attachment between pup and dam by pairing an odor stimulus, in this case lemon, with the dam and then observing which stimulus a pup prefers, the dam ...
THE INFLUENCE OF NATURAL VARIATIONS OF MATERNAL CARE ON THE EMOTIONAL AND BEHAVIORAL REACTIVITY OF OFFSPRING IN THE RODENT MODEL
Ashley M. McFarland
A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of
MASTER OF ARTS August 2008 Committee: H. Casey Cromwell, Ph.D., Advisor Lee A. Meserve, Ph.D. Dara Musher-Eizenman, Ph.D.
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ABSTRACT H. Casey Cromwell, Advisor
Maternal care in rodents is a strong determinate of lifelong stress responsiveness and emotional regulation. The goal of the present study was to observe natural fluctuations of maternal care in rodents and to examine the effects of this variation in care on early affective and behavioral states of offspring. Rodent maternal care was observed for 8 days after birth. Using mean levels of arched back nursing and maternal licking and grooming (MLG) dams were categorized into high, medium, and low MLG mothers. Behavioral tests were then completed to examine levels of social motivation between the different MLG conditions. For isolation distress testing, each pup was isolated at postnatal day (PND) 10 for 2 minutes during which isolation ultrasonic vocalizations (USVs) were recorded. Next, on PND 15, place preference and USVs were measured involving a maternally paired odor. A third test was conducted to examine trends of juvenile play behavior, play suppression, and play USVs during 9 alternating days beginning on PND 24. After the completion of behavioral testing, thyroid hormone analysis (total T3 and T4) was conducted. Results showed an increase in vocalizations and maternal attachment during isolation in the high MLG animals, possibly indicating a greater ability to elicit maternal behaviors by high MLG pups during infancy. In contrast, animals experiencing low MLG displayed reduced preference for maternal cues and low levels of isolation calls. The two extreme levels of MLG diverged during play as well, with high MLG showing less response to isolation and a quicker recovery to baseline levels of play after the introduction of a predator odor. In contrast, low MLG animals showed higher levels of play following isolation and greater suppression after the presentation of the predator odor. Thyroid hormone analysis revealed a significant deficit of T4 in low MLG animals when compared to medium and high MLG
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animals. This decrease was still apparent in T3 analysis; however, it was not significant. Collectively, results suggest that slight, natural variations in maternal care influences early social learning and behavior in rat pups. Animals receiving varying types of maternal care diverge within the first 2 weeks of life in terms of their social motivation to respond to cues and communicate to their primary caregiver and also in their play behavior as juveniles.
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This manuscript is dedicated to my support group of seven: Mom, Dad, Emmy, Aunt Karen, Phill, Grandma, and Googs. Thank you for your ever-present and all-encompassing encouragement and support.
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ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. H. Casey Cromwell for all of his time and efforts during this process. His valuable input and numerous corrections to this research and manuscript were essential to the completion of this project. A large part of my continued involvement in this program was based on Dr. Cromwell’s enthusiasm for current projects and research in general. I cannot thank you enough for this opportunity. This manuscript would also not be nearly as complete without the editing and input of the rest of my committee, Dr. Lee Meserve and Dr. Dara Musher-Eizenman. Thank you for all of your time and advice. Many people assisted me with data collection and scoring during this project. Special thanks goes to Kelly Harmon for guiding me through each behavioral paradigm and USV scoring; Megan Greenwald for her support in the lab and assistance with maternal care observations; Travis Beckwith for running behavioral testing and scoring; and everyone else who assisted with behavioral testing, observations, and data scoring. Thank you for all of your hard work. I would also like to thank the staff at animal facilities and the Department of Psychology at Bowling Green State University for supporting this project. Last, but certainly not least, acknowledgement is due to those who served as a sounding board for my proposal, defense, and other aspects of this thesis. Thank you for your willingness to listen and for your advice and support.
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TABLE OF CONTENTS Page INTRODUCTION..... ...........................................................................................................
1
The Importance of Maternal Care .............................................................................
1
Maternal Care and the Rodent Model .......................................................................
2
Naturally Occurring Variations in Maternal Care.....................................................
4
Additional Evidence for the Importance of Maternal Care: Deprivation Studies .....
9
Ultrasonic Vocalizations and Early Social Motivation in the Rat Pup .....................
14
Conditioned Odor Preference and Pup-Maternal Relationships ...............................
17
Play and Play Suppression as Indicators of Positive and Negative Affect ...............
18
Thyroid Hormone: A Novel Approach to Studying Variations in Maternal Care ....
19
Specific Aims and General Hypotheses ....................................................................
20
METHODS……....................................................................................................................
22
Subjects........... ..........................................................................................................
22
Maternal Care Observations ......................................................................................
22
Behavioral Tests ........................................................................................................
23
Isolation Distress ...........................................................................................
23
Conditioned Odor Preference........................................................................
24
Play................................................................................................................
27
Thyroid Hormone Analysis .......................................................................................
29
Euthanasia .....................................................................................................
29
Hormone Assays............................................................................................
29
ANALYSIS……...................................................................................................................
30
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Maternal Care Behavior ............................................................................................
30
Ultrasonic Vocalizations (USVs)... ...........................................................................
30
Conditioned Odor Preference....................................................................................
32
Play Behavior... .........................................................................................................
32
Thyroid Hormone Assays..........................................................................................
33
RESULTS..............................................................................................................................
34
Maternal Care Behavior ............................................................................................
34
Ultrasonic Vocalizations (USVs)... ...........................................................................
35
Isolation...... ...................................................................................................
35
COP..... ..........................................................................................................
35
COP vocalizations per chamber ....................................................................
36
COP vocalizations per minute.......................................................................
36
Play USVs .....................................................................................................
37
Conditioned Odor Preference....................................................................................
49
Duration.........................................................................................................
49
Entrances... ....................................................................................................
50
Novel Odor Approach ...............................................................................................
50
Play............................................................................................................................
56
Thyroid Hormone ......................................................................................................
65
Correlations.................. .............................................................................................
67
DISCUSSION............. ..........................................................................................................
68
Maternal Care Behaviors...........................................................................................
68
Major Findings for Changes in Pup Affect and MLG Condition..............................
69
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Influence of Maternal Care on Pup Maternal Attachment.. ......................................
72
Juvenile Social Motivation and Fear Reactivity........................................................
73
Examination of Thyroid Hormones...........................................................................
75
Clinical Implications..... ............................................................................................
76
Conclusions and Future Studies ................................................................................
77
REFERENCES......................................................................................................................
79
APPENDIX A: MATERNAL CARE SCORE SHEET............ ............................................
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APPENDIX B........................................................................................................................
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Figure B1: Cohort 1, T3 assay standard curve and linear regression graph..............
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Figure B2: Cohort 1, T4 assay standard curve and linear regression graph..............
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Figure B3: Cohort 2, T3 assay standard curve and linear regression graph..............
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Figure B4: Cohort 2, T4 assay standard curve and linear regression graph..............
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Figure B5: Cohort 3, T3 assay standard curve and linear regression graph..............
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Figure B6: Cohort 3, T4 assay standard curve and linear regression graph..............
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APPENDIX C........................................................................................................................
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Table C1: Low MLG Pearson correlations for USV data............ .............................
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Table C2: Low MLG Pearson correlations for COP data.............. ...........................
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Table C3: Low MLG Pearson correlations for Play data............... ...........................
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Table C4: Medium MLG Pearson correlations for USV data...................................
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Table C5: Medium MLG Pearson correlations for COP data.............. .....................
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Table C6: Medium MLG Pearson correlations for Play data.................................... 100 Table C7: High MLG Pearson correlations for USV data............ ............................ 101 Table C8: High MLG Pearson correlations for COP data......................................... 102
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Table C9: High MLG Pearson correlations for Play data............... .......................... 103
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LIST OF FIGURES Figure
Page
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Spectrogram of 44 kHz Isolation USVs ...................................................................
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2
COP Apparatus..........................................................................................................
25
3
Normal Distribution of Litters...................................................................................
38
4
Maternal Care Behaviors...........................................................................................
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5
Individual Maternal Care Behaviors per MLG Condition ........................................
40
6
Average Number of MLG Behaviors per MLG Condition.......................................
41
7
USVs for Isolation Distress Testing..........................................................................
42
8
USVs for COP Testing................................................................................................
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9
COP USVs over Maternal LG Condition and Associative History. PND 14 ................................................................... ................
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10
A Comparison of Isolation Distress and COP USV...................................................
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11
USVs per Minute of COP Testing..............................................................................
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12
COP USVs per minute and sex...................................................................................
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13
Play USVs during play day 5......................................................................................
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14
Durations for Both Odor Chambers Dependent On Associative History ...................................................................................................
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15
Time Spent in the Conditioned Odor or Neutral Chambers ......................................
52
16
Entrances During COP Testing .................................................................................
53
17
COP Entrances by Associative History and Chamber Odor .....................................
54
18
Novel Odor Approach by Associative History And Maternal LG Condition ................................................................... .................
55
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19
Total Play Behaviors Averaged Across 9 Days ........................................................
59
20
Average Play Behaviors Across 9 Days for MLG Condition ...................................
60
21
Change in Play Behaviors in the High MLG Condition ...........................................
61
22
Change in Play Behaviors in the Low MLG Condition ............................................
62
23
Change in Play Behaviors in the Medium MLG Condition......................................
63
24
Duration of Collar Approach (Day 4) and Collar Contact (Day 5)...........................
64
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Thyroid Hormone Concentrations.............................................................................
66
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LIST OF TABLES Table 1
Page Physiological Differences between Low MLG And High MLG Animals ..........................................................................................
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Natural Variations in Maternal Care 1 INTRODUCTION The Importance of Maternal Care Different levels of parental care and investment can dramatically influence the path of offspring development. From extreme cases of abuse, to those of neglect and even subtler indifference, maternal care has the power to influence the behavior of offspring, not only during childhood and early development, but also throughout adulthood (De Bellis, 2005). Strong and supportive maternal relationships also have a resounding impact on child development (Baumrind, 1991). Self-control, self-reliance, and social competence tend to be greatest in children that experience highly supportive maternal behavior. The importance of maternal care and bonding is not only apparent in the extremes of the maternal care spectrum, but also in the subtler, naturally occurring variations of parenting styles (Baumrind, 1991). A range of animal models has been used to study maternal care and behavioral effects comparable to clinical cases focusing on parent and child relationships. In the rodent model, the importance of maternal care is made apparent by the changes that occur in pup behavior following the disruption of maternal care or from the effects of subtler, naturally occurring variations in maternal licking and grooming. Early life maternal care in rodents is a strong determinate of stress and fear responsiveness, learning and memory development, expression of behavioral defensiveness, along with other hormonal and behavioral effects on the pups (Caldji, Diorio, & Meaney, 2000; Lovic & Fleming, 2004; Oitzl, Workel, Fluttert, Frosch, & de Kloet, 2000; Menard & Hakvoort, 2007; Champagne, Francis, Mar, & Meaney, 2003). In the rat, greater maternal care produces a more exploratory and less emotionally reactive offspring, while
Natural Variations in Maternal Care 2 less maternal care leads to just the opposite: a less exploratory and more emotionally responsive pup (Champagne et al. 2003). The present study was conducted in order to gain a better understanding of the importance of maternal care in determining affective responsiveness and maternal-pup bonding in the rodent model. Many researchers have concentrated their efforts on examining the causes of alterations in maternal care (Champagne, Diorio, Sharma, & Meaney, 2001; Camerons, Champagne, Parent, Fish, Ozaki-Kuroda, & Meaney, 2005; Patin, Lordi, Vincent, Thoumas, Vaudry, & Caston, 2002), the effects of maternal deprivation (Cirulli, Alleva, Antonelli, & Aloe, 2000; Knuth & Etgen, 2007), and the effects of altered maternal care experienced in infancy on adult behavior (Moore & Power, 1992; Francis & Meaney, 1999). The current study is the first to explore the early behavioral impact of natural variations in maternal care on early rat pup social response and USVs. Measures of early social motivation are used to evaluate the emergence of either neophilic or neophobic pup responses associated with maternal attachment, juvenile social behaviors, levels of social communication, and reactions to novel stimuli and environments. Maternal Care and the Rodent Model The rodent model offers several advantages compared to other methods used in the investigation of maternal care, early social interactions, and general development. These include the ability to exert extensive control over environmental and experimental details; a shorter maternal care observation period; the precise measurement of easily quantifiable maternal care variables; the ability to add multiple and more invasive manipulations; and, overall, less genetic, social, and experimental confounds. The longer periods of maternal care that are needed in human and non-human primate studies can make the manipulation and subsequent tracking of
Natural Variations in Maternal Care 3 specific variables difficult. These obstacles make the primate model, although still useful, more difficult as a method for examining maternal care (Champagne et al. 2003). Rodents exhibit considerably less prenatal development as compared to humans, but the rodent model allows for greater control of experimental details, and allows invasive procedures and manipulations that are more potent in their likelihood to change development (Rice & Barone, 2000). The regional development of the rodent brain is comparable to the development of the human brain on a timeline of days versus weeks to months, and the gross regional development of the rodent brain and human brain is similar; however, as with all animal models, findings must be placed into context (Rice & Barone, 2000). Specific details may not be transferable, but, in most cases, general relationships are conserved from rodents to primates, and even to human cases. Rodent maternal care, transmitted from mother to pup can be defined into easily quantifiable and observable variables (Myers, Brunelli, Squire, Shindeldecker, & Hofer 1989; Champagne et al. 2003; Caldji et al. 2000). Maternal cues associated with arched-back nursing and licking and grooming during the first week postpartum regulate the developing systems of the pups (Champagne et al. 2003). This nursing and grooming regimen consists of four main behaviors: 1) arched back nursing, observed when the mother is arched over the pups with legs splayed; 2) maternal licking and grooming, observed when the mother is licking or grooming any pup; 3) blanket nursing, observed when the mom is lying over the pups with no visible arch in her back; and 4) passive nursing, observed when the mother is lying on either her back or side while the pups nurse (Myers et al. 1989, Champagne et al. 2003). These maternal care behaviors play a major part in maternal attachment and bonding and are easily susceptible to environmental factors such as chronic stress and toxin exposure (McEwen, 2007; Simmons, Cummings, Clemens, & Nunez, 2005).
