- Abstract
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- High- and low-yawning rats (HY and LY) were
selectively bred as a function of their
spontaneous yawning frequency with the LY
subline about 2 yawns/hr and the HY 20 yawns/hr.
The HY rats have more grooming bouts and travel
longer distances in an open field. HY dams spent
less time in the nest, retrieved their pups
faster, and show a longer latency to licking and
mouthing the pups than the LY or outbred
Sprague-Dawley (SD) animals. The percentage of
HY dams that had atypical retrieving was higher,
with a lower nest quality, and produced
offspring whose weights were lower than those
from the LY subline. We also showed that the
pregnant HY dams have fewer pups and the
percentage that had lost at least three pups
during lactation was higher than the SD and LY
dams. In conclusion, HY dams are motivated to
take care of their pups, but the "fine tuning"
of maternal care is disturbed.
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- INTRODUCTION
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- Social interactions among members of a
community are important for their organization
and survival. These interactions need the
display of affiliative behaviors such as
maternal care, sexual behavior, and grooming,
compared to the antagonistic ones such as
aggression, isolation, and submissive behaviors.
In altricial species, maternal behavior is the
most important and most studied behavior not
only because it allows the immature offspring to
become independent over time, but it is also a
good model for the offspring's physiological,
neuroendocrine, and cognitive display as adults
(Beach & Jaynes, 1954; Hofer, 1994).
Maternal behavior is the expression of a series
of motor and somatosensory events by the mother
at the end of pregnancy, parturition, and during
the postnatal preweaning period (Rosenblatt,
1967; Rosenblatt & Lehrman, 1963). In
rodents, once the pups are born, the mother
retrieves them to the nest, licks their bodies
and the anogenital region followed by a nursing
posture over them in a highly stereotyped and
defined organization (González-Mariscal
& Poindron, 2002; Rosenblatt & Lehrman,
1963). Thus, the offspring receive warmth,
nutrients, protection, and sensory and social
stimulation in the form of social contact with
the mother and their siblings (Beach &
Jaynes, 1954; Hofer, 1994; Levine, Haltmeyer,
Karas, & Denenberg, 1967).
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- In rats and mice, differences in the timing
and components of maternal behavior have been
described over the course of the nurturing
period, variations that mediate behavioral
transmission of traits and thus
transgenerational or nongenomic transmission to
the offspring (Champagne, Francis, Mar, &
Meaney, 2003; Fleming et al., 2002). There are
variations in mother-infant interactions within
a same strain, that is, natural variations
(Champagne, Curley, Keverne, & Bateson,
2007; Champagne et al., 2003; Meaney, 2001). For
example, by choosing the extremes of the
populations of the Long-Evans rats and comparing
the frequency that the mothers lick their pup's
body and genitals, and their nursing posture,
Meaney and his group had found that there are
mothers that show high levels of licking and
arched-back nursing (HG-ABN) and other mothers
that show low levels of these behaviors (LG-ABN;
Champagne et al., 2003).
-
- Interestingly, lactating rats from HG-ABN
mothers show a low reactivity of the
hypothalamus-pituitary-adrenal (HPA) axis after
exposure to stressful environment, with a small
fear response, a good level of spatial learning,
and mainly spent much time licking and nursing
their pups compared to the LG-ABN dams. These
differences are not caused by the genetic
background because cross-fostering studies have
shown that the offspring phenotypes depend on
the mother that reared it (Champagne et al.,
2003). Experimentally, it is possible to
regulate how the mothers take care of their
offspring. If lactating rats are exposed to a
handling paradigm (pups are removed from the
nest for 15 mm), the dams spend more time
licking the body and genital area of the pup's
when they are returned to the nest (Meaney et
al., 1985; Pryce, Bettschen, & Feldon,
2001). Contrary to the handling paradigm,
mothers who had been isolated from their mothers
during infancy spent less time taking care of
her pups (Gonzalez, Lovic, Ward, Wainwright,
& Fleming, 2001; Melo,
HernándezCuriel, & Hoffman, 2009;
Melo et al., 2006). Different groups of mice
showed remarkable variations in the expression
of maternal behavior (Anisman, Zaharia, Meaney,
& Merali, 1998; Broida & Svare, 1982;
Brown, Mathieson, Stapleton, & Neumann,
1999; Champagne et al., 2007; Ohta, Shirota,
Tohei, & Taya, 2002; Shoji & Kato,
2006).
