Amniotic fluid can act as an appetitive unconditioned stimulus in preweanling rats

Amniotic Fluid can Act as anAppetitive UnconditionedStimulus in Preweanling Rats

Carlos AriasDepartment of Psychology, Binghamton

University—SUNY, P.O. Box 6000Binghamton, New York 13902-6000

E-mail: [email protected];[email protected]

M.G. ChotroFacultad de Psicologıa

Universidad del Paıs Vasco UPV/EHUSpain ABSTRACT: Studies in humans and animals indicate that exposure to flavors in

the amniotic fluid during the later gestational period may induce preferences forthose flavors. Considering that during the last prenatal period the amniotic fluidcontains substances that activate the opioid system, and that this system plays acritical role in the acquisition of olfactory preferences early in life, it has beenhypothesized that the amniotic fluid may acquire appetitive unconditionedproperties during this period. This has been tested in an experiment in whichpreweanling rats were exposed to alcohol odor (CS) paired or unpaired with theintraoral infusion of amniotic fluid (US) collected on gestational day 20. Thepairing of these two stimuli induced an enhanced palatability of alcohol’s flavor aswell an increased intake of the drug. These results support the idea that amnioticfluid acquires appetitive unconditioned properties during the last days of gestationand suggest that associative mechanisms involving the amniotic fluidcould be underlying odor and taste preferences acquired through fetal exposure.� 2007 Wiley Periodicals, Inc. Dev Psychobiol 49: 139–149, 2007.

Keywords: amniotic fluid; rat; ethanol; classical conditioning

INTRODUCTION

In humans, as in other mammals, innate preferences for or

aversions to certain flavors have been observed. In

general, during early ontogeny there is a preference for

sweet flavors, which are generally related to a high caloric

value, and an aversion towards bitter flavors that often

indicate toxicity (Hudson & Distel, 1999). However,

beyond these innate tendencies, there also seems to be a

sensitive period during early development in which

experiences with flavors produce enduring preferences

that ensure the acquisition of important information

about which foods are safe, appropriate, and nutritious

(Mennella & Beauchamp, 1998).

Several studies conducted with both animals

(Altbacker, Hudson, & Bilko, 1995; Arias & Chotro,

2005b; Bilko, Altbacker, & Hudson, 1994; Capretta &

Rawls, 1974; Dominguez, Lopez, & Molina, 1998;

Hepper & Waldman, 1992; Mennella & Beauchamp,

1998; Smotherman, 1982; Sneddon, Hadden, & Hepper,

1998) and humans (Mennella, Jagnow, & Beauchamp,

2001; Schaal, Marlier, & Soussignan, 2000) have found

that exposure to different tastes and odors during early

development can generate subsequent preferences for

these stimuli. In these studies, however, it is not clear

whether this preference is just the result of mere exposure,

or if certain associative processes are also involved. Odor

preferences can be acquired readily in rats without explicit

reinforcement (Caza & Spear, 1984; Hudson, 1993),

which probably helps the pup to become familiarized with

its environmental odors (Hudson & Distel, 1999). In other

cases, different sources of stimulation related to maternal

care strengthen the acquisition of olfactory preferences,

for example, thermo-tactile stimulation (Alberts & May,

1984). Milk and amniotic fluid are maternal-related fluids

that may also participate as unconditioned stimuli in

the acquisition of these preferences. Both fluids seem

able to induce appetitive unconditioned responses such as

mouthing movements and inhibition of fetal aversive

responses to sensory stimuli, like facial wiping. In

Received 11 April 2006; Accepted 10 November 2006Correspondence to: C. AriasPublished online in Wiley InterScience

(www.interscience.wiley.com). DOI 10.1002/dev.20205

� 2007 Wiley Periodicals, Inc.

addition, milk is able to induce an increase in rear limb

activity as part of a general reorganization in motor

behavior, an expression of the fetal stretch response

(Koffman, Petrov, Varlinskaya, & Smotherman, 1998;

Korthank & Robinson, 1998; Robinson & Smotherman,

1994). Furthermore, these fluids serve as vehicles through

which the fetus and the newborn are exposed to a variety of

chemosensorial stimulation (Mennella & Beauchamp,

1996; Mennella et al., 2001). Therefore, it is possible

that the fetus or the infant rat associates the different

odors or flavors presented in the amniotic fluid or milk,

respectively, with the reinforcing properties of such fluids.