Natural Variations in Maternal Care 4 Naturally Occurring Variations in Maternal Care Variations in rodent maternal licking and grooming can be quantified and classified into levels of high or low maternal care. Natural variations in maternal care lead to observable differences in rat pup behavior (Champagne et al. 2003). Examining these natural variations can aid in the identification of the important and necessary aspects of maternal care that lead to behavior differences. This type of research also promotes a better understanding of basic rat behavior, necessary to consider when working with a model to understand human psychology and social interactions. Previous work by Champagne et al. (2003) guided the design of the present study. This group completed observations in order to measure the adaptive, behavioral effects of a normal range of maternal care fluctuation on offspring. Litters were left undisturbed for the first 10 postnatal days and observed for the first 8. Each dam was observed for five, 72 minute periods, 25 observations within every observational time period. Five specific behaviors, as previously described by Myers et al. (1989) were observed: mother licking and grooming any of the pups, mother nursing the pups in an arched-back posture, mother nursing the pups in a blanket posture, mother nursing the pups in a passive posture, and a no contact observation that included any activity not associated with the litter. The assignment of the animals into high or low licking and grooming groups was calculated from the mean and standard deviation of the licking and arched-back nursing behavior for the entire cohort. Arched-back nursing and maternal licking are crucial in the production of major differences between the groups in previous studies. Mothers performing licking and grooming behaviors at least 1 standard deviation above the mean were termed high licking and grooming, arched-back nursing (LG-ABN) dams and animals performing at least 1 standard deviation below the mean were termed low LG-ABN dams. The frequency distribution
Natural Variations in Maternal Care 5 calculated for the high and low mothers nearly significantly matched a normal distribution curve, with fewer high and low LG-ABN dams and an abundance of medium maternal care givers. The detailed observations of the Champagne study (2003) also revealed many factors related to the maternal behaviors themselves, such as; the nursing behaviors in rats occurs more frequently during the day/night cycle’s light phase and that the amount of licking and grooming decreases significantly among groups as the pups aged. Results showed that the amount of maternal licking and grooming received by one pup did not differ significantly from the amount of care received by the entire litter, and that litter size was not correlated with levels of archedback nursing or maternal licking and grooming. Sex differences also did not provoke more or less maternal care during the first 8 days of observations. High and low LG-ABN dams remained distinct and recognizable from each other during most of the observation time periods, specifically during PND 2-4; however, after 6 days of observations, observed behaviors between the two groups were no longer significantly different. This shows that the first week postpartum is crucial in terms of the differences in the type of maternal care received (Champagne et al. 2003). The results of the Champagne study (2003) concur with those of others in finding that maternal care did not differ in the overall contact time the dam spent with the pups, but in the amount and duration of specific behaviors such as licking and grooming bouts: high LG-ABN dams showed a significant increase in duration and number over low LG-ABN dams. The results of this portion of the study support the existence of variations in maternal care that are not necessarily harmful or neglectful since the amount of contact time spent with the pups is not significantly different between the two groups, and litter size and pup weaning weights also did not differ between low and high LG-ABN litters (Champagne et al. 2003). The fluctuations
Natural Variations in Maternal Care 6 found in rodent maternal care could possibly play an adaptive role, preparing the pups for a less or more harsh environment depending on current environmental conditions. These natural variations in maternal care are not considered as “good” or “bad” parenting and tend to lie within the functional range of care and are unrelated to measures of reproductive success (Champagne et al. 2003). This stability in reproduction makes these behaviors more adaptive than harmful. The defensive, adaptive behaviors exhibited by rat pups that have experienced fewer licking and grooming bouts tend to be more emotionally responsive and are more neophobic in nature as a response towards novel stimuli. Also pups that have experienced greater amounts of licking and grooming are less emotionally responsive and tend to be neophilic towards novel stimuli (Champagne et al. 2003). It is plausible that the increase in stress reactivity apparent in the offspring of low MLG mothers is adaptive by evoking more fear and causing the pups to be more cautious of their environment (Francis et al. 1999). Natural variations in maternal licking, grooming, and arched-back nursing have been associated with several effects on the development of several neural systems that govern the hypothalamic-pituitary-adrenal (HPA) axis response and learning and memory (Table 1; Champagne et al. 2003). Variations in maternal care can impact the number of neurochemical receptors involved in the reaction to acute stress, such as glucocorticoid receptors in the hippocampus and oxytocin receptors (Liu, Diorio, Tannenbaum, Caldji, Francis, Freedman, Sharma, Pearson, Plotsky, & Meaney, 1997; Champagne et al. 2001). This occurs more profoundly in the low MLG animals, contributing to their display of neophobic behavior (Champagne et al. 2003). Deficits in memory retention are also more pronounced in the low MLG animals, possibly resulting in a greater display of neophobic behavior (Lovic & Fleming, 2004). The array of changes caused by less licking and grooming, a subtle change in behavior
Natural Variations in Maternal Care 7 that is still considered part of the adequate maternal care spectrum, adds more importance to these maternal behaviors. Individual differences in maternal behavior are transmitted from the dam to her offspring and displayed in the interactions of the offspring with their young (Francis & Meaney, 1999). Behavioral profiles are passed through non-genomic means from generation to generation and are readily manipulated by environmental factors (Champagne et al. 2003). Cross-fostering experiments have linked the rodent model to other animal models in terms of the transmission of maternal care behaviors, by producing similar results. This makes the rodent model useful in studying manipulations and the natural progression of maternal behaviors (Champagne et al. 2003; Gonzalez, Lovic, Ward, Wainwright, & Fleming, 2001; Francis, Diorio, Liu, & Meaney, 1999). Non-genomic behavioral transmission has been displayed in numerous studies including a study by the Gonzalez group (2001), where pups that were artificially reared and experienced maternal deprivation developed into dams that expressed low levels of maternal care to their own pups. Results of cross-fostering studies involving natural variations in maternal care revealed that low LG-ABN pups reared by high LG-ABN mothers behaved like their high LG-ABN littermates. The same behavior was true for the reverse situation: high LG-ABN pups reared by low LG-ABN mothers were indistinguishable from the offspring of the low LG-ABN females (Francis et al. 1999a). Together, these findings suggest that individual differences in maternal behavior can be transferred not only through the alteration of gene expression caused by low maternal licking and grooming (Fish, Shahrokh, Bagot, Caldji, Bredy, Szyf, & Meaney, 2004), but also through a behavioral mode of transmission: maternal behaviors can affect phenotypic expression in the pups.
Natural Variations in Maternal Care 8
Physiology ACTH response to acute stress
Increase In Low MLG Animals
CORT response to acute stress PVNhCRF mRNA expression
Behavior Affects the core of the HPA axis. Increase in CRF expression and receptors lead to an increase in ACTH expression from the pituitary. An increase in the ACTH response leads to an increased CORT response. This leads to increased stress reactivity and hypervigilance to novel surroundings.
CRF receptor in Locus ceruleus
Decrease in Low MLG Animals
Hippocampal GC receptor protein expression Deceasein memory retention CBZ receptors in the amygdale Increase in fearfulness
CBZ receptors in the locus ceruleus NMDA receptors in hippocampus Decrease in learning and memory
Synaptophysin in hippocampus Acetylcholine levels in dorsal hippocampus
Decreasein anxiety modulation
Vasopressin receptorsin the amygdale
Associated with high anxietylevels, poor social interaction, and decrease in maternal behaviors
Oxytocin receptors Estrogen receptor in medial preopticarea
Table 1: Physiological and behavioral differences between low maternal licking and grooming (MLG) and high MLG animals. Adapted from Champagne et al. 2003.
Natural Variations in Maternal Care 9 Additional Evidence for the Importance of Maternal Care: Deprivation Studies The use of animal models in maternal care studies is useful in analyzing the effects of early trauma such as maternal deprivation and environmental or pharmacological stress on the development of offspring. Classic isolation research conducted by Harlow and colleagues (1965) studied the effects of partial and complete maternal and social isolation in young rhesus monkeys. Partial maternal and social isolation, in which monkeys were reared from birth in bare wire cages in close proximity to each other, resulted in abnormal behavior such as repetitive stereotypical movements, detachment from the environment, hostility toward self and others, and difficulty in forming attachments in adolescence and adulthood (Harlow et al. 1965). Total social isolation was achieved by housing monkeys from a few hours after birth for 3, 6, or 12 months in stainless-steel chambers. During this time the monkeys were not allowed contact with any animal or human, but were allowed to still experience what sensory stimulation permeated their cages (Harlow et al. 1965). Isolation and maternal deprivation effects were then observed during social interaction in pairs after the isolation time period of 3, 6, or 12 months expired. The effects of 3 months of isolation proved to be debilitating but reversible. Monkeys that experienced 6 months of isolation were unable to interact socially except with random bouts of aggression, and the monkeys experiencing 12 months of isolation became helpless in social situations prompting aggression from their control counterparts (Harlow et al. 1965). The severe changes in behavior and development caused by extended isolation, further supports the importance of early life maternal care in forming parental and social bonds. Rat studies have also been used to examine the effects of interrupted maternal care on offspring. A study by Oitzl et al. (2000) examined the effects of maternal deprivation on spatial learning and memory in the Brown Norway rat from a juvenile age through older adulthood. The
Natural Variations in Maternal Care 10 Morris water maze was used to evaluate cognitive abilities of rats deprived of maternal interaction and their non-deprived littermates as young adults (3 months), adults (12 months), aged animals (24 months) and senescent animals (30-32 months). Separation of the pups from the dam occurred on postnatal day (PND) 3 and lasted for 24 hours, and after this separation the home cages were not changed or disturbed until weaning at PND 21. Rats were then tested at varying ages with the Morris water maze: a pool filled with warm water made opaque by the addition of chalk. Maternally deprived animals showed a delayed acquisition of knowledge of the position of the platform during youth and adulthood. Adult rats showed impaired reversal learning, the ability to remember a change in platform location, when reintroduced to the Morris water maze. This study also establishes that individual differences in learning ability attributed to the deprivation of maternal care become amplified during aging. The results from this study demonstrate that a traumatic event in early life, such as a 24 hour maternal deprivation, can modify cognitive function and learning ability throughout development and adulthood. Another study used to explore the severe effects of maternal deprivation was conducted by Lovic and Fleming (2004). In this study 3 day old female rat pups were removed from the dam and placed on an artificial rearing system with untouched littermates as controls. Experimental groups consisted of pups that received gastric cannulas and were artificially reared with minimal stimulation mimicking maternal care (AR-MIN); a group of pups who received a gastric cannulae and were artificially reared with maximal maternal-like stimulation (AR-MAX); and a gastric cannulae sham operation group that experienced a sham operation and was then returned to the mother (SHAM). Mock maternal stimulation was received a variable number of times daily depending on the experimental condition of the group. Several experiments were then
Natural Variations in Maternal Care 11 conducted to test the ability of the animals to focus and hold attention, levels of startle response, and subsequent level of maternal behavior. The animals artificially reared with minimal maternal-like stimulation displayed hyperactivity throughout testing. AR-MIN animals required more trials to successfully complete the attention shifting task than controls. AR-MAX animals did not show significant differences from controls. Prepulse inhibition testing was conducted to measure levels of startle response, during which the animals experienced an array of unexpected sound pulses, varying in intensity, frequency, and duration. AR-MIN animals showed significantly less prepulse inhibition than maternally reared animals, indicating a greater startle response with AR-MIN animals. Overall, the animals that were artificially raised and experienced the least maternal care showed the greatest deficits in the learning and memory tasks and also showed more anxiety and a greater fear response during the prepulse inhibition task. AR-MIN pups also showed less maternal care to their own pups, exhibiting the intergenerational transmission of maternal care. This study enforces the importance of maternal care at early stages of development and explores the influence of negative and stress-provoking experiences in the absence of this care. Results also demonstrate that tactile stimulation, the maternal licking and grooming, plays a role as one of the more important maternal care behaviors in determining offspring behavioral phenotypes (Lovic & Fleming, 2004). Environmental stressors also impact the quantity and quality of maternal care received. Alongside social and economic hardships, a toxic environment can lead to stress and altered physiological behavior. Exposures to toxins such as led, mercury, and PCB can commonly occur under concurrent environmental conditions (Shen, Wania, Lei, Teixeira, Muir, & Xiao, 2006). Polychlorinated biphenyls (PCBs) were regularly produced through the 70s as a nonflammable
Natural Variations in Maternal Care 12 industrial lubricant and are still one of the leading persistent environmental toxins in the ecosystem today (Borlakoglu & Haegele, 1991). The combination of the stable molecular structure and level of toxicity make PCB a very good candidate as an environmental stressor. Most PCB exposure occurs through bioaccumulation in food sources, such as fish, lower on the food chain (Delinger, 2004). PCBs can easily be ingested by a mother rat through the consumption of other exposed animals and then transferred to her pups via nursing (Crofton, Kodavanti, Derr-Yellin, Casey, & Kehn, 2000). Not only do the pups experience physiological deficits caused by the ingestion of PCB, litters exposed to this contaminant tend to have lower survival rates in the first 6 days (Borlakoglu & Haegel, 1991). The ingestion of PCB not only affects pup behavior but also alters the maternal behavior of the dam (Simmons et al. 2005). Larger doses, 4 mg/kg of diet, of PCB can cause the dam to spend a greater amount of time at the nest and a greater percent of that time licking and grooming the pups when compared to animals exposed to lower doses, 1.5 mg/kg, and unexposed animals (Simmons et al. 2005). These findings seem to convey an opposite message to the results observed with social status as a stressor. In a situation involving a toxin, the dam attempts to counteract the physiological deficits caused by the PCB in her pups and chooses to become more protective; aiding the pups by increasing her level of maternal care resulting in an adaptive neophilic behavioral change in the pups (Cameron et al. 2005). Aside from harsh chemical pollutants, other more subtle stressors can cause similar adaptive adjustments in maternal care. In a 2000 study by Pryce and colleagues, the effects of early handling of the pups and early maternal deprivation on maternal care were compared. This study consisted of four treatment groups: early-handling (EH); early deprivation (ED); early nonhandling (NH); and a control group. Early handling consisted of removing each pup from the
Natural Variations in Maternal Care 13 home cage for 15 minutes and early deprivation consisted of a separate group of pups isolated for 240 minutes daily for PND 1-21. Pups that received the early handling treatment were licked by the dam more than early deprivation or control animals and also experienced greater levels of arched-back nursing, a critical maternal care behavior, than control or early deprivation pups. Other studies have shown that handled pups have a reduced stress response and decreased fearfulness (Pryce, Bettschen, & Feldon, 2000; Meaney, Diorio, Francis, Widdowson, LaPlante, Caldji, Sharma, Seckl, & Plotsky, 1996; Francis et al. 1999) and enhanced cognitive function (Feldon & Weiner, 1992). Animals that experience prolonged maternal deprivation do not acquire any beneficial behavioral outcomes and can experience an increased stress response (Plotsky & Meaney, 1993). The two behavioral profiles produced by EH and ED are comparable to the behavioral changes resulting from natural fluctuations in maternal licking and grooming. Prenatal stress is another environmental factor that can have a major impact on the quality of maternal care. In a study by Patin and colleagues (2002), prenatal stress not only caused changes in maternal behavior, but also caused abortion and death of several stressed litters. Stressed dams can display decreased levels of maternal licking and also a decrease in the percentage of pups brought back to the nest during a pup retrieval task. These examples of environmental stressors reflect the adaptive ability of maternal care from the normal, anticipated level of demand. Environmental stressors, such as prolonged pup handling and a toxic environment caused by PCBs momentarily increase maternal care, reinforcing the theory that fluctuations in maternal care are not necessarily examples of neglect or abuse, but can be a form of adaptive response to changes or challenges in the environment.
