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piriform cortex
Behavioural Brain Research, 42 (1991) 99-105 Elsevier
99
BBR 01143
Parental behavior in the mouse: effects of lesions in the entorhinal/piriform cortex Michael Koch I and Gtinter Ehret 2 ~Fakult~t fiir Biologie, Universit~t Konstanz, D-7750 Konstanz 1 (F.R.G.) and 2Abteilung fiir vergleichende Neurobiologie, Universit~t UIm D-7900 Uhn (F.R.G.) (Received 23 May 1990) (Revised version received 24 September 1990) (Accepted 24 September 1990) Key words: Anosmia; Entorhinal cortex; Lesion; Olfactory learning; Parental behavior; Piriform cortex
The effects of bilateral radiofrequency lesions in the region of the entorhinal and piriform cortex (ENT/PIR cortex) on pup-retrieving and ultrasound-elicited pup-searching behavior were investigated in ovariectomized estrogen-treated female mice, which were sensitized to pups by co-caring for a litter for 5 days together with the mother (FoEBex), as well as in primiparous lactating females (Flact). A further group of FoEBex were rendered anosmic by an intranasal treatment with zinc sulfate-solution before the sensitization period and tests for parental motivation. Both pup-retrieving and pup-searching behavior were disturbed to the same extent by ENT/PIR lesions and ZnSO4-induced anosmia in FoEBex. In Flact, however, pup-retrieving was not affected by the lesion, while ultrasound-recognition leading to pup-searching was disrupted. The present data suggest sensitization to pups as a case of olfactory learning and thus, the effects of the ENT]PIR lesions are regarded as anterograde olfactory amnesia. From the present data and results reported in the literature ~6, it is suggested to differentiate between the neural substrates processing stimuli relevant for the appetitive component (pup-searching) of parental behavior (among others, the region of the entorhinal and piriform cortex), and the mechanisms regulating the consummatoo, component (pup;retrieving).
INTRODUCTION
Parental experience of adult female mice achieved by co-caring for a litter together with the mother for five days increases parental motivation T M and leads to an increase in the amount ofestrogen receptors in the brain Of ovariectomized estrogen-treated female mice ~~ This effect is likely to be induced by sensory cues from the pups or the mother 2. The increase in the amount of estrogen receptors is most obvious in the region of the entorhinal and piriform cortex. Although a considerable number of studies have been carried out on the neural mechanisms of parental behavior (e.g. pup-retrieving, licking, nest-building, nursing), nothing is known about the role of the entorhinal and/or piriform cortex in the regulation of this behavior ~6. It is therefore necessary to investigate whether this region of the brain is directly involved in the neural control of parental behavior. Correspondence: M. Koch. Present address: Lehrstuhl Tierphysiologie, Universitfit Ttlbingen, Auf der Morgenstelle 28, D-7400 Tfibingcn, F.R.G.
The present study investigates how lesions in that particular part of the entorhinal and piriform cortex, where the estrogen receptor-content of neurons was previously found to be sensitive to parental experience, affect ultrasound-elicited pup-searching and pupretrieving of estrogen-treated females. In addition a group of estrogen-treated females was rendered anosmic by zinc sulfate instillation since it has been shown that lesions in the entorhinal cortex produce anterograde olfactory amnesia in an olfactory discrimination task 22. The prediction in our study was that anosmia and lesions of the entorhinal/piriform cortex (here referred to as 'ENT/PIR lesions') would produce the same kind of effect on parental behavior. Additionally, the effects of ENT/PIR lesions on maternal behavior were tested also in primiparous lactating females. These non-ovariectomized animals served as a control with the natural supply of hormones during the course of pregnancy and lactation. Parental behavior was investigated with a test that allows for the discrimination between the appetitive component (pup-ultrasound elicited pup-searching behavior) and the consumntatory response of the animal,
0166-4328/91/S03.50 9 1991 Elsevier Science Publishers B.V. (Biomedical Division)
100 i.e. pup-retrieving. Here, pup-retrieving is regarded as a consummatory act, because it terminates pup-searching activities in a strikingly fixed way following contact with the pup. Although it might precede some other pupdirected behaviors, it should not be regarded as an appetitive behavior, because it is not followed by a distinct behavior, but instead the animal might engage in a variety of behaviors while staying in the nest with the young, e.g. licking, nest-building, suckling, selfgrooming, eating, drinking, sleeping.
