after hypoxia may become preferred through either simple familiarity or some reinforcing aspect of hypoxia offset, e.g. increased oxygenation. The experiment also demonstrates that stimuli associated with the onset of hypoxia are avoided whereas those associated with the offset of hypoxia are preferred. Such a difference may be expected given the potential life-threatening consequences of prolonged hypoxia in utero. Behaviour patterns or events initiated by the fetus that result in hypoxia, e.g. lying on the cord or vigorous movements disrupting placental blood flow, and that may lead to neurological damage (Mann 1986), should be avoided whereas those that result in the release from hypoxia and may prevent adverse neurological sequelae, should be preferentially learned. Since it appears that the onset and offset of hypoxia can both support learning but with different results, their mechanism of action may be different. Most likely the different effects are caused by the resultant oxygen deficit and reoxygenation associated
480
Animal Behaviour , 41, 3
with the onset and offset of_hypoxia, respectively. In this regard, it is of interest that fetuses are more active during hypoxia and remain somewhat hyperactive after the hypoxic event. Studies of learning during the immediate postnatal period have indicated that behavioural activation accompanies the learning process (Johanson & Hall 1982; Terry & Johanson 1987) and, in other instances activation of pups may be sufficient in and of itself to support learning (Sullivan & Hall 1988). Perhaps behavioural activation is a necessary correlate of fetal learning as well. Previous studies have suggested that uterine exposure to chemical cues can influence postnatal preferences and behaviour (Hepper 1987, 1988, 1989, in press). Experimentally induced aversive conditioning has also been demonstrated in the rat fetus (Stickrod et al. 1982), but although inducing powerful learning (Smotherman 1982b), questions of how such learning may occur naturally have been raised (Hepper, in press). The experiment performed here clearly demonstrates that transient hypoxia, a naturally occurring event, is capable of supporting associative conditioning. Furthermore, a single episode of hypoxia produced a significant effect; in nature, hypoxia may occur much more frequently which suggests that stronger associations would be formed under these conditions and this may be advantageous in the prevention of neurological damage. The study provides further evidence that naturally occurring prenatal experiences may have important consequences for behaviour after birth. ACKNOWLEDGMENTS This research is supported by grants from the NATO Collaborative Research Grants Program 0551-88, The Wellcome Trust, The Nuffield Foundation and The Physiological Society ofGB. I thank Bill Smotherman for his comments on the manuscript.
REFERENCES Blass, E. M. & Teicher, M. H. 1980. Suckling. Science, 210, 15-22. Hepper, P. G. 1983. Sibling recognition in the rat. Anita. Behav., 31, 1177-1191.
Hepper, P. G. 1987. The amniotic fluid: an important priming role in kin recognition. Anim. Behav., 35, 1343-1346. Hepper, P. G. 1988. Adaptive fetal learning: prenatal exposure to garlic affects postnatal preferences. Anim. Behav., 36, 935-936. Hepper, P. G. 1989. Fetal learning: implications for psychiatry. Br. J. Psychiat., 155, 289-293. Hepper, P. G. In press. Fetal olfaction. In: Chemical Senses in Vertebrates 5 (Ed. by D. MacDonald). Oxford: Oxford UniversityPress. Isingrini, M., Lenoir, A. & Jaisson, P. 1985. Preimaginal learning as a basis of colony recognition in the ant Cataglyphis cursor. Proc. natn. Acad. Sci. U.S.A., 82, 8545-8547. Johanson, I. B. & Hall, W. G. 1982.Appetitive conditioning in neonatal rats: conditioned orientation to a novel odour. Devl Psychobiol., 15, 379-397. Mann, L. I. 1986. Pregnancy events and brain damage. Am. J. Obstet. Gynecol., 155, 6-9. Pedersen, P. E. & Blass, E. M. 1982. Prenatal and postnatal determinants of the 1st sucklingepisode in albino rats. Devl Psychobiol., 15, 349-355. Shindler,K. M. 1984. A three year study of fetal auditory imprinting. J. Wash. Acad. Sci., 74, 121-124. Smotherman, W. P. 1982a. Odour aversion learning by the rat fetus. Physiol. Behav., 29, 769-771. Smotherman, W. P. 1982b. In utero chemosensory experience alters taste preferences and corticosterone responsiveness.Behav. neural Biol., 36, 61-68. Smotherman, W. P. & Robinson, S. R. 1986. A method for endoscopic visualization of rat fetuses in situ Physiol. Behav., 37, 663-665. Smotherman, W. P. & Robinson, S. R. 1987. Stereotypic behavioural response of rat fetuses to acute hypoxia is altered by maternal alcohol consumption. Am. J. Obstet. Gynecol., 157, 982-986. Smotherman, W. P. & Robinson, S. R. 1988a. The uterus as environment: the ecologyof fetal behavior. In Handbook of Behavioral Neurobiology. Vol. 9. Developmental Psychobiology and Behavioral Ecology (Ed. by E. M. Blass), pp. 149-196. New York: Plenum Press. Smotherman, W. P. & Robinson, S. R. 1988b. Response