The presence of chemosensory cues transmitted through the mother's milk increases the search and ingestion responses toward bitter and sour solutions
Introduction
Rodents are able to recognize their environment through different cues. Newborns show a preference for their maternal odor. This is determined by previous experiences, that is, it can be either their biological mother or a foster one (Sanchez-Andrade & Kendrick, 2009). Moreover, infant rats display an enhanced capacity for odor-preference learning, accompanied by a decreased capacity for avoidance learning, during postnatal days (PD) 1–9 (Moriceau, Roth, & Sullivan, 2010). During this period, pups even develop a preference for odors paired with mild aversive stimuli (e.g., a 0.5 mA electric shock; Camp & Rudy, 1988). These particular learning capabilities appear to be beneficial for them for two reasons. Firstly, since the mother's odor can be modified by diet (Leon, 1992), this mechanism allows the pups to readily learn approaching behaviors toward her. In accordance to this, newborn babies are able to learn from the flavor compounds they receive through their mother's milk. Studies show that the human milk compound is continuously changing; showing that the babies receive a variation of chemosensory experiences (Hausner, Bredie, Mølgaard, Petersen, & Møller, 2008). These variations will impact their diet choices later in life (Cooke & Fildes, 2011).
It has been shown that, in humans, flavors experienced via the mother's milk can modify the infants' preferences for those flavors. For example, Mennella, Daniels, and Reiter (2017) found that human infants whose mothers drank vegetable juice for one month (beginning at two weeks postpartum) showed an increased intake of carrot-flavored cereal. Similar results were found when the mothers consumed garlic capsules or carrot juice in the nursing context; the babies spent more time breastfeeding when they were re-exposed to this flavor (Cooke & Fildes, 2011; Mennella & Beauchamp, 1993).
Interestingly, this can also be observed in non-human animals. When rat dams were administered onions, the pups showed a preference toward this flavor, which may indicate that there is a transmission of food's taste that the mother consumes through maternal milk to the pup, that determines later food preferences (Wuensch, 1978). Similarly, adult mice whose mothers were exposed to an artificial sweetener during lactation exhibited a greater preference for sweet foods, when compared to the offspring of non-exposed dams (Zhang et al., 2011). Several studies, in which the factors that influence ethanol consumption were evaluated, showed that prenatal pre-exposure to the drug through i.g. administration to the mother can affect postnatal preferences towards it (Arias & Chotro, 2005; Pueta, Abate, Haymal, Spear, & Molina, 2008; Spear & Molina, 2005). This phenomenon was also observed when the flavor of alcohol was detected by the pup through maternal milk, which can influence the acceptance of this solution later in life (Hunt, Kraebel, Rabine, Spear, & Spear, 1993). This begs the question of whether exposing infant rats to a novel odor, through the mother's milk, can modify their reactions to flavors.
Although taste reaction responses comprise an innate component, they are plastic and depend on previous experiences and learning. Studies with numerous species and at various stages of development showed that there is a tendency to accept foods with sweet flavors, and to reject bitter, acidic and salty ones (Berridge, 2000). However, under certain environmental manipulations, reactions to the sweet taste may resemble those that occur when a bitter taste is presented and vice versa (Suárez, Ifran, Pautassi, & Kamenetzky, 2017; Suárez, Pautassi, & Kamenetezky, 2016). Other studies from our lab showed that prenatal exposure to an odor through an i.g. administration can induce a preference for typically-rejected flavors after birth. Kamenetzky, Suárez, Ifran, Nizhnikov, and Pautassi (2018) exposed rats to a lemon scent in-utero in order to assess whether this manipulation would alter their first sucking responses towards a bitter solution -which is usually rejected by the pups-delivered through a surrogate nipple. Three hours after being born, the subjects were tested while being stimulated by the same scent. The authors found an increased intake of quinine in pups that were pre-exposed to the scent in the womb, when compared to a control group that was not previously exposed. Similar effects were found when exposure to the odor was carried out immediately after birth. Kamenetzky, Suárez, Pautassi, Mustaca, and Nizhnikov (2015) exposed newborn rats to a lemon scent during 1 h immediately after birth. Three hours later, they stimulated the pups with a surrogate nipple containing a quinine solution, odorized with this scent. There was an increased intake of this solution when compared with a control group that was not previously exposed to the scent. The increase in consumption did not occur when the artificial nipple contained a sucrose solution, or a higher concentration of quinine. These results suggest that the presence of a familiar odor facilitates the acceptance of usually rejected flavors, as long as it is a moderate concentration.