Natural Variations in Maternal Care 14 Ultrasonic Vocalizations and Early Social Motivation in the Rat Pup Ultrasonic vocalizations (USVs, Figure 1) are important indicators of the emotional state of the rat pup and are used by the pup to elicit certain types of maternal behavior, such as licking and grooming (Barron, Segar Yahr, Baseheart, and Willford, 2000; D’Amato, Scalera, Sarli, Moles, 2005).
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2.4
2.6 s
Figure 1: An example of 44 kHz isolation USVs reduced to an audiablle frequency and visualized through an Avisoft Bioacustic spectrogram. Rats emit USVs during distressful situations, such as isolation from the mother (Brudzynski, Kehoe, & Callahan, 1999) and in positive social situations, such as play (Panksepp & Burgdorf, 2003). Studies have shown that ranges of vocalizations are indicative of certain emotional states in the rat, and that the three different identifiable ranges of vocalizations are each specific to an emotional context (Knutson, Burgdorf, & Panksepp, 2002). Distress calls in adult rats are emitted at a lower frequency, around 20-25 kHz and tend to be longer in duration than social play behavior calls that register around 50 kHz and have a very short duration (Panksepp & Burgdorf, 2003). Isolation calls in younger pups to juveniles register in the 30-44 kHz range (Barron et al. 2000). The high-range USVs fall into three general categories: physiological, where high USVs are an artifact of motor activity; motivational, where high USVs are the by
Natural Variations in Maternal Care 15 product of a motivational or emotional state; and social, where high USVs enable individual rats to communicate (Knutson, Burgdorf, & Panksepp, 1998). Pups produce the highest rates of lower frequency isolation calls when separated from littermates and the home cage and placed alone in novel surroundings at cool temperatures (Shair, Masmela, Brunelli, & Hofer, 1997). Isolation calls are used not only to elicit licking/grooming and nursing from the dam, but also to initiate pup retrieval (Shair et al. 1997). Isolation calls can be seen as a measurement of attachment: the calls, under normal circumstances arrive at mother as a predictable outcome. Lower frequency USVs can also serve as a quantifiable measure of anxiety: the greater the calls, the more anxiety observed. The neuropharmacology of these calls closely represents that found with similar anxiety provoked behaviors in adult rats and anxiety behaviors in humans (Shair et al. 1997). When placed into a novel environment and separated from the dam, we predicted that the neophobic behavior produced by a low licking and grooming (low MLG) would lead to the production of a greater number of isolation distress calls when compared to a control pup or a pup that experienced high licking and grooming (high MLG). As hypothesized above with the isolation distress testing, the neophobic behavior incited by low MLG should also provoke a greater emotional response expressed through increased distress calls compared to the number of calls elicited from a high MLG pup during the isolation experienced during the conditioned odor preference testing. In comparing calls across paradigms and development, it is also feasible that a greater number of calls will be emitted during COP than in isolation distress. A study by Levine and colleagues (1985) compared the response of rhesus monkey infants when totally separated from their
Natural Variations in Maternal Care 16 mothers and those physically separated but still allowed visual, auditory, and olfactory contact (Levine, Johnson, & Gonzalez, 1985). Results showed that infants in total separation called less but did display increased levels of arousal, as measured by cortisol levels. The infants that were still allowed sensory contact called more but maintained a controlled cortisol response. Levine showed that the reaction from these infants, their proximity-seeking behavior, caused an adaptive modulation in arousal when maternal cues were present (Levine et al. 1985). Therefore, the maternal-paired odor used in the COP testing should cause the pups to call more than when in total isolation. The difference in calls might be diminished within the low MLG group, since they display greater levels of reactivity. The USVs found during play in the rat are much higher in pitch than those found during distressful situations. Panksepp and Burgdorf (2003) also investigated the possible emotional context behind these 50 kHz chirps. This “laughter” is emitted by rats during human tickling of the animals and rough and tumble play with conspecifics. Juvenile rats display a desire for human tickling after previous handling, by responding and moving towards a human hand, after only two days of isolation. We hypothesized that a neophobic and more emotionally reactive rat pup would be less likely to engage in play bouts in a novel environment with a novel partner. The introduction of a collar previously worn by a cat into the play environment would suppress the 50 kHz vocalizations, causing an increase in the production of low frequency vocalizations by the low licking and grooming pups. The increase in anxiety and emotionality in these animals should lead to an increase in the amount of lower vocalizations (30-44 kHz) in times of isolation distress and maternal separation, and a decrease in the amount of 50 kHz chirps during play.
Natural Variations in Maternal Care 17 Conditioned Odor Preference and Pup-Maternal Relationships Conditioned odor preference testing (COP) is another way to observe the neurobiological attachment between pup and dam by pairing an odor stimulus, in this case lemon, with the dam and then observing which stimulus a pup prefers, the dam conditioned stimulus or a neutral stimulus. Since rat pups are blind and deaf at birth, this odor preference tests allows for young pup behavior to be explored using a sensory model appropriate for the developmental time period (Nelson & Panksepp, 1996). Similar to the isolation distress paradigm, we predicted that with the COP, in a novel environment a pup is more likely to survive if closer to the dam, and therefore, it can be speculated that those pups more suited for survival search mom out more readily. The greater amount of anxiety exhibited by the low MLG pup, as shown by the increase in the number of maternal separation calls and a greater tendency to search out the mother implies that the low MLG pup would be more attracted to the conditioned stimulus over the neutral stimulus than the high MLG pup. However, as previously demonstrated (Table 1), low licking and grooming pups have fewer oxytocin receptors in several brain areas besides the hypothalamus (Champagne et al. 2003). Manipulations of levels of oxytocin, and therefore oxytocin receptor numbers, result in alterations of infant-dam attachment: less oxytocin results in less maternal attachment between pup and dam. This depression of attachments leads to a decrease in the affinity for the conditioned stimulus (Nelson & Panksepp 1996). When pups were tested after being exposed to a substance known to decrease oxytocin levels, polychlorinated biphenyl (PCB), a decrease in a preference for the maternal conditioned odor was observed (Cromwell, Johnson, McKnight, Horinek, Asbrock, Burt, Jolous-Jamshidi, & Meserve, 2007). This insight poses a counter point that low licking and grooming pups will show less affinity for the
Natural Variations in Maternal Care 18 conditioned stimulus, such as exhibited by the PCB exposed pups, due in part to the subnormal concentration of oxytocin. However, dams exposed to greater doses of PCB exhibit a greater amount of licking and grooming than control dams (Simmons et al. 2005). The toxic nature of PCB causes difficultly in pinpointing the cause of the increase in maternal licking and grooming. Possibly this extra care is given in an attempt to bring the pups back to normal behavior after PCB exposure. This increase in licking and grooming could cause oxytocin deprived pups to exhibit a control to high MLG pup phenotype through a different neurobiological mechanism. This experiment provided a critical test in order to choose from these alternative scenarios. Play and Play Suppression as Indicators of Positive and Negative Affect The introduction of the smell of a predator into the play chamber has been demonstrated to significantly decrease the amount of ongoing non-defensive play behavior (Siviy, Harrison, & McGregor, 2006). Within the context of low licked and high licked pups, the low MLG pups would be expected to react in a highly defensive manner, displaying a greater amount of rearing and cautious behavior and virtually no play. High MLG pups would also be expected to display defensive behavior and a change in play behavior, but less severe than the low licking and grooming pups. The introduction of the novel control stimulus, the unworn cat collar, could also cause an initial decrease in play due to exploration. As the pups begin to incorporate the unworn cat collar into their play bouts, the low MLG pups would tend to interact less with the unworn cat collar caused by the novelty and anxiety provoked by the stimulus. After removing the predator smell of the worn cat collar and continuing the play bouts as they were conducted to determine the baseline, the recovery of play and risk assessment was examined. In an experiment conducted by Siviy et al. (2006), play behavior did not return to
Natural Variations in Maternal Care 19 baseline until several play sessions following the worn cat collar exposure. The number of nape contacts was significantly depressed from baseline until 6 play exposures after the worn cat collar and pins did not reach baseline values within the seven play exposures given to test extinction. Due to the increase of anxiety behaviors attributed to low MLG behavior, we hypothesized that low licking and grooming pups would have a more severe reaction to the presence of the worn cat collar and a slower recovery from play extinction than the high MLG and control pups. Thyroid Hormone: A Novel Approach to Studying Variations in Maternal Care Another aim of this study was to examine a hormone possibly involved in and affected by natural fluctuations in maternal care. Changes in the hypothalamic-pituitary-adrenal (HPA) axis has been pinpointed in many studies as the main physiological activator of the increase fear reactivity and stress response seen in low MLG pups (Huot, Gonzalez, Ladd, Thrivikraman, & Plotsky, 2004; Caldji et al. 2000). Abnormally elevated circulating levels of thyroid stimulating hormone have been associated with HPA axis dysfunction in autism (Nir, Meir, Zilber, Knobler, Hadjez, & Lerner, 1995) and the neurobehavioral characteristics of ADHD (Negishi, Kawasaki, Sekiguchi, Ishii, Kyuwa, Kuroda, & Yoshikawa, 2005). HPA axis dysfunction can lead to an increase of thyroid stimulating hormone which can lead to an increase in circulating thyroid hormones T3 (triiodothyronine) and T4 (thyroxine). Abnormal amounts of T4 have been found in both depressed and schizophrenic patients (Roca, Blackman, Ackerly, Herman & Gregermen 1990). Stepping away from the well studied HPA axis, we examined the little known effects of maternal care on the circulating concentration of thyroid hormone T3 and T4, searching for possible hyperthyroidism or hypothyroidism within the distinctive groups of high MLG and low MLG pups.
Natural Variations in Maternal Care 20 Specific Aims and General Hypotheses Maternal care, displayed through licking/grooming and nursing in the rat, can have widely dispersed effects on offspring (Champagne et al. 2003). Several researchers have studied the causes of variations of maternal behaviors, but few have examined the extent of the effects of natural variations in maternal care on early pup behavior. The affective states of the pups in early social learning and social contact, with the use of ultrasonic vocalization (USV) analysis and a conditioned odor preference paradigm, has yet to be examined. The current study set out to add to the general descriptions of the behavioral profiles attributed to each MLG condition. We expected to see two distinct sets of behavioral reactions within our testing paradigms: 1) Low MLG animals would display more anxiety and less exploration, consistent with the previously identified variables of their neophobic behavioral profile and hyperactive HPA response. Isolation USVs would be greater in these animals than in high MLG pups during the isolation distress and COP paradigms, indicating more pronounced levels of anxiety. The higher levels of anxiety would also appear in the COP behavioral data, causing the low MLG animals to have a great affinity for the familiar, conditioned odor. Less affinity for the conditioned odor could also be seen in the low MLG animals due to a decrease in oxytocin receptors. We also expected to find less play, a slower rate of recovery from play extinction, and an increase in circulating thyroid hormones when compared to our controls, the medium MLG group and also the high MLG animals. 2) High MLG animals would display behaviors opposite those of the low MLG animals, consistent with their neophilic behavioral profile. We expected low MLG animals and high MLG animals to differ significantly throughout all behavioral testing paradigms and the thyroid
Natural Variations in Maternal Care 21 hormone analysis. High MLG animals would possibly display behaviors indicative of an adaptive advantage gained from greater maternal care, differing also from the behaviors of the medium MLG control group.
