PRENATAL COCAINE: EFFECTS ON NEONATAL VOCALIZATIONS, CUE- INDUCED MATERNAL RESPONSE, AND BRAIN DEVELOPMENT
Elizabeth Thomas Cox
A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum of Neurobiology
Chapel Hill 2012
Approved by:
Dr. Josephine Johns
Dr. Karen Grewen
Dr. Clyde Hodge
Dr. Carl J. Malanga
Dr. John Gilmore
Dr. P.S. Zeskind ©2012 Elizabeth Thomas Cox All Right Reserved
ii ABSTRACT
ELIZABETH T. COX: Prenatal Cocaine: Effects on Neonatal Vocalizations, Cue-induced Maternal Response, and Brain Development (Under the direction of Josephine M. Johns)
Cocaine use in women is correlated with child neglect and abuse. Young children prenatally exposed to cocaine are reported to exhibit early signs of neurobehavioral stress and decreased response to caregivers. It is reasonable to suggest that drug-exposed infants seeking comfort from a non-nurturing caretaker might have difficulty responding to their mother/caretaker because of direct neurobehavioral effects following prenatal cocaine exposure (PCE). Animal models of cocaine- induced maternal neglect suggest rodent mothers from both drug-treated and control groups exhibit disrupted offspring-induced maternal behavior towards cocaine-exposed neonates. Results support human studies and suggest cocaine-induced changes in both mother and offspring influence the quality of maternal care and hence developmental outcome of the infant although little is understood about the mechanisms underlying these effects. Work presented here employed parallel translational studies examining behavioral responses of drug-treated mothers, their drug-exposed infants, and neurobiological mechanisms underlying behavioral differences in both human and animal models.
Cries were elicited in cocaine-exposed and control human infants at one month of age and subsequently examined for variations in cry acoustics. Mothers returned to lab at three months postpartum and were tested for perceptual response to naturally occurring cries. An animal model of
PCE was employed examining if PCE results in similar acoustic variations during the neonatal period and if these differential acoustic properties influence a rodent mother’s preference-like behavior.
Additionally, our rodent model was used to explore early brain development in PCE and control rodent offspring during early neonatal and juvenile periods. Findings suggest in human and rodent
iii neonates PCE affects early mother-infant interactions in part through changes in infant-produced vocalizations and that gestational cocaine-exposure in human and rodent mothers affects maternal responsiveness to those stimuli. Additionally results suggest variations in neuronal developmental in the Central Amygdala and Ventral Medial Hypothalamus may contribute to variations in vocalizations in juvenile but not infant rodents. These studies provide both translational value, assessment of human and rodent offspring vocalizations and maternal response, and possible mechanisms underlying behavioral effects in offspring. The objectives serve to determine how cocaine affects development in PCE offspring, nurturing behavior in mothers, and long-term offspring developmental outcome.
iv To my family and friends who have never wavered in their support and encouragement. The destination would be meaningless without you coming along for the journey.
v ACKNOWLEDGMENTS
It would be impossible for me to express the gratitude I feel for all the people who have provided support, encouragement, and advice leading up to this moment. I would first like to thank my advisor, mentor, and friend Dr. Josephine Johns. You have provided me with so much support over the past six years (even when I was being headstrong and stubborn). You’ve provided me an amazing environment in which to learn, make mistakes, and succeed and I will forever be grateful. I would also like to thank the entire Johns’ Lab Crew: Sarah Williams, Matthew McMurray, Abigail
Jamieson-Drake, Caitlin Zoghby, Pooja Makam, Marlana Radcliffe, and Thomas Jarrett. Specifically,
I need to thank Dr. Sarah Williams, Dr. Matthew McMurray, and Abigail Jamieson-Drake. I cannot have imagined surviving the past six years without you three in my life. Sarah and Matt: you have not only been people I look up to and admire (for your scientific contributions and friendly advice) but you have also been my two largest personal motivators. I have been working hard and striving to do my best in a mere attempt to keep up with your brilliance. Princess Abigail, my breath of fresh air in a windowless lab, thank you does not suffice to say how much your friendship has helped me along the way. You’re an amazing person and will undoubtedly succeed at anything you do. This thesis is a product of all three of yours’ mentorship and friendship and I cannot thank you enough. I also want to thank all of my committee members: Drs. Karen Grewen, Clyde Hodge, CJ Malanga, John
Gilmore, and Sandy Zeskind for the support and advice at every step along the way. Clyde, I cannot thank you enough for never closing your office door to me and letting me turn to you during my graduate student woes. I was lucky to have you as a mentor and a friend and I only hope one day I can return the favor.
vi I next need to thank my mom, dad, sister, and nieces. After studying maternal behavior for the past six years I have a new found respect for everything you have done for me in my life. You’re encouragement, support, and unwavering love has given me the confidence to pursue my dreams. I also need to thank two of the best friends a girl could have: Rebekah McSwain and Britt Patterson. I don’t think I could ever voice how much your friendships have shaped my life. Thank you for being there when I needed you and even more for being there when I thought I didn’t. Lastly and maybe most importantly I need to thank Eli Lippard. You are my best friend and the love of my life. I will spend the rest of my life trying to make you proud and giving you the same level of support that you have provided me over the past six years (although I’m afraid this will be impossible). Having you with me along this journey to laugh with, wipe my tears, and ask “whose butt do I need to kick” is the sole reason why I’m a sane and happy person today. The term grateful or appreciative does not even begin to touch on how I feel and for this reason I won’t try and explain it more.
Aside from the individuals mentioned above I also need to acknowledge my financial support of the National Institute of Drug Abuse, through my predoctoral fellowship (F31DA030060), Dr.
Johns’ research grant (P01DA022446), and the UNC NIH Neurobiology Curriculum training grant
(T32NS007431) without whose funding I would not have been able to follow my interests and complete the work reported here and elsewhere.
vii TABLE OF CONTENTS
TABLE OF CONTENTS ...... VIII
LIST OF TABLES ...... X
LIST OF ABBREVIATIONS ...... XIII
CHAPTER 1. INTRODUCTION ...... 1
1.1. GESTATIONAL COCAINE-EXPOSURE AND EFFECTS ON HUMAN MATERNAL BEHAVIOR ...... 1
1.2. IMPACT OF ACOUSTIC CHANGES AND ADULT EMOTIONAL STATE ON ADULT PERCEPTION OF INFANT VOCALIZATIONS………………………..…….2
1.3. PRENATAL DRUG EXPOSURE AND INFANT BEHAVIORAL CHANGES IN HUMANS ...... 4
1.4. GESTATIONAL COCAINE-EXPOSURE AND EFFECTS ON MATERNAL BEHAVIOR IN ANIMAL MODELS……...... 5
1.5. RODENT MATERNAL RESPONSE: IMPORTANCE OF INFANT BEHAVIOR ...... 6
1.6. NEUROBIOLOGY OF ALTERED CRYING BEHAVIOR ...... 8
1.7. PRENATAL COCAINE’S KNOWN EFFECTS ON BRAIN DEVELOPMENT ...... 9
1.8. VALIDITY OF TRANSLATIONAL VALUE OF RODENT EARLY BRAIN DEVELOPMENT: HUMAN VS. RODENT ...... 10
1.9. SUMMARY ...... 11
CHAPTER 2. HUMAN PCE: EFFECTS ON ONE-MONTH OLD INFANT CRIES AND THREE- MONTH POSTPARTUM MATERNAL PERCEPTION OF CRIES ...... 12
2.1. INTRODUCTION ...... 12
2.2. METHODS ...... 15
2.3. RESULTS ...... 22
viii 2.4. DISCUSSION ...... 38
CHAPTER 3. RODENT PCE: EFFECTS ON RODENT PUP VOCALIZATIONS AND RODENT DAM PREFERENCE BEHAVIOR ...... 53
3.1. INTRODUCTION ...... 53
3.2. METHODS ...... 56
3.3. RESULTS ...... 63
3.4. DISCUSSION ...... 69
CHAPTER 4. DELAYED DEVELOPMENTAL CHANGES IN NEONATAL VOCALIZATIONS CORRELATES WITH VARIATIONS IN VENTRAL MEDIAL HYPOTHALAMUS AND CENTRAL AMYGDALA DEVELOPMENT IN THE RODENT NEONATE: EFFECTS OF PRENATAL COCAINE ...... 76
4.1. INTRODUCTION ...... 76
4.2. METHODS ...... 78
4.3. RESULTS ...... 84
4.4. DISCUSSION ...... 97
4.5. CONCLUSIONS ...... 103
CHAPTER 5. GENERAL DISCUSSION ...... 104
5.1. OVERALL CONCLUSIONS, LIMITATIONS, AND FUTURE DIRECTIONS ...... 104
5.2. CONCEPTUAL MODEL FOR FUTURE WORK ...... 110
5.3. GENERALIZABILITY OF FINDINGS ...... 112
REFERENCES……………………………………………………………………………..115
ix LIST OF TABLES
TABLE 1. LIST OF BOTH DRUG DRUG-EXPOSED MATERNAL GROUPS BROKEN DOWN BY DRUG COMBINATION USED DURING PREGNANCY….....16
TABLE 2. AVERAGE ACOUSTIC VARIABLES FROM SIX CRY STIMULI PLAYED TO MOTHERS ...... 18
TABLE 3. CRY ANALYSIS: MANOVA ACOUSTIC MEASURE GROUPING…...... 20
TABLE 4. MEAN DEMOGRAPHIC INFORMATION FOR MOTHERS/INFANTS PARTICIPATING IN STUDY…………. …………………………………...... 23
TABLE 5. LINEAR REGRESSION RESULTS OF EMOTIONAL PERCEPTUAL RATINGS FOR EACH MATERNAL EXPOSURE GROUP ORGANIZED BY SPECIFIC ACOUSTIC FEATURES OF CRY STIMULI AND EMOTIONAL PERCEPTUAL RATINGS …...... 33
TABLE 6. LINEAR REGRESSION RESULTS OF PERCEIVED BEHAVIORAL RESPONSE FOR EACH MATERNAL EXPOSURE GROUP ORGANIZED BY SPECIFIC ACOUSTIC FEATURES OF CRY STIMULI AND PERCEIVED BEHAVIORAL RESPONSE …...... 35
x LIST OF FIGURES
