Establishment of Pheromonal Bonds and Diet Choice in Young Rats

by Odor Pre-Exposure’

Stimulu\ novclty Slolhcr-young interaction

f)..jK f’LKfl:K I’ rats t>picall) emit an odor highly atlracllw

IO their I4 I(, 27 da!, old young (IX]. Thih alfractant.

which has hwn t~~rmed maternal pheromone. is synthe\i/cd

hy caec.~l bactcm and rmittrd in rhc anal cxcreta 01

lactating Icmalc rats during the third and fourth wwk\

t’ollouing part uritmn 1 I S, I H 1 ,Although the cauws of the Initiation and tcrrnination 01

cucrction of maternal pheromone by the parlurlcnt dam

twtc txxn srudictl III some detail. relativctv litrle IS known

ahout the dc\clopriicnt ot rcqww to thjs pheromone in

pups. Availrlt>lC information strongly supgcsts I hat Itic drveloprn~nr ot’ pups’ tcndzncy to approach Ihc ~111~11 01

their mother is dependent on cvposurc lo nndtcrnat odor

c’arly in lit’?. f;or exainplc. pups rcared by darns ni;linfaincd

on 3 dir,1 which fails 10 provide ;I suhs,tratc suft’i~icnt for

coccal riI~crohI;il fun&ion and Lvhich, thercforc. supprcwb

phcrornonc \ynthcsis. do no1 cxhihit any tcnticncy to

approach Itic odor of niothcrs ingesting ;I normal (list which 15 highly aflracllvc to thclr ot\n young [ IS]. ITllrIIIc’rn~c~rt~.

pups i-camI 1)~ Inolhcrs not cinirtin g maternal phcrorllonL~.

hul hou6c‘J In ;i colon! room iontaininf icnlaks cniitting

phcroinonc. will sllbscqucrllly approach the anal cyc‘rcI;i ot 111~ phcromonc producing anilll~ls I 161 It has alw I,scn

tound th;ll phcromcmc producing mother rats cniit quati~;l-

tiv?ly diffcrcnl attractant\ 2s 3 tunction of their diet and

pups shou a sIrmy prefcwncc only for the ;Inal c’xcrcta oi

their own tl;ini or other siriiilarl) Ied t;lctating fem3lr’s ( I (11

388

normally neither attracted to her odor nor to the odor ofoth- er mothers maintained on a diet sufficient to permit maternal

pheromone production [ 15.161

Animals and Procedure

Twelve pregnant Wistar rats were placed in individual sealed containers (30.5 cm dia., 14 cm height; Tupperware) several days prior to parturition and maintained on ad lib water and a diet containing sucrose as its only constituent carbohydrate (diet composition in g/kg: sucrose 584.4, casein 21 1.0. cellulose 104.5. corn oil 50.0, salt mix 40.0. vitamin mix 10.0, Teklad Mills, Madison. Wisconsin). Filtered air (30 mljmin) was passed into each container through a I .5 cm hole and allowed to exit via 3 similar holes to maintain a constant air flow. Bedding (Sani-chips: Animal Laboratory Diets, Derby, K.Y.) was changed and the container washed on Day 20 of gestation. After parturition each litter was culled to 8 pups.

From Day I to Day I9 postpartum pups were taken from their mother for 3 hriday and placed in an cxposurc apparatus. similar to their home unit. but divided inro 8 wedge-shaped sections each of which held a single, isolated pup. Pups from S litters received filtered air while in the exposure apparatus, while those from 7 other litters received filtered air which had passed through a sealed container holding a 16 - 2 I day postpartum Wistar female maintained on Purina Laboratory Chow. Such females havr previously been shown to produce anal excrcta highI> attractive to pups which they rear [ IS.171 On Day 20 postpartum. the mother of each litter was removed from INS home unit. and 3 hr later her pups were tested in an olfactory discrimination apparatus described helow to determine their response to the odor of the anal excreta 01 Day I6 -?I postpartum lactating Wistar rats maintained on Purina Laboratory Chow.