Natural Variations in Maternal Care 22 METHODS The first step in this study was to observe each litter in order to quantify the maternal care received by each litter into the low MLG, medium MLG, and high MLG categories. After this observational period, a battery of behavioral tests was used to examine and evaluate possible behavioral changes mediated by fluctuations in maternal care. Subjects A total of 35 litters were observed across 3 cohorts. The female Long-Evans hooded rats for breeding were ordered from Charles River Canada (St. Constant, Quebec). Approximately a week after arrival, the females were pair-housed with a male breeder from Bowling Green State University animal facilities. In some cases, due to a low male to female ratio, two females were housed per one male breeder. Pregnancy, in most cases, was determined by weight gain: after reaching approximately 290-300 grams, females were individually housed in suspended 50x30.5x24cm plastic, transparent cages that served as a home cage throughout the duration of maternal observations, and until pup weaning. Maternal Care Observations The maternal care observation regimen used in this study is based on the methods found in Champagne et al. 2003. Observation of maternal licking and grooming behaviors occurred during postnatal days 1-8, with the day of birth counting as postnatal day 0. Behaviors were recorded every three minutes within each of five, two hour sessions occurring daily at 6am, 9am, 1pm, 5pm, and 9pm. The 6am and 9pm shifts served as dark period observations and were conducted under red light. The occurrences of six behaviors important in determining the different levels of rodent maternal care were recorded during each observation period: 1) arched
Natural Variations in Maternal Care 23 back nursing (AB), recorded when the mother is arched over the pups with legs splayed; 2) maternal licking and grooming (MG), observed when the mother is licking or grooming any pup; 3) blanket nursing (BL), recorded when the mom is lying over the pups with no visible arch in her back; 4) passive nursing (PN), recorded when the mother is lying on either her back or side while the pups nurse; 5) self-grooming (SG), when the mother is licking or grooming herself and not the pups; 6) and no contact (NC), defines any activity not included above and not involving the pups. After 3 minutes, each litter was briefly observed and checked for the presence of one or more of these behaviors. These spot observations were recorded on a maternal care observation sheet (Appendix A). As soon as the first litter was observed, the three minute time period started over again, so that each litter was observed every three minutes during the 2 hour time period. During the eight days of observation the dam and litter remained untouched and undisturbed besides routine changes of food and water. Behavioral Tests Isolation Distress The isolation chamber consisted of a 500 mL glass beaker placed inside a larger plastic cage located in a room separate from that where the animals were housed. No other animals were present in the room at the time of testing. To record the ultrasonic vocalizations emitted by the pups during isolation, a high frequency bat detector, Pettersson D230 Ultrasound, was placed about 16 cm above the base of the beaker, resting on a metal lid on top of the plastic cage. Habituation to the isolation chamber occurred on postnatal day 9. After the dam was removed, the pups were transported in the home cage to a room other than the housing room and closer to the testing room. Each pup was weighed and placed in the isolation chamber located in
Natural Variations in Maternal Care 24 the testing room for one minute, under red light conditions, while USVs were recorded. After habituation, pups were tattooed on the ventral surface of a paw for identification purposes. Placement of the tattooed dot, which of the four paws was tattooed, and the color of the ink corresponded with a code to indicate the pup number for future recognition and data comparison. After tattooing, the dam and pups were returned to a clean home cage to the housing room. Isolation testing took place on postnatal day 10. The dam was again removed from the home cage and the pups were transported to a separate room. Each pup was weighed and then individually placed in the isolation chamber for two minutes under red light while USVs were recorded. After testing, the pups were returned to the dam in the home cage and returned to the housing room. Conditioned Odor Preference Habituation to the condition odor preference (COP) apparatus (Figure 2) occurred on postnatal day 12. The dam was placed in a separate cage and the pups were transported in the home cage to a different room. Each pup went through a one minute habituation in the COP apparatus under red light conditions. The apparatus consisted of a plexi-glass, boxlike chamber, 23 x 6 x 9 cm in size, marked with lines on the outside walls, visibly dividing the entire space into 3 equal compartments. The bottom of the chamber was made of metal rods, spaced sufficiently close together so that the animal could not fall through, but also allowing the conditioned or neutral scent to enter the apparatus. The chamber was placed on top of two small glass jars situated one at either end, each with two cotton balls held at the top of the containers by a metal mesh tube located inside of the glass containers.
Natural Variations in Maternal Care 25 Conditioning the pups to a lemon scent for this test occurred on postnatal day 13. Two different associative histories were used to control for any effects caused by conditioning, and to insure that the animals were conditioned to associate a lemon sent with the dam: half of each litter was exposed to four cotton balls and the conditioned scent to serve as a control, and the other half of the litter was conditioned with the dam.
Figure 2: COP apparatus: chamber = 18 x 6 x 9 cm; total height of apparatus with jars = 23cm On postnatal day 13, the pups were separated equally into the two different conditions; one exposed to cotton balls with the lemons scent and the other exposed to the dam with the lemon scent. Each group was placed in a separate cage and isolated from the dam for 3 hours before the first conditioning session. Each isolation and conditioning session took place under red light. During each conditioning session, 1 mL of lemon extract was applied to the ventral surface of
Natural Variations in Maternal Care 26 the dam and 0.25 mL of lemon extract was applied to each of the four cotton balls: one positioned in each of the four corners of the conditioning cage. The pups were exposed to either the dam and the lemon scent or the cotton balls and the lemon scent for thirty minutes. After the first thirty minutes of conditioning, another 3 hour isolation period occurred, where the dam was placed in a separate room from the pups, the pups were returned to their respective cages, and the cotton balls were discarded. In total, there were 3 thirty-minute conditioning sessions, each separated by 3 hours of isolation. At the end of the conditioning process, the pups were placed in a clean home cage, and the dam was thoroughly washed free of lemon scent and also returned to the home cage. COP testing occurred on postnatal day 14. The pups were isolated as a group from the dam and placed in another room three hours before testing. During testing, each pup was weighed and then placed in the testing apparatus for 5 minutes under red light conditions. Each session was videotaped and scored at a later date by a trained assistant blind to the MLG condition, position of the lemon scent in the testing apparatus, and associative history condition of the pup. The USV recorder was placed at the front of the apparatus and USVs were recorded. Before testing, two cotton balls were placed inside each of the small glass jars supporting the plexi-glass chamber on either side. One jar contained cotton balls saturated with 1 mL of lemon extract, the conditioning scent, and the other contained cotton balls saturated with 1 mL of tap water, a neutral scent. The location of the lemon scent, the left or right side, was counterbalanced for each litter per every five trials to insure that the location of the lemon scent did not influence the behavior of the pup and to keep those scoring the testing sessions impartial. Cotton balls were refreshed every 5-6 pups depending on litter size.
Natural Variations in Maternal Care 27 After initial COP testing, pups underwent a novel odor approach (NOA) test. Each pup was placed at the start of a 38 cm plastic runway with either a lemon-scented cotton ball or a peppermint-scented cotton ball located at the goal end. The latency of the pup to reach the end of the runway was record with a maximum allowed time of 60 seconds. Pups were tested over 5 trials on each scent and the initial scent was alternated between pups. After testing, the pups were returned to the home cage. Play Pups were weaned on postnatal 21: the litter was separated from the dam according to sex and placed in two separate 45.5 x 23 x 20.5 cm cages. They were then housed in a separate room from the original housing room. The play regimen for each litter began on postnatal day 23 with isolation. This play regimen was adapted from Knutson et al. (1998) and Siviy et al. (2006), with 9 testing days instead of 5 to allow for better observation of play recovery after extinction. Each litter was reduced to 8 animals: 2 pairs of females and 2 pairs of males. The 4 females and 4 males closest in weight from each litter were isolated for twenty four hours. The play sequence began on postnatal day 24: postnatal day (PND) 24, play day 1; PND 26, play day 2; PND 28, play day 3 and baseline play day; PND 30, play day 4 and unworn cat collar; PND 32, play day 5 and worn cat collar; PND 34, play day 6 and extinction 1; PND 36, play day 7 and extinction 2; PND 38, play day 8 and extinction 3; PND 40, play day 9, extinction 4 and final day of play. Before every play session, each pair of animals was weighed and one animal from each pair was marked with a magic marker for identification purposes. Play pairs remained the same throughout the play regimen in most cases, except for a few pairs that were accidentally
Natural Variations in Maternal Care 28 mismatched in cohort 1, in which case the data was removed from the day the pairs were switched. Each pair of animals was placed in the play apparatus, a 31 cm cube with 3 aluminum sides, 1 plexi-glass side facing the camera, and an open top. The bottom of the play apparatus was covered with approximately 2 cm of standard corncob laboratory bedding. Each play session lasted for 5 minutes under red light conditions and was videotaped for scoring by an assistant blind to the MLG condition of the pups. Bedding was changed between litters and within litters, between sexes. The USV recorder rested directly on top of the play apparatus and USVs were recorded for scoring at a later date. After each play session, animals were returned to their individual cages. On play day 4, PND 30, an unworn cat collar was introduced into the play chamber as a control for the worn cat collar that was used on play day 5. The unworn and worn cat collar methods were based on the procedure from Siviy et al. (2006). Four, 2 cm pieces of unworn cat collar were placed one at each corner, slightly covered by bedding. A separate collar was used for each sex to control for extraneous and distracting scents. Each collar was stored in separate bags in a freezer at -20º C. As in the study by Siviy et al. (2006), on the day of testing, each bag was immersed in a beaker of 50º C water without wetting the collars themselves, for approximately 10 minutes. After the play session, the collars were placed back into their separate bags and stored in the freezer until the next play session. On play day 5, PND 32, a collar previously worn by a cat, was introduced into the play chamber. All of the same procedures were followed as with the control collar. A separate, nearly identical, play chamber was used for the worn cat collar play so as not to contaminate the other chamber with scent from the cat collar. Multiple cat collars, worn by different indoor cats for approximately a week, were used throughout testing to better preserve the scent of the cat. The
Natural Variations in Maternal Care 29 final four days of play followed pre-cat collar procedures and were conducted to observe the rate of recovery of play behaviors from the extinction displayed during the presence of the worn cat collar. Thyroid Hormone Analysis Euthanasia After completion of the play regimen, litters were reduced to two males and two females each. These animals were pair-housed by litter and sex until sacrifice. After the play paradigm, animals were used in an additional prepulse inhibition test not included in this study. Euthanasia occurred at approximately 60-80 days of age and was carried out through decapitation after the animals were first anesthetized. Trunk blood was collected from each animal, allowed to clot, and placed in a centrifuge in order to separate formed elements from the blood serum. The serum samples were stored at -20º C before assaying. Hormone Assays We conducted assays for two different thyroid hormones: thyroxine (T4) and triiodothyronine (T3). Since we were looking for smaller fluctuations in the thyroid hormone both total T4 and total T3 assays were preformed. Assays were prepared according to instructions from MP Biomedicals. The absorbencies of the assays were read spectrophotometrically at 450 nm using a microtiter plate reader. To determine the amount of thyroid hormone in each sample, a standard curve was plotted for each assay (Appendix B), and concentration of unknown samples were determined using the best fit regression line to the curve.
Natural Variations in Maternal Care 30 ANALYSIS Maternal Care Behavior Maternal care behaviors were recorded and observed for the first 8 days after birth. Of the 6 behaviors recorded, the 2 primary care-giving behaviors, arched-back nursing and maternal licking and grooming, were used to determine the dam’s maternal licking and grooming (MLG) condition (High, Medium, or Low). The observed numbers of the two behaviors were averaged among the 3 cohorts and dams displaying at least 1 standard deviation above the mean were considered high licking and grooming, and dams displaying at least 1 standard deviation below the mean were considered low licking and grooming. Dams falling between the two extremes were considered medium licking and grooming. A two-way analysis of variance (ANOVA) was used to examine if there was an effect of MLG condition on the number of behaviors observed. This analysis was performed in order to verify that the three MLG groups differed significantly. Post-hoc t-tests were then conducted to determine significant differences between each MLG condition within each MLG behavior. Ultrasonic Vocalizations (USVs) In order to complete the first aim of this study, the observation of the effect of MLG on pup affect, USVs were recorded and analyzed during each of the behavioral tests. To examine the USV, data a 2-factor ANOVA was used for calls from each behavioral paradigm. The ANOVA for the isolation distress paradigm consisted of 2 between-subjects factors: MLG condition (High, Medium, or Low) and sex (Male or Female). The number of distress calls (20-40 kHz) emitted by the pup in a 60 second time frame served as the dependent variable. For the conditioned odor preference (COP) USV analysis, a 4-factor mixed design was used to examine
Natural Variations in Maternal Care 31 the average number of distress calls from 60 seconds of COP testing. There were three betweensubject factors of MLG condition (High, Medium, or Low), sex (Male or Female), and associative history (Dam or Cotton). There was one within-subjects factor of odor of compartment (Conditioned or Neutral). USVs from the play data were also analyzed using a 2-factor ANOVA. The 2 between-subject factors again consisted of MLG condition (High, Medium, or Low) and sex (Male or Female). Due to a loss of data and shortcomings of the experimental design, only day 5 of play was analyzed for 20 kHz and 50 kHz vocalizations. Post hoc t-tests were conducted to determine group differences within each paradigm. Correlations between tests for each MLG condition were also conducted for further exploration and to track the effects of the MLG condition across development. A subset of animals was selected for two-tailed Pearson correlations conducted for each MLG condition and for the three USV tests (Isolation Distress, COP, and Play). The analysis of USVs was also used in completing a second aim of this study: the observation of the effects of MLG on pup social motivation and maternal bonding. During COP testing, the number of USVs emitted by each pup over the conditioned scent or the neutral scent was recorded. For this subset of animals, a mixed-design ANOVA was used to examine the effects of MLG condition, associative history, and sex on the number of vocalizations produced over each odor stimulus (conditioned and neutral) during the five minute testing session. Total vocalizations were recounted for the subset of animals using the same auditory counting technique used to determine the number of vocalizations emitted over each chamber. A mixed-
Natural Variations in Maternal Care 32 design ANOVA was used to examine the effects of the minute of testing (1-5), MLG condition, associative history, and sex on the number of vocalizations produced during each minute. Conditioned Odor Preference The results for the conditioned odor preference (COP) test were analyzed using two 4-factor mixed design ANOVA tests in order to further explore aim 2. The fist 4-factor, mixed-design ANOVA consisted of a within-subjects variable of the lemon or the water side of the testing apparatus with duration as the dependent variable. Three between-subject factors were also used in this analysis. They included the MLG condition (High, Medium, Low), the conditioning group for the testing (Cotton Ball or Dam exposure), and sex (Male or Female). The second 4-factor, mixed-design ANOVA consisted of the same between-subject factors as the first ANOVA, but measured a different dependent variable: the number of entries into either the lemon or water side of the apparatus. T-tests were used for post hoc comparisons. Pearson correlations between COP USVs, the total time spent in the lemon chamber, and associative history were also analyzed. Novel odor approach testing (NOA) was analyzed using a 4-factor, mixed-design ANOVA, with the latency to reach the goal at the end of the runway as the dependent variable. The within subject variable was the scent at the end of the runway (peppermint or lemon), and the 3 between-subject variables included MLG condition, sex, and associative history. Play Behavior To further examine aim 3 of this study; the observation of the effects of MLG on juvenile play behavior, pins and dorsal contacts for each play session were analyzed separately using two 3factor mixed design ANOVAs. A within-subjects repeated measure for testing sessions (play day
Natural Variations in Maternal Care 33 1-9) and 2 between-subjects factors of gender (Male or Female) and MLG condition (High, Medium, Low) will used in the ANOVAs. T-tests were conducted to examine group differences in MLG condition and differences within each condition comparing the baseline day of play (day 3) to play days 4-9. A third and fourth, 2-factor between-subjects ANOVAs will be conducted to calculate the effects of MLG condition and sex on amount of time the animals spent interacting (approaching or touching) the unworn (day 4) or worn (day 5) cat collar. Thyroid Hormone Assays In order to complete aim 4 of this thesis, the observation of the effects of MLG on thyroid hormone, a 2-factor ANOVA was used to examine the thyroid hormone assay data. For each assay, T3 and T4, an ANOVA consisting of 2 between-subject factors of MLG condition (High, Medium, or Low) and gender (Male or Female) was be conducted. The dependent variable was the concentration of hormone (T3 or T4) found in the blood samples with the hormone assay. Ttests will be conducted for post hoc analysis.
