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Trigeminal lesions and maternal behavior in Norway rats: II. Disruption of parturition
Physiology & Behavior, Vol. 60, No. 1, 187-190, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/96 $15.00 + .00
ELSEVIER
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Trigeminal Lesions and Maternal Behavior in Norway Rats: II. Disruption of Parturition JUDITH M. STERN
Department of Psychology, Rutgers University, New Brunswick, NJ 08903 USA Received 26 June 1995 STERN, J. M. Trigeminal lesions and maternal behavior in Norway rats: II. Disruption of parturition. PHYSIOL BEHAV 611(1) 187-190, 1996.--Profound deficits in maternal behavior occur following postpartum bilateral infraorbital denervation (IO-X), with substantial recovery 12-24 h later. In the present experiment, primiparous rats were subjected to IO-X or sham surgery on gestation day 22. Whereas parturition behavior was normal in controls, it was severely disrupted in all IO-X dams. Impairments included absent, abbreviated, or delayed perioral behaviors--nest building, participation in delivery, placentophagia, licking and retrieval of pups--accompanied by increased general activity and delayed onset of nursing. There was increased mortality in the litters of IO-X dams at birth and by postpartum day (PD) 3, and impaired retrieval by IO-X dams on PD1-3. A surgery-birth interval of < 7 h vs. > 22 h was associated only with poor litter growth. Thus, prepartum IO-X disrupts onset of maternal behavior during parturition, with long-term consequences for offspring well-being. Infraorbital nerve Recovery of function
Birth Placentophagia Retrieval of pups Prepartum Postpartum
APPROPRIATE behavior of parturient rats is essential for the successful birth and subsequent well-being of their offspring (9,16,19). The maternal nest is constructed within the day prior to parturition, providing a birthing site as well as warmth and shelter for the young postpartum. Self-licking of the lower abdominal and anogenital regions during labor extends to the emerging fetus: licking and pulling with the teeth facilitate delivery of the pup and the attached birth products (i.e., umbilical cord and placenta). Thorough licking of the neonate cleans it of amniotic fluids and membranes, thereby stimulating its respiration and the mobility needed to begin nursing. Diminished locomotion of the dam during parturition, coupled with gathering pups toward her, result in the grouped pups gaining access to her nipples, from which position the first nursing occurs [cf. (21,22)]. The stimuli that contribute to the rat dam's behavior during parturition and the initial establishment of maternal behavior have not been completely elucidated. Visual stimuli are dispensible (1), a finding consonant with the darkness of rat burrows in nature. The role of auditory stimuli during parturition has not been studied; however, rats deafened prepartum show essentially normal maternal behavior following late-pregnancy Cesarean delivery, except for slight delays in retrieval (8). The role of olfactory stimuli is controversial. Whereas bilateral olfactory bulbectomy severely disrupts parturition behavior, including deficits in licking pups clean, placentophagia, and suppression of general activity (9,13), anosmia of the main olfactory system, induced peripherally by intranasal ZnSO4 lavage, is consistent
Licking of pups
with normal parturition and onset of maternal behavior (2,13), as is destruction of the accessory olfactory system (10,13). Thus, nonsensory effects of bulbectomy may be largely responsible for many behavioral impairments following this insult, including that during parturition (13). During lactation, anaptia of the rostral, upper snout via anesthesic blockade of the infraorbital (IO) nerve--an enormous sensory branch of the trigeminal nerve that innervates the skin of the upper lip, nose and snout, whiskers, and mucous membrane of the mouth--results in profound deficits in retrieval (11,23), licking of pups, and nursing behavior (20,21,23). Bilateral lesions of the IO nerve have similar effects but there is substantial behavioral recovery 12-24 h postsurgery (12,24). In the present study bilateral infraorbital denervation was carried out shortly prior to term and its effects on parturition and postpartum retrieval and litter growth were assessed. Following more extensive trigeminal sensory denervations, mouth opening in response to perioral food or water stimuli is severely impaired but mouth opening and other oral reflexes still occur in response to intraoral or extraoral stimuli (27), indicating that the behavioral deficits are sensory, not motor, in nature. Similarly, whereas licking of pups is suppressed during perioral anesthesia, self-grooming is not (21,23). Thus, insistent uterine, cervical, and vaginal stimuli may be sufficient to trigger the sequence of maternal oral activities during parturition. Alternatively, the deleterious effects of surgical anaptia may be more long-lasting on the establishment of maternal behavior at term than on its maintenance postpartum. 187
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METHOD
All procedures received prior approval from the Rutgers University Animal Care Committee. The subjects were 13 primiparous Long-Evans female rats, Rattus norvegicus, born, reared, and bred in my laboratory [e.g., (23)] and 110-130 days of age at the time of parturition. Clock-controlled lights were on from 0800-2000 h. On gestation day (GD) 19 subjects were housed individually in a clear Plexiglas cage (26 x 35 × 27 high cm; 0.5625 cm wire-mesh flooring above a pan containing wood chips), designed to facilitate videotaping of a rat within it; strips of paper towels were provided for nesting material. Surgery was carried out on GD22. The first two subjects per group were anesthetized with Chloropent, IP (0.3 ml/100 g). Because of the possibility that the IP injection of anesthetic caused a sham-operated dam ($3) to have an abnormal birth, the anesthesia was subsequently changed to the short-acting inhalant, methoxyfluorane (Metofane). Bilateral infraorbital denervation (IO-X; n = 8) caudal to the mystacial pad was carried as described previously (24). Sham surgery (SHM; n = 5) entailed bilateral exposure, but not sectioning, of the IO nerves. Because of possible transient hypophagia following IO-X (27), extra food was provided to encourage ingestion [i.e., liquid diet (Nutrament) in a bottle and soft, semidry cat food (Happy Cat) in a bowl]. Subjects were tested for snout sensitivity and the operation sites examined upon completion of all testing (24). Births were recorded with a low-light-sensitive camera and time-lapse videorecorder with time-date generator; red lights