To a certain extent, this association may play a role in

the development of preferences for the olfactory and

gustatory stimuli included in the mother’s diet during

gestation or lactation found in several studies (Galef &

Henderson, 1972; Galef & Sherry, 1973). Evidence

supporting this hypothesis is that milk can act as an

appetitive US in different procedures of classical

conditioning (Cheslock, Varlinskaya, Petrov, & Spear,

2000; Nizhnikov, Petrov, Varlinskaya, & Spear, 2002;

Robinson, Arnold, Spear, & Smotherman, 1993).

To our knowledge, the possibility that amniotic

fluid can act as an appetitive US has not been clearly

demonstrated. Nevertheless, there is evidence from

studies with humans and animals that indirectly supports

this hypothesis. First, during its intrauterine life the fetus

swallows and inhales large amounts of amniotic fluid

(Printchard, 1965; Ross & Nijland, 1997, 1998). The

intake of this fluid increases in the presence of some

flavors (De Snoo, 1937, cited by Ross & Nijland), which

may facilitate the processing of those stimuli. It is known

that a variety of substances included in the mother’s diet

reach the amniotic fluid (Abate, Pepino, Dominguez,

Spear, & Molina, 2000; Dominguez, Lopez, Chotro, &

Molina, 1996; Mennella, Johnson, & Beauchamp, 1995;

Nolte, Provenza, Callan, & Panter, 1992); and also that

prenatal experience with several chemosensory stimuli

increases their acceptance after birth (Abate, Spear, &

Molina, 2001; Arias & Chotro, 2005b; Bilko et al., 1994;

Capretta, Petersik, & Stewart, 1975; Dominguez et al.,

1998; Mennella et al., 2001; Smotherman, 1982). There is

some evidence to indicate that the amniotic fluid itself

induces unconditioned responses of appetence and

orientation. Human newborns show an orienting response

towards amniotic fluid (Marlier, Schaal, & Soussignan,

1997, 1998; Schaal, Marlier, & Soussignan, 1995, 1998)

and a preference for nipples that have been spread with

this fluid (Varendi, Porter, & Winberg, 1996). It has also

been found that the odor of amniotic fluid can calm crying

induced by maternal separation (Varendi, Christensson,

Porter, & Winberg, 1998).

In studies with rats it has also been found that amniotic

fluid guides the newborn towards the location of the nipple

during the first episode of breast-feeding (Mennella &

Beauchamp, 2002). Furthermore, pups show a preference

for nipples spread with an odor (citral) when this has been

added to the amniotic fluid before birth (Pedersen & Blass,

1982). It is interesting to indicate here that several studies

have shown that amniotic fluid contains substances

capable of activating the opioid system (Korthank &

Robinson, 1998; Kristal, Tarapacki, & Barton, 1990;

Kristal, Thompson, & Abbott, 1986). The amniotic fluid

collected during the last 2 days of gestation of the rat (GD

20–21) has been found to reduce facial wiping responses

induced by lemon (Korthank & Robinson), a behavior

expressed by the fetus in response to novel or aversive

stimuli which is known to be controlled by the opioid

system (Smotherman & Robinson, 1987). This same

effect has been observed when administering other

substances that activate the opioid system, such as milk

or morphine (Smotherman & Robinson, 1992a,b). In

general, there is evidence to indicate that the activation of

this neurochemical system plays an important role in the

acquisition of conditioned preferences both during the last

days of gestation (Arias & Chotro, 2005a; Arnold,

Robinson, Spear, & Smotherman, 1993; Chotro & Arias,

2003), and during the first days of postnatal life (Kehoe,

1988; Roth & Sullivan, 2001, 2003).