Natural Variations in Maternal Care 34 RESULTS Maternal Care Behavior Replicated from Champagne et al. 2003, the fluctuations in maternal care fell along a distribution comparable to a normal curve: of the 35 litters, 9 were calculated as low MLG, 5 were calculated as high MLG, and the 21 in the middle of the distribution were considered medium MLG (Figure 3). Also following along with observations from Champagne et al. 2003, cumulatively, dams spent the most time engaged in the AB, arched back nursing, behavior and the NC, no contact, behavior than other components of maternal care (Figure 4). To determine the maternal care condition of each dam the percent of AB and MG, maternal licking and grooming, were averaged for each litter and those litters at least 1 standard deviation or more below the mean were considered low MLG and the litters at least 1 standard deviation or more above the mean were considered high MLG (Figure 3). A two-way ANOVA was used to examine if there was an effect of MLG condition on the number of behaviors observed. This was done in order to verify that the three groups, low MLG, medium MLG, and high MLG differed significantly. Results showed a significant main effect of maternal care behavior, F(2.032, 65.017) = 131.75, p < 0.001 and a significant interaction between the type behavior and the MLG condition, F(4.062, 65.017) = 8.175, p < 0.001. Prompted by the interaction effect, post hoc t-tests were conducted for each MLG behavior and MLG condition. Results show significant differences between the MLG conditions for the amount of AB behavior, t(34) = -23.526, p < 0.001 and also for the BL, blanket nursing, t(34) = 14.133, p < 0.001. High MLG litters had the greatest amount of AB behaviors, followed by medium MLG litters, with low MLG litters showing the lowest amounts of AB (Figure 5).
Natural Variations in Maternal Care 35 Results for the BL behavior were the exact opposite. The amount of maternal behaviors that involved pup contact, including AB, MG, BL, and PN, were totaled for each MLG condition. As shown in figure 6, the total amount of pup contact did not differ significantly between MLG conditions. These results show that, as stated in Champagne et al. 2003, the difference in maternal care occurs with the different types of maternal care behaviors expressed, not necessarily the amount of overall contact. Ultrasonic Vocalizations Isolation A 2 X 3 between-subjects ANOVA was used to examine the effects of sex (female, male) and MLG condition (low, medium, high) on the number of USVs averaged in a minute during isolation testing. Results indicated a significant effect of MLG condition on number of USVs, F(2,466) = 14.133, p < 0.001. More specifically, high MLG pups emitted the highest number of USVs (M = 77.55, SD = 45.27), followed by low MLG animals (M = 53.62, SD = 40.38), with medium MLG animals emitting the lowest amount of USVs (M = 47.85, SD = 36.79). Pairwise comparisons showed significant differences between low and high MLG conditions, p = 0.001 and also between the high and medium MLG conditions, p < 0.001 (Figure 7). There was not a significant effect of sex, p = 0.118; however, there was a trend of males vocalizing more than females throughout both testing paradigms (Figures 7 & 8). COP A 2 X 2 X 3 between-subjects ANOVA with a covariate of isolation time was used to examine the effects of sex (female, male), associative history (cotton, dam), and MLG condition (low, medium, high) on the average number of USVs emitted during COP testing. Due to testing
Natural Variations in Maternal Care 36 design, some litters were isolated longer than 3 hours before testing; therefore, a covariate of isolation time was added to the analysis. There was a significant main effect for MLG condition and USV during COP, F(2,454) = 7.008, p = 0.001. More specifically, high MLG pups vocalized the most (M = 55.36, SD = 35.09), followed by medium MLG pups (M = 46.75, SD = 37.06), with low MLG pups vocalizing the least (M = 34.18, SD = 28.07). Pairwise comparisons showed a significant difference between low and medium MLG groups, p = 0.008 and also between low and high MLG groups, p = 0.002 (Figure 9). There was no significant effect of sex, p = 0.148, associative history, p = 0.306, and the covariate of isolation time, p = 0.491; however there was a trend of animals conditioned with mom vocalizing more than animals conditioned with cotton, except in the low MLG group (Figure 9). A comparison of vocalizations during isolation testing and vocalizations during COP revealed that more USVs were emitted per minute during isolation than during COP (Figure 10). COP vocalizations per chamber A mixed design ANOVA was used to examine the effect of the odor of the chamber (conditioned or neutral scent) on the number of USVs emitted in that chamber. There was not a significant effect of odor on the number of USVs emitted, p = 0.108. There were also no significant interactions between the within subject variable of odor and the three between subject variables: associative history, MLG condition, and gender. COP vocalizations per minute A 4-factor mixed design ANOVA was used to examine the effect of the minute of COP testing, MLG condition, associative history, and sex on the number of vocalizations per minute. Results showed a significant interaction between the minute of COP testing (1-5) and MLG
Natural Variations in Maternal Care 37 condition, F(5.705, 268) = 4.471, p < 0.001. The assumption of sphericity was violated for the within factor of minute, so Greenhouse-Geisser values are reported. There was also a main effect of MLG condition, F(2, 67) = 6.739, p = 0.002. High MLG pups vocalized the most across each minute of testing, and low MLG pups vocalized the least for each minute of testing (Figure 11). It is also apparent that high MLG pups vocalized the most during the 1st minute and the least during the 5th minute, while the opposite was true for medium MLG pups, and low MLG pups did not differ across minutes. Pairwise comparisons showed that low and high MLG groups were significantly different from each other, p = 0.002. There was also a significant main effect of sex, F(1,67) = 5.486, p = 0.022 and a trend for the interaction between MLG condition and sex, F(2,67) = 3.063, p = 0.053 (Figure 12). Males vocalized less as the testing time increases, while females increased their vocalizations until minute 4 when they leveled off. Play USVs A 2-factor, between-subjects ANOVA was used to examine the effect of MLG condition and sex on the mean number of 20 kHz and 50 kHz USVs produced during the worn cat collar day (day 5) of play. There was not a significant effect of sex, p = 0.120 or MLG condition, p = 0.784; however, low MLG pups produced the most 20 kHz vocalizations (M = 13.45, SD = 40.89) while high MLG pups produced the least amount of 20 kHz vocalizations (M = 6.31, SD = 20.94) (Figure 13). For 50 kHz vocalizations, low MLG pups produced the least amount of vocalizations (M = 2.38, SD = 5.01).
Natural Variations in Maternal Care 38
Figure 3: Histogram of the percentage of total arched back nursing and maternal licking and grooming (ABLG) per litter with a superimposed normal curve. Litters at least 1 SD below the mean are low MLG litters and those at least1 SD above the mean are high MLG litters.
Natural Variations in Maternal Care 39
31% 34%
Arched Back Nursing (AB) Maternal Licking and Grooming (MG) Blanket Nursing (BL) Passive Nursing (PN) Self Grooming (SG)
4% 7%
7%
No Contact (NC)
17%
Figure 4: Cumulative percentages of individual maternal care behaviors across MLG conditions.
Natural Variations in Maternal Care 40
900
Low MLG 800
Medium MLG High MLG
700
Average Occurance
600 500 400 300 200 100 0 AB
MG
BL
PN
SG
NC
Maternal Care Behavior
Figure 5: The analysis of each maternal care behavior per MLG condition showed that MLG conditions differ significantly in the quantity of the various types of maternal behaviors, p < 0.001*.
Natural Variations in Maternal Care 41
Figure 6: The average number of behaviors involving dam-pup contact, including AB, MG, BL, and PN showed that there was no difference in the total maternal care received by the litters among MLG condition.
Natural Variations in Maternal Care 42
100 90 80
USVs per Minute
70 60 50 40 30 20 10 0 Female
Male Low
Female
Male
Medium MLG Condition
Female
Male High
Figure 7: USVs during isolation testing. There was a significant main effect for MLG condition and USVs during isolation distress testing. Post hoc T-tests showed significant differences between low and high MLG conditions, p= 0.001* and also between the high and medium MLG conditions, p< 0.001**, but not between the low and medium MLG conditions.
Natural Variations in Maternal Care 43
70
60
USVs per Minute
50
40
30
20
10
0 female
male Low
female
male Medium
female
male High
MLG Condition
Figure 8: USVs during COP testing. The trend of males vocalizing more than females can also be seen in during COP testing and for increased vocalizing with increased MLG. Pairwise comparisons showed a significant difference between low and high MLG groups, p = 0.008* and also between low and medium MLG groups, p = 0.002**, but not between high and medium MLG conditions.
Natural Variations in Maternal Care 44
70
USVs per Minute
60 50 40 30 20 10 0 mom
cotton
mom
Low
cotton Medium
mom
cotton High
MLG Condition
Figure 9: COP USVs over maternal MLG condition and associative history, PND 14. Pair-wise comparisons showed a significant difference between low and medium MLG groups, p= 0.008* and also between low and high MLG groups, p= 0.002**.
Natural Variations in Maternal Care 45
90
Low MLG 80
Medium MLG
70
High MLG
USVs per Minute
60 50 40 30 20 10 0 Isolation
COP Testing Paradigm
Figure 10: A comparison of isolation USVs and COP USVs.
Natural Variations in Maternal Care 46
Figure 11: USVs per minute of COP. There was a significant main effect of MLG condition, F(2,67) = 6.739, p = 0.002 for USVs emitted per minute of COP testing. High MLG pups vocalized significantly more than low MLG pups until minute 3, when the vocalizations of low MLG pups became significantly less than medium MLG pups until the end of testing.
Natural Variations in Maternal Care 47
Figure 12: COP USVs per minute and sex. There was a significant main effect of sex, F(1,67) = 5.486, p = 0.022 for COP USVs per minute.
Natural Variations in Maternal Care 48
Figure 13: USVs from play day 5. Low MLG pups produced the most 20 kHz vocalizations and the least 50 kHz vocalizations, while high MLG pups produced the least amount of 20 kHz vocalizations.
Natural Variations in Maternal Care 49 Conditioned Odor Preference Duration A 2 X 2 X 2 X 3 mixed-design ANOVA was used to calculate the effect of sex (female, male), associative history (cotton, dam), odor of chamber (lemon, water), and MLG condition (high, medium, low) on the time spent in each side (conditioned, neutral) of the apparatus. There was a significant main effect for the within subject variable of odor (lemon or water), F(1,448) = 12.183, p = 0.001 and a significant interaction between the odor and associative history (dam or cotton balls) condition F(2, 448) = 12.536, p < 0.001 (Figure 14). Post hoc t-tests show that the two associative history groups, dam and cotton, differ significantly in the lemon chamber, t(459) = 2.885, p = 0.005 and in the water chamber, t(459) = -2.530, p = 0.012. A marginally significant interaction between odor, sex, and the MLG condition, F(2, 448) = 2.946, p =0.054 was also present (Figure 15). More specifically, in the high MLG group both associative history groups showed an effect of conditioning: those conditioned with cotton and lemon showed a preference, a longer duration, for the neutral chamber (Neutral: M = 109.5, SD = 40.12; Conditioned: M = 67.21, SD = 43.32), while those conditioned with mom showed a preference for the conditioned scent chamber (Conditioned: M = 94.06, SD = 47.82; Neutral: M = 77.63, SD = 41.37). This trend is seen more prominently in males, and even disappears in the females conditioned with the dam. In the low MLG group, the pups conditioned with cotton show the same conditioning effect as the high MLG pups (Neutral: M = 98.93, SD = 52.55; Conditioned: M = 71.32, SD = 35.02); however, this effect disappears completely and no preference is shown for the low MLG pups conditioned with the dam (Conditioned: M = 83.60, SD = 34.36; Neutral: M = 87.77, SD = 46).
Natural Variations in Maternal Care 50 Entrances A 2 X 2 X 2 X 3 mixed-design ANOVA was used to calculate the effect of sex (female, male), associative history (cotton, dam), odor of chamber (lemon, water), and MLG condition (high, medium, low) on the number of entrances made into each side of the COP apparatus. Results showed a significant main effect for the within variable of odor (lemon or water) for entrances during COP, F(1,448) = 6.108, p = 0.014, (Figure 16). There was also a significant interaction between odor and associative history (conditioning with the dam or with cotton), F(1, 448) = 6.141, p = 0.014 (Figure 17). Novel Odor Approach Novel odor approach testing (NOA) was analyzed using a 4-factor, mixed-design ANOVA, with the latency to reach the goal at the end of the runway as the dependent variable. Results showed a significant main effect of odor, F (1,1856) = 62.068, p < 0.001; a significant interaction between odor and MLG condition, F (2, 1856) = 5.821, p = 0.003; odor and associative history, F (1, 1856) = 16.133, p < 0.001. Pups with an associative history of being conditioned with the dam discriminated more between the two scents, and over all, high MLG animals ran slower towards the goal than low MLG animals (Figure 18).
Natural Variations in Maternal Care 51
120
100
mom associative history cotton associative history
Duration (Sec)
80
60
40
20
0 lemon
water Apparatus Chamber
Figure 14: Duration for each odor chamber dependent on associative history. There was a significant interaction between the odor and associative history (dam or cotton balls) condition, p < 0.001. Post hoc t-tests show that the two associative history groups, mom and cotton, differ significantly in the lemon chamber, p = 0.005* and in the lemon chamber, p= 0.012**.
