Physiology and Behavior, Vol. 8, pp. 683-686. Brain Research Publications Inc., 1972. Printed in Great Britain.

The Development of the Pheromonal Bond in the Albino Rat'

M I C H A E L L E O N A N D H O W A R D M O L T Z

Department of Psychology, The University of Chicago, Chicago, Illinois 60637, U.S.A.

(Received 8 N o v e m b e r 1971)

LEON, M. AND H. MOLTZ. The development of the pheromonal bond in the albino rat. PHYSIOL. B~qAV. 8 (4) 683-686, 1972.---The lactating female rat begins to emit a pheromone at about 14 days postpartum, coincident with the age the young first become responsive to the pheromone. Correlatively, at about 27 days postpartum, the mother ceases to release the pheromone, which corresponds, in turn, to the age at which the young cease to be attracted to the pheromone. What role this olfactory bond might play in synchronizing the mother-young relationship was discussed.

Maternal behavior Pheromone Mother-litter interaction

LEON AND MOLTZ [1] demonstrated recently that 16-day lactating female rats characteristically release an odor capable of attracting 16-day-old young. Since the young involved showed no preference for the odor of their own mother, but instead selectively approached any lactating female who was also 16 days postpartum, it was apparent that the underlying cue was not litter specific. To this cue, clearly olfactory in nature, the term maternal pheromone was applied.

The purpose of the present experiment was to investigate just when, during the breeding episode, this maternal phero- mone characteristically appears and for how many days thereafter it is effectively emitted. Correlatively, we also wanted to determine when the young first begin to respond to the pheromone and the age-range over which they sub- sequently maintain such responding. I t was hoped that these data would provide insight into what role, if any, the pheromone plays in synchronizing the mother-young relation- ship.

METHOD

We used 960 Wistar pups from litters born in our laboratory colony. All litters were reduced routinely to six pups shortly after birth. Each pup was tested in the olfactory discrimina- tion apparatus previously described by Leon and Moltz [1]. In brief, this apparatus was designed to permit approach from a start box, across an open-field, to either of two goal boxes, each containing a stimulus female. F rom a single over-head valve, forced air was made to flow from each of the goal boxes to the start box.

The stimulus animals, lactating females and nulliparae alike, were isolated for three hr prior to being placed in the goal compartments. A single pup, deprived of maternal attention for the same period of time, was subsequently placed in the start box and allowed 15 min for a choice. Since

young of several different ages were used, locomotor capa- bilities had to be considered in defining what in fact constituted a choice. Specifically, Day 1 pups were required to crawl at least 8 in. from the start box along one or another side of the apparatus; pups Day 10 and 12 of age had at least to approach the ledge leading to a particular goal box; and finally pups older than Day 12 had actually to descend into the goal box. After each pup registered a choice, or 15 min had passed, it was removed from the apparatus and the absorbant paper covering the floor of the open field and start box was changed. After three pups of a given litter were tested, the apparatus was washed thoroughly with warm water, following which the goal-box positions of the stimulus females were reversed. The remaining three pups of the litter were then run. It should be emphasized that each pup was tested oaly once, and with only a single paired-combination of stimulus females. Moreover, any one such paired combina- tion was used for only a single litter.

RESULTS

Experiment 1 In Experiment 1, we were interested in obtaining normative

data relevant to the question of when, during the course of the breeding episode, the pheromone first comes to attract the young to their mother. Accordingly, young of 1, 10, 12, 14, 16, 21, 27 and 41 days of age were each presented with a choice between own mother and a nulliparous female.

The results, shown in Fig. 1 and Table 1, indicate that by 14 days of age, the young begin to approach own mother in preference to a nulliparous female. By 16 days, the preference becomes stronger, and in fact at this age is displayed by virtually all young. The mother continues to be chosen overwhelmingly until Day 21, after which time the attraction begins to wane. By Day 41, little evidence of the initial preference remains.

XThis research was supported in part by NSF Grant #GB23943 to H.M.

683

684 LEON AND MOLTZ

TABLE 1

PREFERENCES EXHIBITED BY YOUNO FOR OWN MOTHER OR NULL1PAROUS FEMALE (NF)

Choice Pup age N To mother To NF No choice Sig.*

Day 1 mother vs NF 1 day 30 0 0 30 - -

Day 10 mother vs NF 10 days 30 1 0 29 - -

Day 12 mother vs NF 12 days 30 11 2 17 > 0.05 Day 14 mother vs NF 14 days 30 23 7 0 <0.01 Day 16 mother vs NF 16 days 30 29 1 0 <0.01

Day 21 mother vs NF 21 days 30 28 2 0 <0.01

Day 27 mother vs NF 27 days 30 23 7 0 <0.01 Day 41 mother vs NF 41 days 30 15 10 5 ~>0.05

*Fisher Exact Probability Test was used.