Taking into account all these antecedents, one may

expect the amniotic fluid to act as an appetitive US not

only during the intrauterine life, but also after birth. The

objective of the present experiment is to study this

possibility with a paradigm of classical conditioning in

preweanling rats. Ethanol odor was selected as the CS, in

light of previous studies demonstrating that preweanling

rats readily transfer conditioned learned responses from

the odor to the taste of ethanol (Molina, Hoffmann, &

Spear, 1986; Molina, Serwatka, Spear, & Spear, 1985;

Molina, Serwatka, & Spear, 1984, 1986; Serwatka,

Molina, & Spear, 1986). The hypothesis that guides this

study is that the association between the odor of ethanol

and the intraoral infusion of amniotic fluid will enhance

the palatability of ethanol’s flavor and will also increase

consumption of the drug.

METHOD

Subjects

Subjects for this experiment were 39 preweanling Wistar rats

(19 males and 20 females) derived from 7 litters. Pups were born

and reared in a temperature-controlled vivarium at the University

of the Basque Country (Spain). The colony room was maintained

on a 12-hr light/12-hr dark illumination cycle, with light onset at

8 a.m. Female rats were time-mated to provide subjects for this

study and were housed individually in standard maternity cages

with continuous access to food (Panlab, Barcelona, Spain,

Developmental Psychobiology. DOI 10.1002/dev140 Arias and Chotro

maternity formula) and water. In all experiments the European

regulations for care and treatment of experimental animals

were followed, and procedures were controlled and approved

by the Diputacion Foral de Gipuzkoa (Spain), in compliance

with the European Communities Council Directive (86/609/

EEC).

Procedures

Amniotic Fluid Collection. The US used in this experiment

was amniotic fluid collected on GD 20. To obtain this fluid,

pregnant rats were put down in a CO2 gas chamber.

Subsequently, a ventral laparotomy was performed and the fluid

was extracted from the amniotic sacs, placed in sterilized tubes

and frozen at �20�C until conditioning day. On conditioning

day, the fluid was defrosted to room temperature and placed into

the syringes used for its intraoral administration with an infusion

pump.

Conditioning Trials. On PDs 12 and 13 pups were separated

from their mothers, and distributed randomly into three groups,

Paired (n¼ 14), Unpaired (n¼ 13), and Nonexposed (n¼ 12).

Each group was placed separately in a holding chamber

(15� 8� 15 cm) maintained at 28–30�C with heating pads.

After 15 min, pups from the Unpaired group were exposed to the

ambient odor of ethanol, the CS. To carry out this exposure, a

cotton piece (4� 2 cm) was saturated with 5 mL of ethanol (96%

v/v) and attached to the internal surface of the cover of the testing

chamber. Fifteen minutes later, all pups were intraorally

cannulated using a procedure described in previous studies

(Hall & Rosenblatt, 1977). Cannulae are made from 5-cm

sections of polyethylene tubing (PE 10, Clay Adams) and

one end of the section is heated to form a small flange. A thin wire

attached to the nonflanged end of the cannula is placed on the

internal surface of the pup’s cheek and the wire is then pushed

through the oral mucosa until the flanged end of the cannula is

positioned over the internal surface of the cheek while the

remainder of the cannula exits from the oral cavity. The entire

procedure takes less than 5 s per pup and induces minimal stress.

These cannulae were later used to infuse the amniotic fluid

during the conditioning trials.

Two hours later, the bladders of pups from the Paired and

Unpaired conditions were voided by gentle brushing of the

anogenital area and their body weights were registered.