MATERIALS AND METHODS Anhnals Subjects were female albino house mice (strain NMRI) aged 2-3 months which were kept in plastic cages (16.5 • 22 x 14 cm) in groups of 4 animals under a continuous light-dark cycle (lights on at 06.00 h, off at 20.00 h) at 21-23 ~ Food and water were provided ad libitum. The following experimental groups were used: Estrogen-treated ovariectomized females with bilateral ENT/PIR lesions with parental experience (FoEBex lesion, n = 16), sham-lesioned controls (FoEBex sham, n = 8), estrogen-treated ovariectomized females with intranasal zinc sulfate treatment ('anosmic') and parental experience (FoEBex ZnSO4, n = 12), saline-treated controls (FoEBex saline, n = 8), primiparous lactating females with bilateral ENT/PIR lesions (Flact lesion, n = 17), sham-lesioned controls (Flact sham, n = 8). Surgery and other treatments The subjects of the FoEBex lesion group received their lesions and were ovariectomized at the same time under a combined anesthesia of 80 mg/kg Nembutal and 3 mg/kg Taractan (Hoffmann-La Roche) injected intraperitoneally. The animals were placed in a stereotaxic frame without earbars in order not to destroy the tympanum. It was attempted to destroy those parts of the entorhinal and piriform cortex where a marked change in estrogen receptor content was observed in a previous study ~~ The stereotaxic coordinates for the bilateral ENT/PIR lesions (ap: - 2.5 mm; ml: 4.6 mm; dv: 5.2 mm: relative to bregma) were taken from the atlas of Slotnick and Leonard 2~ Lesions were produced using a radiofrequency lesion generator (Kopf Instruments Tujunga, USA, Model RFG-4). A steel needle inserted into the muscle of the neck served as a reference electrode. The temperature of the lesion electrode (tip size: 0.25 mm) was kept at 70-80 ~ for 60 s. In the FoEBex sham and Flact sham groups the electrode was lowered into the target region in the brain without passing radio-fre-
quency current. The animals were tested 4 weeks after surgery. Estrogen-treatment was started two weeks after surgery by implanting silastic tubing of 8 mm length filled with estradiol benzoate (Sigma) under the skin of the neck as described previously ~. The procedure by which the animals achieved parental experience was the same as described in our previous studies l~ Briefly, animals of the groups FoEBex lesion, FoEBex sham, FoEBex Z n S O 4 and FoEBex saline were placed each in the cage of a pregnant female (day 10 of pregnancy) and remained there during the rest of pregnancy, parturition and for the first 5 days of lactation of the mother. Females of the primiparous mother's group (Flact) were mated 3 days after surgery and were tested when their young were 5 days old, so that the period of time between surgery and test was the same for all experimental groups (4 weeks). Litters were standardized to 12 pups. Mating success of the females was not impaired by the ENT/PIR lesions. Anosmia was produced 8 days before the day of the behavioral tests by intranasal instillation of a 5 ~ Z n S O 4 solution (0.17 M). The procedure of Matulionis 12"~3 was used in a slightly modified way. The animals were anesthetized with Nembutal and Taractan (same dosage as for lesions) and were placed in a supinate position, then a drop of ZnSOa-solution was placed on both nares and was immediately inhaled by the mouse. Swallowing of the toxic ZnSO4-solution was prevented by loosely packing absorbing paper into the upper part of the throat of the animal and continuously aspiring the throat with a small vacuum pump. 30 s after instillation of the first drop, the procedure was repeated with a second drop of zinc sulfate solution. The animals were allowed to recover for two days in a separate cage. All zinc sulfate-treated animals showed normal behavior when they were reintroduced into the cage of the pregnant female they had previously been housed with. Aggressiveness towards the pregnant or lactating female or the pups was not observed in zinc sulfate-treated females. Controls (FoEBex saline) were treated with 0.9~o NaCI, but otherwise handled in the same way. Behavioral tests In order to discriminate between the appetitive and the consummatory component of goal directed parental behavior, we tested both ultrasound-elicited pup-searching and pup-retrieving, which latter might have multiple sensory key stimuli ~. As in our previous studies 7"t~ the experimental mice were placed in the central nest depression of a plastic running board (8 cm wide, 110 cm long) together with 5 pups at least 4 h before the testing began in order to minimize explo-
101 ratory tendencies during the test. One ultrasonic loudspeaker was mounted 65 cm from the nest depression at each end of the board but independent of it. The propensity to retrieve pups was tested by successively placing 5 pups on the board in a random distribution, one at a time at a distance of 30 cm from the nest. The criterion for a mouse being scored as a retriever was to retrieve all 5 pups within i0 min. After retrieving of the last pup the test for appetitive (pup-searching) behavior was started (non-retrievers were not tested for pup-searching behavior). Ultrasound-elicited pupsearching was tested by switching on both loudspeakers simultaneously, so that the animals had to discriminate between neutral 20-kHz tone pulses and 50-kHz tone pulses, known to function as specific releasers of pupsearching 5"6. The sound pressure levels were 89 dB for 50 kHz and 75 dB for 20 kHz which is about 71 dB (SPL re 20/tPa) above the threshold of perception of both frequencies 4. It was recorded towards which loudspeaker the mouse moved. The criterion for a response was leaving the nest for at least 30 cm towards one of the two loudspeakers within i0 min after tone onset. In consecutive tests the two frequencies were randomly assigned to the two loudspeakers. A maximum of 7 responses were taken from one animal: the data of all animals of an experimental group were summed up and percent responses to 50-kHz and 20-kHz stimuli calculated. During the behavioral tests the experimenter was unaware of the treatment (sham or operated) of the animals.
perfused as described above. Fur, skin, eyes and muscles were removed from the skull, the nose was cut off and the lower jaw together with the tongue was removed. This preparation was postfixed for two days in 4~o formalin/PBS, decalcified for 3 days in 0.125~ EDTA (Titriplex III, Merck. pH 7.4; T = 37 ~ The upper incisivi were pulled out and frontal sections (40/tin) of the nose were cut on a cryostat, mounted on gelatine-coated slides and stained with Cresyl violet.
RESULTS
The size and location of the E N T / P I R lesions in FoEBex is shown in Fig. 1; obviously, a large part of the
Statistical evahtation Only non-parametric statistics are used. Differences between the numbers of retrievers in each group are compared with Fisher's exact probabilities test. Retrieving scores are expressed as '~o Retrievers'. Tone discrimination is expressed as '~o Responses to 50 kHz' and statistical significance of tone preference was examined with the binomial test. All tests are two-tailed and a P < 0.05 was considered significant.
Histology
After completion of the behavioral tests the animals were perfused through the left ventricle with 0 . 9 ~ NaCI followed by 4 ~ formalin in 0.02 M phosphate-buffered saline (PBS). The brains were removed and placed in 3 0 ~ sucrose/PBS until they sank. They were cut on a cryostat in 40-#m sections which were mounted on gelatine-coated slides and stained with Cresyl violet. Every second section was drawn with a camera lucida and projected onto representative drawings from the atlas of Sidman et al. t9. For the histological analysis of the effects of ZnSO4treatment on the nasal epithelium the animals were
FoEBex Fig. I. Representative coronal sections (taken from the atlas of Sidman et aL 19711 show the size of ENT/PIR lesions in the FoEBex lesion group (n = 16). Stippled areas indicate lesion size of the group as a whole and lines circumscribe lesions in individual animals. The left part of Fig. 1 shows neuroanatomieal key structures, caudal to rostral from top down. bA, basal amygdala; C, claustrum; cA, cortical amygdala; co, corpus eallosum; Co, Neocortex; ENT, entorhinal cortex; FD, fascia dentata; HPC, hippocampus (proper); IA, lateral amygdala; mA, medial amygdala; PIR, piriform cortex; PP, perforant path; PS, presubieulum; S, subiculum.