FIGURE 1. INFANT EXPOSURE GROUP DIFFERENCES IN ONE MONTH OLD VOCALIZATIONS…...... 24
FIGURE 2. INFANT EXPOSURE GROUP AND SEX DIFFERENCES IN ONE MONTH OLD VOCALIZATIONS ...... 25
FIGURE 3. LINEAR REGRESSION SHOWING THE RELATIONSHIP BETWEEN THE NUMBER OF TIMES MOTHERS USED COCAINE IN THE FIRST TRIMESTER AND THE MINIMUM AMPLITUDE AT THE FUNDAMENTAL FREQUENCY OF ONE MONTH OLD INFANT CRIES...... 26
FIGURE 4. LINEAR REGRESSION SHOWING THE RELATIONSHIP BETWEEN THE AVERAGE NUMBER OF CIGARETTES MOTHERS IN THE POLYDRUG- EXPOSED MATERNAL GROUP SMOKED DURING THE FIRST TRIMESTER...... 27
FIGURE 5. EXPOSURE GROUP DIFFERENCES IN MATERNAL PERCEPTUAL RATINGS…………………..…………………...... 29
FIGURE 6. LINEAR REGRESSIONS OF COCAINE AND CIGARETTE EXPOSURE AND MATERNAL RATINGS OF GIVE PACIFIER…...... 30
FIGURE 7. DIFFERENCES IN MATERNAL PERCEPTUAL RATINGS BASED ON THE NUMBER OF DRUGS USED DURING PREGNANCY...... 31
FIGURE 8. HIERARCHY OF BEHAVIORAL RESPONSE BETWEEN EXPOSURE GROUPS....37
FIGURE 9. LINEAR REGRESSIONS SHOWING THE RELATIONSHIP BETWEEN ONE MONTH CRIES AND MATERNAL PERCEPTION...... 38
FIGURE 10. PREFERENCE BOX APPARATUS...... 58
FIGURE 11. USV ANALYSIS SCHEMATIC…...... 60
FIGURE 12. RODENT DAM PREFERENCE BEHAVIOR FOR AN UN OR CC MALE OFFSPRING………………………...... 65
FIGURE 13. PPD 1 STIMULUS PUP VOCALIZATION COMPARISON...... 66
FIGURE 14. PPD 1 CORRELATIONS BETWEEN DAM PREFERENCE BEHAVIOR AND STIMULUS PUP'S VOCALIZING BEHAVIOR……………...... 68
FIGURE 15. PPD 5 CORRELATIONS BETWEEN DAM PREFERENCE BEHAVIOR AND STIMULUS PUP'S VOCALIZING BEHAVIOR……………...... 69
xi FIGURE 16. CHARACTERIZATION OF VOCALIZATIONS WITH MEDASSOCIATES EQUIPMENT…………….………………………... ………...... 85
FIGURE 16. SCHEMATIC OF REGIONS IMAGED ON PNDS 14 AND 21…...... 87
FIGURE 18. PND 1 VOCALIZATIONS AND PAG DEVELOPMENT IN NEONATAL RAT MALE AND FEMALE OFFSPRING FOLLOWING CC, CS, OR NO PRENATAL EXPOSURE …...... 88
FIGURE 19. PND 14 VOCALIZATIONS AND NAC CORE, CEA, VMH, AND VPAG DEVELOPMENT IN INFANT RAT MALE AND FEMALE OFFSPRING FOLLOWING CC, CS, OR NO PRENATAL EXPOSURE …...... 90
FIGURE 20. PND 21 VOCALIZATIONS AND NAC CORE, CEA, VMH, AND VPAG DEVELOPMENT IN JUVENILE RAT MALE AND FEMALE OFFSPRING FOLLOWING CC, CS, OR NO PRENATAL EXPOSURE …...... 94
FIGURE 21. PND 1-21 DEVELOPMENTAL CHANGES IN FREQUENCY CATEGORIES OF USVS AND RELATIONSHIP BETWEEN 22KHZ USVS AND VMH AND CEA DEVELOPMENT…...... 96
FIGURE 22. SCHEMATIC SHOWING THE COMPLEXITY OF THE MOTHER-INFANT RELATIONSHIP...... 110
FIGURE 23. VALUE OF TRANSLATIONAL STUDIES...... 111
FIGURE 24. PRENATAL AND GESTATIONAL COCAINE-EXPOSURE RESULTS IN BEHAVIORAL AND BRAIN CHANGES IN BOTH MOTHER AND INFANT...... 112
FIGURE 25. SCHEMATIC OF COCAINE'S AND ENVIRONMENT'S RELATIVE IMPORTANCE TO NEUROBEHAVIOR………..……………………………...... 113
xii LIST OF ABBREVIATIONS
PCE Prenatal cocaine-exposure
PPD Postpartum Day
GD Gestational Day
USV Ultrasonic Vocalization dPAG Dorsal Periaqueductal Gray vPAG Ventral Periaqueductal Gray
CeA Central Amygdala
BLA Basal Lateral Amygdala
VMH Ventral Medial Hypothalamus
NAc Nucleus Accumbens
CC Chronic Cocaine
CS Chronic Saline
UN Untreated
TLFB Time Line Follow Back
Hz Hertz dB Decibel kHz Kilohertz
ANOVA Analysis of Variance
ANCOVA Analysis of Covariance
MANOVA Multiple Analysis of Variance
xiii HSD Honestly Significant Test
LSD Least Significant Difference
xiv CHAPTER 1. INTRODUCTION
1.1. GESTATIONAL COCAINE-EXPOSURE AND EFFECTS ON HUMAN MATERNAL BEHAVIOR
Parental substance abuse, cocaine in particular, is a strongly associated with child neglect and abuse (Murphy et al., 1991;Leventhal et al., 1997). According to the National Institute of Drug
Abuse’s National Pregnancy and Health Survey in the early 1990s, more than 45,000 women reported that they used cocaine during some period of their pregnancy (Mathias, 1995). This value has increased over the years a 2005 National Survey estimating that approximately 4% of pregnant women and 10% of non-pregnant, reproductive-aged women (aged 15–44 years) use illicit drugs
(National Survey on Drug Use and Health (NSDUH) Series, 2005). This is an increase in drug use since the early 1990s when the prevalence of illicit drug use among pregnant women was 2.3%, thereby underscoring the need for more research on the effects of cocaine use on maternal-infant interactions.
Observational studies reveal that cocaine-exposed mothers interact less with their infants (Burns et al., 1991), are less sensitive to infant/child cues (Minnes et al., 2005), and less able to focus their attention on their child (Ball et al., 1997). Mothers recovering from cocaine addiction also report greater difficulty with impatience, anger, and emotion control, and are more vulnerable to maladaptive parenting behaviors including neglect, depression, disorganized care taking, infant- directed hostility, abandonment, and abuse (Murphy et al., 1991;Leventhal et al., 1997). Despite drug rehabilitation treatment and abstinence, mothers who have used cocaine during pregnancy often display difficulties in mother-infant interactions and attachment (Mahony and Murphy, 1999;Beeghly
1 et al., 2003). Therefore, even in the absence of obvious abuse or neglect cocaine-exposed mother- infant dyads may be more susceptible to unfavorable developmental outcomes resulting from decreased quality of mother-infant interactions critical for the child’s social, emotional, and cognitive development. In light of the importance of the early environment on infant developmental outcome, a large amount of research has been conducted exploring the underlying mechanism(s) of altered maternal behavior, yet mechanism(s) still remain unclear.
1.2. IMPACT OF ACOUSTIC CHANGES AND ADULT EMOTIONAL STATE ON ADULT PERCEPTION OF INFANT VOCALIZATIONS
Recent studies suggest that cocaine-exposed mothers (mothers that used cocaine during pregnancy but did or did not continue use in the postpartum period) may not be as sensitive to changes in infant cues as are comparison nondrug using mothers (Minnes et al., 2005). Neonatal vocalizations (i.e. cries) in particular, are an important infant cue for the early rearing environment.
The neonatal cry is a graded signal with variations in acoustic characteristics corresponding to parallel changes in infant arousal state, specifically changes in the homeostasis between the sympathetic and parasympathetic nervous systems (Zeskind et al., 2011). For example, expirations emitted by an infant during a painful condition, i.e. circumcision, are initially higher in pitch but quickly become lower in pitch as the pain is resolved. These changes in pitch correspond to parallel changes in vagal tone (a measurement of parasympathetic activity) (Porter et al., 1988). These acoustic changes are a valuable communication tool in the care taking environment. As the cry signal changes (based on a spectrum of internal arousal state in the infant) a synchronous change in caretaker arousal occurs (Porter et al., 1986). It is important that the caregiver’s arousal state matches that of the infant. This synchrony of arousal between infant and parent (i.e. an infant highly aroused emits a high pitched, loud cry that is considered urgent by the care taking environment which leads to increased arousal in the parent and therefore a quick behavioral response) results in better infant care
2 in the short term, and also nourishes the relationship between the infant and caregiver over the long term.
Adult cry perception studies show that the demographics and emotional state of adults are associated with changes in infant cry perception. Perception of infant cries change based on if the adult listener is a parent (Green et al., 1987;Irwin, 2003), the personality of the parent (Ziefman,
2003), and if the mother has other children already (Boukydis and Burgess, 1982;Zeskind, 1980).
Cocaine-using mothers have previously been shown to differentially respond to changes in the acoustic signal of cries (Scheutze et al., 2003). Yet it is still unknown to what extent and how cocaine-exposure specifically alters maternal perception. Women abusing cocaine often fall within higher risk categories because of the secondary effects of drug abuse including depression. It has been shown that depressed women find cries less arousing, aversive, sick, and urgent sounding. They also exhibit an opposite synchrony of arousal in behavioral response to increases in cry pitch. So in essence, as the cry stimuli increases in pitch, depressed mothers do not show parallel increases in perceptual active responses as is reported for non-depressed control mothers but instead they report they would be more likely to wait and see as the pitch increase (Schuetze and Zeskind, 2001). This suggests that depressed mothers may not show the same sensitivity to changes in the fundamental frequency of cries (Donovan et al., 1998). Adults considered at increased risk for child abuse, exhibit an increase in heart rate in response to cries; the opposite pattern as that seen in low risk adults who have a decrease in heart rate to cries (Crowe and Zeskind, 1992). Abusive parents also have been shown to not discriminate between certain cry characteristics as well as non-abusive comparison parents (Zeskind and Shingler, 1991). Differences in perception of cries could have a profound impact on the mother-infant relationship and subsequent infant developmental outcome, thus it is important to determine the role cocaine may play in a mother’s perception of infant cries and their subsequent response.