Natural Variations in Maternal Care 52
160 140
LemonTime male LemonTime female WaterTime male
120
WaterTime female
Duration (sec)
100 80 60 40 20 0 mom
cotton
mom
Low
cotton High
MLG Conditions
Figure 15: Time spent in the conditioned odor (lemon) or neutral odor (water) chambers. There was a significant main effect for the within subject variable of odor (lemon or water) for duration, p = 0.001. There was also a trend for the interaction between odor, sex, and the MLG condition, p = 0.054.
Natural Variations in Maternal Care 53
Mean Number of Entrances
25 20 15 10 5 0 Lemon
Water Chamber of Apparatus
Figure 16: Entrances during COP testing. Results showed a significant effect of odor, lemon (conditioned) or neutral (water), on the average number of entrances into each chamber during COP testing, p = 0.014*.
Natural Variations in Maternal Care 54
Figure 17: COP entrances by associative history and chamber odor. There was a significant interaction between odor and associative history (conditioning with mom or with cotton), F(1, 448)= 6.141, p= 0.014.
Natural Variations in Maternal Care 55
Figure 18: Novel odor approach by associative history and MLG condition. NOA results showed a main effect of odor, p < 0.001; a significant interaction between odor and MLG condition, p = 0.003; odor and associative history, p < 0.001.
Natural Variations in Maternal Care 56 Play The mixed-design ANOVAs conducted for pins and dorsal contacts (dc) during play reveled a significant difference between days of play: pins, F(4.819, 742.193) = 34.351, p < 0.001; dc, F(5.196, 742.193) = 39.301, p < 0.001, and a significant interaction between day and MLG condition: pins, F(9.639, 742.193) = 5.836, p < 0.001; dc, F(10.392, 742.193) = 1.905, p = 0.039. The within-subject variable of day violated sphericity so Greenhouse-Geisser values are reported above. Results also showed a significant main effect of MLG condition for both pins and dorsal contacts: pins, F(2, 154) = 16.101, p < 0.001; dc, F(2, 154) = 9.390, p < 0.001. The average number of dorsal contacts over the entire play period was lowest in the high MLG condition (M = 18.11, SD = 11.06), followed by the low MLG condition (M = 24.31, SD = 11.5), with the medium MLG condition at the top with the highest rates of dorsal contacts (M = 27.73, SD = 11.07) (Figure 19a). The results for pins followed the same trend as those for dorsal contacts: the medium MLG condition had the highest average amount of pins (M = 6.73, SD = 3.89), followed by the low MLG condition (M = 6.11, SD = 3.91), and the high MLG condition with the least amount of pins (M = 2.188, SD = 3.98) (Figure 19b). Pairwise comparisons show a significant difference in dorsal contacts for the high and medium groups, p < 0.001, and a significant difference in pins for the high and medium groups, p < 0.001, and the high and low groups, p = 0.002. Pairwise comparisons were conducted to analyze the differences in dorsal contacts and pins between MLG conditions for each day (Figure 20 a and b). During most days, the high MLG group showed significantly less play behavior than the medium and low MLG groups. This was true for both pins and dorsal contacts during day 1, all p < 0.05; day 3, all p < 0.001; and day 9 for dorsal contacts, p < 0.05. The medium MLG group also showed significant differences in
Natural Variations in Maternal Care 57 play behavior from the low MLG and high MLG groups on day 2 for pins, p < 0.001; day 4 for dorsal contacts, p < 0.05, and pins, p < 0.001; and day 6 for pins, p < 0.005. The high MLG condition differed significantly from the medium MLG condition during day 6 for dorsal contacts, p < 0.001 and day 8 for pins, p < 0.05. The high MLG condition was also significantly different from the low MLG condition on day 7 for dorsal contacts, p < 0.05. Play values were not significantly different between the 3 MLG conditions for dorsal contacts on play days 2, 5, and 8; and for pins on days 5, 7, and 9. Pins and dorsal contacts were averaged from play day 3 to serve as a baseline rate for both behaviors. Paired-sample t-tests were used to compare the rates of play after day 3 to the baseline rates for each MLG group. Play days 6-9 were compared to day 3 in order to examine the rate that play behaviors recovered from the worn cat collar extinction experienced on day 5. Pins and dorsal contacts from each consecutive day for each MLG group were compared to baseline rates. The high MLG group showed the quickest recovery from extinction due to the presence of the worn cat collar on day 5 back to baseline play for pins and dorsal contacts by day 7 (Figure 21). The low MLG group (Figure 22) did not recover to baseline rates for pins, and only showed rates of dorsal contacts not significantly different from baseline rates on play day 9. The medium MLG group (Figure 23) did not return to baseline play rates for pins or dorsal contacts. The interactions between each MLG group with the control and worn cat collars were also examined. A 2-factor, between-subjects ANOVA showed a significant effect of MLG condition for the time spent approaching/sniffing the control cat collar on day 4 of play, F(2,110) = 8.05, p = 0.001. There were differences across MLG conditions for collar approach during day 4 and collar contact during day 5 (Figure 24). Pairwise comparisons showed a significant increase in control collar approach in high MLG animals when compared to low and medium MLG animals,
Natural Variations in Maternal Care 58 p = 0.001 for collar approach. A 2-factor, between-subjects ANOVA was also conducted for the amount of contact made in seconds with the control collar on play day 4. Results showed a significant main effect of sex, F(1,110) = 4.99, p = 0.028, with males showing more collar contact (M = 35.84, SD = 23.86) than females (M = 25.76, SD = 16.43). There were no significant differences in MLG conditions for control collar contact. ANOVAs were also conducted for the worn cat collar introduced the play paradigm on day 5. There were no significant results for worn collar approach, but there was a significant main effect of sex on worn collar contact, F(1,110) = 6.84, p = 0.010, and a significant interaction between sex and MLG condition, F(2,110) = 5.31, p = 0.006. Females from high (M = 14.35, SD = 13.96) and low (M = 5.35, SD = 6.39) MLG conditions showed more collar contact than males; however, in the medium MLG condition, males (M = 5.22, SD = 8.25) showed more collar contact than the females (M = 3.86, SD = 5.02). Overall, the high MLG females showed the collar contact on day 5.
Natural Variations in Maternal Care 59
Figure 19a 8
7
Mean Number of Pins
6
5
4
low medium high
3
2
1
0 low
medium
high
MLG Condition
Figure 19b 35
30
Mean Number of Dorsal Contacts
25
20 low medium
15
high
10
5
0 low
medium
high
MLG Condition
Figure 19: Play behaviors averaged across 9 days. Figure 19a shows the average number of pins across nine days of play, p< 0.001* and p= 0.002**. Figure 19b shows the average number of dorsal contacts for each MLG condition from all 9 days of play, p< 0.001*.
Natural Variations in Maternal Care 60
Figure 20a 16
low 14
medium 12
Mean Number of Pins
high 10
8
6
4
2
0
day1
day2
day3
day4
day5
day6
day7
day8
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‐2
Figure 20b 45
low
Mean Number of Dorsal Contacts
40
medium high
35
30
25
20
15
10
5
0
day1
day2
day3
day4
day5
day6
day7
day8
day9
Figure 20: Average play behaviors across 9 days of play. Figure 20a shows the differences in pins between the 3 MLG conditions, and the Figure 20b shows differences in dorsal contacts.
Natural Variations in Maternal Care 61 40
35
30
Average Number of Occurances
25
20 high pin high dc 15
10
5
0 1
2
3
4
5
6
‐5
7
8
9
Figure 21: Change in play behaviors in the high MLG condition. The high MLG condition made the fastest return to baseline play rates (day 3) from extinction on day 5. Rates of play stop being significantly different from the baseline rate* on play day 7.
Natural Variations in Maternal Care 62 45
40
35
Average Number of Occurances
30
25
low pin
20
low dc
15
10
5
0 1
2
3
4
5
6
‐5
7
8
9
Figure 22: Change in play behaviors in the low MLG condition. Pins never returned to baseline rates (day 3) after extinction on day 5 and rates of dorsal contacts were not significantly different from baseline rates* only on play day 9.
Natural Variations in Maternal Care 63 45
40
Average Number of Occurances
35
30
25 medium pin medium dc
20
15
10
5
0 1
2
3
4
5
6
7
8
9
Figure 23: Change in play behaviors in the medium MLG condition. Play behaviors never returned to baseline rates (day 3) after extinction (day 5). Values are significantly different from the baseline day* throughout testing.
Natural Variations in Maternal Care 64
Figure 24a 40
35
Collar Approach (Sec.)
30
25
low
20
medium high 15
10
5
0 low
medium
high
Figure 24b 18
16
14
Duration (Sec)
12
10
8
6
4
2
0 Male
Female Low
Male
Female Medium MLG Condtion
Male
Female High
Figure 24a: Duration of control collar approach (play day 4). Pairwise comparisons showed a significant increase in control collar approach in high MLG animals when compared to low* and medium** MLG animals, p = 0.001. Figure 24b: Durations of worn cat collar contact. There was a significant interaction between sex and MLG condition for the duration of worn collar contact, p= 0.006.
Natural Variations in Maternal Care 65 Thyroid Hormone Two separate 2-factor ANOVAs were used to analyze the effect of MLG condition and sex on the concentration of thyroid hormones T3 and T4. Results showed a significant main effect of MLG condition on serum concentration of T4, F(2,82) = 6.538, p = 0.002. Pairwise comparisons revealed that the T4 concentrations of low MLG (M = 9.78, SD = 2.05) were significantly lower than high MLG (M = 12.91, SD = 2.12) and medium MLG (M = 12.37, SD = 2.39) T4 concentrations, p = 0.004 and p = 0.003 (Figure 25). An error in the preparation of standard values required removal of the T3 assay for cohort 3 from data analysis. Only analyzing the data from the first 2 cohorts left a small number of high MLG animals in the analysis (n = 4). Results for the ANOVA run on the first 2 cohorts only did not show significant differences for MLG condition or sex. However, the T3 concentrations still followed the same trend as the T4 concentrations: low MLG animals had the lowest values (M = 2.05, SD = 1.16); high MLG animals had the highest values (M = 3.03, SD = 3.08); and concentrations for medium MLG animals feel in between low MLG and high MLG values (M = 3.01, SD = 2.20).
Natural Variations in Maternal Care 66
Figure 25a 16 14
T4 Concentration (ug/dl)
12 10 Low MLG Medium MLG High MLG
8 6 4 2 0
MLG condition
Figure 25b 4.5
4
T3 Concentration (ug/dl)
3.5
Low MLG
3
Medium MLG
2.5
High MLG 2
1.5
1
0.5
0 MLG Condition
Figure 25a: T4 thyroid hormone concentrations. Pairwise comparisons revealed that the T4 concentrations of low MLG were significantly lower than high MLG and medium MLG T4 concentrations, p= 0.004** and p= 0.003*. Figure 25b: T3 thyroid hormone concentrations. There were no significant differences with T3; however, the low MLG animals still showed lower levels of T3 when compared to high and medium groups.
Natural Variations in Maternal Care 67 Correlation Results Six two-tailed Pearson correlations were conducted to measure the effects of variations in maternal care during each testing paradigm and developmental time frame (Appendix C). Correlations were conducted for each MLG condition to investigate the relationships of USVs across paradigms, the various aspects of COP testing and vocalizations, and play behaviors and vocalizations. Results showed surprising little correlation within each data set. For the low MLG animals there was a positive correlation between isolation USVs and COP USVs (r = 0.037, p = 0.004), a negative correlation for the conditioned odor scent duration and associative history (r = -.292, p = 0.020), and positive correlations between the day 3 pins and dorsal contacts (r = 0.619, p < 0.001) and day 5 pins and dorsal contacts (r = 0.686, p < 0.001). There were no USV correlations for the medium MLG animals, and only a negative correlation between the conditioned odor scent duration and associative history (r = -0.300, p = 0.002) for the COP behavioral data. For the medium MLG animals during play, day 3 dorsal contacts and day 5 pins were positively correlated (r = 0.219, p = 0.029). Day 3 pins were also positively correlated with day 3 dorsal contacts (r =0.619, p < 0.001), and day 5 pins were positively correlated with day 5 dorsal contacts (r = 0.696, p < 0.001). The high MLG animals showed a negative correlation between the COP USVs and the 50 kHz play USVs (r = -0.603, p < 0.001), no correlations for COP behavior, and a positive correlation between play day 3 pins and dorsal contacts (r = 0.686, p < 0.001).