Immediately, these pups were placed in individual clear

Plexiglas chambers (15� 8� 15 cm) maintained at 28–30�C

with heating pads. Then, pups received intraorally the infusion of

amniotic fluid (US) delivered through an infusion pump (KD

Scientific, New Hope, PA) connected to the oral cannula of each

pup by a polyethylene catheter (PE 50, Clay Adams). The

volume administered to each subject’s mouth during this trial

was equivalent to 4% of their body weight and was infused with a

continuous flow during 15 min, with pups being able to either

consume or reject the infused solution. During these trials, pups

from group Paired were exposed simultaneously to the odor of

ethanol using a procedure similar to the one described for group

Unpaired. At the end of the session, postinfusion weights were

registered. Immediately after the conditioning trial, all pups

were returned to the home cage.

The intake of amniotic fluid was expressed as a percentage of

body weight gain after the administration of the ethanol solution,

using the following formula:

½ðpostinfusion weight � preinfusion weightÞ=preinfusion weight� � 100

Taste Reactivity Test

On PD 14 all pups were evaluated using an ethanol taste

reactivity test. Pups were separated from their mothers, placed in

heated holding chambers and intraorally cannulated, following

the procedure described previously. Three hours later, pups’

reaction to the taste of a 6% ethanol solution (v/v) was tested

following a procedure already described in previous studies

(Arias & Chotro, 2005a,b). The evaluation was conducted in a

trapezoid-shaped chamber with a front wall (29 cm wide)

made of clear glass and the remaining walls (back 18 cm, sides

11.5 cm) and floor made of mirror, so as to allow observation of

the pups’ orofacial expression and body movements in any

position. The chamber was 12.5 cm high and was divided into

two equal sections. Two pups were evaluated at a time, one per

section of the chamber. Pups remained in the test chamber for

2 min before the start of the intraoral infusions of ethanol; this

was considered the baseline period. Intraoral infusions were

performed using procedures similar to those described for

the conditioning trials, with two exceptions: the total volume

administered to each subject was reduced to 2.5% of their body

weight and it was infused in five 15-s pulses, with the onset of

each pulse separated by 2 min. The duration of the infusion test

was 10 min, preceded by a 2 min baseline period. During this

period pups were videotaped for subsequent analysis of their

behavior. Once the entire litter had been tested, pups were

returned to the maternal cage.

Based on previous studies using taste reactivity tests with

infant rats (Arias & Chotro, 2005a,b, 2006; Chotro, Kraebel,

McKinzie, Molina, & Spear, 1996; Hall & Bryan, 1981; Vigorito

& Sclafani, 1988) and adult rats (Grill & Norgren, 1978; Parker,

1988; Parker, 1995), as well as on our own observation of the

pups’ reaction to the taste of ethanol, the following behavioral

measures were selected as dependent variables under analysis:

general activity and wall climbing, considered here as aversive

reactions, and mouthing and paw licking, considered here as

ingestive/appetitive responses.

The behavioral measures were analyzed from the videotapes

and were scored every 30 s. General activity was rated in the

seven categories described by Hall & Bryan (1981): 0¼ no

movement, except for occasional twitches; 1¼ slight movement,

usually of the head or paw, sustained for 5 s; 2¼ substantial

movement of the head and paws, including grooming, but no

locomotion; 3¼ locomotion involving forelimbs and often

including rooting and probing but with hind limbs motionless

and usually serving as a pivot; 4¼ clear and sustained

locomotion about the test container; 5¼ vigorous locomotion,

often including rolling, kicking, and wall climbing; 6¼ an

extreme, but occasionally observed, form of rating five in which

the pup tumbled about its container for most of the 30 s interval,

locomoting, rolling, probing, wall climbing and jumping.

Developmental Psychobiology. DOI 10.1002/dev Amniotic Fluid as an Unconditioned Stimulus 141

A pup was considered to be wall climbing when standing on

its rear limbs with its forepaws against the wall of the testing

chamber. Time in seconds for wall climbing was registered for

each pup every 30 s during the baseline and test intervals.