102 entorhinal cortex is destroyed, whilst the piriform cortex is affected to a lesser extent. The lesions are confined to the ENT and PIR except for the caudalmost part of the claustrum and fibers of the corpus callosum underneath the cortex, which were also damaged in some animals. General observations: anosmic females and those with ENT/PIR lesions performed very similar to the sham-treated animals with regard to exploratory behavior and general activity. ENT/PIR lesions significantly (P < 0.05) reduce the retrieving scores in FoEBex (Fig. 2a) and impair acoustic key-stimulus recognition (Fig. 2b) compared to sham-operated animals. Non-retrievers did not pay attention to the pups and did not attempt to retrieve any pup back to the nest within the 10-min observation time. In contrast, retrievers were very interested in the pups, sniffed at them, and immediately retrieved all 5 pups to the nest area. Microscopic investigation of the nasal epithelium revealed a considerable degree of destruction of the olfactory epithelium (65-100 ~o), but not of the vomeronasal organ, following zinc sulfate treatment. Fig. 3a,b shows that the destruction of the olfactory epithelium resulted in an impairment of both the retrieving score and key stimulus recognition in FoEBex compared to shams, indicating that anosmia produces the same kind of effect on parental behavior than ENT/PIR lesions in FoEBex. Fig. 4 shows the size of the ENT/PIR lesions in the Flact group. Note that the extent of the lesion is very similar to that shown in Fig. 1 for FoEBex. The effects FoEBex
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Fig. 3. Bars indicate (a) retrieving scores (% Retrievers, criterion see Materials and Methods) and (b)key stimulus recognition ( ~ Responses to 50 kHz, calculation see Materials and Methods) in ZnSO4-treated animals (FoEBex ZnSO4) and saline-treated controls (FoEBex saline). Asterisks indicate significant differences ( a ) P < 0 . 0 5 (Fisher's exact probability test, two-tailed) and (b) P < 0.01 (binomial test, two-tailed). The stippled line in (b) indicates chance level of choice, n.s., statistically non-significant.
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Fig. 2. Bars indicate (a) retrieving scores ( ~ Retrievers, criterion see Materials and Methods) and (b)key stimulus recognition ( ~ Responses to 50 kHz, calculation see Materials and Methods) in ENT/PIR-lesioned animals (FoEBex lesion) and sham-operated controls (FoEBex sham). Asterisks indicate significant differences ( a ) P < 0 . 0 5 (Fisher's exact probability test, two-tailed) and (b) P < 0.05 (binomial test, two-tailed). The stippled line in (b) indicates chance level of choice, n.s., statistically non-significant.
Fiacf Fig. 4. Representative coronal sections (taken from the atlas of Sidman et al? 9) show the size of ENT]PIR lesions in the Flact lesion group (n = 17). Stippled areas indicate lesion size of the group as a whole and lines circumscribe lesions in individual animals. The left part of Fig. 4 shows neuroanatomical key structures, caudal to rostral from top down. Flact, primiparous lactating females; ENT, entorhinal cortex; PIR, piriform cortex.
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Fig. 5. Bars indicate (a) retrieving scores ( ~ Retrievers, criterion see Materials and Methods) and (b)key stimulus recognition (% Responses to 50 kHz, calculation see Materials and Methods) in ENT/PIR-lesioned animals (Flact lesion) and sham-operated controls (Flact sham). Asterisks show significant differences in (b) P < 0.01 (binomial test, two-tailed). The stippled line in (b) indicates chance level of choice, n.s., statistically non-significant.
of those lesions on maternal behavior in Flact are shown in Fig. 5a,b. Retrieving scores are obviously not reduced by ENT/PIR lesions in lactating females in contrast to pup-experienced, non-lactating, estrogentreated females (FoEBex lesion: Fig. 2a). Nevertheless recognition of the acoustic key stimulus is disturbed by the ENT/PIR lesion in Flact. (Fig. 5b)
DISCUSSION
The first aspect of the results concerns the effects of lesions in the ENT/PIR leading to a significant impairment of sound recognition in lactating and sensitized, estrogen-treated females and to a reduction in the number of retrievers in the FoEBex group. Also anosmia produced by zinc sulfate treatment reduced pup-re: trieving and disturbed sound recognition in FoEBex. This clearly shows that olfactory cues in the context of parental care (either from the lactating female or from the pups) are necessary for the increase of parental motivation in cocaring females in the course o f achieving parental experience (sensitization). According to the findings of Staubli et al. 22, who reported disruption of the acquisition of an olfactory discrimination task following lesions in the entorhinal cortex, the similar disrupting effects of ENT/PIR lesions on parental behavior might likewise be interpreted as an effect of anterograde olfactory anmesia. On the other hand, ENT/PIR lesions in lactating females (Flact) only affect the appetitive contponent (pup-searching in response to 50-kHz sound) and not the consummatory