were on for videotaping rats during the dark phase. Tapes were played back on a dynamic-motion analysis videorecorder, which permits analysis of individual frames and variable speeds, and viewed on a black and white monitor. A 24-h tape (2.5 frames/s) is played back in 2 h. During direct observations (when possible) and videotape playback of parturition, the following were noted: presence of a nest; nest building; delivery of pups; ingestion of birth products including placenta; licking of pups; location of pups; gathering a pup by picking it up with the incisors (mouthing), carrying it in this way from one location to another (retrieval), or by some other means [e.g., with paws (rolling)]; general activity (walking and rearing); and nursing [upright crouching (22) for >_ 2 rain] by 1 h after the last pup was born. Litters were examined 2-15 h after parturition; at this time, dead pups were removed and pups cleaned of birth products and attached nesting material, if need be. On postpartum day (PD) 2, one IO-X dam had lost six of her 10 remaining pups and received four pups from a SHM dam, whereas another IO-X dam lost three of her six pups and received three pups of the same age from a colony dam. Otherwise, litter size was not adjusted nor foster pups provided in this experiment. On PD1-3, the dam and litter were weighed each morning (0900-1000 h). After the dam was returned to her cage, all of the pups were scattered in the corner opposite her nesting area. The latency to retrieve each pup was noted during the next 10 rain; pups not retrieved were then returned to the nest. Because one (SHM) dam had only five live pups, and retrievals are normally completed in < 5 min, retrieval of five pups in 5 min was analyzed [cf. (11,12,23,24)]. Groups were compared with Student's unpaired t-test, twoway analysis of variance (ANOVA), Fisher's Exact Probability test, and the chi-square test. Also, within the IO-X group, a simple regression was carried out comparing surgery-birth interval and other variables (litter size at birth; number of dead pups; weights of dam and pups; retrieval number and latency). RESULTS
The parturition and subsequent lactation of $3 (SHM) was abnormal due to retained fetuses (discovered at sacrifice on PD3
after substantial weight loss of dam and failure of the litter to gain weight); therefore, data from this dam on birth duration, litter mortality, and growth were not included in statistical assessments. The nocturnal birth of $9 (IO-X) was not recorded because the red lights inadvertently were not turned on; therefore, data on surgery-birth interval and duration of parturition are missing. $7 (IO-X) lost her entire litter by PD2; therefore, data on litter growth and retrieval were excluded for this rat. Neurological testing indicated that IO-X dams were all anaptic, that is, mystacial pads were insensitive to touch, and nose, rather than paws, touched first when the dam was held by the tail and lowered to a shelf. At necropsy of IO-X dams, all fibers of the IO nerves were found to be severed. Also, the facial nerve was intact, indicated as well by the presence of whisking during neurological testing. The interval between surgery and the onset of birthing (SBI) was > 12 h for three of four SHM and three of seven IO-X subjects; this difference and the overall group differences in SBI (SHM, 17.5 + 3.1 h; IO-X, 13.1 + 4.0 h) are not significant. The duration of parturition was similar for SHM (2.9 + 0.7 h) and IO-X (2.5 ± 0.4 h) dams. Between PD1 and 3, groups did not differ significantly in dam body weights ( p > 0.8) and dams in both groups gained weight (SHM, 310 ± 11 to 314 + 14; IO-X, 304 + 14 to 311 ___13). Within the IO-X group, SBI is significantly related to litter growth (see below), but not any other variable. The SHM dams gave birth normally (Fig. 1A). They displayed anticipatory nest building, which continued intermittently during and after parturition; active participation in the expulsion of the pups with tongue and teeth; rapid and complete cleaning of each pup by licking; ingestion of amniotic fluids, membranes, umbilicus, and placenta; retrieval and grouping of the young at the nest site; and finally nursing the gathered young within 1 h after delivery of the last pup. In contrast, there were deficits in all or most components of parturition behavior in the seven IO-X dams whose parturition was observed (Fig. 1A), including little or no nest building, before, during, or after parturition, and an impairment in facilitating delivery of each fetus and placenta with tongue and teeth. Most astonishing were the instances (in five IO-X dams) of a pup half out of its mother's vagina while the dam moved around the cage in an agitated state, in one instance for 40 rain, or an expelled pup being dragged about because it was still attached to its cord and retained placenta (in all seven IO-X dams). Although initial pup licking and placentophagia occurred, most IO-X dams seemed to "lose steam" as parturition proceeded; in the most severe cases, they walked around, leaving uncleaned pups strewn about, often with cord and placenta still attached. There was little or no normal retrieval; consequently, the onset of nursing was delayed. However, there were also many instances of behavioral plasticity (i.e., use of rhinarium, chin, and paws to gather pups and nest material by rolling or pushing, and, in one dam, use of paws to help pull an emerging fetus out). Initial litter size was similar (SHM, 12.0 + 0.7; IO-X, 10.6 _+ 0.8; p > 0.1), but pup mortality was minimal in SHM litters and substantial in the IO-X group, seven to eight times higher both by the end of parturition (2% vs. 14%, X ~ = 5.04, p < 0.05) and cumulatively by PD3 (4.3% vs. 35.2%, X 2 = 15.55, p < 0.001) (Fig. 1B). There was no evidence of direct physical injury to pups (i.e., bites, either during parturition or subsequently). Of surviving pups, the weight per pup was not significantly different between groups over PD1-3 ( p > 0.2); for example, on PD3, pups of SHM and IO-X dams weighed 8.9 + 0.5 g and 8.0 ± 0.6 g, respectively. Nonetheless, of the four IO-X dams with the shortest SBIs (range 3.0-6.5 h; mean of 4.75 h), one dam lost her
TRIGEMINAL LESIONS AND MATERNAL BEHAVIOR
four times longer in IO-X than SHM dams between PD1-3, F(1, 20) -- 18.89, p < 0.01 (Fig. 1C). One IO-X dam failed to retrieve at all in the formal tests, yet she had a long SBI (26.5 h) and normal litter growth (9.4 g average pup weight, PD3). In some tests with absent or incomplete retrieval of pups in the normal manner (i.e., with the incisors), the dam moved the nest to where the pups were by manipulating nest material with snout and paws, and then succeeded in grouping the pups by mouthing and handling them.
