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Research article

Perinatal undernourishment and handling:


effects on olfactory discrimination in the
newborn rat
Marina Ruiz-Diaz, Carmen Torrero, Mirelta Regalado, Manuel Salas
Departmento de Neurobiologa del Desarrollo y Nuerofisiologia, Instituto de Neurobiologa,
Universidad Nacional Autnoma de Mxico, Campus UNAM Juriquilla, Quertaro, Mxico

The heart rates of rats aged 4, 7, and 12 days were recorded for control (CG), undernourished
(UG), control-handled (CHG), and undernourished-handled (UHG) groups after exposure to amyl
acetate and menthol. Pregnant dams were undernourished and received 50% of a balanced diet
from gestational day (G) 6 to G12, 60% from G13G18, and 100% from G19G21. On postpartum
days 012, pups remained for 12 h with a normal and 12 h with a nipple-ligated dam. The pups
were handled 5 min daily on days 112 of age to ameliorate the stress from undernourishment.
Basal heart rate, heart rate habituation, and the discrimination to a second odor were evaluated.
Body weight of UG and UHG rats was significantly reduced. Basal heart rate gradually increased
with age, especially in the CHG, without changes in the UHG; habituation was present and odor
discrimination was modified by the diet and handling. These data indicate that olfactory
discrimination is present at birth, and undernourishment does not impair but handling modifies its
functionality.
Keywords: undernourishment, handling, olfactory discrimination, habituation, rats

Introduction
In the nest environment, newborn rats may respond to
odors of the mother and nesting cage by reducing their
motor activity or by approaching their mother in a
maze in preference to either a non-lactating female or
a male. Olfactory experience may be critical for
suckling, protection, and warmth; later in development when walking emerges, the olfactory channel
plays a critical role as a basis to refine odor
discrimination and individual or species recognition
by pairing an olfactory stimulus with tactile, electrical
Correspondence to: M. Salas PhD, Departmento de Neurobiologa del
Desarrollo y Neurofisiologa, Instituto de Neurobiologa, Universidad
Nacional Autnoma de Mxico, Campus UNAM Juriquilla, Quertaro,
Qro. 76230, Mxico
Tel: +52 555623 4059; Fax: +52 555623 4001;
E-mail: masal@servidor.unam.mx
Received 11 October 2009, revised manuscript accepted 14 February 2010

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shock, visual or auditory stimuli to promote early


experience.15 This precocious physiological ability of
newly born rats is concurrent with a notorious
immaturity of the olfactory system (e.g. olfactory
glomeruli, mitral/tufted output cells, granule neurons,
and neurons of some amygdala nuclei, etc.), which
achieves its adult characteristics only at the end of the
third postnatal week.510 Olfactory discrimination
early in life coincides with the sensitive period of
enhanced neuronal plasticity in the olfactory bulb.
This activity is initially mediated by the maternal care
and thereafter by nest environmental odors that
induce important basic structural and functional
plasticity for the pups learning.5,1114
Several studies have developed the reliable crosshabituation paradigm to test odor discrimination in
immature animals by using the heart beat response,
where a novel odor is presented repeatedly to habituate
W. S. Maney & Son Ltd 2010
DOI 10.1179/147683010X12611460764408

Ruiz-Diaz et al.