3 1.3. PRENATAL DRUG EXPOSURE AND INFANT BEHAVIORAL CHANGES IN HUMANS
Changes in mother-infant behavioral synchrony on the infant side can also have a profound impact on the mother-infant relationship. It is now well supported that PCE also influences the infant’s behavioral signals, which aid in eliciting maternal attention. Infants exposed to cocaine during pregnancy, even if generally healthy and full term, may show less clarity of infant cues
(Minnes et al., 2005), be more irritable, cry more than non-exposed infants (Reijneveld et al., 2004), and separation from their mother followed by reunion does not elicit normal emotional behavior in the cocaine-exposed child (Goldman-Fraser, 1997). PCE leads to subtle deficits in infant affect, arousal, and attention, coupled with compromised ability to optimally engage with mother in early face-to-face interaction (Tronick et al., 2005;Tronick and Beeghly, 1999;Lester et al., 2004;Frank et al., 2001). As they grow, these children are at greater risk for cognitive (Noland et al., 2003), emotional, behavioral, and physical deficits (Chasnoff, 1987;Claussen et al., 2002;Minnes et al.,
2006), and are more likely to show increased irritability, aggressive behaviors, and poor social attachments as children (Noland et al., 2003). These deficits may hold a clue to mechanisms of an infant’s apparent inability to elicit care from mothers normally and warrant further study.
Changes in the acoustic characteristics of infant cries can have a profound impact on how the infant’s cry is perceived and virtually the way the infant is responded to. Studies examining parent and non-parent adults support that increased fundamental frequency (Schuetze and Zeskind,
2001;Zeskind and Marshall, 1988;Zeskind and Lester, 1978;Scheutze et al., 2003), decreased amplitude and increased amounts of dysphonation (Gustafson and Green, 1989), and changes in the duration of expirations and/or the interval or inspiratory pauses between expirations (Zeskind et al.,
1992) in general correspond to synchronous changes in arousal level of the listener. Changes in the acoustic properties of cries have been observed in many populations of infants who are considered developmentally at risk. Higher fundamental frequency (basic pitch or F0), higher cry threshold
(measured by latency to cry after exposure to a painful stimuli), longer latency to begin crying,
4 shorter duration of the initial expiratory sound, and a shorter overall duration of crying have all differentiated infants with no evident neurological insult, but who were considered at increased risk of developmental challenge due to conditions such as preterm birth, low birth weight, prenatal malnutrition, and/or non-optimal obstetric histories (Michelsson, 1971;Tenold et al., 1974;Lester,
1975;Lester, 1979;Zeskind and Lester, 1978). Spectral frequency analysis of infant cries is also sensitive to prenatal drug exposure (Lester and Dreher, 1989;Lester et al., 1991;Huntington et al.,
1990) though results following PCE are mixed (Keller, Jr. and Snyder-Keller, 2000;Eyler et al.,
1998). Cries of infants prenatally exposed to various drugs are typically marked by a higher peak F0, higher dominant frequency (frequency with the highest energy or amplitude), shorter expiratory sounds, increased dysphonation (sonic turbulence), longer latencies and other spectral features through one month of age (Zeskind et al., 1996b;LaGasse et al., 2005).
The combination of infant's cry characteristics and maternal perception have been related to the infants' language and cognitive development at 18 months (Schuetze and Zeskind, 2001). This and other associated differential outcomes of infants with high-pitched cry sounds have been posited to result from the transactional processes in which the infant and parent's behavioral attributes synergistically interact to produce non-optimal developmental outcomes. In extreme cases, high- pitched, aversive cries may contribute to the development of physical abuse and/or neglect (Frodi and
Lamb, 1980;Zeskind and Ramey, 1981;Zeskind and Shingler, 1991). Studies suggest that PCE is also associated with different acoustic parameters of early neonatal cries (Lester et al., 2002;Lester et al.,
1991;Corwin et al., 1992). More research is needed to understand the impact these changes in behavior have on the mother and the infant and how cocaine disrupts this synergistic relationship.
1.4. GESTATIONAL COCAINE-EXPOSURE AND EFFECTS ON MATERNAL BEHAVIOR IN ANIMAL MODELS
Animal studies generally agree that all forms of cocaine treatment (acute, intermittent, and chronic) disrupt aspects of maternal behavior, the extent of disruption dependent on dose, length and
5 time of testing, and treatment regimen (Kinsley et al., 1994;Peeke et al., 1994;Johns et al., 1994). The greatest deficits in maternal behavior have been suggested to occur during the early postpartum period (Postpartum days (PPDs) 1-5) in rodents. Additionally, maternal behavior deficits have been shown not to simply be a consequence of cocaine withdrawal (Johns et al., 1997) or a byproduct of cocaine induced hyperactivity (Johns et al., 1994;Vernotica et al., 1999;Kinsley et al., 1994;Vernotica et al., 1996) suggesting other mechanisms underlie behavioral effects. Recently, a study examining rodent maternal behavior following treatment with chronic or intermittent cocaine using a cross- fostering paradigm (Johns et al., 2005) found that all rodent mothers or dams, chronic cocaine (CC)- treated and untreated (UN) controls alike, displayed altered maternal behavior towards the CC- exposed pups suggesting that some attribute of the CC-exposed pups may render them less able to elicit normal care taking. Studies have shown when pups are isolated from their dam and littermates they elicit ultrasonic vocalizations (USVs); inaudible to the human ear but highly salient to rodent dams. The effects of PCE on pup-produced cues such as USVs, which might impact the early maternal-infant environment, have not been thoroughly investigated.
1.5. RODENT MATERNAL RESPONSE: IMPORTANCE OF INFANT BEHAVIOR
The onset of maternal behavior is largely dependent on changes in endocrine system function
(Numan, 1994;Numan and Insel, 2003;Keverne, 1988;Rosenblatt, 1990); however, somatosensory input is also important in onset and retention of maternal behavior (Morgan et al., 1992). Pup stimuli are salient contributors to maternal behavior across the postpartum period, with the degree of saliency dependent on the maternal environment and the time of testing (Champagne et al., 2001;Champagne et al., 2003;Brudzynski, 2005;Mattson et al., 2001). Rat dams have also been shown to be able to detect and prefer pups of their own litter versus pups of another litter, implying a difference in the reinforcing properties of familiar versus unfamiliar pup cues (Heyser et al., 1992;Mattson et al.,
2003;Mattson et al., 2001;Mattson and Morrell, 2005;Lee et al., 2000). Of relevance to the work reported here rodent pups’ USVs play an important role in altering both the neurobiology and
6 response of the dam (Stern et al., 1984;Farrell and Alberts, 2002a;Farrell and Alberts, 2002b;Brunelli et al., 1994) and differences in acoustic parameters of USVs have been suggested to directly alter maternal behavior (Noirot, 1972). USVs produced by pups vary by age and gender. Males vocalize more than females, if littermates, with these differences developing later in the postnatal period
(Postnatal day (PND) nine but not PND four) (Hahn and Lavooy, 2005;Naito and Tonoue, 1987).
Calls decrease in mean and peak frequency across the early life period (Naito and Tonoue, 1987), while mean and total duration increase as pups approach weaning (Brudzynski et al.,
1999;Brudzynski, 2005). USVs can be elicited in rat pups by handling, cold temperatures, isolation and depend on social factors in an age-dependent fashion (Branchi et al., 2001;Shair et al.,
1997;Blumberg et al., 1992;Hahn and Lavooy, 2005). It has been proposed that USVs emitted by pups are simply by-products of laryngeal braking that occurs during brown fat thermogenesis, especially during the early neonatal period (Blumberg and Sokoloff, 2001). However, anesthetized hypothermic pups (PND 3-17) do not make as many USVs as conscious hypothermic pups, providing support for additional regulatory mechanisms of USVs (Brunelli et al., 1994). Previous studies suggest social potentiation of vocalizations in individual pups peaks around PND ten to fifteen (Shair et al., 2003a;Hofer et al., 1998;Hofer and Shair, 1980). Whether vocalizations are a by-product or cognitively produced by pups, their communicative properties to the dam are well documented.
Sustained high-rate vocalizations emitted by pups are the most effective for eliciting retrieval from dams (Brunelli et al., 1994;Farrell and Alberts, 2002a;Zimmerberg et al., 2003a). Additionally, it has been shown that USVs can be an important stimulus for consumption of pup excretions during maternal anogential licking (Brouette-Lahlou et al., 1992), indicating their importance in other maternal behaviors following retrieval. The effect of developmental exposure to drugs of abuse on
USVs varies by drug, timing, and dose of exposure. Prenatal alcohol seems to consistently increase
USVs across the neonatal period (Barron and Gilbertson, 2005;Barron et al., 2000;Marino et al.,
2002), though there are mixed results for early cocaine. Neonatal exposure to cocaine (PND 4-11) seems to cause either an increase or decrease in isolation induced USVs on PND 14 (Barron and
7 Gilbertson, 2005;Barron et al., 2000). More research is necessary to clarify the extent and nature of variations in USVs following PCE and the impact these changes have on the early rearing environment.
1.6. NEUROBIOLOGY OF ALTERED CRYING BEHAVIOR
With the importance of the neonatal cry to the mother-infant relationship well established and several decades of studies suggesting that changes in cry acoustics may potentially be a behavioral marker for infants at increased risk for detrimental outcome, it is surprising that few, if any, studies have explored the neurobiology underlying altered crying behavior in a high-risk animal model. Human studies examining spectral measures of cries are limited in their capabilities of exploring the underlying neurobiology of neonatal cries. Animal models have been very useful in exploring what brain regions are involved in normal or typical vocalization control, elicitation, and complexity. Studies on communication in squirrel monkeys have found that the periaqueductal gray
(PAG) is a critical region for cry elicitation (Jurgens and Richter, 1986;Jurgens, 2002). The PAG is responsible for regulating responses to aversive conditions by controlling defensive behavior, analgesia, and vocalizations (Carvalho-Netto et al., 2009). Additionally the PAG receives nociceptive and temperature input (Price et al., 1978;Hayes et al., 1979;Uschakov et al., 2009;de Menezes et al.,
2009) suggesting potential involvement in even early neonatal vocalizations thought to be more autonomic byproducts of sensory regulation (i.e. pain and temperature). Jürgens and colleagues have previously shown that glutamatergic and GABAergic input into the PAG (Jurgens and Lu, 1993a) from limbic structures, including the hypothalamus and amygdala (Jurgens, 1982;Jurgens and Lu,
1993b), play a large role in the emotional control of vocalizations in squirrel monkeys. Rodent studies also support these structures as having a role in vocalizations. As mentioned above human infant cries vary in acoustic properties based on the internal arousal state of the infant (i.e. hunger vs. pain) and in turn rodent infant cries have also been found to vary in acoustic properties somewhat, based on arousal state (i.e. positive/rewarding vs. negative/pain). Positive affect vocalizations are higher in
8 pitch and thought to be associated with the neural circuitry of reward, i.e. the nucleus accumbens
(NAc) (Burgdorf et al., 2007); while negative affect vocalizations are lower pitched and are linked to limbic regions associated with fear and pain including the ventral medial hypothalamus (VMH)
(Borszcz, 2006) and the basal lateral (BLA) and central amygdale (CeA) (Koo et al., 2004). Rodent studies also further support that these limbic regions project to the PAG (Jurgens, 1994;Chen et al.,
2009;Li et al., 1990;Calizo and Flanagan-Cato, 2002). The neonatal limbic system is plastic following birth and continues to mature during the first years (human) or first weeks (rodent) of neonatal life.