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FIG. 1. Comparisonof rats subjected to bilateral infraorbitaldenervation (IO-X) or sham surgery (SHM) on gestation day 22 with respect to (A) percentage showingabnormalparturitionbehavior,(B) proportionof dead pups by the end of parturition (day 0) and cumulatively by day 3 postpartum, and (C) latency to retrieve the fifth pup within 5 min, days 1-3 postpartum, with 300 s assigned in the absence of retrieval. * p < 0.05; * * p < 0.01, all days; * * * p < 0.001.
entire litter of 12 pups by PD2, and the remaining three dams had litters with poor weight gain (PD0-3; e.g., pup weight on PD3 of 6.2-6.7 g). The correlation between SBI and pup weight on PD1-3 among the six IO-X dams with these data available is highly significant on each day (all r > 0.9, all p < 0.01). One possible reason for these results is the weakened condition of pups resulting from the inadequate parturition behavior of IO-X dams. Thus, one IO-X dam with a short SBI (6.5 h) received three foster pups on PD2 to replace three that had died; whereas her own three pups gained an average of only 0.1 g by PD3, the foster pups gained an average of 1.0 g, within the range of weight gains of control pups in the same period. Retrieval occurred in all SHM dams and in all but one IO-X dam between PD1-3. However, whereas four out of five SHM dams retrieved five pups on all three tests, only one out of seven IO-X dams did so ( p < 0.1). Using a latency of 300 s for retrieval failure, the latency to retrieve the fifth pup was two to
DISCUSSION
Bilateral infraorbital denervation of primiparous rats carried out on gestation day 22 resulted in severe deficits in parturition behavior and impairments in pup retrieval lasting at least 3 days. Abnormal parturition behavior included inadequate perioral behaviors, failure to suppress general activity, and delayed onset of nursing, culminating in increased pup mortality and, with surgery-birth intervals of < 7 h, little or no litter growth. Oral facilitation of delivery, licking, and ingestion of birth products were initiated by IO-X dams but were usually not persistent. Stimulation of the uterus, cervix, and vagina, provoked by the emerging fetuses, probably contributes to the occurrence of these behaviors during parturition, as it does to the induction of maternal responsiveness in nonparturient rats (7,26). Indeed, the intense, repetitive self-licking by parturient rats appears to be stimulus bound, perhaps in response to sensations, including pain, from uterine contractions and the passage of fetuses through the cervix and vaginal canal. This interpretation is supported by the recent demonstration that intravesicular instillation of the neurotoxin capsaicin "evoked an intense licking directed toward the lower abdominal and perineal skin which lasted for 15 min," a response mediated by pudendal urethral sensory fibers (14). However, the effects of pelvic-pudendal anesthesia on parturition behavior in rats has not been assessed. Maternal behaviors carried out with the mouth probably require activation of the trigeminal nerves for initiation or normal execution, or both. Although all but one IO-X dam recovered retrieval with the incisors in the present study, most rats did not retrieve consistently or efficiently up to 4 days after IO-X, suggesting that the impairment in retrieval may be more long lasting than that found previously following postpartum IO-X (12,24). Perhaps the postpartum establishment of retrieval contributes to its recovery following an insult such as bilateral denervation of one sensory branch of the trigeminal nerve. Damage to sensory branches of the trigeminal in addition to the infraorbital is likely needed to achieve a more severe and longlasting deficit in maternal oral activities (20,24), as is true of the trigeminal regulation of ingestive behavior (27). Plasticity in the execution of maternal behavior seen in IO-X dams (e.g., use of paws and snout to move pups) also occurs in some dams during perioral anesthesia (23). Similarly, some rats with severely impaired ingestive behavior following extensive trigeminal lesions use their paws to scoop food (27). The proximity of the representations of paws and snout in the cerebellum (17) and cerebral cortex (3) may underlie this plasticity of function. Increased pup mortality in the litters of IO-X dams, without evidence of biting, is most plausibly explained by anoxia due to retention in the vagina for a prolonged time before emergence and inadequate cleaning at birth, resulting in failure or delay of the pup to establish normal respiration and movement. Whereas severe anoxia would lead to rapid death, mild anoxia shortly after birth may weaken pups sufficiently to impair their ability to suckle well, leading to malnutrition. Thus, because foster pups given to one IO-X dam, whose own pups failed to thrive, gained
190
STERN