of the rat fetus to acute umbilical cord occlusion: an ontogenetic adaptation? Physiol. Behav., 44, 131-135. Smotherman, W. P., Robinson, S. R. & Miller, B. J. 1987. A reversiblepreparation for observing the behavior of rat fetuses in utero. Physiol. Behav., 37, 57-60. Stickrod, G., Kimble, D. P. & Smotherman, W. P. 1982. In utero taste/odour conditioning in the rat: effects on nipple preference testing. Physiol. Behav., 28, 5-7. Sullivan,R. M. & Hall, W. G. 1988.Reinforcersininfancy: classical conditioning using stroking or intra-oral infusions of milk as UCS. Devl Psychobiol., 21, 215-223. Terry, L. M. & Johanson, I. B. 1987. Olfactory influences on the ingestivebehavior ofinfantrats. DevlPsychobiol., 20, 313-332. Waldman, B. 1981. Sibling recognition in toad tadpoles: the role of experience. Z. Tierpsychol., 56, 341-358.
Transient hypoxic episodes: a mechanism to support associative fetal learning P E T E R G. H E P P E R School o f Psychology, The Queen's University o f Belfast, Belfast BT71NN, U.K. (Received 16 June 1989; initial acceptance 20 December 1989; final acceptance 20 August 1990; MS. number: 3417)
Abstract. Fetal learning has been demonstrated to play an important role in neonatal rodents' response to chemical stimuli. However, the mechanism of such learning under natural conditions~ unknown. In this experiment, fetal rats, Rattus norvegicus, were subjected to transient hypoxic episodes before or after being exposed to orange essence through the uterine and amniotic membranes. At 12 days after birth, pups that experienced orange followed by hypoxia avoided orange in a two-choice preference test whereas those that experienced orange during recovery from hypoxia preferred it. These results suggest that transient hypoxic episodes present a naturally occurring mechanism that is capable of supporting fetal learning in utero. The importance of hypoxia-supported learning for the avoidance of intra-uterine neurological damage is discussed. Recent research has indicated that experiences gained during the prenatal period may have important consequences for later behaviour (e.g. Hepper 1987). Of particular interest is learning prior to birth or hatching, which is not restricted to mammals but is found in invertebrates (e.g. Isingrini et al. 1985), amphibians (e.g. Waldman 1981) and birds (e.g. Shindler 1984). A number of experiments have demonstrated that the mammalian fetus is capable of learning in the womb (e.g. Smotherman 1982a, b; Hepper 1989). However, many have involved experimental manipulation of the uterine environment (e.g. Smotherman & Robinson 1986; Hepper, in press) and this raises the question of how such learning can take place naturally (Hepper, in press). Recently, experiments have indicated how prenatal exposure learning could occur naturally and influence dietary and social preferences (Hepper 1987, 1988). Associative learning may be a more powerful means of learning, yet to date there has been no indication of how this could occur naturally within the womb. Growth of the fetus, reduction in the volume of amniotic fluid and other changes that occur within the uterine environment increasingly confine the fetus as pregnancy progresses and may result in compression of the umbilical cord during the latter stage of pregnancy (Smotherman & Robinson 1988a). In third trimester human pregnancies transient compression of the umbilical cord is often seen (personal observations) and cord compression is frequently observed in the rat, Rattus norvegicus, 0003-3472/91/030477 + 04 $03.00/0
fetus especially during the last few days of gestation (Smotherman & Robinson 1988a). Experimental occlusion of the umbilical cord in the rat fetus resuits in a stereotyped response consisting of behavioural suppression, activation and then suppression (Smotherman & Robinson 1987), the exact pattern of behavioural change being dependent upon the age of the fetus (Smotherman & Robinson 1988b). Acute hypoxia resulting from compression of the umbilical cord in utero is common during the latter stages of pregnancy. Hypoxia has been suggested as a significant source of brain damage and/or mortality among human fetuses (Mann 1986). Hypoxic events during pregnancy are thus frequent naturally occurring phenomena which may have important consequences for later development. In this paper I investigate whether hypoxic episodes can influence and possibly support learning. METHODS
Subjects I used 24 Sprague-Dawley female rats (supplied by B & K Animal Suppliers) in this experiment. They were 'time mated' with Sprague Dawley males (i.e. the day of conception was known, and this was denoted as day 1 of gestation). Females were housed in groups of three until day 18 of gestation and then housed individually in NKP plastic cages (41 • 18cm). Food and water were freely available throughout the study and subjects were
9 1991 The Association for the Study of Animal Behaviour 477
478
A n i m a l B e h a v i o u r , 41, 3
kept on a 12:12 h light:dark cycle, darkness starting at 1200 hours.