-
- Thus, lactating female mice of CS7BL/6,
CBAIH, C3FIJIco, and CBAIJ strains retrieved
pups faster than BALB/c, NBZ, DBAI2, XLII, NJ,
and AKR strains (Carlier, Roubertoux, &
Cohen-Salmon, 1982). Furthermore, DBAI2J females
built better nests and spent more time crouching
over and nursing pups (Brown et al., 1999) than
CS7BL/6J dams.
-
- A comparison between inbred and outbred mice
has shown that the 129Sv strain had shorter
latencies in nest building, built the nest less
frequently, and spent less time engaged in
licking the pups than outbred dams (Broida &
Svare, 1983; Champagne et al., 2007; Meaney,
2001). In rats, psychogenetic selection has
resulted in at least four different strains
that, besides their own phenotype, have
variations in postpartum maternal behaviors; (1)
The Flinders Sensitive Line (FSL), considered a
genetic animal model of depression, spent less
time licking the
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- pups and nursing them (Lavi-Avnon, Yadid,
Overstreet, & Weller, 2005), a shorter
latency to first pup retrieval, and more
self-directed behavior than controls (Braw et
al., 2009); (2) spontaneously hypertensive rats
(SHR) more often had an arched and
blanket-nursing posture and a lesser
passive-nursing posture, spent less time licking
their pups, and retrieved them more quickly than
the Wistar strain (Myers, Brunelli, Squire,
Shindeldecker, & Hofer, 1989); (3) Roman
high (RHA-Verh)- and low (RLA-Verh)-avoidance
sublines of rats were selected and bred for
their rapid response compared to poor
acquisition in a two-way active-avoidance
response (Steimer, Escorihuela,
Fernández-Teruel, & Driscoll, 1998).
Female rats of RHA-Verh mothers had a high
active avoidance, spent less time with their
young, are more active, and also assumed the
side-nursing position less often than the
RLA-Verh mothers (Driscoll, Fumm, &
Bàttig, 1979); and (4) Hatano high- (HAA)
and low- (LAA)-avoidance selective-breeding
lines from the Sprague-Dawley strain (Ohta,
Matsumoto, Nagao, & Mizutani, 1998) show a
high variation in the expression of maternal
behavior, with the low avoidance (LAA) females
having longer latencies for retrieving the pups,
spent less time with them, showed a decreased
amount of milk ejection, a lesser increase in
blood prolactin, and a greater increase of
adrenocorticotrophic hormone (ACTH) than the HAA
mothers (Ohta et al., 2002).
-
- Although the behavioral differences among
inbred groups of mice or rats are attributed to
genetic variations, it has been reported that
the genetic-environment interactions early in
life, mainly mediated by maternal care and their
siblings, are the main cause of those variations
(Francis, Szegda, Campbell, Martin, & Insel,
2003; Myers et al., 1989; Ohta et al., 1998;
Shoji & Kato, 2009; Steimer & Driscoll,
2005). These data show that, besides the
phenotype used, during inbreeding the process
can generate other changes of the behavioral
display that could be caused by maternal
care.
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- Yawning is a phylogenetically old behavior
and stereotypically shown by reptiles, fish,
birds, and mammals (Walusinski & Deputte,
2004). It consists of a wide opening of the
mouth with a long inspiration, followed by a
short expiration. Yawning can be modulated by
several peptides such as adrenocorticotropin
hormone, alpha-melanocyte stimulating hormone,
and oxytocin, and also by several
neurotransmitters as GABAergic, dopaminergic,
and muscarinic cholinergic systems in several
strains of rats, as well as HY and LY sublines
(for review, see Collins & Eguibar, 2010;
Doger, UrbáHolmgren, Eguibar, &
Holmgren, 1989; Eguibar, Barajas, & Moyaho,
2004; Eguibar, Romero-Carbente, & Moyaho,
2003; Urbá-Holmgren, Santos, Holmgren,
& Eguibar, 1993). The HY males yawned more
and also had more grooming bouts after exposure
to a novel environment than LY rats (Eguibar
& Moyaho, 1997). The HY subline is also more
active in an open-field arena (Moyaho, Eguibar,
& Diaz, 1995). In addition, after wetting
the HY showed a disorganized grooming-chain
sequence compared to the LY animals with a clear
cephalocaudal organization, similar to that
obtained in other strains of rats (Moyaho et
al., 1995). These observations suggest that the
early life experience, such as maternal care and
lactation, could be the cause of the differences
among the sublines. In our experiments, we
analyzed maternal care toward their own
offspring of HY and LY dams and compared them
with outbred Sprague-Dawley dams during the
early-to-middle lactation period. In a second
experiment, we compared the number of pups at
parturition and weaning and the fertility index
of the females of all groups.