the heart beat odor responses; then, a second novel odor


exposure (cross-habituation) induces a heart beat odor
response as a sign of odor discrimination to a new
odor.8,15 The reliability of this learning paradigm is based
on the high sensitivity of heart rate at neonatal ages to
chemosensory cues.1619 The absence of a linkage between
heart rate and motor activity and the fact that the
tachycardia response was not higher than the
bradycardia response until 12 days of age, makes the
latter a useful parameter to evaluate neonatal sensory
function.17,19,2024
Undernourishment throughout gestation and/or
suckling and the associated sensory deprivation in the
newborn rat provoke delayed sensory maturation and
permanent central nervous system (CNS) alterations.
These changes are notorious in structures undergoing
important changes in cell proliferation, migration, and
synaptic connectivity that closely correlate with longlasting deficiencies in discriminative, social, and learning
behaviors.12 Furthermore, these alterations also affect
olfactory structures (glomeruli profiles, mitral/tufted
output, granule neurons, amygdala and hippocampal
neuronal substrate), which delays the electrophysiological
development and interferes with odor discrimination and
long-term olfactory-guided behavior.9,10,2527
During the lactating period, the motherlitter bonds
are essential for the development of the newborn rats
physical, behavioral, cognitive, and endocrine responses to
stress.2830 Maternal care can be disrupted if the lactating
dam is exposed to stressful conditions.3135 Furthermore,
increases in the quality and/or quantity of handling given
to the pups may improve anatomical, electrophysiological,
and long-term adaptive behavioral responses.28,3644 In the
rat, early handling has been used as a tool to attenuate
morphological and behavioral damages associated with
perinatal undernourishment and sensory deprivation.41,45,46
The heart rate-orienting response to a new odor
paradigm has been used to investigate the development
of fine odor discrimination in infant rats.8 Although
odor discrimination performance is well documented in
adult altricial rodents, little is known about its
characteristics in pups undernourished during the preand neonatal periods of life, nor of the role of early
handling on their ability to olfactory learning suggested
by this paradigm.

Materials and methods


Animals
Four groups of 24 female and male Wistar rats (Rattus
norvegicus), descendants of a stock originally purchased
from Harlan Sprague-Dawley (Indianapolis, IN, USA),

Neonatal olfactory discrimination

and maintained in an automatically controlled room


at 23 2C, a 12-h light/dark cycle (lights on at 0700
h), with water and food (Purina chow) ad libitum were
used. For mating, a male was placed in a plastic cage
containing three females (200250 g). Sperm-positive
females were placed individually in plastic maternity
cages (50 40 20 cm) with wood shavings as nesting
material. The day after birth (day 1 of age), pups from
different litters were weighed and sexed, and four
females and four males in each litter were randomly
distributed among dams in order to minimize genetic
and nutritional differences and to give them equal
probability of development. Animal care and protocols
were approved by the University of Mexico INB
according to NIH guidelines. The four experimental
groups were as follows.
Control group (CG)

This group consisted of 24 female and male rats


obtained from 8 well-nourished litters, nursed by wellfed dams with free access to food (Purina chow) and
water. After birth, CG rats were fed by interchanging a
pair of normally lactating dams every 12 h between
litters as previously described.47 This experimental
procedure allows adequate nursing of pups and the
care given by the mother (body licking, retrieving,
thermal and tactile stimulation when crouching,
anogenital stimulation, etc.) when the dams were
interchanged between litters.
Undernourished group (UG)

This group contained a total of 24 both sexes (4


female and 4 male subjects/group) obtained from at
least 8 different litters. Mothers were undernourished
from gestational (G) day 6 to G12 with 50% of the
standard diet (Purina chow), from G13G18 with
60%, and from G19G21 with 100% of the same diet
until parturition to avoid re-absorption of the fetus or
cannibalism by the dam.47 The diet contains lipids
(59.81%), carbohydrates (12.3%), and proteins
(28.04%); L-lysine represents 1.42% of the diet, an
amount that surpasses the normal 1.38% nutritional
requirement for the rat. The calorific value calculated
for this diet (indicated) was 89.31 kcal per day (in the
case of) for a normal adult rat. This feeding schedule
was chosen because most of brain stem neurogenesis
and cardiac autonomic efferent projections occur
prenatally.48 After birth, prenatally undernourished
subjects were nursed for 12 h by a normal lactating
mother and 12 h by a subcutaneously nipple-ligated
foster dam, interchanged every 12 h (0800 and 2000 h)
between litters from postnatal days 112. In the
present study, 80% of the total undernourished rats
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were underfed during the light phase and the


remaining 20% during the dark phase; there were no
differences between these groups in the pups body
weight (F(2,28) = 0.2543; P = 0.77).
Control handled group (CHG)