act (pup-retrieving) of this goal-directed maternal behavior. This interesting difference between experienced but non-lactating females with the same lesions in the ENT/PIR cortex, which normally are highly motivated to retrieve pups (see FoEBex sham in Figs. 2 and 3), and primiparous lactating mothers points to a fundamental difference between the mechanisms of parental motivation in animals 'sensitized '~7 of 'primed '15 for parental behavior and lactating females. Although in lactating females olfactory learning of pup cues seems to be necessary for the recognition of the acoustic key stimulus leading to pup-searching behavior, the consummatory response (retrieving pups) is not affected by the lesion. In sensitized non-lactating females (FoEBex) both responses appear to depend more on higher order learning of olfactory cues from pups. Second, we suggest to differentiate between the corticolimbic structures of the brain such as the entorhinal and piriform cortex which are shown to be involved in the appetitive responses to pup cues, requiring some sort of cognitive processes such as learning, and those brain regions involved in the regulation of the consummatory response (retrieving), like basal forebrain structures such as the M P O A and some of its connections (more detailed in Numan16). This idea is supported by recent studies of Everitt s on the sexual behavior of male rats, where lesions in the amygdala disrupt appetitive responses, whereas MPOA,lesions disrupt the consummatory sequence of male sexual behavior, i.e. copulatioh and ejaculation. Recent studies of Dickinson and Keverne 3, Kaba etal. 9, as well as Selway and Keverne TM, indicate that olfactory cues perceived and processed by the vomeronasal system in the context of both maternal behavior and pregnancy block by strange males are stored within the accessory olfactory bulb, while olfactory discrimination learning is mediated by the hippocampus. It should be noted here that the ZnSO4-treatment did not affect the vomeronasal organ, so that the perception of pheromones was not influenced in FoEBex. Since the entorhinal and piriform cortex are part of the main olfactory system, the present data suggest a somewhat different type of olfactory learning underlying the 'cognitive' aspects ofparental care compared to learning pheromonal cues perceived by the vomeronasal organ. Third, anosmic animals and those with bilateral lesions in the ENT/PIR perform very poorly on the auditory discrimination task. The notion that lesions in the higher centers of the olfactory system as well as anosmia disrupt the response to acoustic stimuli appears to be somewhat strange at fiirst glance. If, however, parental motivation is regarded as a central state of the brain, in which certain sensory stimuli can trigger
104 a behavioral response, then olfactory stimuli influencing this central motivational state can certainly lower the response threshold to stimuli of a different sensory domain. In fact, Smotherman et al. 2~ have shown that the responsiveness of female mice to calls from their young is enhanced if they receive olfactory cues at the same time. In addition Witter et al. 23 in a recent review article provided evidence that the entorhinal cortex is the most important corticolimbic relay station conveying sensory information from different parts of the sensory and associational fields of the neocortex, together with olfactory information from the piriform cortex to the hippocampus. It follows that ENT/PIR lesions may have disrupted the polysensory-associative information flux to the hippocampus. Although non-lactating females may perceive olfactory cues (from the lactating female, as suggested by Menella and Moltz ~4, or from the pups) from both the main and accessory systems as sensitizing stimuli, those processed by the main olfactory system appear to depend more heavily on estrogen, as suggested by the striking increase of estrogen receptors in the ENT/PIR in the course of sensitization ~~ This process can therefore be regarded as a case of estrogen-dependent olfactory learning. In lactating females, on the other hand, olfactory input to the hippocampus is less important for parental motivation, since these animals appear to depend more on olfactory cues processed by the vomeronasal system 3. It should be noted that both ENT/PIR lesions as well as anosmia reduce the retrieving scores to a level observed in gonadectomized estrogen-treated inexperienced females and males, and in intact, inexperienced females TM ~, thus a basal level of parental motivation is observed in inexperienced animals in the presence of estrogen as well. In summary, the present study shows that lesions in those parts of the entorhinal and piriform cortex, where the amount of estrogen receptors increases in ovariectomized estrogen-treated females during sensitization ~~ disrupt both pup-retrieving and pup-searching behavior. Anosmia produces the same kind of effect in those animals. It is therefore suggested to interpret the effect of the ENT/PIR lesions as anterograde olfactory amnesia. In lactating females, however, ENT/PIR lesions impair only acoustically released pup-searching behavior but not retrieving behavior. Therefore it is suggested to differentiate between the neural mechanisms underlying parental motivation in lactating females and sensitized, non-lactating animals. The adaptive significance of these differences is obvious, since it is certainly more important for a lactating female to retrieve her own pups.
The present study also shows that it is reasonable to use tests enabling discrimination between the appetitive and consummatory components of goal-directed behaviors in the study of their neural mechanisms because both components are shown to be regulated by different structures of the corticolimbic and basal forebrain.
ACKNOWLEDGEMENTS
We thank Dr. Barry Keverne (Cambridge) for helpful suggestions on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft Eh 53/9-2 and 10-1.
REFERENCES