I" 2O

J i i : I Ii lil i • i i i . i d

10 12 14 16 21 27 41

Age of pups in days

FIG. l. Approach behavior of young of different ages to own mother as opposed to nulliparous female (NF).

The appearance of the kind of pheromonal bond just described depends of course, not only on the ability of the young to discriminate and respond to a critical olfactory stimulus but on the capacity of the mother to emit that stim- ulus. Testing young, then, of different chronological ages with mothers of corresponding lactational ages, confounds mother with young in the development of the pheromonal bond. Obviously, for a complete ontogenetic picture the two must be traced separately--the young to determine when they first respond to the pheromone and the mother to determine when she emits the pheromone. The experiments described below were designed with this aim.

Experiment 2 Here we used in paired stimulus combination lactating

females, 16-days postpartum, and nulliparous females of comparable chronological maturity. Lactating animals of this particular postpartum age were chosen since they are known to emit the pheromone in a concentration that overwhelmingly attracts not only own young (Experiment 1)

but strange young as well [1]. The question, of course, was at what age such a standard stimulus female will first be approached and, correlatively, at what age she will cease to be approached. As before, the young selected for testing were, respectively i, 10, 12, 14, 16, 21, 27 and 41 days old.

Figure 2 and Table 2 present essentially the same picture as Fig. 1 and Table 1. That is, as with own mother, the strange, 16-day lactating female was preferred beginning when

:il i, Ill,.., I j'°ll I , I I I I [111

I 10 12 14 16 21 27 41

Age of pups in days

FIG. 2. Approach behavior of young of different ages to a standard 16-day lactating female (LF) when opposed to a nulliparous

female (NF).

the young were 14 days of age. Young at 16 days and at 21 days, of course, also showed a significant preference for the lactating female. By Day 27, however, choice behavior declined, and by Day 41, little evidence of it remained.

Experiment 3 This experiment was essentially the converse of Experiment

2. Here we took young at 16 days of age--young known to respond strongly to the pheromone--and tested them with

MATERNAL PHEROMONE 685

TABLE 2

PREFERENCES EXHIBITED BY YOUNG FOR 16-DAY LACTATING FEMALE (LF) OR NULLn~AROUS FEMALE (NF)

Choice Pup age N To 16 day LF To NF No choice Sig.*

1 30 0 0 30 - - 10 30 2 0 28 - - 12 30 8 5 17 > 0.05 14 30 27 2 1 < 0.01

16-day LF vs NF 16 30 29 1 0 < 0.01 21 30 24 6 0 < 0.01 27 30 18 12 0 > 0.05 41 30 12 17 1 > 0.05

*Fisher Exact Probability Test was used.

females of different lactational ages, namely, 1, 10, 12, 14, 16, 21, 27 and 41-days postpartum. With each, nulliparous females were once again used in paired combination.

Figure 3 and Table 3 show that the lactating female is

I I0 12 14 16 21 27 41

Postpartum age of mot lw~ in days

FIG. 3. Approach behavior of 16-day young to lactating females (LF) of different lactational ages when opposed to nulliparous

females (NF).

first chosen when she reaches about 14 days postpartum and remains the stimulus of choice until 21 days postpartum. Thereafter, her attraction wanes, to disappear completely by 41 days.

The results just described, together with those of Experi- ments 1 and 2, reveal a striking synchrony in the development of the pheromonal bond. They show, specifically, that the lactating female begins to emit the pheromone at about the time the young begin responding to it, and ceases to emit the pheromone at about the time the young cease responding to it.

Experiment 4

To this point we have used lactating females in combination with only nulliparous females, and under such test conditions we were able t o show the synchrony just referred to. However, there remains the possibility that the pheromone is not emitted in what thus far appears to be virtually an all-or-none fashion, but that once present, is emitted at different con- centrations by females of different lactational ages. To the extent that this is true, one might expect our standard 16-day lactating female, for example, to be chosen over a lactating female either 21-days or 14-days postpartum. Accordingly, we once again used females 1, 10, 12, 14, 16, 21, 27 and 41 days postpartum, but this time paired each with a 16-day lactating female rather than with a nulliparous female. The young against which such pairs were tested were in every case themselves 16 days of age.

