Maternal behavior induced in male rats by bilateral lesions of the bed nucleus of the accessory olfactory tract

Physiology&Behavior,Vol. 52, pp. 707-712, 1992 Printed in the USA.

0031-9384/92 $5.00 + .00 Copyright© 1992 PergamonPressLtd.

Maternal Behavior Induced in Male Rats by Bilateral Lesions of the Bed Nucleus of the Accessory Olfactory Tract M. A N G E L E S P. I Z Q U I E R D O , P A L O M A C O L L A D O , S A N T I A G O S E G O V I A , A N T O N I O G U I L L A M O N A N D M. C R U Z R. D E L C E R R O t

Departamento de Psicobiolog{a, Universidad Nacional de Educaci6n a Distancia, Ciudad Universitaria, P.O. Box 50487, 28040 Madrid, Spain Received 4 N o v e m b e r 1991 IZQUIERDO, M. A. P., P. COLLADO, S. SEGOVIA, A. GUILLAMON AND M. C. R. DEL CERRO. Maternal behavior induced in male rats by bilateral lesions of the bed nucleus of the accessory olfactory tract. PHYSIOL BEHAV 52(4) 707-712, 1992.--In the present study, we investigate the effect of bilateral electrolytic lesions of the bed nucleus of the accessory olfactory tract (BAOT) in male Wistar rats that did not have care-pups experience, using a test of induced maternal behavior. Consistent with our previous findings in virgin female rats (10), there was a significantly shorter sensitization (3 days) and retrieval (2 days) latencies in the BAOT-lesioned group than in the sham-lesioned and intact-control male groups (12 days for both). Based on these findings, we propose that BAOT, a sexually dimorphic nucleus of the vomeronasal system, exerts an inhibitory modulation in the expression of parental behavior in male and female virgin rats. It may do so by maintaining an olfactory-based tonic inhibition of maternal behavior, thereby resulting in the adults' tonic avoidance of the pups until this inhibition is abolished by lesion, or reduced or overridden by appropriate hormonal and/or sensory influences. Maternal behavior

Male adult rats

Bed nucleus of the accessory olfactory tract

IF nonpregnant female rats are exposed continuously to young foster pups, they begin to show nurturant behaviors toward them, after latencies of from 6 to 15 days, depending on the strain (21). This procedure is known as concaveation (17,49) or sensitization (39). Mature male rats can also be sensitized, but their latencies to initiate parental behavior are significantly longer than those of females. Moreover, some mature males may not become parental within the time limits employed in induction experiments (13). Furthermore, the incidence of pup killing during sensitization is highly variable among both males and females, and depends on strain and age of the pups, the newborns being the most vulnerable (37). However, in general, more males than females kill test pups (28). Different authors have suggested that virgin female rats find the pups aversive during the early phases of sensitization (14,15,27). In support of this, nonmaternal females made anosmic by olfactory bulbectomy (eliminating main and accessory olfactory sensory input) did not avoid pups and became maternal within 2 days (14,15). Females with intact main and accessory olfactory systems, in contrast, generally require significantly longer exposure to pups than anosmic females, before they behave maternally. Both the main and accessory olfactory systems are involved in mediating the effects of these hypothesized aversive odors

Vomeronasal system

Lesion

(16). It is known that lesions in structures that are considered to be components of the vomeronasal system (VNS) can facilitate or disrupt parental behavior. The period of active avoidance is markedly diminished in both males and females by vomeronasal organ (VNO) deafferentiation (16,26), and VNO remotion decreases infanticide behavior in male rats (28). Facilitation of maternal behavior in virgin female rats can also be seen after lesioning different vomeronasal structures or tracts (17,22). In contrast, lesions of the medial preoptic area (MPA), that receives vomeronasal input, disrupt maternal behavior (32-35) and prevent the facilitation observed after amydaloid lesions (l 8). MPA is considered essential to the induction of maternal behavior in both virgins and postparturient females (30,31). These findings suggest that the absence of maternal behavior in virgin females is normally due to vomeronasal sensory input tonically inhibiting the MPA. As we reported in a previous study (10), the BAOT is a cell group of the forebrain that is a component of the VNS. It is associated with the accessory olfactory tract (2,8,42), located rostral and ventral to the nucleus of the lateral olfactory tract, and its caudal portion is located ventrolaterally and adjacent to the anterior medial amygdala (8,23,25,42) (Fig. 1). Histologically, the BAOT is composed of a small group of medium sized cells which have a high affinity for cresyl violet stain, and it has been