weight normally, postpartum mortality and morbidity of pups probably stemmed from the mother's inadequate behavior during birth rather than postpartum maternal neglect or lactation failure. Similarly, the poor outcome of pups born to bulbectomized rats is due to their birth experience (9). Because nursing behavior of rats requires sufficient perioral and ventral somatosensory stimuli received from pups (20-25), delayed onset of nursing behavior by IO-X dams probably resulted from inadequate perioral stimuli from pups leading to delays in grouping and hovering over them, and impaired suckling ability of some pups. Although it would have been valuable to test retrieval with healthy foster pups, retrieval deficits were evident even in the IO-X dams with the heaviest litters. Also, prior maternal experience may not be relevant to the severity and persistence of maternal behavior deficits following late-term IO-X. One pair of biparous rats was subjected to IO-X or sham surgery on GD21; whereas normal behavior was seen in the control dam, a similar pattern of parturition behavior and retrieval impairments was seen in the IO-X dam as that seen in the maternally inexperienced primiparous IO-X dams herein, despite successful rearing of a litter several weeks earlier and a much longer surgery-birth interval (43 h vs. maximum of 26.5 h). Nonetheless, the effects of prior maternal experience and daily provision of healthy foster
litters on responses to late-term IO-X during and after parturition require more careful investigation. Severe deficits in parturition behavior, including decreased oral facilitation of births, incomplete cleaning of pups and ingestion of birth products, increased general activity, and little or no nest building or retrieval of pups, have been seen following lesions of the neocortex (1), cingulate cortex (18), and medial preoptic area (4,6,15). In addition, a similar pattern of deficits occurred after lesions of the septum (5) and bilateral removal of the olfactory bulbs (9,13), except that retrieval was disorganized instead of absent, and nest building was increased after bulbectomy (9). The overlap in effects caused by these different insults may be due to "decreased perception a n d / o r processing of the powerful sensory stimuli provided by the emerging fetuses, neonates, and birth products" (19). ACKNOWLEDGEMENTS This work was supported by MH-40459. The author thanks C. McDonald, G. Gronlund, and S. Johnson for their help in carrying out the study, and J. Lonstein for his comments on the manuscript. A portion of these results was presented at the 1987 meeting of the International Society for Developmental Psychobiology, New Orleans, LA, Nov. 12-15.
REFERENCES 1. Beach, F. A. The neural basis of innate behavior. I. Effects of cortical lesions upon the maternal behavior patten in the rat. J. Comp. Psychol. 24:393-436; 1937. 2. Benuck, I.; Rowe, F. A. Centrally and peripherally induced anosmia: Influence on maternal behavior in lactating female rats. Physiol. Behav. 1:439-447; 1975. 3. Chapin, J.; Lin, C-S. The somatic sensory cortex of the rat. In: Kolb, B.; Tees, R. C., eds. The cerebral cortex of the rat. Cambridge, MA: MIT Press; 1990:341-380. 4. Cohn, J.; Gerall, A. A. Pre- and postpuberal medial preoptic area lesions and maternal behavior in the rat. Physiol. Behav. 46:333-336; 1989. 5. Fleischer, S.; Slotnick, B. M. Disruption of maternal behavior in rats with lesions of the septal area. Physiol. Behav. 21:189-200; 1978. 6. Franz, J. R.; Leo, R. J.; Steuer, M. A.; Kristal, M. B. Effects of hypothalamic knife cuts and experience on maternal behavior in the rat. Physiol. Behav. 38:629-640; 1986. 7. Graber, G. C.; Kristal, M. B. Uterine distension and the onset of maternal behavior in pseudopregnant but not in cycling rats. Physiol. Behav. 19:133-137; 1977. 8. Herrenkohl, L. R.; Rosenberg, P. A. Exteroceptive stimulation of maternal behavior in the naive rat. Physiol. Behav. 8:595-598; 1972. 9. Holloway, W. R.; Dollinger, M. J.; Denenberg, V. H. Parturition in the rat: Description and assessment. In: Bell, R. W.; Smotherman, W. P., eds. Maternal influences and early behavior. New York: SP Med. Sci. Books; 1980:1-26. 10. Jirik-Babb, P.; Manaker, S.; Tucker, A. M.; Hofer, M. A. The role of the accessory and main olfactory systems in maternal behavior of the primiparous rat. Behav. Neural Biol. 40:170-178; 1984. 11. Kenyon, P.; Cronin, P.; Keeble, S. Disruption of maternal retrieving by perioral anesthesia. Physiol. Behav. 27:313-321; 1981. 12. Kenyon, P.; Cronin, P.; Keeble, S. Role of the infraorbital nerve in retrieving behavior in lactating rats. Behav. Neurosci. 97:255-269; 1983. 13. Kolunie, J. M.; Stem, J. M. Maternal aggression in rats: Effects of olfactory bulbectomy, ZnSO4-induced anosmia, and vomeronasal organ removal. Horm. Behav. 29:492-518; 1995. 14. Lecci, A.; Ginlian, S.; Lazzeri, M.; Benaim, G.; Turini, D.; Maggi, C. A. The behavioral response induced by intravesical instillation of capsaicin in rats is mediated by pudendal urethral sensory fibers. Life Sci. 55:429-436; 1994.