Mouthing was defined as any obvious movement of the mouth

and jaws, and the total time (seconds) was also registered during

the testing trials. Mouthing and paw licking were independent

measures, that is, mouthing movements displayed during

paw licking were not measured as mouthing. Since mouthing

and paw licking are behaviors directly related to the intraoral

infusion of the liquid, no data were obtained during baseline

periods for these variables, with baseline data therefore

only being analyzed for general activity and wall climbing. As

with general activity, all these behaviors were scored every 30 s.

So, for general activity and wall climbing there were data for four

30-s baseline bins followed by twenty 30-sec infusion bins,

whereas for mouthing and paw licking there were data for only

twenty 30-sec testing bins. All behavioral observations were

performed by a trained researcher blind to the experimental

conditions.

Ethanol Intake Test

On the 2 days following the taste reactivity test (PDs 15 and 16)

all pups were tested in terms of ethanol intake. In the morning of

the corresponding day, pups were separated from their mothers,

placed in a holding chamber (15� 8� 15 cm) and intraorally

cannulated, as described above. The procedures used for this

ethanol intake test were similar to those described for the

intraoral infusion of amniotic fluid, except for the solution

infused (ethanol 6% v/v) and the volume administered to each

subject (5.5% of their body weight). The dependent variable

analyzed was ethanol intake expressed as a percentage of body

weight gained during the test.

Data Analysis

The effect of conditioning (Paired, Unpaired, and Nonexposed)

on body weights on PD 14 was analyzed with a one-way analysis

of variance (ANOVA).

Consumption data from the conditioning trials were analyzed

with a 2 (Conditioning: Paired and Unpaired)� 2 (Day) mixed

ANOVA, while data from the ethanol intake test were analyzed

with a 3 (Conditioning: Paired, Unpaired, and Nonexposed)�2 (Day) mixed ANOVA. Wall climbing data during baseline

trials were analyzed separately from the remaining infusion

trials with a 3 (Conditioning: Paired, Unpaired and Non-

exposed)� 4 (Bin) mixed ANOVA. Mouthing, paw licking, and

wall climbing data collected during the 20 test bins were

analyzed with a 3 (Conditioning: Paired, Unpaired, and

Nonexposed)� 20 (Bin) mixed ANOVA. Significant effects

were further explored using Tukey HSD post hoc tests and

repeated measures were compared using t-test for dependent

samples. Since general activity scores are ordinal data,

nonparametric tests (Kruskal–Wallis ANOVA and Mann–

Whitney U-test) were used. A rejection criterion of p< .05

was adopted for all the analyses presented in this study.

RESULTS

Body Weights

No differences between groups were detected when

comparing body weights on PD 14 (mean 29.18 g,

standard deviation¼ 1.54).

Amniotic Fluid Intake

The consumption of amniotic fluid during both condition-

ing trials is presented in Figure 1. As can be observed, the

intake of amniotic fluid increased on the second day of

conditioning. However, this effect was observed mainly in

group Paired, which showed a higher intake of amniotic

fluid when compared to group Unpaired. The ANOVA

showed a significant effect of the main factors Condition-

ing [F(1,25)¼ 6.15, p< .05] and day [F(1,25)¼ 25.53,

p< .001], indicating that pups ingested more amniotic

fluid on the second conditioning trial, and that pups from

group Paired consumed more than those from group

Unpaired. The interaction between both factors was

nearly significant [F(1,25)¼ 4.22, p¼ .051]. Planned

comparisons indicated that during the second trial, group

Paired consumed significantly more amniotic fluid than

group Unpaired [F(1,25)¼ 7.61, p< .05].

Taste Reactivity Test

Figure 2 presents the mean duration of mouthing response

to ethanol during the twenty 30-sec scoring intervals as

a function of the conditioning treatment. As can be

observed in this figure, groups Paired and Unpaired

displayed more mouthing than group Nonexposed during

all infusion bins. During the last ones, however, group

Paired showed more mouthing than the other two groups.