Preparation of the Pregnant Female Females on day 20 of gestation were placed under ether anaesthesia, and were injected with 70 ~tl of 2% lidocaine plus 1% adrenaline between T1 and T2 vertebrae, to produce a reversible chemical transection of the spinal cord (Smotherman et al. 1987). This eliminates afferent and efferent stimuli from the lower part of the body without the use of general anaesthesia and lasts for approximately 1 h. The females were supported in a holding apparatus and the uterus was externalized, onto sterile pads, by midline laparotomy. Endoscopic observations of the fetus in situ (e.g. Smotherman & Robinson 1986) indicate that externalization of the uterus exerts little effect on fetal behaviour (personal observations). The females were then left for 10 min to recover fully from the ether anaesthesia.
Preparation of the Fetus I used four fetuses from each female and randomly assigned them to one of four conditions. (1) Stimulus Hypoxia: 15 ~tl pure orange essence was injected into the mouth of the fetus through the uterine and amniotic membranes. After 30 s, the umbilical cord was clamped using a specially designed clamp, which consisted of a modified needle and hook. This was inserted through the uterus and amnion and placed around the umbilical cord. The hook was then pulled up against the body of the needle, trapping the umbilical cord and effectively stopping circulation of fetal blood. The clamp remained in place for 79 s and was then removed. The fetus was checked to ensure normal blood flow resumed. (2) Hypoxia Stimulus: the umbilical cord was clamped as described above and 79 s of hypoxia were induced. Within 5 s of removal of the clamp 15 ~1 pure orange essence was injected into the mouth of the fetus. (3) Hypoxia control: the clamp was fixed and 79 s of hypoxia followed. No chemical stimuli were introduced into the amniotic cavity. An orange only control was not used since the comparison was between stimuli associated with hypoxia onset and hypoxia offset. Also the effects of orange only exposure have previously been reported (Hepper, in press).
(4) Control: the clamp was inserted into the uterine cavity and left without clamping the umbilical cord for 79 s. The above conditions were undertaken in a random order across females. After this procedure I marked the fetuses by subcutaneous injection of ink for identification after birth. The uterus was replaced in the abdominal cavity and the midline laparotomy sutured.
Care of Mothers The whole procedure from ether anaesthesia to replacing the mother in her cage took approximately 18 min. During the time in the holding apparatus the mothers did not appear to be stressed and made no attempts to 'escape' from the situation. Furthermore recording of maternal heartbeat during this procedure indicates that heart rate does not deviate significantly from unoperated controls. The mothers were administered analgesics (Tengesic; Reckitt & Coleman; 0.035 mg/kg by subcutaneous injection every 12 h for 48 h after the operation) and antibiotics (one injection of Pen. & Strep.; Norbrook; 0-25ml/kg by intramuscular injection immediately after surgery followed by Terramycin; Pfizer; 50mg/100ml added to the drinking water for 5 days following surgery). I paid close attention to the mothers after surgery and all gave birth naturally within 30-36 h after the operation. I observed two mothers giving birth and none exhibited any signs of distress or differences in behaviour from the normally parturient rat. Furthermore no sutures were broken before or during the birth of the pups. After birth all mothers successfully reared their litters with no pup deaths.
Testing The pups and mothers were left undisturbed until day 12 when testing took place. The pups were given a choice between orange essence and water in a two-choice preference test. They were tested singly in an aluminium box (30 • 15 • 15 cm) with mesh floor, and one side of clear Perspex. The apparatus stood 2 cm above the ground and orange and water stimuli were placed under the floor at either end of the box in glass petri dishes. The pups were left for 2 min in the box and the time spent over each side recorded. I noted the side over which the pups spent the most time and number of pups preferring the orange and water for each condition.