Natural Variations in Maternal Care 68 DISCUSSION Maternal Care Behaviors The outcome of this study hinged upon the appearance of natural variations in maternal care within the 3 cohorts. Maternal care results paralleled those from Champagne et al. (2003) in showing that the distribution of low, medium, and high MLG litters closely followed a normal distribution. However, results from the present study found a significant difference in AB, but not in MG, as reported in previous research (Champagne et al. 2003). MG behavior still followed the same trend, low MLG animals receiving less than high MLG animals, with medium MLG animals in the middle; however, differences were not significant. Unfortunately, a cohort effect was found in the distribution of MLG conditions in this study: all of the low MLG litters were from the first cohort, and all but one of the high MLG litters were from the third cohort. This cohort effect does not necessarily indicate a flaw in the observation regimen, but possibly the influence of extraneous environmental influences on the maternal behavior of the dams. Levels of maternal care are very susceptible to environmental variables (Patin et al. 2002; Simons et al. 2005; Champagne et al. 2003). Each of the three cohorts was observed at a different time period during the year: the first cohort during the summer, the second cohort during the fall, and the third cohort in the winter. The animals were housed in an indoor facility where the environment was kept relatively consistent, but in the summer the humidity increases and in the winter, the facility tends to be colder. Observers also differed per cohort and, even though they were similarly trained, could have brought with them different scents or made uncontrolled noises during observations that affected the maternal behaviors of the litters. The present results indicate that our observations of MLG behaviors were
Natural Variations in Maternal Care 69 still sufficiently accurate, despite cohort effects, to produce the behavioral profiles of each MLG condition found in previous research. Major Findings for Changes in Pup Affect and MLG Condition The first aim of this study was to examine the impact of variations in maternal care on the affective states of offspring throughout development. This was accomplished through the recording and analysis of ultrasonic vocalizations (USVs) during each behavioral paradigm: isolation distress (PND 10), COP (PND 14), and play (PND 24-40). The results from these two paradigms were diametrically opposed to previous predictions made in this study: high MLG animals produced the most 30-44 kHz vocalizations when compared to low and medium MLG animals. Rates of calls produced by low MLG animals were also significantly less than the other conditions. Since isolations USVs can be viewed as indicators of anxiety (Barron et al. 2000) it was hypothesized that low MLG animals would produce greater amounts of isolation USVs due to the increase in activity in their HPA axis, leading to higher levels of anxiety and emotional reactivity (Champagne et al. 2003). The results are now pointing towards the different conclusion that isolation calls are produced in order to provoke maternal care behaviors and pup retrieval; and if the vocalizations indicate anxiety, perhaps more of these anxious calls aid in moving the dam into action more efficiently. Studies have examined the nature of sensory regulation of pup-induced maternal behavior in virgin female rats (Stern, 1996). Results show that dam deafened through eardrum puncture initiated significantly more instances of infanticide than those blinded through enucleation and control animals. Deafened animals were 10 times more likely to cause pup injury than the other two groups. Deafened animals also exhibited greater latencies to the onset of full maternal
Natural Variations in Maternal Care 70 behavior when compared to blind and control animals. These data suggest that an auditory component of pup behavior is important in the initiation and regulation of maternal behaviors. The number of USVs produced by pups in each MLG condition is possibly indicative of the high MLG pups’ ability to elicit maternal care and the low MLG condition’s inability to prompt maternal behaviors as efficiently. The variable of sex was not significant in either testing paradigm; however, there was a trend during both isolation and COP for males to vocalize more than females across conditions. This suggests, contrary to the findings of Champagne et al. (2003) that variations in maternal licking and grooming persist even between the sexes. Also contrary to the results was the prediction that USVs emitted during complete isolation would be fewer in number than USVs emitted during COP. In both high and low MLG conditions, USVs per minute were greater during isolation than COP testing. This finding is in contrast to the study by Levine et al. (1985) which showed that monkeys vocalized more when isolated with access to maternal cues as opposed to monkeys subjected to total isolation. The absence of this finding in the present study is possibly due to the effects of conditioning. Associative history was not a significant predictor of the number of isolation calls; however, a trend was present across MLG conditions for pups conditioned with the dam to vocalize more than those conditioned with cotton. This is also an indication that conditioning was successful, and the lemon scent did not have an aversive effect as to inhibit successful conditioning in the pups exposed to the lemon scent and the dam. The two testing paradigms were also run at different developmental time periods: isolation distress testing took place first on PND 10, while COP testing occurred second at PND 14. By this later PND day and age, the pups had already experienced periods of isolation, and this could have decreased COP vocalizations when compared to isolation USVs.
Natural Variations in Maternal Care 71 The limited analysis, caused by loss of data, of the 20 kHz and 50 kHz vocalizations during play did not result in any significant findings. However, low MLG animals produced the greatest number of 20 kHz vocalizations during play day 5 (PND 32) when play was reduced with the introduction of a collar previously worn by a cat, and vocalizations were countable. All MLG conditions showed a reduction in play behavior during day 5, along with the 20 kHz distress vocalizations. 20 kHz vocalizations have been previously labeled as distress calls (Barron et al. 2000). Data from the current study coincide with this label and further indicate the transformation of the purpose of the lower vocalizations throughout development: from prompts to the onset of maternal behaviors in infancy to anxiety provoked distress and possibly warning calls in adolescence to adulthood. Fifty kHz vocalizations, USVs indicative of joy and comparable to laughter (Panksepp & Burgdorf, 2003), were also counted during play day 5. Lower rates of these joyful calls were seen in the low MLG animals, corresponding with their greater numbers of 20 kHz vocalizations during the same day. Overall, it is difficult to distinguish whether the varying levels of maternal care have an influence on the affective states and rates of vocalizations of the pups or if the pups partially determine the amount of maternal care they received dependent upon the amount of USVs they emit. The data presented here, along with previous findings in favor of both situations, suggests that there is interplay between the two scenarios. Under conditions unaffected by other variables such as environmental containments (Simons et al. 2005) or random stressful events (Patin et al. 2002), pup USVs possibly serve as a main determinate of the maternal care they receive, and the amount of care received then predicts effects on emotional reactivity, level of anxiety during stress, and social motivation in adolescents and adulthood.
Natural Variations in Maternal Care 72 The Influence of Maternal Care on Pup Maternal Attachment The second aim of this study was to examine the influence of natural variations in maternal care between litters on early pup social motivation and levels of maternal attachment. This aim was investigated by measuring preference for maternal-associated odor in a conditioned odor preference paradigm. Examined together, all litters in the study showed an effect of conditioning, indicating that the conditions regimen was successful in paring the conditioned scent with the dam. Two possible scenarios were predicted for this paradigm: 1) The increased anxiety and HPA activity (Liu et al. 1997) in the low MLG animals would lead to a greater need for comfort, higher levels of attachment, and a stronger preference for the conditioned scent when paired with the dam; 2) The decrease in oxytocin receptors in low MLG animals (Champagne et al. 2001) would lead to a lack of preference for the maternally conditioned scent, or even a possible aversion to the odor due to novelty. Results support the second hypothesis. A marginally significant interaction between odor, sex, and the MLG condition reveled that low MLG females conditioned with the dam showed a complete lack of preference, measured through duration, for either the conditioned or neutral scent. A preference for the lemon was shown by the high MLG pups conditioned with the dam; however, this effect was lost with the females of that group. Both low MLG and high MLG animals conditioned with the cotton balls showed the expected preference for the neutral scent during testing. These results are consisted with the findings in a study by Nelson and Panksepp (1996). This study provides the evidence for a direct relationship between levels of oxytocin and pup maternal attachment. Their results showed that animals treated with an oxytocin antagonist displayed no preference for the conditioned scent; similar to the results for the low MLG females.
Natural Variations in Maternal Care 73 Behavioral results for the COP paradigm parallel the results from the USV recordings. The sex differences found in the USV data and in the COP behavioral data indicates the possibility of differences in MLG within litters between the sexes. Champagne et al. (2003) states that sex differences in maternal care are not found in the first 8 PND, but they are possibly present after the first 8 observation days. However, a study by Richmond and Sachs (1984) indicates that greater rates of anogenital licking are directed towards male offspring, but this observation was made following brief periods of pup handling. Further analysis of maternal observations from our study would be needed in order to identify when the changes in maternal behavior that result in sexual dimorphism of care-giving occurred. If males and females are not differentiated from each other in terms of maternal care until after PND 8, sex effects in the results of the present indicate that maternal care received after PND 8 still affects the developing attachment behaviors of offspring. Juvenile Social Motivation and Fear Reactivity The third aim of this study set out to observe the effects the MLG condition on juvenile social motivation and fear reactivity. Dorsal contacts and pins followed the same pattern as the USVs: high MLG animals showed significantly less play behavior. We had predicted that the high MLG animals would have the greatest number of pins and dorsal contacts, possibly attributed to their highly social, more fearless behavioral profile (Champagne et al. 2003). We suggest here that the lower rates of play seen in the high MLG animals are not hallmarks of a deficit, but a sign of continued social efficiency. The play paradigm used in this study consists of pup isolation since PND 23 with weaning at PND 21. This type of isolation directly after weaning can still cause irreversible disturbances in pup behavior (Hol, Van den Berg, Van Ree, & Spruijt, 1999). If measured in approach and avoidance behaviors, isolated animals displayed fewer approach and
Natural Variations in Maternal Care 74 more avoidance behaviors when compared to control rats (Hol et al. 1999). The lower rates of play for the high MLG animals are a sign of quicker adaptation to the play apparatus and the preplay isolation. Results also showed that play behaviors in high MLG animals also peaked at day 2, before the low or medium MLG animals, whose play behaviors peaked at day 3 on average. Overall the results for the number of play pins across conditions are much fewer than those found in the controls of other studies (Siviy et al. 2006). The number of dorsal contacts however, seem to be similar to controls in the medium and low MLG groups. A play study using early handled (EH), early deprivation (ED), and control animals found that male ED animals, experiencing lower amounts of anogenital maternal grooming, showed significantly greater amounts of play behaviors. As mentioned previously, ED animals have a very similar behavioral profile to low MLG animals (Pryce et al. 2000). It would be very interesting to conduct the play protocol again, without isolation. Possibly, without the effect of isolation, play rates would be less in low MLG animals when compared to high MLG animals. The play paradigm was also used to examine play extinction in the face of a naturally fearprovoking stimulus and recovery from that extinction. Baseline levels of play were taken from play day three, giving the animals adequate time to adapt to the play apparatus, play partner, and play regimen (Siviy et al. 2006). After the implementation of a collar previous worn by a cat, play rates for each behavior collapsed over MLG conditions; bringing average numbers of pins and dorsal contacts very low, except in the medium MLG animals for dorsal contacts. By comparing play behaviors from play day 3 to days after the introduction of the worn cat collar, the rates of recovery from extinction were examined for each MLG condition. Low MLG and medium MLG conditions did not return to baseline rates of play by day 9, except for low MLG dorsal contacts on day 9. It is not unusual to see a more rapid return of dorsal contacts as
Natural Variations in Maternal Care 75 compared to pins; however, in other play studies dorsal contacts usually return to baseline levels much more quickly (Siviy et al. 2006). High MLG animals experienced a quicker return to baseline rates, and were back to the same rate as play day 3 by play day 7 in both pins and dorsal contacts. This could be due to the lower amounts of these behaviors seen on day 3 as opposed to the two other conditions, but levels of pins seem to be very consistent across all play days for the high MLG group and the slope for the dorsal contacts after extinction much steeper than that of low MLG and medium MLG animals. Examination of Thyroid Hormones In investigation of our 4th aim, venturing away from the HPA-axis proved to be beneficial in the analysis of thyroid hormone differences between MLG conditions. Low MLG animals had significantly lower levels of circulating T4 when compared to medium and high MLG groups and also lower levels of T3, however not significantly lower. This underlying hormonal difference could account for many of the behavioral changes seen in the low MLG animals, such as depressed learning and memory (Donahue, Dougherty, & Meserve, 2004) and possible auditory damage (Uziel, Rabie, & Marot, 1980). These two deficits would help explain the low MLG pups lack of attachment to the dam during COP conditioning, a possible deficit in learning the conditioned stimulus, and reduction of USV calls caused by a reduction in appropriate feedback. PCBs have been shown to cause hypothyroidism in rats, significantly decreasing the amounts of total T4 found in blood plasma (Morse, Wehler, Wesseling, Koeman, & Brouwer, 1995). Previous studies have also indicated that PCBs affect the maternal behavior of rats, increasing the licking and grooming behavior, but significantly decreasing the arched-back nursing behavior
Natural Variations in Maternal Care 76 (Simmons et al. 2004). Results from a conditioned odor preference study conducted by Cromwell and colleagues, showed a similar lack of preference for the conditioned scent in animals conditioned with the dam and exposed to 25 ppm of PCB as seen in the low MLG animals (Cromwell et al. 2007). It is possible that the reduced calls and maternal preference found in both the low MLG animals and PCB animals (unpublished manuscript, McFarland, 2008) can be attributed to lower levels of thyroid hormones partially caused by the lower levels of maternal AB nursing. It would be beneficial to explore these thyroid hormone differences at various ages to determine when the deficit in low MLG animals is present. Clinical Implications Even though the differences between human and rodent development are dramatic, it is still possible that variations in maternal care in the rodent model can lead to a better understanding of the effects of different levels of parental care in humans. Abuse and neglect are considered outside the normal range of parenting and are maladaptive in nature; whereas natural fluctuations in parenting styles tend to serve an adaptive purpose and also have far reaching and pronounced effects on child development and attachment. Parenting styles encompass two major factors: parental responsiveness, also considered parental warmth and supportiveness, and parental demandingness, which consists of behavioral and psychological control (Baumrind, 1991; Barber, 1996). Four main sub-types of parenting styles result from observations of high and low parental responsiveness and demandingness and each style reflects naturally occurring patterns of parental behaviors. These parenting behaviors can result from environmental variables such as stress and social support and can also be transmitted from parent to child (Van Ijzendoorn, Goldberg, Kroonenberg, & Frenkel, 1992). Four main parenting styles include the indulgent parent, the authoritarian parent, the authoritative parent, and the uninvolved parent (Maccoby &
Natural Variations in Maternal Care 77 Martin, 1983). The balance achieved with the authoritative parenting style between demanding and permissive, along with the inclusion of high levels of nurturing, allows children to be more independent and develop as individuals (Baumrind, 1971). Levels of self-reliance, self-control (Baumrind, 1991), social competence, academic performance (Darling, 1999) positive coping, lower anxiety levels (Wolfradt, Hempel, & Miles, 2003) tend to be the greatest in children raised under this parenting style, serving as a great example for the need of a balance between supportiveness and demandingness (Baumrind, 1991). Using the variations in maternal care rodent model, could perhaps give a closer insight into these effects by utilizing longer, more detailed and less disturbed observations. Low levels of thyroid hormone have also been implicated in the development of Autism (Nir et al. 1995). Low MLG animals share many traits found on the Autism spectrum, such as communication disruption and withdrawal stereotypes. Animals exposed to PCBs and other toxins have been previously used as models for Autism and with further research, perhaps the rodent model derived from variations in maternal care could be used to represent and study higher functioning levels of the Autism spectrum. Conclusions and Future Studies Results from this study have contributed to the knowledge of basic rodent maternal care behaviors and their affects on offspring development. The animals used in this study were standard, unmanipulated, Long-Evans rats that would be used collectively as control animals in other studies. The effects of the natural variations in maternal care on offspring behavior and development displays the need for the continuation of basic animal behavioral research in order to better understand results obtained from our control animals.
Natural Variations in Maternal Care 78 For future studies, it would be interesting to further divide the animals into subgroups representing more extreme ends of the variations in maternal care. This could possibly emphasize results already found in this and other studies, and also assist in examining the slight sex differences found in this study. It would also be beneficial to run a similar MLG observation paradigm to examine the separation of low, medium, and high MLG within the PCB treated animals to further the comparisons between the two paradigms. Hopefully, with further research, different MLG conditions can be used as a model to further the research into developmental disorders such as Autism and possibly ADHD.