Animals of this group consisted of 24 pups of both


sexes (4 female and 4 male pups/age group) obtained
from eight, well-nourished litters, nursed by a pair of
well-fed dams with free access to food and water as the
CG subjects; however, they also received a daily
handling massage for a 5-min period from days 112,
to evaluate the effects of the handling as a reference
for statistical comparisons. After 5 min of handling,
the pups were gently returned to their home cage.
Undernourished handled group (UHG)

This group included a total of 24 both sexes (4 female


and 4 male rats/age group) obtained from 8 different
litters experimentally treated as the UG pups but also
receiving experimental handling for a daily 5-min span
from days 112 of age under the same schedule and
environmental conditions as the CHG animals and
then returned to their home cage.
Early handling
Neonatal handling of pups was done by separating
them from their mothers 5 min prior to give them
gentle, individual handling that consisted of touching,
holding, shaking, and rubbing the pups for a 5-min
period from days 1 to 12 of age. These handling
sessions were carried out once a day between 1000 and
1200 h in a small plastic cage (30 23 15 cm) with 3
cm of wood shavings on the floor, which was
maintained at 28C with a circulating water heating
pad; the cage was in a sound-proof room separated
from the ambient noise of the main laboratory. At the
end of the handling treatment, the pups were returned
to their home cage, where they frequently received
extra stimulation from the dam (anogenital and body
licking, retrieving, and touching).49 Pups under all of
the different experimental treatments were maintained
with the mother in their habitat for at least 23 h
before the olfactory discrimination test began.
For all experimental groups, after parturition the
mother in each litter was kept on an ad libitum diet of
Purina chow and water at a temperature of 24 2C
and with 12 h of light per cycle (lights on at 0800 h).
Heart beat recordings and cross-discrimination to odors
Odor habituation-evoked heart rate responses of pups
under different experimental treatments were recorded
on postpartum days 4, 7, and 12. For this purpose,

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each pup was gently placed in a small (2.89-l), opaque


plastic cage (17.0 17.0 10 cm), and two round,
silver electrodes (3 mm in diameter) embedded in
electrolytic gel were placed on the ventrolateral
thoracic skin surface and held with adhesive tape
without interfering with the respiratory rhythm. The
electrodes and attached wire leads did not appear
impede the animals movements or cause any
discomfort. The entire placement of electrodes lasted
less than 2 min in the odor-testing cage. The electrodes
were connected to a model 79-C Grass polygraph and
to an oscilloscope (model Tektronix 502-A). The
recordings were analyzed by using a window
discriminator (model WPI 121) and an analoguedigital converter (CED Micro 1401) to be processed
and analyzed by spike 2 software. The heart period
interval between R-spikes of the EKG signal was
measured in ms (inter-beat interval; IBI). In all cases
after placing the electrodes, the pups were gently
transferred into the recording cage for a 10-min
adaptation period before the odor exposure sessions
began. Under these conditions, pups of different ages
spent most of their time in a passive posture with few
body movements. When movement activity of pups
did occur, it consisted of lateral head movements and
discrete crawling episodes. Movement artifacts were
recognized when the IBI deviated by more than 15%
from the surrounding scores, and the corrected score
was substituted if an error had occurred.
Odors were placed in a second or a third different
small plastic cage (17 17 10 cm), each containing a
circular glass container of 3-cm diameter with 1 ml each
of a volatile dilution of amyl acetate or menthol (diluted
1:10 in glycerol; approximately 2 ppm) 2 min before
testing. In all cases, the same small plastic cage was used
repeatedly to conduct a particular odor to the pup. The
three plastic cages were interconnected by two metallic
valves with 8-mm diameter plastic tubes to an artificial
respiratory pump (Harvard Instruments, model 681).
The pump generated a continuous air flux (300 ml/min)
along the plastic tube that reached the cage containing
the subject with a single odor (Fig. 1A). In accordance
with previous studies to permit statistical comparisons,
basal heart beat samples were taken from subjects (10 s
in duration) and were compared against three heart beat
samples (S1, S2, and S3 lowest bradycardia response,
selected from 15 experimental sessions of 20-s exposures
to a particular odor, three heart beat samples with the
lowest bradycardia responses were selected: sample S1
from session 1, S2 from session 7, and S3 from session 15
(Fig. 1B).8 For the heart beat habituation, the first odor
trial of 20 stimuli (each 1 s in duration) were alternated
with 100-s intervals of clean air flow. The procedure for