Hepper: Hypoxia andfetal learning 24
E R ~6 12
=
itimulus
Hypoxio
Hypoxio Stimulus
f
Hypoxia Control
Control
Figure 1. Number of pups in the four groups showing a preference for orange odour. Stimulus Hypoxia: the pups experienced orange followed by hypoxia; Hypoxia Stimulus: the pups experienced hypoxia followed by orange; Hypoxia Control: the pups experienced hypoxia only; Control: the pups experienced neither hypoxia nor orange. RESULTS Exposure to a hypoxic event before or after experiencing orange essence significantly affected the pups' later response to the stimulus (Fisher's exact test: P < 0.01; see Fig. 1). Pups experiencing orange followed by hypoxia avoided the orange (,~=7, N=24, P=0.032 one-tailed, binomial) whereas pups experiencing orange during recovery from hypoxia preferred the orange (.Y= 6, N = 24, P=0'011, one-tailed, binomial). In both control conditions, no preference for orange or water was found (Hypoxia Control: number that preferred orange 11, water 13; Control: number that preferred orange 12, water 12). DISCUSSION The results of this study indicate that prenatal hypoxia significantly affects the pup's response to the stimuli associated with this hypoxia. Stimuli associated with the onset of hypoxia become aversive while stimuli associated with the offset of hypoxia become attractive. The study demonstrates the existence of a naturally occurring circumstance capable of supporting associative learning by the fetus. Chemical stimuli assume importance for rodents in the neonatal period for the initiation of' suckling and attachment to the nipple (see Blass & Teicher 1980). Furthermore neonatal rats are responsive to odours produced by their conspecifics (Hepper
479
1983, 1987) and this is important for social interactions, present and future (Hepper, in press). Evidence suggests that the prenatal period is important for learning about these stimuli (Pedersen & Blass 1982; Hepper 1987, in press) although the exact mechanism has yet to be determined. This study demonstrated that a single hypoxic event is capable of supporting learning by the rat fetus. Since such transient hypoxic events are known to occur naturally during pregnancy (Smotherman & Robinson 1988a), experience of stimuli in conjunction with hypoxia may be one means through which the fetus can learn prenatally. Chemical stimuli used for both the initiation of suckling and in social interactions are present in the amniotic fluid throughout pregnancy (Hepper 1987) and thus could be associated with both the onset and offset ofhypoxia. This presents an apparent problem for learning by the fetus in that the same stimulus would be associated with both the onset and offset of hypoxia. The results from this experiment, however, suggest that a stronger effect is established by associating the stimulus with the offset of hypoxia than with the onset. Thus stimuli associated with both the onset and offset ofhypoxia may become preferred rather than avoided due to the greater 'reinforcing' association with hypoxia offset. It should be noted that prenatal exposure to chemical stimuli with no hypoxia also results in a postnatal preference for the familiar chemical (Hepper, in press). Thus stimuli presented after hypoxia may become preferred through either simple familiarity or some reinforcing aspect of hypoxia offset, e.g. increased oxygenation. The experiment also demonstrates that stimuli associated with the onset of hypoxia are avoided whereas those associated with the offset of hypoxia are preferred. Such a difference may be expected given the potential life-threatening consequences of prolonged hypoxia in utero. Behaviour patterns or events initiated by the fetus that result in hypoxia, e.g. lying on the cord or vigorous movements disrupting placental blood flow, and that may lead to neurological damage (Mann 1986), should be avoided whereas those that result in the release from hypoxia and may prevent adverse neurological sequelae, should be preferentially learned. Since it appears that the onset and offset of hypoxia can both support learning but with different results, their mechanism of action may be different. Most likely the different effects are caused by the resultant oxygen deficit and reoxygenation associated
480
Animal Behaviour , 41, 3
with the onset and offset of_hypoxia, respectively. In this regard, it is of interest that fetuses are more active during hypoxia and remain somewhat hyperactive after the hypoxic event. Studies of learning during the immediate postnatal period have indicated that behavioural activation accompanies the learning process (Johanson & Hall 1982; Terry & Johanson 1987) and, in other instances activation of pups may be sufficient in and of itself to support learning (Sullivan & Hall 1988). Perhaps behavioural activation is a necessary correlate of fetal learning as well. Previous studies have suggested that uterine exposure to chemical cues can influence postnatal preferences and behaviour (Hepper 1987, 1988, 1989, in press). Experimentally induced aversive conditioning has also been demonstrated in the rat fetus (Stickrod et al. 1982), but although inducing powerful learning (Smotherman 1982b), questions of how such learning may occur naturally have been raised (Hepper, in press). The experiment performed here clearly demonstrates that transient hypoxia, a naturally occurring event, is capable of supporting associative conditioning. Furthermore, a single episode of hypoxia produced a significant effect; in nature, hypoxia may occur much more frequently which suggests that stronger associations would be formed under these conditions and this may be advantageous in the prevention of neurological damage. The study provides further evidence that naturally occurring prenatal experiences may have important consequences for behaviour after birth. ACKNOWLEDGMENTS This research is supported by grants from the NATO Collaborative Research Grants Program 0551-88, The Wellcome Trust, The Nuffield Foundation and The Physiological Society ofGB. I thank Bill Smotherman for his comments on the manuscript.