l Request for reprints should be addressed to M. Cruz R. del Cerro.

707

708

I Z Q U I E R D ( ) t:l AI .

A

described as free ofacetylcholinesterase and cholecystokinin activity (8). Embryologically, this nucleus has a biphasic neurogenesis in the rat: the larger growth surge (74%) occurs around the 12th and 13th embryonic days (E): a later surge (17'~) occurs about E 15 ( 1). The BAOT is a major target for tibets projecting from the accessory olfactory bulb (AOB) (7) and is reciprocally connected with the AOB (6,7,42) and the posteromedialcortical amygdaloid nucleus (C3) (23,36). Moreover, it receives afferents from the medial amygdaloid group (23) and is one of the main VNS cellular groups sending vomeronasal input to the MPA (8). In addition, we have proposed that the VNS is a sexually dimorphic network (43,46). There is sexual dimorphism in the volume and number of neurons, controlled by gonadal steroids early after birth, in the vomeronasal organ (VNO) (43,44,46), the AOB (45,47,48), the bed nucleus of stria terminalis (9,20), and the BAOT (4). Other authors have shown morphological sex differences in the MPA (19) and amygdala (12,29). Since in our previous work (10) we found an inhibitory function for the BAOT over maternal behavior, in this study our purpose was to investigate whether the morphological sexual dimorphism found in this nucleus (4) could be reflected in the induction of maternal behavior in adult male rats. METttOD

Subject.s

C

Forty male rats (90 days old, 300 g weight) of the Wistar strain (Alin, S.A., Madrid, Spain), 2 weeks prior to the experiment, were individually housed in clear Plexiglas cages (47 × 22 × 15 cm) with wood shavings provided for nesting material, and ad lib water and food pellets on a reverse light-dark (day/ night) cycle (lights on from 2000 to 0800 h). Additional animals of the same strain were bred to provide donor pups. During the procedures the subjects were removed to a separate observation room in the same cages and similar conditions. Room temperature was thermostatically regulated at 22 _+ I°C. The subjects were randomly assigned to the following three groups: males with BAOT lesions (MBAOT, n = 20): males sham lesioned (MSH, n = 10) and control males (MC. n - 10).

Procedures.]or Brain Lesion.s'

FIG. 1. Reconstruction of representative locations of the bed nucleus of the accessory olfactory tract in rostral (A); medium (B) and caudal (C) sections. ACo: anterior cortical amygdaloid nucleus; BAOT: bed nucleus of the accessory olfactory tract; LOT: nucleus of the lateral olfactory tract; MeA: medial amygdaloid nucleus, anterior; MEAD: medial amygdaloid nucleus, anterodorsal; MeAV: medial amygdaloid nucleus, anteroventral. Bregma levels for A = -1.80 mm; B = -2.12 ram; C = -2.30 mm. Schematic illustrations from Paxinos and Watson atlas (1986).