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Beach, F.A. and Jaynes, J., Studies of maternal retrieving in rats. III. Sensory cues involved in the lactating female's response to her young, Behaviour, 10 (1956) 104-125. Blaustein, J.D. and Olster, D.H., Gonadal steroid hormone receptors and social behaviors. In J. Balthazart (Ed.), Advances in Comparative and Environmental Physiology, Vol.3, Springer, Berlin, 1989, pp. 31-104. Dickinson, C. and Keverne, E.B., Importance of noradrenergic mechanisms in the olfactory bulbs for maternal behaviour of mice, PhysioL Behav., 43 (1988) 313-316. Ehret, G., Age-dependent hearing loss in normal hearing mice, Natunrissenschaften, 61 (!974) 506. Ehret, G., Left hemisphere advantage in the mouse for recognizing ultrasonic communication calls, Nature, 325 (1987) 249-251. Ehret, G. and Haack, B., Ultrasound recognition in house mice: key stimulus configuration and recognition mechanism,./. Comp. Physiol. 148 (1982) 245-251. Ehret, G. and Koch, M., Ultrasound-induced parental behavior in house mice is controlled by female sex hormones and parental experience. Ethology, 80 (1989) 81-93. Everitt, B., Sexual motivation: a neural and behavioral analysis of the mechanisms underlying appetitive and copulatory responses of male rats, Neurosci. Biobehav. Rev., 14 (1990) 217-232. Kaba, H., Rosser, A. and Keverne, E.B., Neural basis of olfactory memory in the context of pregnancy block, Neuroscience, 32 (1989) 657-662. Koch, M., Effects of treatment with estradiol and parental experience on the number and distribution of estrogen-binding neurons in the ovariectomized mouse brain, Neuroendoctinology, 51 (1990) 505-514. Koch, M. and Ehret, G., Estradiol and parental experience, but not prolactin are necessary for ultrasound recognition and pupretrieving in the mouse, PhysioL Behav., 45 (1989) 771-776. Matulionis, D.H., Ultrastructural study of the mouse olfactory epithelium following destruction by ZnSO4 and its subsequent regeneration, Am. J. Anat., 142 (1975) 67-90. Matulionis, D.H., Light and electron microscopic study of the degeneration and early regeneration of olfactory epithelium in the mouse, Am. J. Anat., 145 (1976) 79-100. Mennella, J.A. and Moltz, It., Pheromonal emission by pregnant
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rats protect against infanticide by nulliparous conspecifics, PhysioL Behav., 46 (1989) 591-595. Noirot, E., The onset of maternal behavior in rats, hamsters, and mice. In D.S. Lehrman, R.A. Hinde and E. Shaw (Eds.), Advances in the Study of Behavior, VoL4, Academic Press, New York, 1972, pp. 107-140. Numan, M., Maternal behavior. In E. Knobil and J. Neill (Eds.), The Physiology of Reproduction, Raven, New York, 1988, pp. 1569-1645. Rosenblatt, J.S., Non-hormonal basis of maternal behavior in the rat, Science, 156 (1967) 1512-1514. Selway, R. and Keverne, E.B., ttippocampal lesions are xHthout effect on olfactory memory formation in the context of pregnancy block, PhysioL Behav., 47 (1990) 249-252. Sidman, R.L., Angevinc, J.B. and Pierce, E.T., Atlas of the Mouse
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brah~ and Sphzal Cord, Harvard University Press, Cambridge, 1971. Slotnick, B.M. and Leonard, C.M., A Stereotaxic Atlas of the Albbzo Mouse Forebrain, DttEW Publication, Rockville, 1975. Smotherman, W.P., Bell, R.W., Starzec, J., Elias, J., Zachman, T.A., Maternal responses to infant vocalizations and olfactory cues in rats and mice, Behav. BioL, 12 (1974) 55-66. Staubli, U., Fraser, D., Kessler, M., and Lynch, G., Studies on retrograde and anterograde amnesia of olfactory memory after denervation of the hippocampus by entorhinal cortex lesions, Behav. Neural BioL, 46 (1986) 432-444. Witter, M.P., Groenewegen, H.J., Lopes da Silva, F.tI. and Lohman, A.tt.M., Functional organization of the extrinsic and intrinsic circuitry of the parahippocampal region, Prog. Neurobiol., 33 (1989) 161-254.
99
BBR 01143
Parental behavior in the mouse: effects of lesions in the entorhinal/piriform cortex Michael Koch I and Gtinter Ehret 2 ~Fakult~t fiir Biologie, Universit~t Konstanz, D-7750 Konstanz 1 (F.R.G.) and 2Abteilung fiir vergleichende Neurobiologie, Universit~t UIm D-7900 Uhn (F.R.G.) (Received 23 May 1990) (Revised version received 24 September 1990) (Accepted 24 September 1990) Key words: Anosmia; Entorhinal cortex; Lesion; Olfactory learning; Parental behavior; Piriform cortex
The effects of bilateral radiofrequency lesions in the region of the entorhinal and piriform cortex (ENT/PIR cortex) on pup-retrieving and ultrasound-elicited pup-searching behavior were investigated in ovariectomized estrogen-treated female mice, which were sensitized to pups by co-caring for a litter for 5 days together with the mother (FoEBex), as well as in primiparous lactating females (Flact). A further group of FoEBex were rendered anosmic by an intranasal treatment with zinc sulfate-solution before the sensitization period and tests for parental motivation. Both pup-retrieving and pup-searching behavior were disturbed to the same extent by ENT/PIR lesions and ZnSO4-induced anosmia in FoEBex. In Flact, however, pup-retrieving was not affected by the lesion, while ultrasound-recognition leading to pup-searching was disrupted. The present data suggest sensitization to pups as a case of olfactory learning and thus, the effects of the ENT]PIR lesions are regarded as anterograde olfactory amnesia. From the present data and results reported in the literature ~6, it is suggested to differentiate between the neural substrates processing stimuli relevant for the appetitive component (pup-searching) of parental behavior (among others, the region of the entorhinal and piriform cortex), and the mechanisms regulating the consummatoo, component (pup;retrieving).
INTRODUCTION
Parental experience of adult female mice achieved by co-caring for a litter together with the mother for five days increases parental motivation T M and leads to an increase in the amount ofestrogen receptors in the brain Of ovariectomized estrogen-treated female mice ~~ This effect is likely to be induced by sensory cues from the pups or the mother 2. The increase in the amount of estrogen receptors is most obvious in the region of the entorhinal and piriform cortex. Although a considerable number of studies have been carried out on the neural mechanisms of parental behavior (e.g. pup-retrieving, licking, nest-building, nursing), nothing is known about the role of the entorhinal and/or piriform cortex in the regulation of this behavior ~6. It is therefore necessary to investigate whether this region of the brain is directly involved in the neural control of parental behavior. Correspondence: M. Koch. Present address: Lehrstuhl Tierphysiologie, Universitfit Ttlbingen, Auf der Morgenstelle 28, D-7400 Tfibingcn, F.R.G.