The olfactory discrimination apparatus. described in detail elsewhere [ 171. consists of a start box, leading to a triangular open field. ending in a shallow cliff beyond which lies two visually concealed goal compartments. Filtered air (55 literslmin) passing from a central source through each goal compartment permits delivery of olfac- tory stimuli from the goal compartment to the subject and provides a 72 db masking noise. Individual pups were placed in the start box of the olfactory discrimination device and allowed I5 min to choose between goal compartments. A choice was defined as a descent of the cliff to the entrance of one of the two goal compartments. In the present experiment. anal excreta were collected from a I6 -2 1 day postpartum female maintained on Purina Laboratory Chow after the female had been isolated in a stainless steel cage for 3 hr. The material was thrn transferred to a disposable plastic weighing dish and placed in one goal compartment: an empty plastic weighing dish was placed in the other.

For purposes of statistical analysis, the proportion 01 each litter choosing the compartment containing anal excreta was calculated and these proportions formed the basis for comparisons between groups of litters f I 1.

IIFSIJLW .ANI) I)IS(‘CSSION

Figure 1 describes the percentage of pups choosing the goal box containing maternal excreta. those choosing tht‘

I I,O\. c,.ILt:t. :\Sl) Bt:tlSt

MATERNAL EMPTY PHEROMONE GOAL

BOX

I

cl NO

CHOICE

WWW~ COWROL PRE-EXPOSED

FIG. I. Pcr~enl of pups prec\poscl 10 the &or 01 rnatcrndt pheromone or preexposed 111 ii clean 3it stream choosing maternal pheromone or ~111 empty goal bol in the olfactory discrimination

.ipparatus.

cmply goal box, and those making no choler: in the olfactory discrimination apparatus. AS is clear from exami- nation of the figure. pups which had been prr-exposed to the odor of a lactating female were far more likely to choose the goal box containing maternal excreta than pups

lacking such pre-exposure (Mann-Whitney I! Test. (7 = 0: p<O.OOS). The proportion of pups exposed tv maternal pheromone in Isolation which approached the odor of the anal excrcta of a lactating female in the olfactory discrim- ination apparatus (0.79) was comparable to the proportion of pups (0.83) actually reared hy dams maintained on Purina Laboratory Chow. previously found IO choose the odor of the anal excreta of a lactating female in the test situation (cf: 1 151 ). These data suggest that simple expo- sure of pups to the maternal pheromone in the absence of any cues related to the mother art’ sufficient to support the development of normal responsiveness to maternal odor.

EXPERIMENI 2

While the naturally occurring odors synthesized by dams are limited to the odors produced by caecal micro- organisms, the possibility exists that the weanling rat will respond to any pre-exposed odor by approaching it. Previous investigators have shown that the young of a number of alrricial and precocial rodent species will exhiblr increased responsiveness to non-biologically significant odors following early exposure 14. 5, 7; I’). 20. 3 I. 231.

80 1

389

I R cl PEPPERMINT EMPTY NO

ODOR GOAL CHOICE BOX

390

Pups from 6 litters had been exposed, in isolation, to the odor of peppermint extract 3 hr/day in the exposure apparatus and those from 5 litters to a clean airstream under the same conditions.

On Day 21 postpartum 24 pups which had been exposed to an air-stream containing peppermint odor and 20 pups previously exposed to the control air-stream were individu- ally offered samples of lemon-flavored and peppermint- flavored sucrose based diet in a test apparatus for 24 hr.

Lemon flavored diet was prepared by spraying a solution of I2 ml pure lemon extract (Bowes Co. Ltd.. Toronto) in I2 ml of ethyl alcohol into I kg sucrose based diet with an atomizer and thoroughly mixing the combined ingredients. Peppermint flavored diet was similarly prepared by mixing a solution of I5 ml peppermint extract (Reckitt and Coleman Ltd., Lachine, Quebec) in I? ml of alcohol with I kg of sucrose based diet.

The test apparatus, designed so as to minimize diet spillage, consisted of a plastic dish. 22 cm dia.. 6.6 cm deep. with two detachable food cups mounted 180” apart on the exterior of the plastic dish. Detailed description of the test chamber is to be found in Galef and Henderson [ 12 I. Water was available throughout testing in a shallow dish placed in the center of the apparatus. Intake of each diet was determined hy weighing food cups.