Bilateral BAOT lesions were made with unipolar stainless steel insulated electrodes of 0.2 m m contact diameter (Rhodes Medical Instruments, USA) using a Narishige SR-5 stereotaxic instrument and an electrolytic lesion maker Cibertec GL-2 (Cibertec, Spain) device. The lesion procedure was carried out under 2,2,2, tribromoethanol anesthesia (10 ml/kg, administered IP). Electrode coordinates were based on Paxinos and Watson (38) and upon our previous data. M B A O T and MSH coordinates were: A - P = - 2 . 2 5 ram; L = 3.1 mm; D - V = 9.8 ram. DV coordinate in the SH group was decreased 0.5 m m (for SH, DV = 9.3 mm), in order to avoid lesioning the BAOT. Bilateral lesions (MBAOT group) (Fig. 2) were produced by passing a 0.5 mA anodal direct current for 10 s. In MSH subjects, no current was passed through the electrode. Following recovery from the surgery, the animals were returned to their home cages. To assess the general health of M B A O T and MSH animals, all groups were weighed at the time of surgery and on the day of sacrifice. Finally, subjects of MC group were unexperienced males taking in care of pups.

MATERNAL BEHAVIOR IN MALE RATS

709

FIG. 2. Photomicrograph showinga representative electrolytic lesion of the bed nucleus of the accessory olfactory tract (BAOT). ACo: anterior cortical amygdaloid nucleus; MeA: medial amygdaloid nucleus, anterior. Bar = 200 um.

Maternal Behavior Induction Test The maternal behavior induction test was carried out as follows: after at least 15 postoperative recovery days, all groups were tested for maternal responsiveness to pups. Maternal behavior tests began each day at 1000 h. At this time, four freshly nourished Wistar pups 3- to 8-days-old were placed opposite the male in his cage. The occurrence of the following four behavior patterns were recorded during the next 10 rain: 1. nest building quality, 2. physical contact with pups measured as time (duration in seconds of this behavioral pattern, 600 s maximum) and frequency (total number of contacts throughout 10 min), 3. nursing posture, also recorded as frequency and time and 4. pup retrieving. Pups were left with the males for a 24-h period after which they were removed and returned to their mothers. Each day, four new recently fed pups were presented to the experimental animals and behavioral observations were again begun. The quality of nest built was rated according to the our own following scale: 0 = no nest: no nest shape nor any nest delimitation and pups dispersed in the cage. 1 = poor nest: initial nest structure with at least one pup within. 2 = fair nest: more clear nest shape with at least two pups in the nest site. 3 = good nest: the completed structure but with low walls and at least three pups in it. 4 = excellent nest: the nest structure has high and well-defined walls. The whole litter was located in it. Observations were continued until the males exhibited pa, rental behavior on 2 consecutive days, or for a maximum of 12 days. A male was considered to be parental if he retrieved four pups to a common nest site on 2 successive days (sensitization

criteria). In the control group, one male cannibalized young and was discarded from the study.

Histology Following completion of behavioral observations, all animals were anesthesized with an overdose oftribromoethanol and perfused intracardially with 0.9% saline followed by 10% formalin. Brains were removed and placed in 10% formalin for 4 days, and then embedded in paraffin. Afterwards, the brains were serially cut in 20 um coronal sections through the BAOT with a 1516 Leitz microtome. All of the sections were stained with a 0.1% solution of cresyl violet (Merck). The histological evaluation was done blind. Another researcher unaware of the results of behavior, microscopically inspected all brain sections. The procedure for estimating the degree of damage to the BAOT was done according the following criteria: in 3-month-old male rats, the BAOT approximately measures 800 um along the antero-posterior axis, and its medial part approximately is 650 um. When the medial part of the BAOT was totally damaged, we considered that the whole nucleus was lesioned at least in an 80%. On the basis of histological examination, the behavior of animals that did not reach these criteria was not statistically analized. According to this procedure, the final composition of the groups was as follows: MBAOT (n = 7); MSH (n = I0) and MC (n = 9).