The present study investigates how lesions in that particular part of the entorhinal and piriform cortex, where the estrogen receptor-content of neurons was previously found to be sensitive to parental experience, affect ultrasound-elicited pup-searching and pupretrieving of estrogen-treated females. In addition a group of estrogen-treated females was rendered anosmic by zinc sulfate instillation since it has been shown that lesions in the entorhinal cortex produce anterograde olfactory amnesia in an olfactory discrimination task 22. The prediction in our study was that anosmia and lesions of the entorhinal/piriform cortex (here referred to as 'ENT/PIR lesions') would produce the same kind of effect on parental behavior. Additionally, the effects of ENT/PIR lesions on maternal behavior were tested also in primiparous lactating females. These non-ovariectomized animals served as a control with the natural supply of hormones during the course of pregnancy and lactation. Parental behavior was investigated with a test that allows for the discrimination between the appetitive component (pup-ultrasound elicited pup-searching behavior) and the consumntatory response of the animal,
0166-4328/91/S03.50 9 1991 Elsevier Science Publishers B.V. (Biomedical Division)
100 i.e. pup-retrieving. Here, pup-retrieving is regarded as a consummatory act, because it terminates pup-searching activities in a strikingly fixed way following contact with the pup. Although it might precede some other pupdirected behaviors, it should not be regarded as an appetitive behavior, because it is not followed by a distinct behavior, but instead the animal might engage in a variety of behaviors while staying in the nest with the young, e.g. licking, nest-building, suckling, selfgrooming, eating, drinking, sleeping.
MATERIALS AND METHODS Anhnals Subjects were female albino house mice (strain NMRI) aged 2-3 months which were kept in plastic cages (16.5 • 22 x 14 cm) in groups of 4 animals under a continuous light-dark cycle (lights on at 06.00 h, off at 20.00 h) at 21-23 ~ Food and water were provided ad libitum. The following experimental groups were used: Estrogen-treated ovariectomized females with bilateral ENT/PIR lesions with parental experience (FoEBex lesion, n = 16), sham-lesioned controls (FoEBex sham, n = 8), estrogen-treated ovariectomized females with intranasal zinc sulfate treatment ('anosmic') and parental experience (FoEBex ZnSO4, n = 12), saline-treated controls (FoEBex saline, n = 8), primiparous lactating females with bilateral ENT/PIR lesions (Flact lesion, n = 17), sham-lesioned controls (Flact sham, n = 8). Surgery and other treatments The subjects of the FoEBex lesion group received their lesions and were ovariectomized at the same time under a combined anesthesia of 80 mg/kg Nembutal and 3 mg/kg Taractan (Hoffmann-La Roche) injected intraperitoneally. The animals were placed in a stereotaxic frame without earbars in order not to destroy the tympanum. It was attempted to destroy those parts of the entorhinal and piriform cortex where a marked change in estrogen receptor content was observed in a previous study ~~ The stereotaxic coordinates for the bilateral ENT/PIR lesions (ap: - 2.5 mm; ml: 4.6 mm; dv: 5.2 mm: relative to bregma) were taken from the atlas of Slotnick and Leonard 2~ Lesions were produced using a radiofrequency lesion generator (Kopf Instruments Tujunga, USA, Model RFG-4). A steel needle inserted into the muscle of the neck served as a reference electrode. The temperature of the lesion electrode (tip size: 0.25 mm) was kept at 70-80 ~ for 60 s. In the FoEBex sham and Flact sham groups the electrode was lowered into the target region in the brain without passing radio-fre-
quency current. The animals were tested 4 weeks after surgery. Estrogen-treatment was started two weeks after surgery by implanting silastic tubing of 8 mm length filled with estradiol benzoate (Sigma) under the skin of the neck as described previously ~. The procedure by which the animals achieved parental experience was the same as described in our previous studies l~ Briefly, animals of the groups FoEBex lesion, FoEBex sham, FoEBex Z n S O 4 and FoEBex saline were placed each in the cage of a pregnant female (day 10 of pregnancy) and remained there during the rest of pregnancy, parturition and for the first 5 days of lactation of the mother. Females of the primiparous mother's group (Flact) were mated 3 days after surgery and were tested when their young were 5 days old, so that the period of time between surgery and test was the same for all experimental groups (4 weeks). Litters were standardized to 12 pups. Mating success of the females was not impaired by the ENT/PIR lesions. Anosmia was produced 8 days before the day of the behavioral tests by intranasal instillation of a 5 ~ Z n S O 4 solution (0.17 M). The procedure of Matulionis 12"~3 was used in a slightly modified way. The animals were anesthetized with Nembutal and Taractan (same dosage as for lesions) and were placed in a supinate position, then a drop of ZnSOa-solution was placed on both nares and was immediately inhaled by the mouse. Swallowing of the toxic ZnSO4-solution was prevented by loosely packing absorbing paper into the upper part of the throat of the animal and continuously aspiring the throat with a small vacuum pump. 30 s after instillation of the first drop, the procedure was repeated with a second drop of zinc sulfate solution. The animals were allowed to recover for two days in a separate cage. All zinc sulfate-treated animals showed normal behavior when they were reintroduced into the cage of the pregnant female they had previously been housed with. Aggressiveness towards the pregnant or lactating female or the pups was not observed in zinc sulfate-treated females. Controls (FoEBex saline) were treated with 0.9~o NaCI, but otherwise handled in the same way. Behavioral tests In order to discriminate between the appetitive and the consummatory component of goal directed parental behavior, we tested both ultrasound-elicited pup-searching and pup-retrieving, which latter might have multiple sensory key stimuli ~. As in our previous studies 7"t~ the experimental mice were placed in the central nest depression of a plastic running board (8 cm wide, 110 cm long) together with 5 pups at least 4 h before the testing began in order to minimize explo-
101 ratory tendencies during the test. One ultrasonic loudspeaker was mounted 65 cm from the nest depression at each end of the board but independent of it. The propensity to retrieve pups was tested by successively placing 5 pups on the board in a random distribution, one at a time at a distance of 30 cm from the nest. The criterion for a mouse being scored as a retriever was to retrieve all 5 pups within i0 min. After retrieving of the last pup the test for appetitive (pup-searching) behavior was started (non-retrievers were not tested for pup-searching behavior). Ultrasound-elicited pupsearching was tested by switching on both loudspeakers simultaneously, so that the animals had to discriminate between neutral 20-kHz tone pulses and 50-kHz tone pulses, known to function as specific releasers of pupsearching 5"6. The sound pressure levels were 89 dB for 50 kHz and 75 dB for 20 kHz which is about 71 dB (SPL re 20/tPa) above the threshold of perception of both frequencies 4. It was recorded towards which loudspeaker the mouse moved. The criterion for a response was leaving the nest for at least 30 cm towards one of the two loudspeakers within i0 min after tone onset. In consecutive tests the two frequencies were randomly assigned to the two loudspeakers. A maximum of 7 responses were taken from one animal: the data of all animals of an experimental group were summed up and percent responses to 50-kHz and 20-kHz stimuli calculated. During the behavioral tests the experimenter was unaware of the treatment (sham or operated) of the animals.