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- DISCUSSION
-
- In this study we compared the maternal and
nonmaternal behaviors of primiparous female rats
of the HY and LY inbred sublines compared to
outbred Sprague-Dawley rats during
early-to-middle lactation. Our results showed
that HY mothers express different patterns of
maternal and nonmaternal behaviors compared to
the SpragueDawley and LY dams. In addition, the
number of pups per litter at birth and weaning
from the HY mothers was lower compared to the
other groups of rats.
-
- The results showed that the time engaged in
nursing and licking the pups did not
significantly differ among the groups, but the
HY and LY mothers spent less time inside the
nest than the Sprague-Dawley dams. In contrast,
the HY dams retrieve all pups to the nest more
quickly than the Sprague- Dawley mothers. These
results are similar to that obtained in mice
from the C57BL/6, CBAIH, C3W Ico, and CBAIJ
strains, which retrieved pups more quickly than
the BALB/c, NBZ, DBAI2, XLII, NJ, and AKR
strains (Carlier et al., 1982). The HY dams not
only retrieve the pups more quickly, but most of
them made atypical retrievings and also
displayed reretrieving and built the nest more
rapidly, but with lower quality. These results
match with those found in the 129Sv inbred
strain that had shorter latencies in nest
building, built the nest less frequently, and
spent less time in licking the pups (Champagne
et al., 2007). The above data suggest that
mothers are maternally motivated but the "fine
tuning" of the expression of all maternal
characteristics are disturbed, similar to that
already reported in the organization of grooming
bouts in HY rats (Eguibar & Moyaho, 1997;
Moyaho et al., 1995). Interestingly, when we
compared the latency of each maternal component
in the early compared to the middle lactation,
we showed that mothers from all groups showed
similar latencies during the early lactation.
During the middle lactation the latency to
retrieve the pups, mouthing them, and building
the nest by the HY dams was shorter, but the
latencies to begin licking the body and genital
areas of the pups were longer than that of the
LY and Sprague-Dawley dams.
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- The HY, the LY, and the Sprague-Dawley dams
showed normal maternal motivation because once
they get cues from the pups, they established
contact with them (appetitive component), then
they walk around the maternal cage with the pups
in their mouth and put them in a different place
and later on reretrieve them to the nest. This
shows that they have enough motivation to engage
in a behavioral interaction with a specific goal
object, and they retrieve them to the nest
(consummatory component), but their ability to
show a specific behavior, that is, properly
retrieve the pups is not adequate after putting
them outside the nest, reretrieve them and also
with atypical retrieving suggesting a
disorganized pattern in the global organization
of maternal care in HY rats (Everitt, 1990;
Numan, Fleming, & Levy, 2006; Numan &
Insel, 2003; Timberlake & Silva, 1995). The
HY males also have a disorganized sequence of
their grooming bouts because they showed
caudocephalic or lateralcaudal sequences,
instead of the cephalocaudal sequences shown by
the LY rats and other rodent species (Berridge,
1990; Moyaho et al., 1995). These alterations
could be caused, at least in part, to a greater
number of D1 dopaminergic receptors in the
ventral striatum in the HY compared to the LY
animals (Diaz-Romero, Arias-Montaflo, Eguibar,
& Flores, 2005). Matell, Berridge, and
Wayne-Aldridge (2006) showed that the grooming
syntactic chains can be altered after a lesion
of the striatum or changing the dopaminergic
transmission in the nigrostriatal pathway. It is
well known that the basal ganglia play a crucial
role in the organization, timing, and
coordination of motor sequences including
grooming (Cromwell & Berridge, 1996).