This plasticity, is thought to play a pivotal role in human infant oral behavior, i.e. infants seek social stimuli and have a peak period of vocalizing, but upon amygdala maturation, they cease this behavior and begin showing signs of anxiety from maternal separation and fear of strangers (Joseph, 1999).
Few studies exist exploring the relationship between variations in neonatal vocalizations and altered development in these brain regions. Developmental differences in the PAG, VMH, Amygdala, and/or
NAc, induced by PCE could therefore impact vocalization production and control.
1.7. PRENATAL COCAINE’S KNOWN EFFECTS ON BRAIN DEVELOPMENT
Infant PCE is associated with reduced overall growth and head circumferences (Bandstra et al., 2001;Behnke et al., 2002), and central nervous system (CNS) abnormalities such as periventricular cysts, subependymal hemorrhage, pachygyria and schizencephaly (Bellini et al.,
2005). However, little is known about the effect of PCE on more subtle aspects of brain structure, such as neuronal development, which could underlie more subtle abnormalities in cognitive and emotional development. Many studies have found that adult cocaine-exposure decreases neural proliferation in the hippocampus, specifically the dentate gyrus, subgranular zone, and subventricular zone (Dominguez-Escriba et al., 2006;Yamaguchi et al., 2004;Noonan et al., 2008), a potential mechanism speculated to be involved in cocaine addiction (Venkatesan et al., 2007). Studies have also found that cocaine withdrawal can result in a reversal or rebound in neurogenesis deficits
9 between two to four weeks (Noonan et al., 2008;Xie et al., 2009). While cocaine has been shown to consistently decrease adult neurogenesis, the consequences of PCE on neonatal neurogenesis, a time when cocaine-exposure would co-occur with the brain’s critical growth and development stages have not been fully examined. In rhesus monkeys, cocaine-exposure in utero reduces density of neurons, total numbers of neurons, and total volume of cortex (Lidow et al., 2001;Lidow and Song, 2001b), and decreases cellular proliferation in the cerebral wall (Lidow and Song, 2001a). Additionally, fetal
MRIs in sheep prenatally exposed to cocaine show enlarged lateral ventricles and abnormal gray/white matter contrasts, suggestive of abnormal brain maturation (Akoka et al., 1999). However, research has yet to provide a systematic evaluation of neuronal development following PCE, and the impact these changes have on behavior, or if neuronal developmental deficits normalize during the neonatal period in PCE offspring.
1.8. VALIDITY OF TRANSLATIONAL VALUE OF RODENT EARLY BRAIN DEVELOPMENT: HUMAN VS. RODENT
Neural development in rodents differs from humans, mainly because rodents have a large amount of postnatal development while humans have more prenatal maturation of their nervous system. However, neural development is relatively parallel between the two (Bayer et al., 1993), although the time scale is different (days for rodents vs. weeks to months for humans) validating the rodent model as a useful translational tool in studying early neural development. Neurogenesis is a highly regulated process and occurs at different times depending on the location of structure, in a posterior to anterior gradient of the neuroaxis. A large body of research has investigated and discovered the peak times of rodent neurogenesis for most brain regions (Bayer et al., 1993;Rice and
Barone S Jr, 2000) and found that these peak times are relatively parallel to regional development in humans with the amount and timing of neurogenesis varying depending on brain region. Cortical neurogenesis takes place predominantly during the first and second trimester in humans (gestational period in rodents), during which proliferation, migration, and organization of cells occur. During the
10 third trimester in humans (early neonatal period in rodents) cellular differentiation takes places (for a review see (Krageloh-Mann, 2004). In the rat, the ventricular zone is distinctly recognizable on GD
12 (Takahashi et al., 1995), immediately preceding the most extensive proliferation phase in the rat ventricular and subventricular zones which occurs between GD 13-18 (Bayer and Altman,
1991;Rakic and Caviness, Jr., 1995). The dentate gyrus in rodents and humans continues to show proliferation of granular cells well into the postnatal period (Bayer, 1980). Prenatal insults during these critical time points of brain growth result in various cortical malformations and disorders depending on time and length of exposure (Barkovich et al., 2001). Additionally, antimitotic agent administration during active time points of proliferation has demonstrated the vulnerability of the brain at these critical growth periods and the resiliency of the brain to these agents once cellular proliferation attenuates (Mignone and Weber, 2006;Whitney et al., 1995;Campbell et al., 1997). A vast array of literature now exists on the effects of in utero drug exposure on proliferation (Guerri,
1998;Whitney et al., 1995;Campbell et al., 1997;Roy and Sabherwal, 1998;Miller, 1996;Rodier et al.,
1984;Ponce et al., 1994;Choi, 1989); however, the consequences following PCE are still widely unknown and warrant more attention.
1.9. SUMMARY
Many conflicting results found in neonatal vocalization studies may in part derive from the vast number of elicitation protocols employed, variability in vocalization analysis strategies, postnatal age of vocalization testing, sex of offspring, exposure duration and timing of prenatal insult (Zeskind et al., 2011). The studies reported in this dissertation investigates whether PCE alters normal vocalizations in infants in parallel human and rodent experimental models and the impact these acoustic changes have on maternal perception or preference of vocalizations in human and rodent mothers respectively. Additionally, the rodent is a useful model to explore the neural developmental changes which underlie differential vocalizations in PCE male and female offspring.
11 CHAPTER 2. HUMAN PCE: EFFECTS ON ONE-MONTH OLD INFANT CRIES AND THREE-MONTH POSTPARTUM MATERNAL PERCEPTION OF CRIES
2.1. INTRODUCTION
2.1.1. Cocaine Effects on the Mother-Infant Relationship
While drug use during pregnancy poses a direct threat to maternal and fetal health, studies now strongly support that maternal drug use, especially cocaine, is associated with increased rates of child neglect and abuse (Strathearn and Mayes, 2010;Shankaran et al., 2007). Observational studies suggest cocaine-exposed mothers may show decreased enjoyment and responsivity in their interactions with their infants (Burns et al., 1991), are less sensitive to infant/child cues (Minnes et al.,
2005), and less able to focus their attention on their child (Ball et al., 1997). Mothers recovering from cocaine addiction also report greater difficulty with impatience, anger, emotion control, and are vulnerable to maladaptive parenting behaviors including neglect, depression, disorganized care taking, infant-directed hostility, abandonment, and abuse (Murphy et al., 1991;Leventhal et al., 1997).
PCE has also been shown to correspond with variations in the infant’s behavioral signals which elicit maternal attention. Infants exposed to cocaine during pregnancy, even if generally healthy and full term, may show less clarity of infant cues (Minnes et al., 2005), be more irritable, cry more than non-exposed infants (Reijneveld et al., 2004), and separation from their mother followed by reunion does not elicit normal emotional behavior in the cocaine-exposed child (Goldman-Fraser,
1997). It is likely that these deficits may be negatively impacting the mother-infant relationship via
12 affecting an infant’s behavior which is associated with eliciting care from mothers normally, especially in less favorable care giving environments (i.e. neglectful or abusive environments).
2.1.2. Neonatal Vocalizations and Impact on Environment
Neonatal vocalizations are one of the first forms of communication that emerges in the infant and is capable of signaling the care taking environment. Even at an early age when cries are more of an autonomic response to changes in homeostasis (i.e. hunger, thirst, temperature change, pain, etc.) cries vary in acoustic features depending on the internal arousal state of the infant (Porter et al.,
1986;Porter et al., 1988). The cry can thus affect the quality of care the infant receives and therefore contribute to the infant’s development (Zeskind and Lester, 2001). Altered acoustic properties in infant cries have been shown following prenatal exposure to cigarettes, marijuana, opiates, methadone, alcohol, selective serotonin reuptake inhibitors (SSRIs) and cocaine (Lester and Dreher,
1989;Lester et al., 1991;Corwin et al., 1992;Huntington et al., 1990;Zeskind et al., 1996a). Cries of prenatal drug-exposed infants are typically marked by a higher peak fundamental frequency, higher dominant frequency (frequency with the highest energy or amplitude), shorter expiratory sounds, increased dysphonation (sonic turbulence), longer latencies and other spectral features through one month of age (LaGasse et al., 2005). Studies have found newborns exposed in utero to cocaine specifically also have altered cry characteristics, including significantly fewer cry utterances, shorter cry sounds, more energy (amplitude), and higher fundamental frequency than control infants (Corwin et al., 1992;Lester et al., 2002). However because of discrepancies in cry studies, i.e. prenatal cocaine has been suggested to both decrease (Corwin et al., 1992) and increase neonatal crying and irritability
(Keller, Jr. and Snyder-Keller, 2000;Eyler et al., 1998), more research is warranted on prenatal cocaine’s effects on neonatal vocalizations. Additionally, variations in cry behavior could be altering mother-infant interactions possibly a potential factor in neglectful behavior by mothers.
In humans, high-pitched infant cries (greater than 1000 Hertz) are typically perceived as being more aversive, urgent, arousing, and “sick sounding” to caregivers compared to more typical
13 infant cry sounds with average frequencies between 400-500 Hertz. These high-pitched cries also elicit greater arousal and immediate care giving by adults (Zeskind and Lester, 1978;Crowe and
Zeskind, 1992;Frodi et al., 1978;Zeskind P. and Collins V., 1987;Zeskind, 1980). Responses are also directly associated with the personality and emotional characteristics of the adult listener (Scheutze et al., 2003;Boukydis, 1985;Murray, 1985). As such, adults who have physically abused their infants are more likely to find high-pitched cry sounds to be similar to the cry of their own abused infant. They also respond to the cry with heightened heart rate (HR) and skin conductance (Crowe and Zeskind,
1992;Zeskind and Shingler, 1991), suggesting an increased emotional response compared to adults rated as low-risk of child abuse. Scheutze and colleagues (2003) found that when a single 400 Hz hunger cry of one newborn infant with no cocaine-exposure was digitally altered to increase the fundamental frequency to 500 or to 600 Hertz, cocaine-using mothers rated all cries as less arousing, aversive, urgent and sick, and also rated the higher pitched cries as requiring less ameliorative attention, a response pattern opposite to that of drug-free control mothers (Scheutze et al., 2003).
