PRISM Research Paper
Introduction
“Parental care, including feeding and protection of young, is essential for the survival as well as mental and physical well-being of the offspring” (Dulac, 2014). Cross-sectional studies on parent-child relationships have shown that adverse and traumatic childhood experiences including neglect, abuse, or parental loss correlate to mood and anxiety disorders later in life (Bodensteiner, 2014). Moreover, such individuals have tendencies to form antisocial personalities and impulsive aggression. In girls lacking proper parent-child relationships create ‘decreased age at menarche, earlier onset of sexual behavior, and increased number of sexual partners, all of which may lead to social, psychological, and physical repercussions” (Bodensteiner, 2014). “In humans, the quality of parental care is affected by stress and mental illnesses such as postpartum depression (PPD), which affects more than 10% of mothers in the United States” (Dulac, 2014). That being said, early life experiences, though brief, are critical for molding the future of an infant’s success and overall fitness to survive the demands of life (Anacker, 2013).
Evolution
Maternal care is not cheap. Maternal characteristics “exhibit species-characteristic ways of transporting, holding, feeding, and grooming the young, and of protecting them from the predators and other dangers. For instance, the prospective mother rat constructs a nest before giving birth. She aids the birth by pulling individual pups from the vaginal canal, severs the umbilicus, eats the placenta, cleans the pups, and carries them one-by-one in her mouth to the nest. In the nest, she continues to lick the pups, especially their anogenital regions, and eventually adopts a nursing posture over them. In return, mammalian neonates exhibit behavior that is even more stereotyped and rigid than maternal behavior.” (Fleming,1999). “Lactation represents the most energetically expensive cost of breeding in rodents and mammals (Gittleman & Thompson 1988; Thompson 1992) and lactating females may pay a fitness cost in terms of subsequent survival and reproductive success (Clutton- Brock et al. 1989; Huber et al. 1999)” (Ebensperger, 2006). Mammals have a long gestation period and also do not have large litters of offspring at once. Therefore, the mother puts all her energy into feeding, nurturing, and protecting that single offspring. In contrast, rodents such as the Sprague Dawley rat used in this experiment have several offspring at once with short gestation periods. This allows for the mother to choose between protecting the lives of her offspring or herself. In the case of retrieval, if the mother senses danger, she will be less likely to retrieve and choose to save herself instead. This seemingly selfish act is actually evolutionarily beneficial, because the mother conserves her metabolic activity to reproduce another litter and potential genetic succession.
Maternal Care Effects
The quality of maternal care on its offspring is critical in the short term nurturing affects such as development and epigenetic expression, but also expands long term into future maternal behaviors. “Short-term, rat pups develop an attraction to the chemosensory characteristics of mother’s ventrum and first attaches to the nipples based on prior intrauterine experience with the amniotic fluid” (Fleming, 1999). Long term, infants who receive high licking and grooming interaction as juveniles develop to become high licking and grooming mothers (Fleming, 1999). “Reciprocal mother–infant behaviors increase the probability that the young will survive and, beyond survival, will mate and successfully rear their own offspring” (Fleming, 1999). Mother care such as high or low licking and grooming correlates to future maternal rat characteristics. When an offspring is raised in a high-licking environment, the offspring will also become a high-licking mother (Champagne, 2008). Maternal care was once thought to be rigid; however, plasticity is more accepted. Plasticity allows for an offspring of a low-licking mother to become a high-licking mother in the future if place in a litter with a high-licking mother (Champagne, 2008). “If expression of maternal behaviors were narrowly locked to invariant neonatal characteristics, then the diversity of offspring nurtured and thus surviving would also be narrow. Hence, plasticity in mammalian mother–infant behavior: (a) allows for offspring to be nurtured in widely varying environments and circumstances, and (b) provides for preservation of genetic/behavioral diversity in those offspring.” (Fleming, 1999).
Preclinical Drug Testing
With medicine and drug improvements improving, safety protocols in approval methods must be revised as well. Research is always changing; therefore, preclinical trials for drug testing must also ensure that the procedures are up to date. Most preclinical trials utilize rodents to determine the safety and approval of a drug for human use. In experiments where postpartum rats are tested, preclinical studies have been conducted to determine maternal and offspring’s effects have been randomly assigned a postpartum rat and a litter of pups. These experiments include but several such as: Vitamin D present in rat pups (O’Loan, 2007), hyperactivity in rats separated from pups (Aisa, 2007), teratogenic effects of maternal antidepressant (Forcelli, 2007), and contraceptive usage on maternal rats ( Liu, 2010). This study leads to possible questioning of certain drug approvals as well as disapprovals since a lactating rat discriminates between Own and Alien pups leading to different outcomes in preclinical trials.