TABLE 3

PREFERENCES EXHIat~O BY STANDARD 16-DAY YOUNG FOR A LACTATING FEMALE (LF) OF DIFFERENT LACTATIONAL AGES OR A NULLIPAROUS FEMALE (NF).

Choice Pup age N To LF To NF No choice Sig.*

Day 1 LF vs NF Day 10 LF vs NF Day 12 LF vs NF Day 14 LF vs NF Day 16 LF vs NF Day 21 LF vs NF Day 27 LF vs NF Day 41 LF vs NF

30 10 10 10 >0.05 J 30 16 14 0 >0.05

30 12 3 15 >0.05 30 19 7 4 <0.05

16 days 30 29 1 0 <0.01 30 30 0 0 <0.01 30 18 12 0 >0.05 30 12 15 3 >0.05

*Fisher Exact Probability Test was used.

686 LEON AND MOLTZ

TABLE 4

PREFEI~NCES EXHIBrrED BY 16-DAY YOUNG TO EITHER g 16-DAY LACTATING FEMALE (16-DAY LF) OR TO LACTATING FEMALES OF D I ~ , r r LACTATIONAL AGES (OTHER LF).

Choice Pup age N To 16 day LF To other LF Sig.*

Day 16 LF vs Day 1 LF "~ 30 29 1 <0.01 Day 16 LF vs Day 10 LF } 30 24 6 <0.01 Day 16 LF vs Day 12 LF 30 19 11 >0.05 Day 16 LF vs Day 14 LF 30 17 13 >0.05 Day 16 LF vs Day 16 LF 16 days 30 14 16 >0.05 Day 16 LF vs Day 21 LF 30 14 16 >0.05 Day 16 LF vs Day 27 LF 30 21 9 <0.01 Day 16 LF vs Day 41 LF 30 24 6 <0.01

*Fisher Exact Probability Test was used.

I t is hardly surprising that the 16-day lactating female was chosen over lactating females of 1, 10 and 12 days postpartum and over those 27 and 41 days postpartum (Fig. 4 and Table 4). The important point, however, is that the 16-day lactating female was not selected in preference to either the 14- or the 21-day lactating female. This suggests that during the period of the breeding episode that the pheromone is emitted, it is emitted, not at different concentrations by females of different lactational ages, but rather in all-or-none fashion, as suggested previously.

~ t Illlululll 16 DAY L F 3 0 ~ or.~n t ,

i: t., ,illi Ii Ii iii I I0 12 14 16 21 27 41

Pos t partum age of mother in days

FIG. 4. Approach behavior of 16-day young to either a standard 16-day lactating female (16-day LF) or to lactating females of

different lactational ages (other LF).

DISCUSSION

The present series of studies has revealed the existence of a striking synchrony in the development and subsequent dissolution of the pheromonal bond in the albino rat. On the one hand, this synchrony is expressed in the fact that the pheromone is first released by the mother at about 14 days postpartum, coincident with the age the young first become responsive to the pheromone. On the other hand, it is seen in the fact that at about 27 days postpartum, the mother ceases to release the pheromone, which corresponds, in turn, to the age at which the young cease to be attracted to the pheromone.

Given these parameters relevant to the initial appearance and subsequent disappearance of the pheromonal bond, it is of interest to speculate about the role such a bond might play in synchronizing the mother-young relationship.

Moltz and Robbins [2], among other investigators, have pointed out that the young begin to leave the nest when they reach about 14 days of age at which time there occurs also a sharp decline in the incidence of maternal retrieving. Since nursing, however, does not decrease at 14 days post- partum, but indeed continues for yet another two weeks, mother and young have still to be reunited periodically. The release at this time of a maternal pheromone and the appear- ance, in turn, of a responsiveness to that pheromone on the part of the young would seem eminently suited to assure such a reunion. On the other hand, the young need eventually to be weaned, with their attraction to the mother terminated accordingly. Weaning occurs characteristically at about 27-30 days postpartum [2], and it may be more than coincidental that this is precisely the age at which the mother ceases to emit the pheromone and the young cease to be attracted to it.

REFERENCES

1. Leon, M. and H. Moltz. Maternal pheromone: Discrimination by pre-weanling albino rats. Physiol. Behav. 7: 265-267, 1971.

2. Moltz, H. and D. Robbins. Maternal behavior of primiparous and multiparous rats. J. comp. Physiol. Psychol. 60: 417-421, 1965.