While this study suggests that maternal cocaine use may be associated with less responsiveness to infant cries varying in pitch, the use of a single hunger cry of a newborn, nondrug exposed infant, limits the generalizability of these findings. Additionally, it is reasonable to suggest that altered crying behavior at home can directly alter perceptual response patterns to cries, i.e. a mother with an infant that cries a lot at home might have a different reaction to cries compared to a mother whose infant rarely cries as a result of experience. No prior studies have provided a descriptive analysis of the acoustic and temporal characteristics of cries of cocaine-exposed infants aimed at determining the demand characteristics of those cries nor have they examined responses of cocaine-exposed and nondrug exposed control mothers to naturally elicited cries of drug-exposed and nondrug exposed infants. The present study examined effects of PCE on one month infant vocalizations following a non-pain stimulus (to elicit cries potentially more like what the mother would hear at home). Maternal perception of natural infant vocalizations from recorded nondrug exposed controls and polydrug- exposed infants (not their own infant) at three months postpartum and the relationship between the
14 two measures (cries and perception) were also assessed. Understanding the transactional processes in which the infant and the parent's behavioral attributes synergistically interact to produce non-optimal behavioral and developmental outcomes can hopefully lead to early intervention and better long term outcome.
2.2. METHODS
2.2.1. Subjects
Participants were 87 mothers and their newborn infants. Participants fell into three exposure groups: 38 nondrug exposed controls (mothers who did not take drugs during pregnancy), 34 polydrug-exposed mothers (mothers who had used nicotine, alcohol, marijuana, SSRIs and/or other drugs during pregnancy but not cocaine), and 15 cocaine-exposed mothers (mothers who had used cocaine during pregnancy with or without nicotine, alcohol, marijuana, or SSRIs and to our knowledge were not continuing cocaine use in the postpartum period). Maternal age range was from
18 to 44 years, however, there was no difference between maternal groups in mean maternal age. The nondrug exposed control maternal group consisted of 65.8% Caucasians, 29.0% African Americans, and 5.2% other; the polydrug-exposed maternal group consisted of 44.1% Caucasians, 44.1% African
Americans, and 11.8% other; and the cocaine-exposed maternal group consisted of 66.7%
Caucasians, 26.7% African Americans, and 6.6% other. For a detailed listing of how many mothers in each group took various drug combinations see Table 1. All mothers in both drug-exposure groups were included in the original analysis. Additionally, mothers were given a Drug Time Line Follow
Back (TLFB) Survey as previously reported (Hjorthoj et al., 2011b) at one month postpartum to assess duration and timing of drug exposure during pregnancy. A secondary analysis was conducted to assess quantity of cocaine-exposure in experimental measures of infant cry elicitation and maternal perception (see statistical section below).
15 Table 1. A list of both drug drug-exposed maternal groups broken down by drug combination used during pregnancy. Number following drugs listed represents the number of mothers in each group that used that drug combination. Cocaine-exposed mothers are defined as mothers who used cocaine with or without using other drugs. Polydrug-exposed mothers are defined as mothers who used drug(s) during pregnancy but NOT cocaine. Cocaine‐Exposed (15) Polydrug‐Exposed (34) Cocaine only (2) Cocaine, Nicotine, SSRI (3) Nicotine, SSRI (3) Cocaine, Nicotine (6) Nicotine only (12) Cocaine, Nicotine, Alcohol (2) Nicotine, Alcohol (3) Cocaine, SSRI (2) SSRI (1) Nicotine, Marijuana (5) Alcohol only (2) Nicotine, Marijuana, SSRI (1) Alcohol, Marijuana (2) Nicotine, Alcohol, Marijuana (2) Nicotine, Methadone, TCA (1) Nicotine, Methadone (2)
2.2.2. Infant Cry elicitation/analysis procedure
Cries were elicited and recorded following a standardized procedure in a quiet room.
Elicitation of cries occurred by placing the infants on a cold metal scale used to weigh the infant.
Temperature of the cold scale was measured before each cry elicitation and never dropped below
68ºF. We felt the cold-scale elicited cry would provide more ecologically valid responses with regard to the cry’s value as a social signal and would be more similar to what mothers would hear naturally at home. An audible tone indicated the exact moment that the infant was placed on the scale (for latency measure). An Olympus DM-20 digital recorder (44.1 kHz sampling rate) was held at a standard distance of 20 centimeters vertically and approximately three to four inches horizontally
(mid-sternum) from the infant’s mouth. Recording lasted for 35 seconds following the audible tone after which infants (even if they did not emit a cry) were picked up and soothed. Lack of infant crying provides important data in itself, often reflecting poor neurobehavioral regulation and hence these infants were noted, but not included for analyses of temporal and spectral acoustic variables. Cry
16 characteristics were subsequently analyzed by researchers blind to infant exposure group as previously described (Zeskind et al., 2011). Multi-Speech Lab (KayPentax) software program was used for analysis of infant cry sounds. The first 30-seconds (following the audible tone) of each cry recording was analyzed for latency to the first expiration, temporal morphology, and spectral characteristics. Latency was defined as the duration (in seconds) from the tone indicating infant placement on the cold scale until the first cry expiratory sound. Expiration duration was defined as the duration of the expiratory sound, excluding breath holding before inspiration. The inter-cry-interval was defined as the duration of time from the end of one expiratory cry sound to the beginning of the next expiratory cry sound, so as to measure the duration of pauses between expiratory periods (along with the very short inspiratory period). The duration of pauses has been shown to reflect both the intensity of infant arousal, and to have strong social and perceptual effects on the intensity of adult arousal. To obtain measures of the selected spectral characteristics, a Fast-Fourier Transform was conducted on the 25-msec sample at which the fundamental frequency reached its highest point (in
Hertz) in each expiratory cry sound (peak fundamental frequency). Four measures are obtained from the resulting power spectrum: (1) the frequency (Hertz) and (2) relative amplitude (decibels) of the fundamental frequency and (3) the frequency (Hertz) and (4) relative amplitude (decibels) of the dominant frequency (the peak with the highest power in the power spectrum at the peak fundamental frequency). The amount of dysphonation in each cry expiratory sound was noted based on a scale of 0
– 4: 0 = none, 1 = slight, 2 = moderate, may occur at Peak F0, 3 = harmonic structure mostly obscured, 4 = harmonic structure totally obscured, unable to obtain frequency measures. An energy contour analysis of each expiratory cry sound can also be conducted to provide a digital display of the amplitude across the cry sound from which the maximum amplitude (decibels) was obtained. All cry expiratory sounds were analyzed that occurred during the 30-second recording period following the audible tone. The average of each cry variable was determined for each infant along with the minimum and maximum value to assess the range of acoustic variables.
17 2.2.3. Maternal perception of infant cry stimuli procedure
All mothers listened to a digital recording of six different cry stimuli while in a quiet room free from distractions. Each cry stimuli was 10 seconds in length and were obtained from cry elicitation and recording from one-month old human infants placed on the cold-scale. Cry stimuli used for mothers came from infants that were not their own infant. Each stimulus varied in several acoustic attributes (see Table 1) and was not digitally altered in any way so as to assess maternal response to naturally occurring cries. Participants first listened to three cry stimuli to familiarize themselves with rating procedures. The three practice stimuli used were control cry 2, polydrug cry 1, and control cry 3 (see Table 2). These three stimuli were used for practice based on their acoustic differences, i.e. so all mothers would be exposed to a high pitched cry and a low pitched cry stimuli before testing. Following this practice session the six randomly ordered cry sounds were separated by
20 seconds of silence during which mothers were asked to rate the preceding cry on four 5-point
Likert-type rating scale items used in previous cry-perception studies (Scheutze et al., 2003;Zeskind and Marshall, 1988) including: aroused- not aroused; urgent- not urgent; sick- not sick; and aversive- not aversive. Cries were then played again in a different order separated by 20 seconds of silence during which mothers were asked to rate the immediate proceeding cry on six 5-point Likert-type rating scale items on their perceived care giving responses elicited by the cry sound including how likely mothers were to: pick up- not pick up; cuddle- not cuddle; feed- not feed; give pacifier- not give pacifier; clean- not clean; wait and see- not wait and see. The mothers were all unaware of any attributes of the babies used for cries or how cries were elicited.
Table 2. Average Acoustic Variables from Six Cry Stimuli Played To Mothers
F0 Amplitude Dominant Freq Dom Freq Amp Expiration Duration Inter-cry Interval Max Amplitude STIMULI TYPE # Cries F0 (Hz) Dysphonation (dB) (Hz) (dB) (sec) (sec) (dB) CONTROL CRY 1 7 418.36 51.41 1193.56 59.29 1.00045 0.66316 75.17 0.71 CONTROL CRY 2 9 435.45 52.68 868.51 63.19 0.84796 0.26310 77.87 0.89 CONTROL CRY 3 5 732.13 40.29 3105.09 67.27 1.79848 0.22884 82.54 1.20 POLYDRUG CRY 1 6 509.62 53.89 1676.00 61.43 1.10439 0.61624 76.26 0.17 POLYDRUG CRY 2 9 581.40 47.76 2586.38 59.34 0.72502 0.41643 80.89 2.11 POLYDRUG CRY 3 7 541.41 51.26 2703.96 62.69 0.99757 0.46054 83.30 2.00
18 2.2.4. Statistical Analysis
2.2.4.1. Demographic comparisons: Demographics reported were analyzed by one-way analysis of variance (ANOVA) (for drug-exposure group) for continuous data or using a Fisher’s exact test (for binary measures including if mothers worked, if mothers had a male or female infant, and if infants emitted a cry expiration at the one month cry elicitation visit).
2.2.4.2. Cry characteristics: A multiple analyses of variance (MANOVA) was used to assess exposure group differences on selected cry variables controlling for infant age (in gestational days) at the time of cry elicitation. We controlled for infant age following a Pearson correlation suggesting a positive relationship between infant age at the time of cry elicitation and the mean amplitude at the maximum dominant frequency (χ=4.083, p<0.05) in our study infants. A MANOVA allowed for control of multiple comparisons on acoustic measures of interest. Acoustic measures were grouped into rhythm of cry sound, spectral frequency acoustic measures, spectral amplitude acoustic measures, and lastly for other acoustic measures including dysphonation (For complete grouping details see
Table 3). Following overall significance, Tukey Honestly Significant Difference (HSD) Post hoc tests were used for between group comparisons. A two-way MANOVA was not originally used to assess sex-dependent changes since only five females in the cocaine-exposed infant group had been tested at the time of this original cohort analysis. However, based on variability in cry measures, we examined whether sex-dependent differences existed and were in part contributing to the variability in vocalizations with a two-way (drug exposure group by sex) MANOVA, with acoustic measures grouped as previously mentioned, controlling for infant gestational age.