Survival Costs
“In mammals, the attraction between mother and young, and their mutual recognition depends on olfactory and somatosensory experiences” (Fleming, 1999). “In humans and animals alike, the olfactory system is an instigator of change in the brain circuitry associated with maternal behavior, and is, for the rat, its primary sensory input. For example, olfactory cues from young elicit maternal responses from the new mother, physically and emotionally” (Kinsley, 2010). The ability to recognize a pup is different than retrieving a pup where there is a choice to be made. “Evolutionary theory predict that mothers will direct care preferentially to their own offspring…Discriminative care of own offspring is common in mammals (Gubernick & Klopfer 1981; Holmes 1990; Clutton-Brock 1991), which makes adaptive sense for mothers given the high cost of lactation (Hanwell & Peaker 1977; Ko¨nig 1997) and other forms of maternal care.” (Jesseau, 2008). Maternal rats will retrieve and care for your Own pups and Alien pups; however, in Beach and Jaynes study on pup retrieval, mother rats did not discriminate between pups, but rather retrieved their own pups’ significantly faster (Beach, 1956). In our study, each mother rat is given various ratios of Own to Alien litters and monitored for latency to retrieve.
Current Study
Based on evolutionary outcomes and Beach’s findings, all rats should retrieve pups regardless of litter. We have found that there is significance between Own and Alien trials. This current study is designated to determine the threshold maternal rats discriminate between Own and Alien pups.
Materials and Methods
Animals
A total of thirty (30) 65-75 day old, pregnant Sprague-Dawley rats (Taconic Biosciences, US) were singly-housed in plastic cages with ALPHA-Dri bedding (Innovive, San Diego, CA). Rats were provided access to food (Teklad 2018, Harlan Laboratories, US) and tap water ad libitum on a 12/12 hour light cycle under standard housing conditions. Mothers were allowed tome t give birth and care for her pups. Experimental testing took place 6-9 days postpartum per pup litter.
Behavioral Protocol
All thirty rats underwent 6 trials consisting of different OWN to ALIEN pup ratios. The OWN:ALIEN ratio trials used in this experiment were 8:0, 4:4, 3:5, 2:6, 1:7, and 0:8. Each mother rat was randomly assigned an order to undergo all 6 trials with a minimum of 3 hours between each trial. Likewise, each mother was assigned a novice ALIEN litter of pups per trial to complete the ratios of OWN:ALIEN. The mother rat being tested was removed from her home cage, taken to a testing only room, and placed in her new testing cage environment. The mother rat then had 20 minutes to acclimate to her new cage which had the exact set up as her home cage. While the mother rat was removed, her pups remained in the home cage and placed under a heat lamp to maintain proper pup body temperature. During the acclimation period, the ratio of OWN:ALIEN pups for the specified trial were marked with an odorless marker. The OWN pups were distinguishable with parallel lines (II) while the ALIEN pups received a cross (X). Once marked, pups were placed in a Pyrex “pup cup” and kept under a heat lamp to ensure poikilothermic pup temperatures were maintained. After the 20 minute acclimation period, the “pup cup” was taken to the testing room and placed into the testing cage. Once the proper pup ratio is placed in the cage, 20 minutes of Pup Exposure begins and is recorded for behavioral analysis. After the 20 minutes of testing time, the mother rat and testing pups were returned to their respective home cages. The testing cage was removed and returned to the animal storage room. The Pyrex cups were sprayed with 70% ethanol, wiped with a paper towel, and died in order to remove the presence of odor from previous pups and utilize the cup again for future trials.
Behavior tests were controlled for time of day, age of pup, and source of alien pups. All alien pups were used on days their mothers were not testing. All animal procedures were approved by Longwood University’s Institutional Animal Care and Use Committee.
Behavioral Analysis
The mother rat’s behaviors were recorded for 20 minutes during the Pup Exposure Stage in the testing cage to observe interactions with the pups, including latency to retrieve the 1st, 4th, and 8th pup; time spent interacting with the pups, including grooming, sniffing, nursing, and nesting; time spent self-grooming; and time spent not interacting with the pups, including sleeping, drinking water, and other non-interacting behaviors (exploring cage, sitting, laying, etc.). In some cases, a mother would perform both an interaction activity as well as a non-interacting activity (e.g., some mothers would self-groom while nursing the pups). In this instance, the non-interacting activity (self-grooming, sleeping) was counted as the primary activity. Based on these criteria, mothers were grouped as “good” or “bad” mothers.