15. Noonan, M.; Kristal, M. B. Effects of medial preoptic lesions on placentophagia and on the onset of maternal behavior in the rat. Physiol. Behav. 22:1197-1202; 1979. 16. Rosenblatt, J. S.; Lehrman, D. S. Maternal behavior of the laboratory rat. In: Rheingold, H., ed. Maternal behavior in mammals. New York: Wiley; 1963:8-57. 17. Shambes, G. M.; Gibson, J. M.; Welker, W. I. Fractured somatotypy in granule cell tactile areas of rat cerebellar hemispheres revealed by micromapping. Brain Behav. Evol. 15:94-140; 1978. 18. Slotnick, B. M. Disturbances of maternal behavior in the rat following lesions of the cingulate cortex. Behaviour 29:204-236; 1967. 19. Stem, J. M. Maternal behavior: Sensory, hormonal, and neural determinants. In: Brush, F. R.; Levine, S., eds. Psychoendocrinology. New York: Academic Press; 1989:105-226. 20. Stem, J. M. Somatosensation and maternal care in Norway rats. In: Slater, P. J.; Rosenblatt, J. S.; Milinski, M.; Snowden, C. T., eds. Advances in the study of parental behavior. New York: Academic Press; 1996. 21. Stem, J. M.; Johnson, S. K. Perioral somatosensory determinants of nursing behavior in Norway rats. J. Comp. Psychol. 103:269-280; 1989. 22. Stem, J. M.; Johnson, S. K. Ventral somatosensory determinants of nursing behavior in Norway rats. I. Effects of variations in the quality and quantity of pup stimuli. Physiol. Behav. 47:993-1011; 1990. 23. Stem, J. M.; Kolunie, J. M. Perioral anesthesia disrupts maternal behavior during early lactation in Long-Evans rats. Behav. Neural Biol. 52:20-38; 1989. 24. Stem, J. M.; Kolunie, J. M. Trigeminal lesions and maternal behavior in Norway rats. I. Effects of cutaneous rostral snout denervation on maintenance of nurturance and maternal aggression. Behav. Neurosci. 105:984-997; 1991. 25. Stem, J. M.; Dix, L.; Bellomo, C.; Thramann, C. Ventral trunk somatosensory determinants of nursing behavior in Norway rats. II. Role of nipple and surrounding sensations. Psychobiology 20:71-80; 1992. 26. Yeo, J. A. G.; Keverne, E. B. The importance of vaginal-cervical stimulation for maternal behavior in the rat. Physiol. Behav. 37:2326; 1986. 27. Zeigler, H. P.; Jacquin, M. F.; Miller, M. G. Trigeminal orosensation and ingestive behavior in the rat. Prog. Psyehobiol. Physiol. Psychol, 11:63-196; 1985.
ELSEVIER
PlI 0031-9384(96)00016-3
Trigeminal Lesions and Maternal Behavior in Norway Rats: II. Disruption of Parturition JUDITH M. STERN
Department of Psychology, Rutgers University, New Brunswick, NJ 08903 USA Received 26 June 1995 STERN, J. M. Trigeminal lesions and maternal behavior in Norway rats: II. Disruption of parturition. PHYSIOL BEHAV 611(1) 187-190, 1996.--Profound deficits in maternal behavior occur following postpartum bilateral infraorbital denervation (IO-X), with substantial recovery 12-24 h later. In the present experiment, primiparous rats were subjected to IO-X or sham surgery on gestation day 22. Whereas parturition behavior was normal in controls, it was severely disrupted in all IO-X dams. Impairments included absent, abbreviated, or delayed perioral behaviors--nest building, participation in delivery, placentophagia, licking and retrieval of pups--accompanied by increased general activity and delayed onset of nursing. There was increased mortality in the litters of IO-X dams at birth and by postpartum day (PD) 3, and impaired retrieval by IO-X dams on PD1-3. A surgery-birth interval of < 7 h vs. > 22 h was associated only with poor litter growth. Thus, prepartum IO-X disrupts onset of maternal behavior during parturition, with long-term consequences for offspring well-being. Infraorbital nerve Recovery of function
Birth Placentophagia Retrieval of pups Prepartum Postpartum
APPROPRIATE behavior of parturient rats is essential for the successful birth and subsequent well-being of their offspring (9,16,19). The maternal nest is constructed within the day prior to parturition, providing a birthing site as well as warmth and shelter for the young postpartum. Self-licking of the lower abdominal and anogenital regions during labor extends to the emerging fetus: licking and pulling with the teeth facilitate delivery of the pup and the attached birth products (i.e., umbilical cord and placenta). Thorough licking of the neonate cleans it of amniotic fluids and membranes, thereby stimulating its respiration and the mobility needed to begin nursing. Diminished locomotion of the dam during parturition, coupled with gathering pups toward her, result in the grouped pups gaining access to her nipples, from which position the first nursing occurs [cf. (21,22)]. The stimuli that contribute to the rat dam's behavior during parturition and the initial establishment of maternal behavior have not been completely elucidated. Visual stimuli are dispensible (1), a finding consonant with the darkness of rat burrows in nature. The role of auditory stimuli during parturition has not been studied; however, rats deafened prepartum show essentially normal maternal behavior following late-pregnancy Cesarean delivery, except for slight delays in retrieval (8). The role of olfactory stimuli is controversial. Whereas bilateral olfactory bulbectomy severely disrupts parturition behavior, including deficits in licking pups clean, placentophagia, and suppression of general activity (9,13), anosmia of the main olfactory system, induced peripherally by intranasal ZnSO4 lavage, is consistent