19 Table 3. Cry Analysis: MANOVA Acoustic Measure Grouping Table 2. Cry Analysis: MANOVA Acoustic Measure Grouping mean duration mean interval Rhythm of Cries number of expirations maximum duration maximum interval mean peak fundamental frequency Spectral Frequency mean dominant frequency Acoustic Measures maximum peak fundamental frequency maximum dominant frequency mean amplitude at peak fundamental frequency maximum amplitude at peak fundamental frequency Spectral Amplitude mean amplitude at dominant frequency Acoustic Measures maximum amplitude at dominant frequency mean overall amplitude of cries latency Other Acoustic mean dysphonation Measures minimum dysphonation maximum dysphonation
2.2.4.3. Secondary Analysis of Infant Cries Based on Drug-Exposure Timing and Duration:
To assess whether drug combination used, or quantity and timing of cocaine use was associated with variability observed in one month cry measures, linear regressions for total number of times mothers used cocaine during the first, second, third trimester, and across all three trimesters were performed for acoustic measures of cocaine-exposed infants. Since most of our mothers in the cocaine-exposed maternal group also smoked cigarettes, we also assessed the relationship between nicotine-exposure in the first, second, third, and over all three trimesters in the polydrug-exposed maternal group related to acoustic measures of polydrug-exposed infants.
2.2.4.4. Maternal Perception of Infant Cries: A general linear model (GLM) ANOVA was used to assess effects of exposure group on overall average ratings of cries for each maternal perceptual measure. Tukey HSD tests were used for between exposure group comparisons when overall significance was determined. Since demographic differences existed among maternal groups, data was also analyzed a second time for perceptual ratings using a GLM covariate analysis
(ANCOVA) controlling for postpartum age of mothers at testing (in days since baby’s date of birth), percentage of time breastfeeding, and number of other children mothers already had.
20 2.2.4.5. Secondary Analysis of Maternal Perception of Infant Cries Based on Drug-Exposure
Timing and Duration: To assess if quantity or timing of cocaine use was in part associated with variability observed in three month postpartum maternal perception of cries, linear regressions were used to account for group variability in the total number of times mothers used cocaine during the first, second, third trimester, and over all three trimesters related to average maternal perceptual ratings of cry stimuli. Additionally, quantity or timing of nicotine-exposure was in part associated with perceptual results by use of linear regressions to measure the relationship between the average number of cigarettes per week mothers smoked during the first, second, third, and over all three trimesters and average maternal ratings of cries. The sample size in cocaine and polydrug-exposed maternal groups was not large enough to assess differences in individual drug combinations at the time of this original cohort analysis. Therefore, differences in a multi-hit hypothesis, was used with a
GLM to compare mothers who used no drugs during pregnancy (n=31), mothers who used one drug during pregnancy (n=11), mothers who used two drugs during pregnancy (n=17), and mothers who used three drugs during pregnancy (n=8) to determine if the number of drugs exposed to during pregnancy were related to the dependent maternal perceptual ratings.
2.2.4.6. Synchrony of Arousal based on Stimuli Acoustic Properties: To examine if mothers in different groups differ in their synchrony of arousal to specific acoustic changes in cry stimuli we employed linear regressions in each maternal group to assess relationships between average acoustic qualities of cry stimuli and maternal perceptual ratings.
2.2.4.7. Maternal Hierarchical Behavioral Patterns of Response: Differential patterns of perceived actions mothers would take towards an infant producing the cry sounds were analyzed with a one-way ANOVA assessing category ratings (for each group) followed by Tukey HSD post hoc tests. We had hypothesized that cocaine-exposed mothers would show an opposite response pattern in their reactions to cries than control mothers. Specifically, that control nondrug using mothers would say they were more likely to pick up and cuddle infants than to clean them and give them a pacifier, showing a priority for a ‘high-bonding’ maternal behavioral response. Conversely, cocaine-exposed
21 mothers would be more likely to clean and give a pacifier to the baby than to pick up and cuddle infants indicating a priority for a ‘low-bonding’ maternal behavioral response.
2.2.4.8. One Month Infant Cry Measures Correlations to Three Month Postpartum Maternal
Perception of Infant Cries: To determine if cries emitted at one month correlated with maternal perception at three months postpartum, individual subject ratings were averaged on each perceptual item (i.e. aroused, urgent, etc.) and the relationship between average maternal perception of each mother with acoustic measures of the mother’s infant at one month (elicited on the cold scale) using linear regression models.
2.3. RESULTS
2.3.1. Demographic Comparisons
Demographic differences are summarized in Table 3. More mothers in the nondrug exposed control maternal group worked (53.1%) compared to the cocaine-exposed maternal group (7.1%,
Fisher Exact, p<0.005) but not to the polydrug-exposed maternal group (27.2%, Fisher Exact, p=0.193). Polydrug-exposed mothers had a higher BMI than did control mothers (ANOVA, F=3.488, p<0.05; Tukey HSD p<0.05) at delivery, but did not differ from controls on other demographic measures. Cocaine-exposed mothers (ANOVA, F=6.953, p<0.005) had a greater number of other children at time of testing than both control (Tukey HSD, p<0.001) and polydrug-exposed maternal groups (Tukey HSD, p<0.05). There was no significant difference in birth weight of infants among treatment groups however, cocaine-exposed infants did show a non-significant trend on this measure compared to control infants (ANOVA, F=2.862, p=0.063, Tukey HSD p=0.056).
Cocaine-exposed infants had a younger gestational birth age (ANOVA, F=5.895, p<0.005) than both control (Tukey HSD, p<0.01) and polydrug-exposed infant groups (Tukey HSD, p<0.005).
There were no gender differences (assessed with Fisher exact nonparametric test) in infants born to any maternal group. There were no significant differences between maternal groups and the
22 TableTable 3.4. Mean Demographic Demographic Inforamtion Information for forMothers/Infants Mothers/Infants Participating Participating in in Study.Study. (*p<0.05,(*p<0.05, **p<0.01, **p<0.01, ***p<0.005) ***p<0.005) Control n=38 Polydrug n=34 Cocaine n=15 Mean SE Mean SE Mean SE
Maternal Age (years) 27.868 0.99 26.206 0.992 28.6 1.57
Number of Other Chldren 0.737 0.145 1.029 0.166 1.933 0.396***
Maternal BMI 26.684 1.114 30.848 1.27* 27.8 1.184
Infant Gestational Age at Birth 279.342 1.157 280.088 1.17 272.4 2.543*** (days)
Infant Birthweight (lbs) 7.7 0.144 7.417 0.172 7.053 0.179
% of Time Breastfeeding in First 69.73 7.026 49.844 8.133 40 13.093 Month Postpartum (self-reported) Infant Gestational Age at Cry 302.158 1.376 305.265 1.643 304.667 4.35 Elicitation (days) Infant Weight at Cry Elicitation 4492.65 92.892 4347.36 104.415 4614.36 171.49 (grams) Calculated Ponderal Index at Cry 3.07 0.077 3.26 0.1 3.24 0.125 Elicitation Infant Head Circumference at Cry 37.07 0.182 36.47 0.357 36.51 0.777 Elicitation (cm) Maternal Postpartum Age at Cry 87.09 1.349 85.19 1.665 84.71 2.039 Perception (days) percentage of time mothers spent breastfeeding during the first month following delivery, but there was a trend for cocaine-exposed mothers to breast feed less than controls (ANOVA, F=2.896, p=0.061). At the one-month cry elicitation visit, there were no significant differences observed in the temperature of the room or the temperature of the cold-scale at the time of cry elicitation. There were also no group differences in infant weight, length, head circumference, or ponderal index (infant weight (g) *100/ body length3) at the one-month cry elicitation visit. Additionally, there was no difference in maternal age (postpartum days since delivery) at time of testing for cry perception. For more detailed demographic information see Table 4.
23 Average Interval Between A. B.Minimum Amount of C. Average Dysphonation CONTROL MINIMUM AMOUNT OF DYSPHONATION Expirations AVERAGE INTERVAL Dysphonation Observed Observed OBSERVED AVERAGE DYSPHONATION POLYDRUG 6.5 1.75 COCAINE 6.0 1.00 5.5 1.50 (sec)
5.0 4.5 0.75 4=harmonic 1.25 4=harmonic obscurred)
obscurred)
4.0 3.5 1.00 Interval
3.0 0.50 2.5 0.75 totally totally
2.0 (0=none; QUANTITY 0.50
(0=none; 1.5 0.25 1.0 0.25 Average AMOUNT OBSERVED
Average Interval (sec) 0.5 0.0 0.00 structure 0.00 CONTROL POLYDRUG COCAINE structure CONTROL POLYDRUG COCAINE Quantity Control Polydrug Cocaine CONTROL POLYDRUG COCAINE Quantity Control Polydrug Cocaine Group Control Polydrug Cocaine GROUP Group Group Group Figure 1. Infant Exposure Group Differences in One Month Old Vocalizations. While non‐significant trends were observed for cocaine‐exposed infants to have (A) a greater interval in between their expirations and (B) a greater amount of minimum dysphonation. No other significant differences were observed between groups including (C) the average amount of dysphonation.
2.3.2. One Month Old Infant Cry Analysis
2.3.2.1. Exposure Group Differences in Vocalization Measures: No overall significant differences were observed between infants in the three exposure groups (See figure 1), however univariate trends were observed in the rhythm of cry sounds (Wilks’s Lambda F=1.050, p=0.406) for main effect of exposure group in average interval between cry expirations (Univariate, F=3.349, p=0.041) and the maximum interval observed between cry expirations (Univariate, F=2.794, p=0.068). Additionally, a univariate trend was observed in the minimum amount of dysphonation observed (Wilks’s Lambda F=0.787, p=0.615; Univariate, F=2.608, p=0.084). Hypothesis driven
Tukey HSD Tests revealed that cocaine-exposed infants show a strong trend for having a greater interval between expirations than do nondrug exposed control infants (Tukey HSD, p=0.051), a greater maximum interval than do nondrug exposed control infants (Tukey HSD, p=0.076), and a greater minimum dysphonation compared to polydrug-exposed infants (Tukey HSD, p=0.065) but not nondrug exposed controls. However, we noticed cocaine-exposed infants showed a greater amount of variability in acoustic measures which could be explained in part from sex-dependent differences or differences in duration or timing of cocaine-exposure.