Statistical Analysis The behavioral data were analyzed with nonparametric tests (Kruskai-Wallis and Mann-Whitney U-tests). To analyze the differences in the percentages of animals that rised sensitization criteria Z-test were used. RESULTS Table 1 and Fig. 3 show latencies of retrieval (the average number of clays in which pup-retrieving behavior appeared for

IZQI. IERI)() I!1 ,~1

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TABLE 1 1 ATL:NCY IN DAYS TO THE ONSET OF PATERNAL ('ARE IN LESIONED, SHAM, ,AND CONTROL MALE RATS

Similar findings were obtained by us (10) in vugin female rats after BAOT lesions. BAOT appears to plax ma inhibilory role in the control of maternal behavior because bilateral lcsitms in this vomeronasal structure also facilitate the display or" pup

Groups

Behavior Patterns Retrieval latencies Sensitization latencies Percentage sensitization

Control (n = 9)

Sham (n 10)

Bilateral Lesion (n :: 7)

12 (12-12)

12 (8.7-12)

2 (0-12)*+

12 (12-12)

12 (12-12l

3 (0-12)~§

0

10

71 $§

RETRIEVAL LATENClES 12 10

D A

II

Y Note: Data of retrieval and sensitization latencies are medians interquartile range (IQR) and were analyzed with the Mann-Whitney/_,'-test. *tz~§ Significant differences with respect to the control group (*p < 0.02, ~p < 0.01), and with respect to the sham group (fp < 0.03: §p < 0.01).

I

S

4 2 O CONTROL

the first time) and sensitization (the average number of days prior to 2 consecutive days of retrieval) and percentage of animals that developing parental behavior, in lesioned (MBAOT), shamlesioned (MSH) and intact-control (MC) male rats. KruskalWallis analysis revealed significant differences between the three groups in retrieval, H(2) = 8.43, p < 0.01, and sensitization latencies, H(2) = 13.42, p < 0.001. M a n n - W h i t n e y U-tests comparing these latencies in group M B A O T and each of the MSH and MC groups indicated significantly shorter retrieval latencies in group M B A O T than in groups MSH, U = 13, p < 0.03, or MC, U = 11.5, p < 0.02, and significantly shorter sensitization latencies in the experimental group (MBAOT) than in sham, U = 11, p < 0.01, or intact control groups, U = 9, p < 0.01. Consistent with these data, we found statistically significant differences in the percentage of BAOT-lesioned male rats who became parental (71%) in contrast to MSH (10%) and MC (0%) groups (p < 0.01 for both groups). The maternal behavior patterns of the differents groups are reflected in Table 2. According with the data presented in Table l, the Kruskal-Wallis analysis indicated significant differences between the three groups in nest-building quality, H(2) = 14.98, p < 0.0006. M a n n - W i t n e y U-test showed that M B A O T group raised significantly higher quality scores in nest building pattern than MC and M S H groups, U = 3, p < 0.001, and U = 1.5. p < 0.0002, respectively. There were also statistically significant differences in nursing behavior in time, H(2) = 9.24, p < 0.009, and frequency, H(2) = 9.10, p < 0,01. The experimental group raised higher scores in contrast with the almost nonexistent nursing pattern in control, U = 13, p < 0.04, and sham groups, U -- 11, p < 0.01. Although the time and frequency of physical contacts with the pups shown by M B A O T group was quantitatively higher than in the sham-lesioned and intact-control male rats, these differences did not reach significance in time, H(2) = 4.59, p > 0.10, nor in frequency, H(2) = 1.70, p > 0.42. DISCUSSION

BAOT bilateral electrolytic lesions induced a significant reduction of retrieval and sensitization latencies, as well as a statistically significant increases of nest building and time and frequency of nursing posture patterns and physical contact in inexperienced male rats exposed to pups. These results indicate that bilateral electrolytic lesions of the BAOT are able to facilitate the induction of maternal behavior in the virgin male rat.

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BAOT L E I M O N I

SENSITIZATION LATENCIES 1| 10

O A Y S

I (I 4 2 0

CONTROL

IIHAM

BAOT L EliiON8

PERCENTAGE SENSITIZATION 100

p e r c

6°"

•n

60-

t

a

g

40-

o 20-

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mHAM

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FIG. 3. Graphic representation of retrieval and sensitization latencies and percentage of sensitization in the tree groups of males. Bars indicate medians from total test period (12 days) for each behavioral criteria pattern represented (retrieval, sensitization, and percentage of sensitization).