Tissue Collection
Following behavioral trials, we use a standard protocol to, preserve, isolate, and store maternal rat brains prior to sectioning. First, ensure that all material and supplies needed are accounted for as well as proper safety regulations are being followed such as clovers, lab coat, and glasses. Once ready, section the brain on trim mode until ideal brain regions are identified. Utilize a brain atlas to ensure proper sectioning of the prefrontal cortex, nucleus accumbens, amygdala, and hippocampus. These regions are important in maternal rat behavior, including roles in decision making, learning, memory, emotion, and inter-individual experiences. During sectioning, place each section in a 30-well Costar Tray with approximately 300 microliters of Phosphate Buffered Saline (PBS).
Immunocytochemistry and Neuroquantification
In addition to the behavioral experiment, we will also be analyzing the neural tissue of mother rats. For the previous experiment between strictly 100% Own, 50% Own, and 0% Own, brains were stained for c-fos protein. C-fos is expressed in response to exposure to novel stimuli in mammalian brains. By studying protein expression, we can determine which brain regions are responsible for the observed behaviors. To count neurons, we must first be able to visualize protein expression. This can be done via immunocytochemistry. Once visualized, we can then use software for neuroquantification and eventually data analysis. This method can help further data analysis in Neuroscience by testing for certain proteins in specified brain regions to determine which neurons are active during a specific behavior. The figures below represent the findings of previous experiments conducted.
The neuroquantification has not been completed for this current study of 100% Own, 50% Own, 37.5% Own, 25% Own, 12.5% Own, and 0% Own; the 30 rats used in this experiment, with the same protocol and procedure as the previous 100% Own, 50% Own, and 0% Own experiment which led to this current study of in depth Own ratios, will be stained for Estrogen, Oxytocin, and Fos B. These receptors have all been linked to maternal rat behaviors and will have determine the possibility of ‘Good’ and ‘Bad’ mothers.
After all sectioning is complete, move onto immunohistochemistry. Wash the brain sections with PBS 3-5 times, shaking for 5 minutes after each wash. Next, add the primary antibody to each well and store in the fridge overnight. On Day 2, wash the sections again with PBS to remove unbound primary antibody. After washing, secondary antibody is added for 10 minutes. After 10 minutes, again wash 3X in PBS for 5 minutes each. Then add Avidin-Biotin Complex (ABC) to tag the secondary antibody. Finally, DAB. This chemical – which is a mutagen to our cells, so extra precaution must be taken – causes the color change that allows us to visualize the protein. Lastly, wash tissue in PBS and store in 20 degrees Celsius in fridge until ready to place on microscope slides. When creating the wet mound slides of brain tissue, place a brain section on the slide and drop distilled water to remove folding and allow for the brain to spread out across slide, using a paintbrush to aid in the process. Once on the slide, remove water, add a drop of Permount, and cover with a coverslip. Store the slide in a slide box for further analysis.
The next step in neuron counting is neuroquantification which uses computer software to output neuron counting and neuron size. Now that the slides are ready to be analyzed, set up the microscope, camera, and computer to start recording the data. Here, Q Capture Paint, and Image J software is used to count the number and size of the neurons in a specified brain region including the Prefrontal Cortex, Amygdala, Hippocampus, and Nucleus Accumbens. Counting active neurons in these brain regions will allow us determine, statistically, if there are differences in maternal responses to own and alien pups.
Results
Behavior
After recording and analyzing behavioral videos, the ratio maternal rats distinguish between own and alien pup litters is at the 25% marker (Chi Square p<0.05) as shown in Figure 7. Maternal rats retrieve pups significantly faster than all trials at the 100% own and 50% own ratios at the p<0.05 level (Figure 6: Multivariate ANOVA). The 37.5% threshold is significantly different than the 12.5% ratio of own pups; however, not significant to the 0% level (Figure 6: Multivariate ANOVA). At the 25% own pup threshold, latency to retrieve pups is not significantly different from 12.5% and 0% levels (Figure 6: Multivariate ANOVA). The threshold is 25%, because that is where the mother will treat the ratio of pups as all alien and significantly slower than 100% and 50%. In Figure 7, the graph demonstrates the distribution of mothers who participated in the study. Occasionally, some lactating rats would partake in non-interacting behavior such as sleeping, self-grooming, and cage exploration. These non-interaction times increased as the percentage of own pups dismantled. Concluding that, in a litter of 100% own pups, more rats retrieve or interact with their pups. As the percent of own pups diminishes, as does the number of interacting mothers.