Licking of pups
with normal parturition and onset of maternal behavior (2,13), as is destruction of the accessory olfactory system (10,13). Thus, nonsensory effects of bulbectomy may be largely responsible for many behavioral impairments following this insult, including that during parturition (13). During lactation, anaptia of the rostral, upper snout via anesthesic blockade of the infraorbital (IO) nerve--an enormous sensory branch of the trigeminal nerve that innervates the skin of the upper lip, nose and snout, whiskers, and mucous membrane of the mouth--results in profound deficits in retrieval (11,23), licking of pups, and nursing behavior (20,21,23). Bilateral lesions of the IO nerve have similar effects but there is substantial behavioral recovery 12-24 h postsurgery (12,24). In the present study bilateral infraorbital denervation was carried out shortly prior to term and its effects on parturition and postpartum retrieval and litter growth were assessed. Following more extensive trigeminal sensory denervations, mouth opening in response to perioral food or water stimuli is severely impaired but mouth opening and other oral reflexes still occur in response to intraoral or extraoral stimuli (27), indicating that the behavioral deficits are sensory, not motor, in nature. Similarly, whereas licking of pups is suppressed during perioral anesthesia, self-grooming is not (21,23). Thus, insistent uterine, cervical, and vaginal stimuli may be sufficient to trigger the sequence of maternal oral activities during parturition. Alternatively, the deleterious effects of surgical anaptia may be more long-lasting on the establishment of maternal behavior at term than on its maintenance postpartum. 187
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STERN
METHOD
All procedures received prior approval from the Rutgers University Animal Care Committee. The subjects were 13 primiparous Long-Evans female rats, Rattus norvegicus, born, reared, and bred in my laboratory [e.g., (23)] and 110-130 days of age at the time of parturition. Clock-controlled lights were on from 0800-2000 h. On gestation day (GD) 19 subjects were housed individually in a clear Plexiglas cage (26 x 35 × 27 high cm; 0.5625 cm wire-mesh flooring above a pan containing wood chips), designed to facilitate videotaping of a rat within it; strips of paper towels were provided for nesting material. Surgery was carried out on GD22. The first two subjects per group were anesthetized with Chloropent, IP (0.3 ml/100 g). Because of the possibility that the IP injection of anesthetic caused a sham-operated dam ($3) to have an abnormal birth, the anesthesia was subsequently changed to the short-acting inhalant, methoxyfluorane (Metofane). Bilateral infraorbital denervation (IO-X; n = 8) caudal to the mystacial pad was carried as described previously (24). Sham surgery (SHM; n = 5) entailed bilateral exposure, but not sectioning, of the IO nerves. Because of possible transient hypophagia following IO-X (27), extra food was provided to encourage ingestion [i.e., liquid diet (Nutrament) in a bottle and soft, semidry cat food (Happy Cat) in a bowl]. Subjects were tested for snout sensitivity and the operation sites examined upon completion of all testing (24). Births were recorded with a low-light-sensitive camera and time-lapse videorecorder with time-date generator; red lights were on for videotaping rats during the dark phase. Tapes were played back on a dynamic-motion analysis videorecorder, which permits analysis of individual frames and variable speeds, and viewed on a black and white monitor. A 24-h tape (2.5 frames/s) is played back in 2 h. During direct observations (when possible) and videotape playback of parturition, the following were noted: presence of a nest; nest building; delivery of pups; ingestion of birth products including placenta; licking of pups; location of pups; gathering a pup by picking it up with the incisors (mouthing), carrying it in this way from one location to another (retrieval), or by some other means [e.g., with paws (rolling)]; general activity (walking and rearing); and nursing [upright crouching (22) for >_ 2 rain] by 1 h after the last pup was born. Litters were examined 2-15 h after parturition; at this time, dead pups were removed and pups cleaned of birth products and attached nesting material, if need be. On postpartum day (PD) 2, one IO-X dam had lost six of her 10 remaining pups and received four pups from a SHM dam, whereas another IO-X dam lost three of her six pups and received three pups of the same age from a colony dam. Otherwise, litter size was not adjusted nor foster pups provided in this experiment. On PD1-3, the dam and litter were weighed each morning (0900-1000 h). After the dam was returned to her cage, all of the pups were scattered in the corner opposite her nesting area. The latency to retrieve each pup was noted during the next 10 rain; pups not retrieved were then returned to the nest. Because one (SHM) dam had only five live pups, and retrievals are normally completed in < 5 min, retrieval of five pups in 5 min was analyzed [cf. (11,12,23,24)]. Groups were compared with Student's unpaired t-test, twoway analysis of variance (ANOVA), Fisher's Exact Probability test, and the chi-square test. Also, within the IO-X group, a simple regression was carried out comparing surgery-birth interval and other variables (litter size at birth; number of dead pups; weights of dam and pups; retrieval number and latency). RESULTS