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Neonatal olfactory discrimination

growth were evaluated by taking the body weight of


pups at 4, 7, and 12 days of age 1 h before the
olfactory habituation test was performed. Because
male and female rats did not show statistically
significant differences in body weight, in figures the
data for both sexes were pooled together for each
experimental treatment.

Figure 1 (A) Schematic representation of the odorant


stimulation device. (B) The heart beat response
sampling procedure. S1, S7, and S15 samples
taken from 15 experimental sessions

olfactory habituation to a second odor was similar to


that for the first odor. The total duration of the heart
beat habituation to each odor required approximately 30
min. Olfactory discrimination was defined as the score
difference between the last bradycardia sample (S3) of
the first odor and the first bradycardia sample (S1) of
the second odor. In all cases, the experimenter was not
present in the chamber. To ensure blind observations
with respect to odor habituation and olfactory
discrimination to a new odor, the different treatments
and odor exposure sequences were randomly modified
from one olfactory test to the other. At the end of each
recording session, the plastic pup cage and the circular
glass containers for odor solutions were washed with
neutral soap, rinsed with clean water, and dried to avoid
odor impregnation. Moreover, to minimize the odor
remaining from the previous exposure, a fan was turned
on to remove surrounding environmental material. All
olfactory discriminative tests were performed between
1400 and 1600 h in a sound-proof chamber illuminated
with red light (60 W) under controlled temperature (28
2C) and separated from the ambient noise of the main
laboratory.
Physical growth
The effects of experimental treatments on physical

Statistical analysis
Experimental data were analyzed with the Systat
Statistical Package v7.0. To compare score differences
between the various experimental groups on physical
growth and the basal heart beat, heart beat habituation
to different odors, and olfactory cross-discrimination
between different odors, the following separate statistical
analyses were used: (i) body weight score differences were
compared in a four-way ANOVA, 2 (dietary conditions)
2 (handling condition) 3 (ages) 2 (sexes); (ii) the
basal heart rate values taken during the last 10 s before
odor presentation in each experimental group were
compared in a four-way ANOVA, 2 (dietary conditions)
2 (handling condition) 3 (ages) 2 (sexes); (iii) the
heart beat habituations to each odor were compared in a
four-way ANOVA, 2 (dietary conditions) 2 (handling
condition) 3 (ages) 2 (sexes); and (iv) score differences in olfactory cross-discrimination were compared
with a four-way ANOVA, 2 (dietary conditions) 2
(handling condition) 3 (ages) 2 (sexes). Additionally,
post hoc Fisher, least square differences (LSD) tests were
conducted for significant differences between groups at
each developmental age. A probability of 0.05 was
considered statistically significant.