24 Figure 2. Infant Exposure Group and INTERVAL OF TIME BETWEEN Sex Differences in One Month Old EXPIRATIONS Vocalizations . Females exposed to 20 cocaine had a significantly greater *** mean and maximum interval in 15 between their expirations compared *** to all other infants. 10
(p<0.001 for all comparisons; N= SECONDS control males: 19, control females: 5 10, polydrug males: 14, polydrug 0 females: 18, cocaine males: 8, CONTROL POLYDRUG COCAINE CONTROL POLYDRUG COCAINE cocaine females: 5). MALES FEMALES
2.3.2.2. Sex-Dependent Differences in Vocalization Measures: To determine sex-dependent differences and their relationship to the variability in vocalizations, a two-way (exposure group by sex) MANOVA controlling for infant gestational age at cry elicitation was employed. There was a significant group by sex interaction on measures of the rhythm of cry sound (Wilks’s Lambda,
F=2.261, p<0.05, See Figure 2). Specifically, differences were found on measures of the mean
(Univariate ANOVA, F=6.531, p<0.005) and maximum interval (Univariate ANOVA, F=7.358, p<0.001) between expirations. Tukey HSD post hoc tests indicated cocaine-exposed female infants had a greater mean interval of silence between expirations than other infants including cocaine- exposed males (p<0.001), control males and females (p<0.0005), and polydrug-exposed males and females (p<0.001). Additionally, cocaine-exposed females were also found to have a greater maximum interval of silence between expirations than cocaine-exposed males (p<0.0005), control males (p<0.0005) and females (p<0.001), and polydrug-exposed males and females (p<0.0005).
While interesting, this finding should be interpreted with care as only five cocaine-exposed females were analyzed compared to eight cocaine-exposed males. Two cocaine-exposed males did not emit a cry during our elicitation protocol; however, when assessing the number of infants that did or did not emit a cry using the Fisher Exact test, we found no significant sex or group differences. Data supports further investigation into sex-dependent differences following PCE. There were no significant sex differences in the spectral frequency or amplitude acoustic measures.
25 2.3.3. Secondary Analysis of Infant Cries based on Drug Exposure (TLFB)
2.3.3.1. Quantity and Timing of Cocaine-exposure: To assess if quantity or timing of cocaine- exposure was in part associated with variability observed in one month cry results regressions were run for the total number of times mothers used cocaine during the first, second, third trimester, and over all three trimesters correlated with acoustic measures of infants prenatally exposed to cocaine.
Linear regressions revealed a negative relationship (See Figure 3) between the number of times mothers used cocaine in the first trimester and the minimum amplitude at the peak fundamental frequency (r2=0.325, β =-0.121 + 0.053, F=5.301, p<0.05). No other acoustic measures were found to be related with the number of times mothers used cocaine in the first, second, third, or over all three trimesters.
LEGEND 60 * Mothers who used cocaine and nicotine only Mothers who used cocaine, nicotine, and another drug ● Mothers who used cocaine but NOT nicotine 50 Figure 3. Linear Regression showing the relationship between the number of times mothers used cocaine during the first trimester and the minimum 40 amplitude at the fundamental frequency of one month old infant cries. The more cocaine mothers used in the first trimester the lower 30 the minimum amplitude of their infant’s expirations. (r2=0.325,
Min Amp at the Peak Fo (dB) the Amp at Min p<0.05; Infants are color coded according to drug exposure (see 20 Legend above) N= 13 cocaine‐ 0 102030405060708090100 exposed infants and their mother’s # Times Used Cocaine (1st Trimester) TLFB response).
2.3.3.2. Quantity and Timing of Nicotine Exposure: Since most of our cocaine-exposed infants were also exposed to nicotine we assessed the relationship with cigarette smoking (average number of cigarettes smoked per week in the first, second, third, and overall three trimesters) with
26 acoustic measures of infant cries. We found several significant correlations between the average number of cigarettes mothers smoked per week in the first trimester and acoustic measures.
Specifically, we found (See Figure 4) a positive correlation between the amount of smoking in the first trimester and the average amplitude at the fundamental frequency (r2=0.504, β =0.936, F=6.108, p<0.05), the minimum amplitude at the fundamental frequency (r2=0.517, β =1.309, F=6.415, p<0.05), the average interval between cries (r2=0.496, β =0.808, F=5.911, p<0.051), and the minimum interval between cries (r2=0.511, β =0.842, F=6.270, p<0.05). We found a negative correlation between the amount of smoking and the average dominant frequency (r2=0.490, β =-
75.921, F=5.759, p=0.053) and the maximum dominant frequency (r2=0.617, β =-204.759, F=9.655, p<0.05).
A. 70 B. 3,000
60
50 2,000
40
30 1,000
20 Min Amp at the Peak Fo the (dB) at Amp Min Average Dominant Frequency (Hz) Frequency Dominant Average 10 0 0 5 10 15 20 25 0 5 10 15 20 25 Average Cigarettes Per Week (1st Trimester) Average Cigarettes Per Week (1st Trimester) Figure 4. Linear Regression showing the relationship between the average number of cigarettes mothers in the polydrug‐exposed maternal group smoked during the first trimester with (A) the minimum amplitude at the fundamental frequency and (B) the average dominant frequency of one month old infant cries. The more polydrug‐exposed mothers smoked during the first trimester the higher the minimum amplitude of their infant’s expirations. (r2=0.517, p<0.05), while the more mothers smoked the lower the average dominant frequency (r2=0.490, p=0.053). (N= 8 polydrug‐exposed infants and their mother’s TLFB response for nicotine).
27 2.3.4. Three Month Postpartum Maternal Perception of Infant Cries.
2.3.4.1. Exposure Group Differences in Perceptual Ratings: A General Linear Model (GLM) indicated a significant main group effect on overall ratings of how aroused the cries made mothers feel (F=3.422; p<0.05, See Figure 5). Tukey HSD post hoc tests revealed that cocaine-exposed mothers (p<0.05) felt less aroused after listening to cries than did nondrug exposed control mothers
(See Figure 5). There were no main group effects on how sick or urgent mothers perceived cries.
However, there was a trend for a group effect in how aversive the mothers perceived cries to be
(F=2.628, p=0.073). Specifically cocaine-exposed mothers showed an interesting trend for perceiving cries as less aversive than nondrug exposed control mothers (p=0.06, Tukey HSD).
There were also significant main group effects (See Figure 5) in the action mothers perceived they would take toward the cry stimuli. Main effects were observed on ratings for give pacifier
(F=13.386; p<0.0005), clean (F=3.421; p<0.05), and wait and see (F=5.889; p<0.005). Post hoc tests showed cocaine-exposed mothers were more likely to give a pacifier in response to cries compared to control and polydrug-exposed mothers (p<0.0005) and also more likely to clean the infant in response to cries compared to polydrug-exposed mothers (p<0.05) and control mothers (p=0.085). Conversely, polydrug-exposed mothers were less likely to respond that they would wait and see compared to both cocaine-exposed and nondrug exposed control mothers (p<0.01).
28 A. B. Average Maternal Perception to Cry Stimuli Average Perceived Action to Cry Stimuli CONTROL CONTROL 4.5 4.5 POLYDRUG POLYDRUG *** 4.0 COCAINE 4.0 COCAINE * * 3.5 3.5 T 3.0 3.0 2.5 2.5 2.0
2.0 Rating **
Rating 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 AROUSED URGENT SICK AVERSIVE E D E KUP EE SE DDL C F LEAN U C D C PI PACIFIER AN E IT IV G WA Figure 5. Exposure Group Differences in Maternal Perceptual Ratings. (A) Emotional percpetual ratings showing cocaine‐exposed mothers reported they felt less aroused when listening to cries than controls and had a trend for felling cries were less aversive than controls. (B) Beahvioral response perceptual ratings showing cocaine‐exposed mothers reported they would be more likely to clean infants producing cry sounds than polydrug‐exposed mothers and more likely to give a pacifier than both control and polydrug‐exposed mothers. Polydrug mothers also reported they would be more likely to wait and see than control and cocaine‐exposed mothers. (* p<0.05, **p<0.01, ***p<0.005; N= 31 Controls, 22 Polydrug‐exposed, and 14 Cocaine‐exposed mothers).
2.3.4.2. Exposure Group Differences in Perceptual Ratings Covaried for Demographic
Differences: Since differences existed amongst maternal groups in the number of other children mothers already had and mothers exposed to cocaine breastfeed somewhat less, we used a GLM
ANCOVA to control for postpartum day of testing, breastfeeding, and number of other children at home to determine if there were differences in perception when those differences were factored in.
The main group effect of how aroused mothers felt after listening to cries barely lost significance
(ANCOVA, F=2.830, p=0.06) with these tests. Other results remained significant as cocaine-exposed mothers were still more likely to give a pacifier than either nondrug exposed control or polydrug- exposed mothers (ANCOVA, F=10.255, p<0.0005; Tukey HSD, p<0.0005) and were still more likely to say they would clean infants than controls (ANCOVA, F=3.539, p<0.05; Tukey HSD, p<0.05).
Polydrug-exposed mothers were also still less likely to wait and see (ANCOVA, F=7.348, p<0.001) compared to cocaine-exposed (Tukey HSD, p<0.001) and nondrug exposed control mothers (Tukey
HSD, p<0.05) suggesting these differences are not a consequence of differences in parity, breastfeeding, or postpartum age.
29 2.3.5. Secondary Analysis of Maternal Perception based on Drug Exposure (TLFB)
2.3.5.1. Quantity and Timing of Cocaine-exposure: When assessing the relationship with number of times mothers used cocaine in the first, second, third, or overall three trimesters and perceptual ratings we found no significant relationships between cocaine-exposure and maternal perceptual ratings of cries (See Figure 6).
2.3.5.1. Quantity and Timing of Nicotine Exposure: When assessing the relationship with average number of cigarettes mothers per week mothers smoked during the first, second, third, or overall three trimesters and perceptual ratings we found a significant positive relationship between the amount of smoking in the first trimester and perceptual ratings of give pacifier (r2=0.453,
β=0.124+0.045, F=7.459, p<0.05), specifically the more mothers smoked during the first trimester the more likely they were to say they would give a pacifier in response to standardized cries (See Figure
6).