perfused as described above. Fur, skin, eyes and muscles were removed from the skull, the nose was cut off and the lower jaw together with the tongue was removed. This preparation was postfixed for two days in 4~o formalin/PBS, decalcified for 3 days in 0.125~ EDTA (Titriplex III, Merck. pH 7.4; T = 37 ~ The upper incisivi were pulled out and frontal sections (40/tin) of the nose were cut on a cryostat, mounted on gelatine-coated slides and stained with Cresyl violet.
RESULTS
The size and location of the E N T / P I R lesions in FoEBex is shown in Fig. 1; obviously, a large part of the
Statistical evahtation Only non-parametric statistics are used. Differences between the numbers of retrievers in each group are compared with Fisher's exact probabilities test. Retrieving scores are expressed as '~o Retrievers'. Tone discrimination is expressed as '~o Responses to 50 kHz' and statistical significance of tone preference was examined with the binomial test. All tests are two-tailed and a P < 0.05 was considered significant.
Histology
After completion of the behavioral tests the animals were perfused through the left ventricle with 0 . 9 ~ NaCI followed by 4 ~ formalin in 0.02 M phosphate-buffered saline (PBS). The brains were removed and placed in 3 0 ~ sucrose/PBS until they sank. They were cut on a cryostat in 40-#m sections which were mounted on gelatine-coated slides and stained with Cresyl violet. Every second section was drawn with a camera lucida and projected onto representative drawings from the atlas of Sidman et al. t9. For the histological analysis of the effects of ZnSO4treatment on the nasal epithelium the animals were
FoEBex Fig. I. Representative coronal sections (taken from the atlas of Sidman et aL 19711 show the size of ENT/PIR lesions in the FoEBex lesion group (n = 16). Stippled areas indicate lesion size of the group as a whole and lines circumscribe lesions in individual animals. The left part of Fig. 1 shows neuroanatomieal key structures, caudal to rostral from top down. bA, basal amygdala; C, claustrum; cA, cortical amygdala; co, corpus eallosum; Co, Neocortex; ENT, entorhinal cortex; FD, fascia dentata; HPC, hippocampus (proper); IA, lateral amygdala; mA, medial amygdala; PIR, piriform cortex; PP, perforant path; PS, presubieulum; S, subiculum.
102 entorhinal cortex is destroyed, whilst the piriform cortex is affected to a lesser extent. The lesions are confined to the ENT and PIR except for the caudalmost part of the claustrum and fibers of the corpus callosum underneath the cortex, which were also damaged in some animals. General observations: anosmic females and those with ENT/PIR lesions performed very similar to the sham-treated animals with regard to exploratory behavior and general activity. ENT/PIR lesions significantly (P < 0.05) reduce the retrieving scores in FoEBex (Fig. 2a) and impair acoustic key-stimulus recognition (Fig. 2b) compared to sham-operated animals. Non-retrievers did not pay attention to the pups and did not attempt to retrieve any pup back to the nest within the 10-min observation time. In contrast, retrievers were very interested in the pups, sniffed at them, and immediately retrieved all 5 pups to the nest area. Microscopic investigation of the nasal epithelium revealed a considerable degree of destruction of the olfactory epithelium (65-100 ~o), but not of the vomeronasal organ, following zinc sulfate treatment. Fig. 3a,b shows that the destruction of the olfactory epithelium resulted in an impairment of both the retrieving score and key stimulus recognition in FoEBex compared to shams, indicating that anosmia produces the same kind of effect on parental behavior than ENT/PIR lesions in FoEBex. Fig. 4 shows the size of the ENT/PIR lesions in the Flact group. Note that the extent of the lesion is very similar to that shown in Fig. 1 for FoEBex. The effects FoEBex
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Fig. 2. Bars indicate (a) retrieving scores ( ~ Retrievers, criterion see Materials and Methods) and (b)key stimulus recognition ( ~ Responses to 50 kHz, calculation see Materials and Methods) in ENT/PIR-lesioned animals (FoEBex lesion) and sham-operated controls (FoEBex sham). Asterisks indicate significant differences ( a ) P < 0 . 0 5 (Fisher's exact probability test, two-tailed) and (b) P < 0.05 (binomial test, two-tailed). The stippled line in (b) indicates chance level of choice, n.s., statistically non-significant.
Fiacf Fig. 4. Representative coronal sections (taken from the atlas of Sidman et al? 9) show the size of ENT]PIR lesions in the Flact lesion group (n = 17). Stippled areas indicate lesion size of the group as a whole and lines circumscribe lesions in individual animals. The left part of Fig. 4 shows neuroanatomical key structures, caudal to rostral from top down. Flact, primiparous lactating females; ENT, entorhinal cortex; PIR, piriform cortex.