Results
Effects of the age, nutritional and handling schedules
on the body weight
As shown in Figure 2, the body weight of subjects
gradually increased with age. The ANOVA comparisons
yielded significant affects of the dietary conditions (F(1,72)
= 542.80; P < 0.0001), handling condition (F(1,72) = 4.93;
P < 0.02), and age (F(2,72) = 736.02; P < 0.0001), but not
of sex (F(1,72) = 0.41; P = 0.52). Moreover, there were
significant interactions between diet and age (F(2,72) =
53.14; P < 0.0001), age and handling condition (F(2,72) =
13.36; P < 0.0001), and sex and handling condition (F(1,72)
= 5.33; P < 0.02). Post hoc comparisons at each developmental age showed significant body weight reductions (P
< 0.05) in the UG and UHG groups compared to their
controls. Furthermore, the UHG exhibited consistently
lower body weights compared to the CHG of rats.
Animals of the CG and CHG did not differ significantly

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Neonatal olfactory discrimination

Figure 2 Mean values SEM of body weight scores of


control (CG; n = 8), undernourished (UG; n = 8),
control handed (CHG; n = 8), and undernourished
handled (UHG; n = 8) animals. Note that UG and
UHG rats showed consistent reductions of body
weight compared to their controls throughout the
study while at 7 and 12 days of age the CHG was
increased compared to the CG. *Comparisons of
each pair indicate significant differences (CG vs
UG, CHG vs UHG; filled diamond CG vs CHG;
double filled diamonds UG vs UHG) P < 0.05

in weight at 4 days of age, but the CHG was significantly


increased (P < 0.05) at 7 and 12 postnatal days (Fig. 2).
Effects on the basal heart rate
The ANOVA analysis showed that the basal heart rate
of pups was affected by the age (F(2,72) = 123.78; P <
0.0001), the diet, (F(1,72) = 46.94; P < 0.0001), and
handling condition (F(1,72) = 20.89; P < 0.0001), with
no effects of sex (F(1,72) = 1.54; P = 0.21). Furthermore,
there were significant interactions between age and
dietary treatment (F(2,72) = 4.06; P < 0.02), dietary
treatment and handling condition (F(2,72) = 21.55; P <
0.0001), handling condition and sex (F(1,72) = 8.11; P <
0.005), and age and dietary treatment and handling
condition (F(2,72) = 5.32; P < 0.006). The basal heart
rate of the various experimental groups increased
gradually with age (Fig. 3). Post hoc comparisons in at
4 and 12 days of age indicated that the basal heart rate
of the CHG was significantly greater (P < 0.05) than that
of CG, UG, and UHG rats, while on postnatal day 7 the
heart rates of UG and UHG rats were significantly lower
(P < 0.05) than their controls (Fig. 3).
Effects of repeated odor exposure to the heart rate
The ANOVA comparisons of samples S1, S2, and S3
of heart rate habituation taken during the exposure to
each odor indicated significant effects associated only

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Figure 3 Basal heart rates (mean SEM) of neonatal rats as


a function of age in CG, UG, CHG and UHG
subjects. Note in all groups the gradual increase in
their values with age, and that the CHG showed
higher values compared to the other experimental
groups. Furthermore, the CHG was consistently
higher (P < 0.05) than the CG throughout the study
(n = 8 per group). *Comparisons of each pair
indicate significant differences (P < 0.05)

with age (F(2,72) = 6.36; P < 0.002), and dietary


treatment (F(1,72) = 4.33; P < 0.04), without sex effects
(F(1,72) = 3.35; P = 0.06). Additionally, significant
interactions were found between dietary treatment and
handling condition (F(1,72) = 16.34; P < 0.001), and age
and dietary treatment and handling condition (F(2,72) =
4.06; P < 0.02). In general, the temporal course of
heart beat habituation to the first and the second odor,
at different ages and with different dietary treatments
in non-handled and handled animals, followed the
typical progressive decline of responses (taken only at
times S1, S2, and S3) along odor stimulations in
response to odor 1 and 2 at the three experimental ages
(Fig. 4). However, at day 12, habituation did not occur
in the UHG to odors 1 and 2.
Effects of a change of the stimulating odorant
The ANOVA analysis of olfactory discrimination to a
second odor showed significant effects of age (F(2,72) =
3.30; P < 0.04), with no sex effects (F(1,72) = 1.72; P =
0.19), and an interaction of age and sex (F(2,72) = 3.11;
P < 0.05). Post hoc comparisons between sample S3 of
the first odorant versus sample S1 of the second
odorant in non-handled and handled groups showed
significant differences at 4 days of age only in the UG
and CHG of animals. At 7 days of age, all
experimental groups had significant differences in
response to the second stimulation. Moreover, at 12