To control for any litter effects, data from all four pups in each litter were combined and statistical analysts carried out using the total intake of each litter as a single data point [ I ]

KESUI.TS AND 1)ISCUSSION

The main results of Lxperiment 3 arc presented in Fig. 3. As is evident from examination of the figure, litters 01 pups pre-exposed to the smell of peppermint extract showed an enhanced intake of peppermint-flavored sucrose- based diet in comparison with control animals (Mann- Whitney U Test, U = 0, p<O.O05). All 6 litters of pups pre-exposed to the smell of peppermint exhibited a preference for peppermint flavored food during a 24-hr diet selection test, while all five control litters preferred the lemon flavored diet. Thus, simple exposure to the smell ot a diet is sufficient to produce a strong preference for that diet at weaning.

GENERAL DISC‘IJSSIOS

A problem of long standing in the study of behavioral development concerns the mechanisms underlying the ontogeny of the approach and avoidance responses which young organisms exhibit with respect to external sources of stimulation. As in the present studies, particular attention has been focussed on the development of approach re- sponses to conspecifics. a possibly necessary precurser to the occurrence of normal social behavior in vertebrates. Previous reviews of the field have indicated the importance in the emergence of approach behavior in the neonate, of phylogenetically evolved sensory-affective systems, of em- bryonic sensory stimulation, and of postnatal associative and perceptual learning of various kinds 13, 14, 27. 28. 291.

The results of the present series of experiments indicate that simple postnatal exposure of young mammals to either a naturally occurring or arbitrary stimulus, in the absence of any obvious reinforcement, is sufficient to enhance

LEMON FLAVORED DIET

FIG. 3. Mean grams of peppermint- anti lemon-tlavored diet eatr~r hy litters of pups me-exposed to peppermint odor or 3 clean air

ttrcarn.

markedly the attractiveness of that stimulus in both affiliative and feeding situations. Extrapolation from these findings suggests a simple non-associative mechanism sup- porting the development of approach responses in juvenile mammals. We would propose that, as a general rule, simple familiarity with a given stimulus will enhance the relative attractivrness of that stimulus to a young animal.

Similar generalizations have been proposed previously both to explain neonatal behavior and certain aspects of diet selection by adults (See for example, [8, 28, 291) but in the case of neonatal altricial mammals, at least. have received little direct experimental study. In the usual paradigm for the investigation of the formation of attrac- tion to stimuli as a result of exposure, stimuli are presented to the neonate in the presence of the dam and litter mates. Thus, the possibility of conditioned positive affective responses to an introduced stimulus typically have not been excluded 161.

The results of the present study support the contention that exposure to a stimulus in the absence of any reinforcement is sufficient to enhance the relative attrac- tiveness of that stimulus. Described in these terms. the observed behavior of our subjects IS not easily interpreted within the framework of conventional models of behavior.

I’lIt~RMo\‘:~L HOSl)S ,\I\I) l)lF:‘f’ (‘110lU:

;llft’rnatJvcly. it is possihlc that for youllf man~mals. 35 for

~IdUllS 13.22 I . nt)vcI stimuli arc inherently awrsivc. I’lac~‘-

mcnt 01 ;I suhjcct in 3 test situation, to which it has not

Ikxn previously ruposrd. introduces that individual to 2

wcaltli 01‘ nwcl. and rhL%rcforc. avcrsiw stimuli. In such 3

5iJualioJJ. rhaJ portion of the tc’st environment confaining 5alicnt. t’;imiliar cuts would bc lcasr novel and consrqucntly

least ;i\cr\i\:c. I‘hc young organism Jriighl hc espcctcd lo

5cck out the souric 01‘ tliniiliar cues in the novel CJlviroJl-

mcnl ot the tcsl situation to rcducc ils cxposurc to

unfclnilliar anA. hcnL,c, aversi\c cxtcroccptiw stiJllulalioJ~.

In Lcrrn$ 01’ [his niodcI. llis apparc‘nt attraclivenrs\ ot lhr

tamiliar 2tiniulu.s is rclnlt,rprctLd 3s a rctluccd un;itlrilcli\c-

nc5’r ot th,~r stimulw in dn t,nvironnient cc~~~~posc‘tl 01’

util’;iJilili;Jr. crnd thcrcforc 3vcr5ivc ~~lcrncnts. Such r-cdu~,cd

REFERENCES