103
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Fig. 5. Bars indicate (a) retrieving scores ( ~ Retrievers, criterion see Materials and Methods) and (b)key stimulus recognition (% Responses to 50 kHz, calculation see Materials and Methods) in ENT/PIR-lesioned animals (Flact lesion) and sham-operated controls (Flact sham). Asterisks show significant differences in (b) P < 0.01 (binomial test, two-tailed). The stippled line in (b) indicates chance level of choice, n.s., statistically non-significant.
of those lesions on maternal behavior in Flact are shown in Fig. 5a,b. Retrieving scores are obviously not reduced by ENT/PIR lesions in lactating females in contrast to pup-experienced, non-lactating, estrogentreated females (FoEBex lesion: Fig. 2a). Nevertheless recognition of the acoustic key stimulus is disturbed by the ENT/PIR lesion in Flact. (Fig. 5b)
DISCUSSION
The first aspect of the results concerns the effects of lesions in the ENT/PIR leading to a significant impairment of sound recognition in lactating and sensitized, estrogen-treated females and to a reduction in the number of retrievers in the FoEBex group. Also anosmia produced by zinc sulfate treatment reduced pup-re: trieving and disturbed sound recognition in FoEBex. This clearly shows that olfactory cues in the context of parental care (either from the lactating female or from the pups) are necessary for the increase of parental motivation in cocaring females in the course o f achieving parental experience (sensitization). According to the findings of Staubli et al. 22, who reported disruption of the acquisition of an olfactory discrimination task following lesions in the entorhinal cortex, the similar disrupting effects of ENT/PIR lesions on parental behavior might likewise be interpreted as an effect of anterograde olfactory anmesia. On the other hand, ENT/PIR lesions in lactating females (Flact) only affect the appetitive contponent (pup-searching in response to 50-kHz sound) and not the consummatory
act (pup-retrieving) of this goal-directed maternal behavior. This interesting difference between experienced but non-lactating females with the same lesions in the ENT/PIR cortex, which normally are highly motivated to retrieve pups (see FoEBex sham in Figs. 2 and 3), and primiparous lactating mothers points to a fundamental difference between the mechanisms of parental motivation in animals 'sensitized '~7 of 'primed '15 for parental behavior and lactating females. Although in lactating females olfactory learning of pup cues seems to be necessary for the recognition of the acoustic key stimulus leading to pup-searching behavior, the consummatory response (retrieving pups) is not affected by the lesion. In sensitized non-lactating females (FoEBex) both responses appear to depend more on higher order learning of olfactory cues from pups. Second, we suggest to differentiate between the corticolimbic structures of the brain such as the entorhinal and piriform cortex which are shown to be involved in the appetitive responses to pup cues, requiring some sort of cognitive processes such as learning, and those brain regions involved in the regulation of the consummatory response (retrieving), like basal forebrain structures such as the M P O A and some of its connections (more detailed in Numan16). This idea is supported by recent studies of Everitt s on the sexual behavior of male rats, where lesions in the amygdala disrupt appetitive responses, whereas MPOA,lesions disrupt the consummatory sequence of male sexual behavior, i.e. copulatioh and ejaculation. Recent studies of Dickinson and Keverne 3, Kaba etal. 9, as well as Selway and Keverne TM, indicate that olfactory cues perceived and processed by the vomeronasal system in the context of both maternal behavior and pregnancy block by strange males are stored within the accessory olfactory bulb, while olfactory discrimination learning is mediated by the hippocampus. It should be noted here that the ZnSO4-treatment did not affect the vomeronasal organ, so that the perception of pheromones was not influenced in FoEBex. Since the entorhinal and piriform cortex are part of the main olfactory system, the present data suggest a somewhat different type of olfactory learning underlying the 'cognitive' aspects ofparental care compared to learning pheromonal cues perceived by the vomeronasal organ. Third, anosmic animals and those with bilateral lesions in the ENT/PIR perform very poorly on the auditory discrimination task. The notion that lesions in the higher centers of the olfactory system as well as anosmia disrupt the response to acoustic stimuli appears to be somewhat strange at fiirst glance. If, however, parental motivation is regarded as a central state of the brain, in which certain sensory stimuli can trigger
104 a behavioral response, then olfactory stimuli influencing this central motivational state can certainly lower the response threshold to stimuli of a different sensory domain. In fact, Smotherman et al. 2~ have shown that the responsiveness of female mice to calls from their young is enhanced if they receive olfactory cues at the same time. In addition Witter et al. 23 in a recent review article provided evidence that the entorhinal cortex is the most important corticolimbic relay station conveying sensory information from different parts of the sensory and associational fields of the neocortex, together with olfactory information from the piriform cortex to the hippocampus. It follows that ENT/PIR lesions may have disrupted the polysensory-associative information flux to the hippocampus. Although non-lactating females may perceive olfactory cues (from the lactating female, as suggested by Menella and Moltz ~4, or from the pups) from both the main and accessory systems as sensitizing stimuli, those processed by the main olfactory system appear to depend more heavily on estrogen, as suggested by the striking increase of estrogen receptors in the ENT/PIR in the course of sensitization ~~ This process can therefore be regarded as a case of estrogen-dependent olfactory learning. In lactating females, on the other hand, olfactory input to the hippocampus is less important for parental motivation, since these animals appear to depend more on olfactory cues processed by the vomeronasal system 3. It should be noted that both ENT/PIR lesions as well as anosmia reduce the retrieving scores to a level observed in gonadectomized estrogen-treated inexperienced females and males, and in intact, inexperienced females TM ~, thus a basal level of parental motivation is observed in inexperienced animals in the presence of estrogen as well. In summary, the present study shows that lesions in those parts of the entorhinal and piriform cortex, where the amount of estrogen receptors increases in ovariectomized estrogen-treated females during sensitization ~~ disrupt both pup-retrieving and pup-searching behavior. Anosmia produces the same kind of effect in those animals. It is therefore suggested to interpret the effect of the ENT/PIR lesions as anterograde olfactory amnesia. In lactating females, however, ENT/PIR lesions impair only acoustically released pup-searching behavior but not retrieving behavior. Therefore it is suggested to differentiate between the neural mechanisms underlying parental motivation in lactating females and sensitized, non-lactating animals. The adaptive significance of these differences is obvious, since it is certainly more important for a lactating female to retrieve her own pups.
The present study also shows that it is reasonable to use tests enabling discrimination between the appetitive and consummatory components of goal-directed behaviors in the study of their neural mechanisms because both components are shown to be regulated by different structures of the corticolimbic and basal forebrain.
ACKNOWLEDGEMENTS
We thank Dr. Barry Keverne (Cambridge) for helpful suggestions on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft Eh 53/9-2 and 10-1.
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