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Neonatal olfactory discrimination

Figure 4 Habituation of the heart beat response to amyl acetate (Odor 1) or menthol (Odor 2) in 4, 7, and 12-day-old rats in
non-handled and handled groups. The cardiac response to both odors initiates with an accelerated response
followed by a gradual deceleration except in the UHG at 4 and 12 days of age. *S1 vs S3, **S2 vs S3, S1 vs S2,
indicate significant differences (P < 0.05)

days of age, only the substitution of the first odorant


by the second elicited significant increases of the heart
rates excepting in the CG and the UHG (Fig. 5).

Discussion
The data showed that body weight was consistently
reduced in the UG and UHG rats when compared to
their controls, and that the body weight of UHG pups
was significantly reduced at 4 and 7 days of age, with a
small increase at postnatal day 12 when compared to
the UG of pups; there were differences between the
CG and CHG animals. The current findings agree
with previous studies showing that perinatal
undernourishment consistently interferes with the
physical development of newborns and adults, and
that food deprivation, which is usually associated with

a deficiency of sensory stimulation, reduces body


weight gain.4446,50 However, the significant increase in
body weight at 7 and 12 days of age between CG and
CHG rats indicates that early sensory stimulation here
used was able to modify the body weight of subjects.
Pups of all experimental groups received sensory
stimulation during the motherlitter encounter in the
living cage,49 and it is known that this maternal
stimulation has an ameliorating effect on adverse
behavioral responses to stress in the offspring;
however, its role in physical development and early
olfactory discrimination is under investigation.5157
The basal heart rate increased steadily with age in
all experimental groups. In the CHG, the basal cardiac
activity was consistently higher than in the other
groups. This suggests that handling in control subjects
promotes the maturation and function of the cardio-

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Figure 5 Heart beat rate as the differences between sample


S3 of Odor 1 (O1) vs the sample S1 of Odor 2 (O2),
along the days of the study in non-handled and
handled groups. Subjects of the UG non-handled
and CHG handled at the three ages were able to
discriminate the substitution of the first odor by
the second. At 7 days of age, the four experimental
groups were able to discriminate the second odor.
*Comparisons at each pair indicate significant
differences (P < 0.05)

acceleratory system, as described in the cerebral cortex


and in a number of segregated CNS nuclei and their
functioning.28,38,47 The present results also indicated
that early handling did not modify the basal heart rate
values in the UHG compared to the CG and UG of
pups. Moreover, in the CG and UG of subjects, the
basal heart rate was similar, except on day 7 when UG
values were significantly reduced (Fig. 3). At present,
we have no clear explanation why this CG and UG
basal heart beat reduction occurred as a consequence
of undernutrition upon the cardiac autonomic system
maturation. Regarding the basal cardiac rate, the
current data are in line with pioneer studies showing
that placing pups alone in an environment of low
intensity, novel stimulation had little effect on motor
activity and heart rate at 1 and 7 days of age, but
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significantly increased in both parameters at later