Furthermore, systemic administration of
SCH23390, a specific dopaminergic D1 antagonist,
produced a disruption of maternal care causing
the mother to leave the pups outside the nest,
so reretrieving them (Byrnes, Rigero, &
Bridges, 2002). This also happens with
intraaccumbens injection of cis-flupenthixol,
which inhibits maternal retrieving and licking
the pups but enhances nursing behavior in
lactating Long-Evans rats (Keer & Stern,
1999). The maternal-care deficits caused by
haloperidol can be restored by demanding pups
(12-hr deprived), showing that pups can reverse
the effects produced by the dopaminergic
antagonist and by bromocriptine, a dopaminergic
agonist that produced an opposite effect
(Pereira & Ferreira, 2006). Because the HY
rats showed an increase of D1 receptors in the
ventral striatum (Diaz-Romero et al., 2005) and
a decrease in the dopamine levels in the nucleus
accumbens (unpublished data), we suggest that
dopamine changes could be responsible for the
alterations in maternal care in the HY
dams.
-
- Recently, it has been reported that the
mother not only gives somatosensorial
stimulation but also gives growth factors such
as prolactin and growth hormone through the milk
that could act in concert to aid growth, weight
gain, and glucose homeostasis in the perinatal
period (Fleenor et al., 2005). Prolactin plays a
fundamental role not only to support milk
production but also in the developmental and
maturation of the pups (Melo et al., 2009). The
above data suggest that because that HY
offspring never gain normal weight during
lactation it could be that these dams produce
milk of lower quantity or quality. It is also
possible that humoral factors such as growth,
oxytocin, and thyroid hormones could be
responsible for the lower rate of body-weight
gain in the HY offspring (Bautista, Boeck,
Larrea, Nathanielsz, & Zambrano, 2008;
Glinoer, 1997; Hapon, Simoncini, Via, &
Jahn, 2003; Valdez, Penissi, Deis, & Jahn,
2007).
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- A relationship between high emotionality and
a deficit in the expression of maternal licking
and grooming as well as arched-back nursing
posture has been demonstrated (Francis, Diorio,
Liu, & Meaney, 1999; Gonzalez, Lovic, Ward,
Wainwright, & Fleming, 2001; for review, see
Numan & Insel, 2003). Female rats that are
isolated early
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- in life had a high stress response and show
a deficit in the expression of maternal
behavior, and these characteristics are
transmitted to the next generation (Gonzalez et
al., 2001).
-
- Because the HY offspring have lower weights
during lactation that persists until adulthood,
it is possible that behavioral disturbances
found in these rats could be caused by
differences in the maternal care, including
grooming and the arched-back posture. There are
some reports that partially support this
hypothesis, that is, mothers that were
undernourished during early life had an abnormal
maternal care, as we found in the HY mothers.
Thus, these dams show a decrease in nest quality
and nursing time, an increase in the latency to
retrieve pups, and atypical retrievings that can
even produce sonic distress in the pups
(Regalado, Torrero, & Salas, 1999; Salas,
Torrero, Regalado, & Perez, 2002; Salas,
Torrero, & Pulido, 1984; Smart, 1976). The
HY mothers also had a lower nest-building rating
and showed atypical retrievings. Rosenblatt and
Lehrman (1963) reported that when a female
cannot maintain a stable nest, she retrieves the
pups too many times and deposits them anywhere
in the cage, similar to that made by HYand LY
dams, suggesting that the sublines had a
disorganized pattern of maternal care.
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- It is important to emphasize that body
weights of undernourished pups from 4 to 20 days
old were about 20-50% lower than well-nourished
rats (Bautista et al., 2008; Salas et al.,
1984,2002; Wiener, Fitzpatrick, Levin,
Smotherman, & Levine, 1977; Zambrano et al.,
2005). We found that body weights in the
HYoffspring were 16-25% lower during lactation
compared to the Sprague-Dawley and LY animals.