Neurological Underpinnings
In Figure 1, the Basal Lateral Amygdala shows maternal rat’s retrieval instinct is triggered more significantly (T-Test p <0.05) in a Mixed condition (Numan, 2010). This may indicate that quick retrieval to alleviate the anxiety of an ambiguous ratio of pups. In Figure 2, the Medial Amygdala fired the most neurons in the Mixed ratios when the mother’s anxiety is heightened (Gary, 2010). The most stressful scenario being a Mixed ratio of pups where the presence of Own or Alien is unclear and therefore, the mother builds in anxiousness and stress. In figure 3, the Hippocampus, learning and memory are not playing a key role in a mother’s decision making process (Weaver, 2004). There is no statistical difference found in the hippocampus w/in or among groups. In figure 4, the Prefrontal Cortex is crucial for using learned rules to control behavior such as complex decision making skill to decide whether to retrieve pups (Parent, 2015). In our study, the medial, dorsal, and lateral aspects of the Prefrontal Cortex were measured with the same outcome: Mixed significantly higher than both Own and Alien (Figure 4). The medial PFC is crucial for the maintenance of persistent licking and the expression of learned feeding strategies (Parent, 2015). The Prefrontal Cortex activation is higher in Mixed groups than in Own or Alien (T-Test p < 0.05), perhaps because the decision is clear in Own and Alien groups. This same trend occurs in the Amygdala. When presented with a concise, instinctual, no stress induced decision, there is less neurons recruited in completely the task at hand: pup retrieval. However, when the decision is hard and stressful, the maternal rat must fire more neurons to create connections and determine an answer to the problem. In figure 5, the Nucleus Accumbens stimulates goal-oriented behavior such as pup retrieval pathways (The Journal of Neuroscience, 2004). As seen in Mixed (T-Test p<0.05) and Alien (T-Test p< 0.01) conditions, the Nucleus Accumbens must recruit and therefore increase neurons to achieve the goal.
Discussion
In our study, we found that in litters of 25% or fewer Own pups the lactating rats will perceive the litter as Alien and retrieve pups slower. Knowing that maternal rats discriminate between Own and Alien pups is crucial in understanding the neuropathology of pup retrieval. In decision making processes where the mother can easily identify Own vs Alien pups, there is less neurons recruited to trigger the retrieval response; however, in cases where a difficult and unclear decision must be made, the number of neurons activated spikes as the neuronal connections increase to initiate a behavioral response. These neurobiological behaviors parallel the threshold of discrimination between Own and Alien pups as the 25% Mixed litter is perceived as Alien more often than the 50% and 37.5% Mixed ratios because the retrieval is clear.
Conclusion
This study provides real world applications in maternal behavior. With more studies conducted on maternal behavior per year, our understandings continue to improve. Maternal latency to retrieve allows one to determine the decision making behind maternal behavior. The effect of maternal care on pups significantly affects the pups innate behavior as parents in the future. “Based on our understanding of experience effects on brain in other animals, it is also possible that children who have been severely neglected or abused have experienced neurologic changes which result in altered affective, perceptual and cognitive function during development. Such changes are more than likely to affect how they perceive and respond maternally to their own offspring” (Fleming, 2008).
These results also become important when creating proper preclinical protocols on animal testing for future drugs. Based on our findings, one would suggest and preclinical scientist to ensure all pups remained with their mothers rather than randomly discharges mothers with random pups. For the most accurate testing, the litters of pups the mother interacts with must have at least 25% own pups for the best maternal behavior and care. If the litter used contained fewer than 25% Own pups, then the maternal rat would not interact properly and skew results. These findings lead to type I and Type II Error and could qualify disqualify a drug for human use. Previous approved drugs may have passed preclinical trials without proper mother –pup relationships and now the human race contains such potentially harmful drugs. In contrast, drugs that were not approved for human use may now be accepted due to a change in experimental protocol.
Future Directions
With this study’s findings, in the future we hope to equate ‘Good’ versus ‘Bad’ mother behavior by staining for Estrogen, Fos B, and Oxytocin. Such receptors for on cell membranes will provide implications of neurological evidence for ‘Good’ or ‘Bad’ mothers. This can this become a predictive method used in determining the likelihood of an offspring’s maternal behavior. In studies, Estrogen, Fos B, and Oxytocin have been linked to maternal rat behavior. We hypothesize that as the number of receptors or a gene expression shown increases, as does the percentage of being a ‘Good’ mother.
Moreover, in regards to animal behavior testing done at Longwood University, we must alert those working in and around the Chichester building that behavior testing is in progress. The slightest sound or smell can alter the maternal rat’s behaviors and therefore skew results. The cause of human error cannot be eliminated; however, efforts to improve experimental protocols and awareness of such experiments are always in need of improvements.
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