ages.8,18,20,58 The data also suggest that, in our
experimental conditions during the first days of life,
the parasympathetic influence to the heart is already
activated and that, concurrently, there is a gradual, but
delayed, increase in the sympathetic input to the heart,
possibly as an adaptive homeostatic mechanism of the
young to the gradual changes in body composition
(adipose and muscle tissues, brain development,
increased metabolic and motor activity) in response to
environmental demands to promote the cardiosomatic coupling until an autonomic balance is
achieved to maintain homeostasis.19,20,59
Current olfactory discrimination indicates that, at
all developmental ages, the UG and the CHG rats
were able to display significant decreases in the heart
beat rate in response to a second novel olfactory cue.
Furthermore, at 7 days of age, all experimental groups
had similar significant decreases in the heart beat
responses (Fig. 5). These findings are partly in line
with previous studies using both separate or olfactory
cues paired with tactile, auditory, visual, chemical and
electric shock cues in order to investigate how early
experience follows the temporal sequence of sensory
development to induce plastic neuronal changes in the
newborn that promote its survival.18,19,22,60 Our data
also indicate that, in the UG and CHG rats at all ages
tested, the olfactory discrimination was preserved,
while, in the CG and UHG pups, the discriminatory
responses were inconsistent (Fig. 5). These findings call
attention to the fact that, over the course of the study,
perinatal undernutrition did not affect novel olfactory
discrimination, and that the effects of early handling on
olfactory discrimination were worse in the UHG than
CG rats. The association of perinatal undernutrition with
early handling (UHG pups) did not improve olfactory
discrimination of newborn rats as recently described in
repeatedly handled 7-day-old rats.61 The fact that odor
discrimination was significantly increased at 4, 7, and 12
days of age in the UG and CHG rats suggests that
perinatal undernutrition and handling each independently interfere with the development of inhibitory
mechanisms during this period of life, without modulating novel olfactory signals.6,62,63 Later, these environmental influences may have negative consequences for the
olfactory channel maturation and its long-term effects
on the stress response and social behavior.25,55,6467 The
present data were obtained in underfed pups placed in
small individual cages, separated from the mother and
nest, and exposed to novel odors to induce adaptive
bradycardia responses to unfamiliar odors with
altered amygdala organization in underfed pups that
may interfere with bradycardia responses.27 In this

Ruiz-Diaz et al.

regard, our data are partly supported by previous data


suggesting that, in control rats, the maternal presence
potentiates the pups survival, learning and attenuated
aversion responses without amygdala involvement.5,57
Our data indicate that early handling as a non-specific
stimulation associated to the licking, retrieving and
maternal grooming of pups may improve in the CHG
rats the olfactory discrimination as occurred by using
specific odorants.13,68 The effects of handling increase
noradrenaline (NA) release in the olfactory bulb of
pups by centrifugal fibres from the locus coeruleus as
an influence necessary for learning.51,53 When perinatal
undernutrition was associated with early handling
(UHG), odor discrimination of our subjects did not
improve consistently because they could have opposite
maturational influences or because early handling was
partly ameliorating the noxious effects of perinatal
undernutrition.46,61 Another point of interest is that
our study analyzes discrimination between artificial
odors that in general is effective with modulations by
age. Moreover, the lack of discrimination at 12 days of
age in the UHG to both odorants, could be the result
of a stronger trigeminal activation that suppress the
odorant activation as previously described.69,70

Conclusions
The olfactory discrimination between maternal and
novel, non-maternal odors is already functional
during the first 12 postnatal days in both UG and
CHG rats. By contrast, it was inconsistent in the CG
and UHG pups suggesting that, during this period of
life, reciprocal granule and mitral inhibitory processes
are emerging, and undernutrition associated to
handling and possible altered attentional mechanisms
interfere the olfactory discrimination by unclear
mechanisms.9,65,71 Undernourishment and handling
can increase this sensory ability, but the combination
of the two factors as an unexpected effect is consistent
with the idea that ontogenetic changes of the
underlying mechanisms are involved. However,
further studies are needed to determine the
neurophysiological levels altered by the treatments.

Acknowledgements
This work was partly supported by DGAPA/UNAM,
IN210903, IN207307. CONACyT No. 184899 and
DGAPA/UNAM No. 50410914 to MR. The authors
thank Dr Dorothy D. Pless for editorial assistance and
P. Galarza and R. Silva for collecting data.

Neonatal olfactory discrimination

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