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Natural Variations in Maternal Care 84 brain. Physiology Review, 87, 873-904. Meaney M.J., Diorio J., Francis D., Widdowson J., LaPlante P., Caldji C., Sharma S., Seckl J.R., & Plotsky, P.M. (1996). Early environmental regulation of forebrain glucocorticoid receptor gene expression: implications for adrenocortical responses to stress. Developmental Neuroscience, 18 (1-2), 49-72. Menard, J.L. & Hakvoort, R.M. (2007). Variations of maternal care alter offspring levels of behavioural defensiveness in adulthood: Evidence for a threshold model. Behavioural Brain Research, 176, 302-313. McFarland, A.M. (2008). The Effects of Polychlorinated Biphenyls on Ultrasonic Vocalizations in Rat Pups. Unpublished manuscript, Bowling Green State University, Bowling Grenn, OH. Moore, C.L. & Power, K.L. (1992). Variation in maternal care and individual differences in play, exploration, and grooming of juvenile Norway rat offspring. Developmental Psycholbiology, 25(3): 165-182. Morse, D.C., Wehler, E.K., Wesseling, W., Koeman, J.H., & Brouwer, A. (1996). Alterations in rat brain thyroid hormone status following pre- and postnatal exposure to polychlorinated biphenyls (Aroclor 1254). Toxicology and Applied Pharmacology, 136(2): 269-279. Myers, M.M., Brunelli, S.A., Squire, J.M., Shindeldecker, R.D., & Hofer, M.A. (1989). Maternal
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Natural Variations in Maternal Care 86 Plotsky, P.M. & Meaney, M.J. (1993). Early, postnatal experience alters hypothalamic corticotrophin-releasing factor (CRF) mRNA, medium eminence CRF content and stressinduced release in adult rats. Molecular Brain Research, 18, 195-200. Pryce, C.R., Bettschen, D., Feldon, J. (2000). Comparison of the effects of early handling and early deprivation on maternal care in the rat. Developmental Psychobiology, 38, 239251. Rice, D. & Barone Jr., S. (2000). Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environmental Health Perspective, 108(3): 511-533. Richmond, G. & Sachs, B.D. (1984). Maternal discrimination of pup sex in rats. Developmental Psychobiology, 17(1): 87-89. Roca, R.P., Blackman, M.R., Ackerley, M.B., Herman, S.M., & Gregermen, R.I. (1990). Thyroid hormone elevations during acute psychiatric illness; relationship to severity and distinction from hyperthyroidism. Endocrine Research, 16, 415-447. Shair, H.N., Masmela, J.R., Brunelli, S.A., & Hofer, M.A. (1997). Potentiation and inhibition of ultrasonic vocalization of rat pups: regulation by social cues. Developmental Psychobiology, 30, 195-200. Shen, L., Wania, F., Lei, Y.D., Teixeira, C., Muir, D.C., Xiao, H. (2006). Polychlorinated
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Natural Variations in Maternal Care 88 Appendix A: MLG behavioral observation score sheet Maternal Licking and Grooming Score Sheet
PND: Litter:
Date:
3 Minute intervals with-in 120 minute time periods MG: Maternal Grooming SG: Self Grooming PN: Passive Nursing NC: No Contact 6am
9am
AB: Arched Back Nursing
1pm
5pm
BL: Blanket Nursing
9pm
Natural Variations in Maternal Care 89
Appendix B1: Cohort 1, T3 assay standard curve and linear regression graph
3
2.5
Absorbances (450nm)
2
1.5
y = -0.207x + 2.038 R² = 0.851
1
0.5
0 0
2
4
6
8
-0.5 T3 Concentration (ng/ml)
10
12
Natural Variations in Maternal Care 90
Appendix B2: Cohort 1, T4 assay standard curve and linear regression graph 2.5
Absorbances (450nm)
2
1.5
y = -0.0706x + 1.8538 R2 = 0.8936
1
0.5
0 0
5
10
15 T4 Concentration (ug/dl)
20
25
30
Natural Variations in Maternal Care 91
Appendix B3: Cohort 2, T3 assay standard curve and linear regression graph 3.5
3 y = -0.197x + 2.907 R² = 0.908
Absorbances (450nm)
2.5
2
1.5
1
0.5
0 0
2
4
6 T3 Concentration (ng/ml)
8
10
12
Natural Variations in Maternal Care 92
Appendix B4: Cohort 2, T4 assay standard curve and linear regression graph 3
Absorbances (450nm)
2.5
y = -0.061x + 2.245 R² = 0.663
2
1.5
1
0.5
0 0
5
10
15 T4 Concentration (ug/dl)
20
25
30
Natural Variations in Maternal Care 93
Appendix B5: Cohort 3, T3 assay standard curve and linear regression graph 3.500
3.000
y = -0.042x + 3.091 R² = 0.719
Absorbances (450nm)
2.500
2.000
1.500
1.000
0.500
0.000 0
2
4
6 T3 Concentration (ng/ml)
8
10
12
Natural Variations in Maternal Care 94
Appendix B6: Cohort 3, T4 assay standard curve and linear regression graph 3.000
2.500
y = ‐0.087x + 2.494 R² = 0.961
Absorbances (450nm)
2.000
1.500
1.000
0.500
0.000 0
5
10
15
20
25
30
T4 Concentration (ug/dl)
Natural Variations in Maternal Care 95
Appendix C1: Pearson Two-Tailed Correlation for the low MLG condition USV data across isolation, COP, and play.
COPusvS IsolationUSVs PlayUSVs20kHz PlayUSVs50kHz
Pearson Correlation COPusvS
.370(**)
.106
‐.113
.004
.407
.377
65
59
63
63
Pearson Correlation .370(**)
1
.187
‐.149
.163
.267
Sig. (2‐tailed) N
IsolationUSVs
Sig. (2‐tailed) N
PlayUSVs20kHz
.004 59
59
57
57
Pearson Correlation
.106
.187
1
‐.137
Sig. (2‐tailed)
.407
.163
63
57
64
64
Pearson Correlation
‐.113
‐.149
‐.137
1
Sig. (2‐tailed)
.377
.267
.280
63
57
64
N
PlayUSVs50kHz
1
N
.280
** Correlation is significant at the 0.01 level (2‐tailed).
64
Natural Variations in Maternal Care 96
Appendix C2: Pearson Two-Tailed Correlation for the low MLG condition COP data .
Conditioned Scent Duration
Pearson Correlation Conditioned Scent Duration
Sig. (2‐tailed)
1
Pearson Correlation Sig. (2‐tailed)
COP USVs
.015
.020
.907
63
63
63
‐.292(*)
1
‐.175
.020
N
COP USVs
‐.292(*)
N
Associative History
Associative History
.169
63
63
63
Pearson Correlation
.015
‐.175
1
Sig. (2‐tailed)
.907
.169
63
63
N
* Correlation is significant at the 0.05 level (2‐tailed).
65
Natural Variations in Maternal Care 97
Appendix C3: Pearson Two-Tailed Correlation for the low MLG condition Play data
PlayDay3DC PlayDay3Pin PlayDay5DC PlayDay5Pin PlayUSVs20kHz PlayUSVs50kHz
Correlation PlayDay3DC
Sig. N Correlation
PlayDay3Pin
PlayDay5Pin
PlayUSVs20kHz
.219(*)
‐.002
‐.010
.150
.000
.029
.984
.928
.155
100
100
100
100
92
92
.619(**)
1
.007
.011
.138
.081
.948
.917
.191
.442
.000
N
100
100
100
100
92
92
.219(*)
.007
1
.696(**)
‐.067
.042
Sig.
.029
.948
.000
.520
.685
N
100
100
104
104
96
96
Correlation
‐.002
.011
.696(**)
1
‐.025
‐.083
Sig.
.984
.917
.000
.809
.420
N
100
100
104
104
96
96
Correlation
‐.010
.138
‐.067
‐.025
1
‐.130
Sig.
.928
.191
.520
.809
92
92
96
96
96
96
Correlation
.150
.081
.042
‐.083
‐.130
1
Sig.
.155
.442
.685
.420
.208
92
92
96
96
96
N
PlayUSVs50kHz
.619(**)
Sig.
Correlation PlayDay5DC
1
N
.208
** Correlation is significant at the 0.01 level (2‐tailed). *Correlation is significant at the 0.05 level (2‐tailed).
96
Natural Variations in Maternal Care 98
Appendix C4: Pearson Two-Tailed Correlation for the medium MLG condition USV data across isolation, COP, and play.
PlayUSVs20kHz PlayUSVs50kHz IsolationUSVs COPusvS
Pearson Correlation PlayUSVs20kHz
‐.130
‐.178
‐.102
.208
.085
.330
96
96
95
94
Pearson Correlation
‐.130
1
.063
.077
Sig. (2‐tailed)
.208
.546
.460
Sig. (2‐tailed)
1
N
PlayUSVs50kHz
N
IsolationUSVs
96
96
95
94
Pearson Correlation
‐.178
.063
1
.175
Sig. (2‐tailed)
.085
.546
95
95
103
101
Pearson Correlation
‐.102
.077
.175
1
Sig. (2‐tailed)
.330
.460
.079
94
94
101
N
COPusvS
N
.079
102
Natural Variations in Maternal Care 99
Appendix C5: Pearson Two-Tailed Correlation for the medium MLG condition COP data
COP USVs Associative History Conditioned Scent Duration
Pearson Correlation COP USVs
Associative History
Conditioned Scent Duration
Sig. (2‐tailed)
1
.021
.043
.832
.672
N
102
100
100
Pearson Correlation
.021
1
‐.300(**)
Sig. (2‐tailed)
.832
N
100
102
102
Pearson Correlation
.043
‐.300(**)
1
Sig. (2‐tailed)
.672
.002
N
100
102
.002
** Correlation is significant at the 0.01 level (2‐tailed).
102
Natural Variations in Maternal Care 100
Appendix C6: Pearson Two-Tailed Correlation for the medium MLG condition Play data
PlayDay3DC PlayDay3Pin PlayDay5DC PlayDay5Pin PlayUSVs20kHz PlayUSVs50kHz
Correlation PlayDay3DC
Sig. N Correlation
PlayDay3Pin
PlayDay5Pin
PlayUSVs20kHz
.219(*)
‐.002
‐.010
.150
.000
.029
.984
.928
.155
100
100
100
100
92
92
.619(**)
1
.007
.011
.138
.081
.948
.917
.191
.442
.000
N
100
100
100
100
92
92
.219(*)
.007
1
.696(**)
‐.067
.042
Sig.
.029
.948
.000
.520
.685
N
100
100
104
104
96
96
Correlation
‐.002
.011
.696(**)
1
‐.025
‐.083
Sig.
.984
.917
.000
.809
.420
N
100
100
104
104
96
96
Correlation
‐.010
.138
‐.067
‐.025
1
‐.130
Sig.
.928
.191
.520
.809
92
92
96
96
96
96
Correlation
.150
.081
.042
‐.083
‐.130
1
Sig.
.155
.442
.685
.420
.208
92
92
96
96
96
N
PlayUSVs50kHz
.619(**)
Sig.
Correlation PlayDay5DC
1
N
.208
** Correlation is significant at the 0.01 level (2‐tailed). *Correlation is significant at the 0.05 level (2‐tailed).
96
Natural Variations in Maternal Care 101
Appendix C7: Pearson Two-Tailed Correlation for the high MLG condition USV data across isolation, COP, and play. Correlations
IsolationUSVs PlayUSVs20kHz PlayUSVs50kHz COPusvS
Pearson Correlation IsolationUSVs
‐.274
‐.094
‐.047
.128
.607
.799
32
32
32
32
Pearson Correlation
‐.274
1
‐.220
.163
Sig. (2‐tailed)
.128
.225
.374
32
32
Sig. (2‐tailed)
1
N
PlayUSVs20kHz
N
PlayUSVs50kHz
32
32
Pearson Correlation
‐.094
‐.220
Sig. (2‐tailed)
.607
.225
32
32
32
32
Pearson Correlation
‐.047
.163
‐.603(**)
1
Sig. (2‐tailed)
.799
.374
.000
32
32
32
N
COPusvS
N
** Correlation is significant at the 0.01 level (2‐tailed).
1 ‐.603(**)
.000
32
Natural Variations in Maternal Care 102
Appendix C8: Pearson Two-Tailed Correlation for the high MLG condition COP data.
COP USVs Associative History Conditioned Scent Duration
Pearson Correlation COP USVs
‐.308
.104
.086
.570
32
32
32
Pearson Correlation
‐.308
1
‐.308
Sig. (2‐tailed)
.086
Sig. (2‐tailed)
1
N
Associative History
N
Conditioned Scent Duration
.086
32
32
32
Pearson Correlation
.104
‐.308
1
Sig. (2‐tailed)
.570
.086
32
32
N
32
Natural Variations in Maternal Care 103
Appendix C9: Pearson Two-Tailed Correlation for the high MLG condition Play data
PlayUSVs20kHz PlayUSVs50kHz PlayDay3DC PlayDay3Pin PlayDay5DC PlayDay5Pin
Correlation PlayUSVs20kHz
‐.220
‐.135
‐.077
‐.115
‐.110
.225
.463
.674
.531
.548
32
32
32
32
32
32
Correlation
‐.220
1
‐.093
‐.030
‐.263
‐.288
Sig.
.225
.611
.869
.145
.110
Correlation
‐.135
‐.093
1
.698(**)
.347
.115
Sig.
.463
.611
.000
.052
.530
Correlation
‐.077
‐.030
.698(**)
1
.076
‐.145
Sig.
.674
.869
.000
.679
.427
Correlation
‐.115
‐.263
.347
.076
1
.320
Sig.
.531
.145
.052
.679
Correlation
‐.110
‐.288
.115
‐.145
.320
Sig.
.548
.110
.530
.427
.074
Sig. N
1
PlayUSVs50kHz
PlayDay3DC
PlayDay3Pin
PlayDay5DC
.074 1
PlayDay5Pin
** Correlation is significant at the 0.01 level (2‐tailed).