It is important to note that there was a peak in mouthing

response during bins 2, 6, 10, 14, and 18. These bins

correspond to the 30 s immediately following the offset of

the intraoral infusions of ethanol. This peak in mouthing

response was observed mainly in groups Paired and

Unpaired, and seems to be habitual across the session for

groups Unpaired and Nonexposed, although not for group

Paired. The ANOVA confirmed these observations with a

significant effect of Conditioning [F(2,26)¼ 5,77,

p< .01], Bin [F(19,494)¼ 9.13, p< .001], as well as the

interaction between both factors [F(38,494)¼ 1.70,

p< .01]. Post hoc analyses of the main factor Condition-

ing revealed that pups from group Paired showed

significantly more mouthing than those from group

Nonexposed, while group Unpaired did not differ from

the other groups. Post hoc analysis of the interaction

indicated that during bin 1, groups Paired and Unpaired

showed more mouthing than group Nonexposed; during


Developmental Psychobiology. DOI 10.1002/dev

FIGURE 1 Amniotic fluid intake (percentage of body weight gain) during the two conditioning

trials as a function of the conditioning treatment (Paired or Unpaired). Vertical bars represent standard

error of the mean.

FIGURE 2 Mean mouthing data (in seconds) in response to the ethanol’s taste as a function of the

conditioning treatment (Paired, Unpaired or Nonexposed). Vertical bars represent standard error of the

mean.

Amniotic Fluid as an Unconditioned Stimulus 143

bin 14, group Paired showed more mouthing than group

Nonexposed; and that during bin 18, group Paired differed

from both groups. The post hoc t-test was only conducted

on those bins in which a peak in the response was observed,

since this analysis provides important information about

changes in mouthing response across the entire TRT

session. These analyses revealed that group Nonexposed

displayed more mouthing during bin 2 than during bins 10,

14, and 18, and also during bin 6 than during bins 14 and 18.

Group Unpaired only showed significant differences when

comparing bins 2 and 6 to bin 18, which seems to indicate a

lower habituation rate to ethanol in this group than in group

Nonexposed. However, when analyzing the response of

group Paired, it appears that even less habituation occurs if

we consider that only during bin 2 did pups engage in more

mouthing than during bin 14.

As was the case with mouthing, in some bins group

Paired showed more paw licking than the other two groups

(Figure 3). However, the analysis of this variable did not

reveal significant differences between groups. It is also

interesting to note that in some bins, pups displayed a peak

in this behavior, as was the case with mouthing. However,

with this index the higher levels are displayed during

the second and third bins after each infusion pulse. The

ANOVA revealed only a significant effect of Bin

[F(2,19)¼ 4.78, p< .001] and the Conditioning�Bin

interaction was close to significance [F(38,684)¼ 1.41,

p¼ .054].

Figure 4 summarizes the mean general activity score

for this experiment. No clear changes across bins were

observed in this index. However, in some, group

Nonexposed showed higher activity levels than the

other two groups. The analysis indicated differences

between groups only during bin 6 [H(2, n¼ 39)¼ 6.34;

p< .05]. Group Nonexposed showed more general

activity during this bin than the Paired and Unpaired

groups (U¼ 44.5, p< .05 and U¼ 37.5, p<.05, re-

spectively).

The same pattern observed for general activity scores

was observed also when analyzing wall climbing in

response to the taste of ethanol (see Fig. 5). The ANOVA

indicated no significant effects on baseline, but when

analyzing wall climbing during the 20 infusion bins a

significant effect of Bin [F(19,684)¼ 3.43, p< .001] and

the interaction Conditioning�Bin [F(38,684)¼ 1.49,

p< .05.] was observed. The main factor Conditioning

produced an effect close to significance [F(2,36)¼ 2.94,

p¼ .066]. Further analysis of the interaction revealed

that group Nonexposed engaged in more wall climbing

than group Unpaired during bins 6, 9, 17, and 19, and than

group Paired during bins 6, 17, 18, and 19.