REFERENCES Blass, E. M. & Teicher, M. H. 1980. Suckling. Science, 210, 15-22. Hepper, P. G. 1983. Sibling recognition in the rat. Anita. Behav., 31, 1177-1191.
Hepper, P. G. 1987. The amniotic fluid: an important priming role in kin recognition. Anim. Behav., 35, 1343-1346. Hepper, P. G. 1988. Adaptive fetal learning: prenatal exposure to garlic affects postnatal preferences. Anim. Behav., 36, 935-936. Hepper, P. G. 1989. Fetal learning: implications for psychiatry. Br. J. Psychiat., 155, 289-293. Hepper, P. G. In press. Fetal olfaction. In: Chemical Senses in Vertebrates 5 (Ed. by D. MacDonald). Oxford: Oxford UniversityPress. Isingrini, M., Lenoir, A. & Jaisson, P. 1985. Preimaginal learning as a basis of colony recognition in the ant Cataglyphis cursor. Proc. natn. Acad. Sci. U.S.A., 82, 8545-8547. Johanson, I. B. & Hall, W. G. 1982.Appetitive conditioning in neonatal rats: conditioned orientation to a novel odour. Devl Psychobiol., 15, 379-397. Mann, L. I. 1986. Pregnancy events and brain damage. Am. J. Obstet. Gynecol., 155, 6-9. Pedersen, P. E. & Blass, E. M. 1982. Prenatal and postnatal determinants of the 1st sucklingepisode in albino rats. Devl Psychobiol., 15, 349-355. Shindler,K. M. 1984. A three year study of fetal auditory imprinting. J. Wash. Acad. Sci., 74, 121-124. Smotherman, W. P. 1982a. Odour aversion learning by the rat fetus. Physiol. Behav., 29, 769-771. Smotherman, W. P. 1982b. In utero chemosensory experience alters taste preferences and corticosterone responsiveness.Behav. neural Biol., 36, 61-68. Smotherman, W. P. & Robinson, S. R. 1986. A method for endoscopic visualization of rat fetuses in situ Physiol. Behav., 37, 663-665. Smotherman, W. P. & Robinson, S. R. 1987. Stereotypic behavioural response of rat fetuses to acute hypoxia is altered by maternal alcohol consumption. Am. J. Obstet. Gynecol., 157, 982-986. Smotherman, W. P. & Robinson, S. R. 1988a. The uterus as environment: the ecologyof fetal behavior. In Handbook of Behavioral Neurobiology. Vol. 9. Developmental Psychobiology and Behavioral Ecology (Ed. by E. M. Blass), pp. 149-196. New York: Plenum Press. Smotherman, W. P. & Robinson, S. R. 1988b. Response of the rat fetus to acute umbilical cord occlusion: an ontogenetic adaptation? Physiol. Behav., 44, 131-135. Smotherman, W. P., Robinson, S. R. & Miller, B. J. 1987. A reversiblepreparation for observing the behavior of rat fetuses in utero. Physiol. Behav., 37, 57-60. Stickrod, G., Kimble, D. P. & Smotherman, W. P. 1982. In utero taste/odour conditioning in the rat: effects on nipple preference testing. Physiol. Behav., 28, 5-7. Sullivan,R. M. & Hall, W. G. 1988.Reinforcersininfancy: classical conditioning using stroking or intra-oral infusions of milk as UCS. Devl Psychobiol., 21, 215-223. Terry, L. M. & Johanson, I. B. 1987. Olfactory influences on the ingestivebehavior ofinfantrats. DevlPsychobiol., 20, 313-332. Waldman, B. 1981. Sibling recognition in toad tadpoles: the role of experience. Z. Tierpsychol., 56, 341-358.