This novel phenomenon is apparently robust, since it was replicated under various conditions. Animals that were evaluated in the presence of maternal odor increased the ingestion responses of a quinine solution and decreased the latency until the first intake response, which suggests that the presence of the mother's odor facilitates the acceptance of a naturally rejected flavor (Ifrán, Suárez, Pautassi, & Kamenetzky, 2018). Previous results suggest that this phenomenon occurs within a sensitive period, given that at Postnatal day (PD) 15 the phenomenon is no longer observed (Ifran, Suárez, Pautassi, & Kamenetzky, 2020).
In order to assess whether this phenomenon could be replicated using other aversive solutions, we conducted an experiment with newborn rats, in which the first sucking experience toward an artificial nipple was evaluated. Half of the rats were pre-exposed to a lemon scent immediately after birth. This was achieved by placing the pups near an embedded cotton swab for an hour. Three-hours later all the animals were evaluated in the presence of the lemon odor, and independent groups received different solutions through the surrogate nipple: quinine (bitter), citric acid (sour), saline solution (salty) or distilled water. The pre-exposed animals showed an increase in the grasp frequency toward the artificial nipple, containing the quinine and citric acid solutions, in comparison to the animals that were not pre-exposed or received the saline solution (unpublished data).
The present study assessed whether 3 day-old rat pups would increase their ingestive behavior towards commonly rejected solutions if presented with a lemon scent, previously exposed through the dam's milk. The objectives of this study were: 1. To determine whether the effect of increased intake of a bitter solution in the presence of a pre-exposed odor generalizes to other solutions with non-palatable flavors (i.e., sour and salty); 2. To evaluate whether breast milk functions as a means of propagation of odors, and whether this results in a modification of the ingestion responses of the offspring. The hypothesis was that subjects presented with a familiar odor, previously perceived during nursing, would increase their intake of quinine, citric acid, and saline solution, when compared to subjects exposed to distilled water or who had not been previously fed breast with milk contaminated with the scent.
Section snippets
Subjects
A total of 95 Wistar, 3-day-old rats (48 males and 47 females), derived from 17 mothers, served as subjects. One male and one female were used per experimental condition to avoid litter overrepresentation. The animals were born and reared at the vivarium of the Instituto de Investigaciones Médicas Dr. Alfredo Lanari (IDIM-CONICET, Argentina) under a 12/12hs light/dark cycle, with lights on at 7 a.m., and controlled temperature (22-24 °C) and humidity. PD 0 was defined as the day of parturition.
Results
Fig. 1 shows the percentage of body weight gained, number of grasps, total time of grasp, mean grasp duration, and latency to grasp across odor conditions. The figure suggests that the animals that had been pre-exposed to the lemon essence through breast milk showed an increased response to the artificial nipple, when it contained the quinine or citric acid solution. Variance analyzes revealed that for percentage of body weight gained, a significant interaction among Pre-exposure solution and
Discussion
The pups pre-exposed to the scent showed an increase in seeking and ingesting behavior toward an artificial nipple containing quinine or citric acid, when compared to pups receiving saline or control (non-pre-exposed) pups. Strikingly, we observed high variability in the citric acid and quinine groups in the pre-exposed groups’ response that could be explained by the in-between-litter variations; while the grasp response is usually homogeneous within the control groups. Although we found high
Author contribution
All authors participated in the design, interpretation of the studies, and analysis of the data and review of the manuscript. MI, AS, and GK conducted the experiments and analyzed the data. MI, AS, MA, and GK wrote the manuscript.
Funding
This work was supported by the PIP-CONICET-2012 [grant number 11220110100228], BID-PICT 2014[grant number 3212] and the Universidad Abierta Interamericana, awarded to Giselle Kamenetzky.
Ethical statement
This research was conducted with full compliance of research ethics norms, and more specifically the codes and practices established by the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The protocol was approved by the Animal Care and Use Committee of IDIM-CONICET at Instituto de Investigaciones Médicas Alfredo Lanari (Approval number 039-16).
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