Ethanol Intake

Consumption of ethanol during both testing days is

depicted in Figure 6. As can be observed, pups from group


FIGURE 3 Mean paw licking data (in seconds) in response to the ethanol’s taste as a function of the

conditioning treatment (Paired, Unpaired, or Nonexposed). Vertical bars represent standard error of

the mean.

144 Arias and Chotro

Paired consumed more ethanol than those from the other

groups, and the intake of the drug for all groups was lower

on the second than on the first day. This descriptive

analysis was fully confirmed by the results of the ANOVA,

which indicated a significant main effect of Conditioning,

[F(2,36)¼ 11.50, p< .001], and Day, [F(1,36)¼ 4.41,

p< .05]. Post hoc analyses revealed that the ethanol intake

of group Paired was higher than that of the remaining


FIGURE4 Mean general activity scores of the pups in response to the ethanol’s taste as a function of

the conditioning treatment (Paired, Unpaired, or Nonexposed). Vertical bars represent standard error

of the mean.

FIGURE5 Mean duration (in seconds) of wall climbing of pups in response to the ethanol’s taste as a

function of the conditioning treatment (Paired, Unpaired, or Nonexposed). Vertical bars represent

standard error of the mean.

Amniotic Fluid as an Unconditioned Stimulus 145

groups, and that all groups reduced alcohol intake on the

second testing day.

DISCUSSION

The results of the present experiment demonstrate that

amniotic fluid can act as an appetitive US for infant rats.

Paired presentations of the odor of ethanol and intraoral

infusions of amniotic fluid produced a clear increase in

consumption and some changes in the palatability of

the drug. The unpaired presentation of both stimuli

also induced more mouthing and less wall climbing in

response to intraoral infusions of ethanol, when compared

to pups not exposed to these stimuli before the test. This

difference seems to indicate that mere exposure to the

odor of ethanol was enough to generate some preference

for the drug. The lack of an explicit US contingent with the

odor of ethanol in the Unpaired group during the

conditioning trials indicates that this preference results

from a nonassociative process (increase in the familiarity

or habituation of the neophobia), an effect that has been

described previously in infant rats (Caza & Spear, 1984).

Nevertheless, we cannot discard the possibility that

differences between groups Paired and Unpaired with

respect to group Nonexposed in the taste reactivity test

were due in part to groups Paired and Unpaired’s previous

experience with the intraoral infusion procedures during

conditioning.

In any case, neither lack of experience with the

procedure nor mere exposure to the odor of ethanol

account for the differences observed between groups

Paired and Unpaired, during both tests and conditioning.

During conditioning, the presence of the odor of ethanol

induced an increase in the ingestion of amniotic fluid in

group Paired; this effect could be explained in terms of

the results of studies that found that the presence of

familiar or preferred odors produces an increase in

ingestive responses during early ontogeny, both in humans

(Marlier & Schaal, 2005) and in animals (Kraebel et al.,

1993). The increase in consumption of amniotic fluid was

mainly observed during the second conditioning trial,

when the odor of ethanol may have acquired a certain

appetitive value after the contingent presentation of both

stimuli during the first conditioning trial. Nevertheless,

the lack of taste reactivity measures during the condition-

ing trials prevents us from drawing conclusions regarding

the cause of this increased consumption of amniotic fluid.

Another important difference between groups Paired

and Unpaired was also observed during the taste reactivity

test. In those measures considered as aversive indexes

(wall climbing and general activity), no differences

were observed between the groups, and both showed

lower scores than group Nonexposed. Nevertheless, when


FIGURE 6 Mean ethanol intake (percentage of body weight gain) as a function of the conditioning

treatment (Paired, Unpaired, or Nonexposed). Vertical bars represent standard error of the mean.