The parturition and subsequent lactation of $3 (SHM) was abnormal due to retained fetuses (discovered at sacrifice on PD3
after substantial weight loss of dam and failure of the litter to gain weight); therefore, data from this dam on birth duration, litter mortality, and growth were not included in statistical assessments. The nocturnal birth of $9 (IO-X) was not recorded because the red lights inadvertently were not turned on; therefore, data on surgery-birth interval and duration of parturition are missing. $7 (IO-X) lost her entire litter by PD2; therefore, data on litter growth and retrieval were excluded for this rat. Neurological testing indicated that IO-X dams were all anaptic, that is, mystacial pads were insensitive to touch, and nose, rather than paws, touched first when the dam was held by the tail and lowered to a shelf. At necropsy of IO-X dams, all fibers of the IO nerves were found to be severed. Also, the facial nerve was intact, indicated as well by the presence of whisking during neurological testing. The interval between surgery and the onset of birthing (SBI) was > 12 h for three of four SHM and three of seven IO-X subjects; this difference and the overall group differences in SBI (SHM, 17.5 + 3.1 h; IO-X, 13.1 + 4.0 h) are not significant. The duration of parturition was similar for SHM (2.9 + 0.7 h) and IO-X (2.5 ± 0.4 h) dams. Between PD1 and 3, groups did not differ significantly in dam body weights ( p > 0.8) and dams in both groups gained weight (SHM, 310 ± 11 to 314 + 14; IO-X, 304 + 14 to 311 ___13). Within the IO-X group, SBI is significantly related to litter growth (see below), but not any other variable. The SHM dams gave birth normally (Fig. 1A). They displayed anticipatory nest building, which continued intermittently during and after parturition; active participation in the expulsion of the pups with tongue and teeth; rapid and complete cleaning of each pup by licking; ingestion of amniotic fluids, membranes, umbilicus, and placenta; retrieval and grouping of the young at the nest site; and finally nursing the gathered young within 1 h after delivery of the last pup. In contrast, there were deficits in all or most components of parturition behavior in the seven IO-X dams whose parturition was observed (Fig. 1A), including little or no nest building, before, during, or after parturition, and an impairment in facilitating delivery of each fetus and placenta with tongue and teeth. Most astonishing were the instances (in five IO-X dams) of a pup half out of its mother's vagina while the dam moved around the cage in an agitated state, in one instance for 40 rain, or an expelled pup being dragged about because it was still attached to its cord and retained placenta (in all seven IO-X dams). Although initial pup licking and placentophagia occurred, most IO-X dams seemed to "lose steam" as parturition proceeded; in the most severe cases, they walked around, leaving uncleaned pups strewn about, often with cord and placenta still attached. There was little or no normal retrieval; consequently, the onset of nursing was delayed. However, there were also many instances of behavioral plasticity (i.e., use of rhinarium, chin, and paws to gather pups and nest material by rolling or pushing, and, in one dam, use of paws to help pull an emerging fetus out). Initial litter size was similar (SHM, 12.0 + 0.7; IO-X, 10.6 _+ 0.8; p > 0.1), but pup mortality was minimal in SHM litters and substantial in the IO-X group, seven to eight times higher both by the end of parturition (2% vs. 14%, X ~ = 5.04, p < 0.05) and cumulatively by PD3 (4.3% vs. 35.2%, X 2 = 15.55, p < 0.001) (Fig. 1B). There was no evidence of direct physical injury to pups (i.e., bites, either during parturition or subsequently). Of surviving pups, the weight per pup was not significantly different between groups over PD1-3 ( p > 0.2); for example, on PD3, pups of SHM and IO-X dams weighed 8.9 + 0.5 g and 8.0 ± 0.6 g, respectively. Nonetheless, of the four IO-X dams with the shortest SBIs (range 3.0-6.5 h; mean of 4.75 h), one dam lost her
TRIGEMINAL LESIONS AND MATERNAL BEHAVIOR
four times longer in IO-X than SHM dams between PD1-3, F(1, 20) -- 18.89, p < 0.01 (Fig. 1C). One IO-X dam failed to retrieve at all in the formal tests, yet she had a long SBI (26.5 h) and normal litter growth (9.4 g average pup weight, PD3). In some tests with absent or incomplete retrieval of pups in the normal manner (i.e., with the incisors), the dam moved the nest to where the pups were by manipulating nest material with snout and paws, and then succeeded in grouping the pups by mouthing and handling them.
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FIG. 1. Comparisonof rats subjected to bilateral infraorbitaldenervation (IO-X) or sham surgery (SHM) on gestation day 22 with respect to (A) percentage showingabnormalparturitionbehavior,(B) proportionof dead pups by the end of parturition (day 0) and cumulatively by day 3 postpartum, and (C) latency to retrieve the fifth pup within 5 min, days 1-3 postpartum, with 300 s assigned in the absence of retrieval. * p < 0.05; * * p < 0.01, all days; * * * p < 0.001.