Because of the ad libitum disposition of rodent
food pellets the disturbed expression of
maternal behavior in HY dams are not because
they are being underfed during neonatal period,
but it is probably generated by maternal care.
These deficits in birth weight can be corrected
by improving postnatal nutrition, as reported in
Wistar Kyoto rats, a good model of anxiety
responses (Romano, Wark, Owens, & Wlodek,
2009). In our experiments the animals have free
access to food (Zeigler) with 22% protein, but
they did not reach weights similar to
Sprague-Dawley pups surely because of their
genetic background (Moyaho et al., 2009). The
"fetal origin hypothesis" proposes that prenatal
environmental exposures, including maternal
stress, could have sustained effects across the
lifespan (Kinsella & Monk, 2009). A positive
correlation of food ingestion during pregnancy
and low body weight in their offspring has been
demonstrated (Massaro, Levitsky, & Barnes,
1974; Passos, Ramos, & Moura, 2000),
including women with a poor diet before and
during pregnancy who had babies with a low birth
weight (Lechtig et al., 1975), and where the
frequency of infant mortality is four times
higher than normal birth weight babies (Habicht,
Yarbrough, Lechtig, & Klein, 1973).
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- Comparisons among inbred strains of rodents
are important to determine the effect of
environmental factors over behavioral traits
made under laboratory conditions. Thus,
environmental manipulations such as maternal
separation early in life, handling, and enriched
environments clearly affect subsequent juvenile
or adult performances (Fleming et al., 2002). As
in many other inbred strains of rodents, work
has focused on the participation of genetic and
epigenetic factors involved in the development
of specific behaviors (Francis et al., 2003).
The HY offspring had fewer contacts with their
mothers, receive less grooming, and were
retrieved carelessly (atypical retrievings and
reretrievings), similar to that reported in
stressed mothers (Salas et al., 2002). The HY
mothers have fewer pups per litter at
parturition, most of them lost pups during
lactation, and the mean weight of their pups is
lower from birth to weaning compared to the LY
and Sprague-Dawley offspring. These behavioral
differences could be caused by some metabolic,
hormonal, or emotional issues during pregnancy
or lactation (Fleenor et al., 2005; Glinoer,
1997; Hapon et al., 2003; Ozzane & Hales,
1999; Shono, Imagima, Zakaria, & Suita,
1999). Pups, exposed to dexamethasone by its
injection into their mothers during pregnancy,
produced an offspring with lower weight and
chronic hyperactivity of the FIPA gland axis.
These pups had higher plasma-corticosterone
levels with an upregulation of hepatic
gluconeogenesis and insulin resistance
suggesting that glucocorticoids levels are a key
factor for metabolic activity as adults (Buhl et
al., 2007; Burlet et al., 2005).
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- The release of oxytocin in the
paraventricular nucleus (PVN) of the
hypothalamus at parturition probably facilitates
a positive feedback in both parvocellular and
magnocellular neurons to coordinate the high
levels of oxytocin release that are important
for the generation of maternal behavior, infant
recognition, and bonding (Carter & Keverne,
2002). This is also true for yawning expression
because the release of oxytocin in the PVN is a
key factor for the generation of yawning, not
only by this peptide, but also in this part of
the brain the dopaminergic, excitatory amino
acids, nitric oxide, GABA, and opioid receptors
converge to increase yawning frequency,
suggesting that neural mechanisms in the
hypothalamus are important regulators for
yawning and pair bonding (for review, see
Collins & Eguibar, 2010). Preliminary
results showed that HY rats yawned more after
the i.c.v. injection of oxytocin than the LY
animals, but with similar grooming scores
(unpublished data), suggesting different
sensitivities in the neural pathways that
mediate these behaviors. In future experiments,
we will address oxytocin levels during
parturition and lactation in both sublines.
-
- In conclusion, our results were that the HY
dams showed a different organization of maternal
care with a reduced litter size and lower
weights of pups at parturition and weaning.
These changes can be caused by hormonal or
neural mechanisms, which are able to alter
somatosensory stimulation of the pups and also
can produce hormonal and metabolic changes that
ultimately are responsible for different
behavioral characteristics of HY rats, such as
yawning and grooming sequences in the
adults.
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