146 Arias and Chotro

analyzing the appetitive measures, group Unpaired,

although not group Paired, showed a decrease in mouthing

by the end of the test. This decreased mouthing, indicating

a habituation process, has been previously observed in

response to intraoral infusions of ethanol and other

solutions using this same procedure (Arias & Chotro,

2005b). The absence of this habituation effect in group

Paired allowed us to observe differences at the end of the

test between this group and group Unpaired. Furthermore,

as indicated above, group Paired showed not only

more mouthing in response to ethanol, but also a higher

intake of the drug. These differences in consumption and

ingestive responses can only be explained by the specific

treatment of group Paired, that is, the paired presentations

of the odor of ethanol and the intraoral infusion of

amniotic fluid, and the consequent appetitive conditioning

induced by this treatment. Considering all these results,

we can conclude that the amniotic fluid collected on GD

20 may act as an appetitive US in preweanling rats.

In this study, it is not possible to know if the amniotic

fluid acted as a primary or secondary reinforcer. As

mentioned before, amniotic fluid contains substances that

can activate the opioid system, and this neurochemical

system may be involved in the rewarding properties of

amniotic fluid (Korthank & Robinson, 1998). However,

we cannot be sure whether these substances act during

PDs 11 and 12 in the same way as during the fetal period,

considering the changes that occur in the opioid system

around PD 10 (Roth & Sullivan, 2003). It is possible also

that these opioid-activating substances may be degraded

after the procedure we used to collect and preserve this

fluid. If this were the case, in our experiment amniotic

fluid would most probably be acting as a secondary

reinforcer; in other words, its flavor may have acquired

rewarding properties in utero. Further research needs to be

done in order to investigate these possibilities. With the

present data we can conclude that amniotic fluid may

acquire appetitive unconditioned properties during the

last days of gestation.

It could be hypothesized that the preference for several

prenatally experienced substances described in several

studies (Abate et al., 2001; Arias & Chotro, 2005b; Bilko

et al., 1994; Dominguez et al., 1998; Mennella et al., 2001;

Molina, Chotro, & Dominguez, 1995; Schaal et al., 2000;

Smotherman, 1982), may be mediated by an associative

process. The presence of certain flavors in the amniotic

fluid seems to increase its ingestion (De Snoo, 1937, cited

by Ross & Nijland, 1997), which in turn may increase the

perception of the chemosensory aspects of these flavors.

During the last days of gestation it seems that the

composition of the physical characteristics of amniotic

fluid undergoes several physical and chemical

changes, and that during this period, this fluid may

activate the opioid system (Korthank & Robinson, 1998).

Considerable evidence indicates that this neurochemical

system plays an important role in the acquisition of

appetitive conditioned responses during early ontogeny

(Kehoe, 1988; Roth & Sullivan, 2001, 2003) as well as

during the last gestational days (Arias & Chotro, 2005a;

Arnold et al., 1993; Chotro & Arias, 2003). Therefore,

during this period of gestation, the fetus may associate the

flavors perceived in utero with the rewarding effects

produced by the ingestion of amniotic fluid, the latter

being mediated by the activation of the opioid system.

This hypothesis has not been supported in this study, and

remains open to future research.

The results of this experiment may also have implica-

tions for the analysis of the results found in other studies in

which the prenatal administration of a moderate ethanol

dose increased the preference for the chemosensory

properties of the drug (Arias & Chotro, 2005a,b; Chotro

& Arias, 2003; Dominguez et al., 1996, 1998; Molina

et al., 1995). This preference seems to be a conditioned

response mediated by the activation of the opioid system

(Arias & Chotro, 2005a; Chotro & Arias, 2003). In other

words, the fetus perceives the chemosensory properties of

ethanol in utero and, apparently, associates them with its

rewarding effects, mediated by the activation of the opioid

system. Nevertheless, it is not yet clear whether the

activation of this neurochemical system is induced by

ethanol, by amniotic fluid or by both.

NOTES

Carlos Arias was supported by the doctoral fellowship from

Gobierno Vasco (Programa de Formacion de Investigadores).

We thank Ana Medina and Oscar Vegas for their technical

assistance.

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Amniotic fluid can act as an appetitive unconditioned stimulus in preweanling rats