entire litter of 12 pups by PD2, and the remaining three dams had litters with poor weight gain (PD0-3; e.g., pup weight on PD3 of 6.2-6.7 g). The correlation between SBI and pup weight on PD1-3 among the six IO-X dams with these data available is highly significant on each day (all r > 0.9, all p < 0.01). One possible reason for these results is the weakened condition of pups resulting from the inadequate parturition behavior of IO-X dams. Thus, one IO-X dam with a short SBI (6.5 h) received three foster pups on PD2 to replace three that had died; whereas her own three pups gained an average of only 0.1 g by PD3, the foster pups gained an average of 1.0 g, within the range of weight gains of control pups in the same period. Retrieval occurred in all SHM dams and in all but one IO-X dam between PD1-3. However, whereas four out of five SHM dams retrieved five pups on all three tests, only one out of seven IO-X dams did so ( p < 0.1). Using a latency of 300 s for retrieval failure, the latency to retrieve the fifth pup was two to
DISCUSSION
Bilateral infraorbital denervation of primiparous rats carried out on gestation day 22 resulted in severe deficits in parturition behavior and impairments in pup retrieval lasting at least 3 days. Abnormal parturition behavior included inadequate perioral behaviors, failure to suppress general activity, and delayed onset of nursing, culminating in increased pup mortality and, with surgery-birth intervals of < 7 h, little or no litter growth. Oral facilitation of delivery, licking, and ingestion of birth products were initiated by IO-X dams but were usually not persistent. Stimulation of the uterus, cervix, and vagina, provoked by the emerging fetuses, probably contributes to the occurrence of these behaviors during parturition, as it does to the induction of maternal responsiveness in nonparturient rats (7,26). Indeed, the intense, repetitive self-licking by parturient rats appears to be stimulus bound, perhaps in response to sensations, including pain, from uterine contractions and the passage of fetuses through the cervix and vaginal canal. This interpretation is supported by the recent demonstration that intravesicular instillation of the neurotoxin capsaicin "evoked an intense licking directed toward the lower abdominal and perineal skin which lasted for 15 min," a response mediated by pudendal urethral sensory fibers (14). However, the effects of pelvic-pudendal anesthesia on parturition behavior in rats has not been assessed. Maternal behaviors carried out with the mouth probably require activation of the trigeminal nerves for initiation or normal execution, or both. Although all but one IO-X dam recovered retrieval with the incisors in the present study, most rats did not retrieve consistently or efficiently up to 4 days after IO-X, suggesting that the impairment in retrieval may be more long lasting than that found previously following postpartum IO-X (12,24). Perhaps the postpartum establishment of retrieval contributes to its recovery following an insult such as bilateral denervation of one sensory branch of the trigeminal nerve. Damage to sensory branches of the trigeminal in addition to the infraorbital is likely needed to achieve a more severe and longlasting deficit in maternal oral activities (20,24), as is true of the trigeminal regulation of ingestive behavior (27). Plasticity in the execution of maternal behavior seen in IO-X dams (e.g., use of paws and snout to move pups) also occurs in some dams during perioral anesthesia (23). Similarly, some rats with severely impaired ingestive behavior following extensive trigeminal lesions use their paws to scoop food (27). The proximity of the representations of paws and snout in the cerebellum (17) and cerebral cortex (3) may underlie this plasticity of function. Increased pup mortality in the litters of IO-X dams, without evidence of biting, is most plausibly explained by anoxia due to retention in the vagina for a prolonged time before emergence and inadequate cleaning at birth, resulting in failure or delay of the pup to establish normal respiration and movement. Whereas severe anoxia would lead to rapid death, mild anoxia shortly after birth may weaken pups sufficiently to impair their ability to suckle well, leading to malnutrition. Thus, because foster pups given to one IO-X dam, whose own pups failed to thrive, gained
190
STERN
weight normally, postpartum mortality and morbidity of pups probably stemmed from the mother's inadequate behavior during birth rather than postpartum maternal neglect or lactation failure. Similarly, the poor outcome of pups born to bulbectomized rats is due to their birth experience (9). Because nursing behavior of rats requires sufficient perioral and ventral somatosensory stimuli received from pups (20-25), delayed onset of nursing behavior by IO-X dams probably resulted from inadequate perioral stimuli from pups leading to delays in grouping and hovering over them, and impaired suckling ability of some pups. Although it would have been valuable to test retrieval with healthy foster pups, retrieval deficits were evident even in the IO-X dams with the heaviest litters. Also, prior maternal experience may not be relevant to the severity and persistence of maternal behavior deficits following late-term IO-X. One pair of biparous rats was subjected to IO-X or sham surgery on GD21; whereas normal behavior was seen in the control dam, a similar pattern of parturition behavior and retrieval impairments was seen in the IO-X dam as that seen in the maternally inexperienced primiparous IO-X dams herein, despite successful rearing of a litter several weeks earlier and a much longer surgery-birth interval (43 h vs. maximum of 26.5 h). Nonetheless, the effects of prior maternal experience and daily provision of healthy foster
litters on responses to late-term IO-X during and after parturition require more careful investigation. Severe deficits in parturition behavior, including decreased oral facilitation of births, incomplete cleaning of pups and ingestion of birth products, increased general activity, and little or no nest building or retrieval of pups, have been seen following lesions of the neocortex (1), cingulate cortex (18), and medial preoptic area (4,6,15). In addition, a similar pattern of deficits occurred after lesions of the septum (5) and bilateral removal of the olfactory bulbs (9,13), except that retrieval was disorganized instead of absent, and nest building was increased after bulbectomy (9). The overlap in effects caused by these different insults may be due to "decreased perception a n d / o r processing of the powerful sensory stimuli provided by the emerging fetuses, neonates, and birth products" (19). ACKNOWLEDGEMENTS This work was supported by MH-40459. The author thanks C. McDonald, G. Gronlund, and S. Johnson for their help in carrying out the study, and J. Lonstein for his comments on the manuscript. A portion of these results was presented at the 1987 meeting of the International Society for Developmental Psychobiology, New Orleans, LA, Nov. 12-15.
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