MATERNAL BEHAVIOR IN MALE RATS

711

TABLE 2 EFFECTSOF LESIONSIN THE BED NUCLEUSOF THE ACCESSORY OLFACTORYTRACTON PARENTALBEHAVIOR PATI'ERNSIN MALERATS Groups BehaviorPatterns Nest building Nursing posture Time Frequency Physical contact Time Frequency

Control (n = 9) 0 (0-1.25) 0 (0-2.1)

Sham (n = 10) 0 (0-1) 0 (0-0)

BilateralLesion (n = 7) 4 (3-4)*I 10.5 (0-31.8)~§

0 (0-1) 11.8 (8.5-15)

0 (0-0) 11 (6.4-13.7)

9 (0-23)I§ 30.4 (9.8-44)

68 (53-91.5)

71.5 (58-96)

89 (58-132)

Note: These data are medians interquartile range (IQR) and were analysed with the Mann-Whitney U-test, two-tailed. *i'~t§Significantdifferenceswith respect to the control group. (*p < 0.001; ~p < 0.04) and with respect to the sham group (tp < 0.0002; §p < 0.01).

care in virgin females, reducing both retrieval and sensitization latencies and increasing nest building and time and frequency of grooming, licking and, crouching (10). The BAOT, a component of the VNS, receives monosynaptic projections from the AOB (7), is connected with the medial amygdala (23), and sends efferents to MPA (8) for which it has been described to play a facilitatory function in the control of the expression of maternal behavior (31). Moreover, VNO removal (11,40,41) or deafferentiation facilitate maternal behavior in both male and female rats. The removal of olfactory bulbs, that includes AOB removal, or lateral olfactory tract lesions ( 14,15) as well as corticomedial amydala lesions (17), produce similar facilitatory effects in the expression of maternal care. Since damage in vomeronasal structures, including BAOT, appears to induce facilitation of maternal behavior, while lesions in MPA disrupt it, we have hypothesized (10) that the VNS participates in the neural control (inhibition/desinhibition)of maternal care to the pups. Furthermore, it is possible to suggest that the VNS regulation on the maternal behavior could be exerted upon the MPA, because MPA receives vomeronasal input from several VNS structures (8), The BAOT is a sexually dimorphic nucleus with males presenting a greater volume and neuron number than female rats, and this sexual dimorphism depends on the neonatal gonadal environment (4,5). We have also described that electrolytic BAOT lesions are able to facilitate maternal behavior in virgin female rats and, as data of present work indicates, also in inexperienced male rat. Besides, virgin male present longer latencies than virgin female rats to become parental (26) and can-

nibalize more pups than females (28). BAOT bilateral lesions in male rats significantly reduce latencies to display parental cares at similar levels than those found by us in virgin female rats after the same kind of lesions (10). Regarding together all these facts, we could suggest that the inhibitory role that BAOT plays in the control of the expression of parental cares is greater in male than in the virgin female rats, and that this functional sexual dimorphism could be related to the sex differences found in the BAOT volume and number of neurons (4). Similar results indicating relationships between structural and functional sexual dimorphism with respect to parental care were reported by Kinsley and Bridges (24). These authors found that prenatal stress, which disrupt sexual differentiation of the MPA-sexually dimorphic nucleus, induces a female-like pattern of parental care in male rats (24). Finally, this study and our earlier one (10) are unique, showing that the BAOT is involved in the neural control of parental behavior not only in virgin female but also in male rats. So, we can suggest that the BAOT maintains an inhibitory role in virgin male and female rats in the display of this reproductive behavior which is relevant for the species survival. ACKNOWLEDGEMENTS This work was supported by a DGICYT Grant No. PB90-0878. Thanks are due to professors B. R. Komisaruk and J. S. Rosenblatt from the Institute of Animal Behavior(IAB), Rutgers University,NJ, for their suggestionsand editorialhelp. Thanks are due to LuisTroca, LuisCarrillo, and Gonzalo Moreno for their technical assistance.

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