B. A. 5.5 5 r
5.0 4
4.5 3
4.0
2 3.5
1 3.0 Perceptual Rating of Give Pacifie Give of Rating Perceptual PerceptualRating for Give Pacifier 2.5 0 0 102030405060708090100 0 5 10 15 20 25 # of Times Used Cocaine (1st Trimester) Avg. Cigarettes Per Week (1st Trimester)
Figure 6. Linear Regressions showing the relationship between the (A) total LEGEND for Cocaine‐Exposed Mothers number of times mothers in the cocaine‐exposed group used cocaine during * Mothers who used cocaine and nicotine only the first trimester with their perceptual ratings of give pacifier showing no significant relationship (r2=0.003, p=0.858); and (B) the average number of Mothers who used cocaine, nicotine, and another drug cigarettes per week polydrug‐exposed mothers smoked during the first ● Mothers who used cocaine but NOT nicotine trimester with their perceptual ratings of give pacifier showing a significant positive relationship (r2=0.453, p<0.05), (N= 14 cocaine‐exposed mothers perceptual ratings with TLFB for cocaine, 11 polydrug‐exposed infants and their mother’s TLFB response for nicotine).
30 2.3.5.2. Drug Combination: In comparing mothers who used no drugs during pregnancy
(n=31), mothers who used one drug during pregnancy (n=11), mothers who used two drugs during pregnancy (n=17), and mothers who used three drugs during pregnancy (n=8) for average perceptual ratings of cry stimuli, we found significant effects (See Figure 7) of number of drugs taken on maternal perceptual ratings of aroused (F=2.966, p<0.05), urgent (F=2.866, p<0.05), and sick
(F=3.080, p<0.05). Mothers who used three drugs during pregnancy reported they were less aroused by cries (Tukey HSD, p<0.05) and thought cries sounded less urgent (Tukey HSD, p<0.05) than mothers who used no drugs during pregnancy. We also found that mothers who used three drugs during pregnancy reported cries sounded more sick sounding compared to mothers who used one drug during pregnancy (Tukey HSD, p<0.05). There was also a trend for mothers who used three drugs during pregnancy to report they would be more likely to give a pacifier compared to those mothers that used no drugs during pregnancy (ANOVA, F=2.553, p=0.063; Tukey HSD p<0.05).
Number of Drugs Used on Perceptual Ratings 0 drugs 4.5 T 1 drug 4.0 2 drugs 3.5 * * * 3 drugs 3.0 2.5 2.0 1.5 1.0 Perceptual Rating 0.5 0.0
D ICK _UP USE S CK URGENT PI ACIFIER ARO P
GIVE_ Figure 7. Differences in Maternal Perceptual Ratings based on the number of drugs used during pregnancy (regardless of drug type). Mothers who used three drugs during pregnancy reported they felt less aroused and thought cries were more urgent than mothers who used no drugs during pregnancy. Mothers who used three drugs also said cries sounded more sick than mothers who used one drug. A strong trend was observed for mothers who used three drugs to report they were more likely to give a pacifier than mothers who used no drugs. (* p<0.05,; N= 31 no drug exposure, 11 one drug exposure, 17 two drug exposure, and 8 with three drug exposure).
31 2.3.6. Synchrony of Arousal to Acoustic Changes in Cry Stimuli Differences By Exposure Group.
2.3.6.1. Emotional Perceptual Rating Differences (Summarized in Table 4):
Control and polydrug-exposed mothers had significant positive relationships between the average duration of cry stimuli and ratings of urgency (p<0.0005), and between the average dysphonation of cry stimuli and ratings of sick (p<0.0005). These two positive relationships were not observed in the cocaine-exposed maternal group. Conversely, the cocaine-exposed maternal group showed significant negative relationships between the number of expirations in acoustic stimuli and ratings of sick (p<0.05) and between the number of expirations in cry stimuli and ratings of aversive
(p<0.05). Neither of these relationships was observed in either the control or polydrug-exposed maternal groups suggesting cocaine-exposed mothers may be attending to different acoustic attributes of cries than control and polydrug-exposed maternal groups.
32 Table 5: Linear regression results of emotional perceptual ratings for each maternal exposure group organized by specific acoustic features of cry stimuli and emotional perceptual ratings. Green highlighted areas indicate a difference between groups in relationship between acoustic stimuli and perceptual ratings.
AROUSED URGENT SICK AVERSIVE
control F=55.367, β=0.005 + 0.001, F=104.967, β=0.008 + 0.001, F=32.789, β=0.005 + 0.001, F=60.839, β=0.006 + 0.001, r2=0.226, p<0.0005 r2=0.356, p<0.0005 r2=0.147, p<0.0005 r2=0.243, p<0.0005 average peak polydrug F=35.666, β= 0.006 + 0.001, F=94.013, β= 0.009 + 0.001, F=29.086, β=0.006 + 0.001, F=37.014, β=0.006 + 0.001, fo r2=0.223, p<0.0005 r2=0.431, p<0.0005 r2=0.190, p<0.0005 r2=0.230, p<0.0005 cocaine F=13.979, β= 0.005 + 0.001, F=28.208, β= 0.007 + 0.001, F=7.077, β= 0.004 + 0.001, F=18.272, β= 0.006 + 0.001, r2=0.146, p<0.0005 r2=0.256, p< 0.0005 r2=0.079, p<0.01 r2=0.182, p<0.0005 control F=44.508, β= -0.106 + 0.016, F=80.357, β= -0.161 + 0.018, F=29.525, β= -0.104 + 0.019, F=46.816, β= -0.128 + 0.019, r2=0.190, p<0.0005 r2=0.297, p<0.0005 r2=0.134, p<0.0005 r2=0.198, p<0.0005 average dB at polydrug F=25.967, β= -0.123 + 0.024, F=58.450, β= -0.176 + 0.023, F=28.039, β= -0.133 + 0.025, F=25.441, β= -0.126 + 0.025, peak Fo r2=0.173, p<0.0005 r2=0.320, p<0.0005 r2=0.184, p<0.0005 r2=0.170, p<0.0005 cocaine F=9.524, β= -0.097 + 0.031, F=13.920, β= -0.128 + 0.034, F=4.259, β= -0.066, r2=0.049, F=11.045, β= -0.104 + 0.031, r2=0.104, p<0.005 r2=0.145, p<0.0005 p<0.05 r2=0.119, p<0.001 control F=8.144, β= 0.652 + 0.229, F=13.852, β= 1.019 + 0.274, F=10.282, β= 0.849 + 0.265, F=11.192, β= 0.896 + 0.268, r2=0.041, p<0.005 r2=0.068, p<0.0005 **** r2=0.051, p<0.005 r2=0.056, p<0.001 average polydrug F=7.283, β= 0.921 + 0.341, F=14.711, β= 1.337 + 0.349, F=8.906, β= 1.058 + 0.354, F=9.287, β= 1.07 + 0.351, duration r2=0.055, p<0.01 r2=0.106, p<0.0005 **** r2=0.067, p<0.005 r2=0.07, p<0.005 cocaine F=4.084, β= 0.864 + 0.428, F=2.902, β= 0.824 + 0.484, F=7.333, β= 1.123 + 0.415, F=9.234, β= 1.268 + 0.417, r2=0.047, p<0.05 r2=0.034, p=0.092 r2=0.082, p<0.01 r2=0.101, p<0.005 control F=50.158, β= -3.119 + 0.440, F=90.509, β= -4.708 + 0.495, F=28.661, β= -2.878 + 0.538, F=41.756, β= -3.420 + 0.529, r2=0.209, p<0.005 r2=0.323, p<0.0005 r2=0.131, p<0.0005 r2=0.180, p<0.0005 average polydrug F=20.779, β= -3.144 + 0.690, F=43.079, β= -4.423 + 0.674, F=17.732, β= -3.067 + 0.728, F=28.339, β= -3.707 + 0.696, interval r2=0.144, p<0.0005 r2=0.258, p<0.0005 r2=0.125, p<0.0005 r2=0.186, p<0.0005 cocaine F=9.165, β= -2.669 + 0.882, F=25.768, β= -4.622 + 0.911, F=5.307, β= -2.050 + 0.890, F=8.693, β= -2.617 + 0.888, r2=0.101, p<0.005 r2=0.239, p<0.0005 r2=0.061, p<0.05 r2=0.096, p<0.005 control F=77.93, β= 0.864 + 0.098, F=136.219, β= 1.257 + 0.108, F=39.379, β=0.773 + 0.123, F=70.232, β=0.982 + 0.117, r2=0.291, p<0.0005 r2=0.418, p<0.0005 r2=0.172, p<0.0005 **** r2=0.270, p<0.0005 average polydrug F=37.232, β= 0.936 + 0.153, F=87.089, β= 1.313 + 0.141, F=39.191, β= 0.997 + 0.159, F=35.274, β= 0.949 + 0.160, dysphonation r2=0.231, p<0.0005 r2=0.413, p<0.0005 r2=0.240, p<0.0005 **** r2=0.221, p<0.0005 cocaine F=11.559, β= 0.694 + 0.204, (F=39.659, β= 1.267 + 0.201, F=1.222, β= 0.237 + 0.214, F=10.607, β= 0.671 + 0.206, r2=0.124, p<0.001 r2=0.326, p<0.0005) r2=0.015, p=0.272 r2=0.115, p<0.005 control F=0.131, β= -0.02 + 0.055, F=0.307, β= -0.037 + 0.067, F=1.594, β= -0.08 + 0.064, F=1.181, β= -0.070 + 0.065, r2=0.001, p=0.717 r2=0.002, p=0.580 r2=0.008, p=0.208 r2=0.006, p=0.278 Number of polydrug F=1.308, β= -0.094 + 0.082, F=2.899, β= -0.146 + 0.086, F=1.984, β= -0.121 + 0.086, F=1.735, β= -0.112 + 0.085, Expirations r2=0.01, p=0.255 r2=0.023, p=0.091 r2=0.016, p=0.162 r2=0.014, p=0.190 cocaine F=1.086, β= -0.107 + 0.102, F=0.083, β= -0.033 + 0.116, F=4.390, β= -0.208 + 0.099, F=5.037, β= -0.225 + 0.1, r2=0.013, p=0.30 r2=0.001, p=0.774 r2=0.051, p<0.05 * r2=0.058, p<0.05 * control F=75.879, β= 0.001 + 0.000, F=144.4, β= 0.001 + 0.000, F=44.258, β=0.001 + 0.000, F=84.441, β=0.001 + 0.000, r2=0.285, p<0.0005 r2=0.432, p<0.0005 r2=0.189, p<0.0005 r2=0.308, p<0.0005 average polydrug F=50.063, β= 0.001 + 0.000, F=149.684, β=0.001 + 0.000, F=45.058, β= 0.001 + 0.000, F=49.128, β= 0.001 + 0.000, dominant 2 2 2 2 frequency r =0.288, p<0.0005 r =0.547, p<0.0005 r =0.267, p<0.0005 r =0.284, p<0.0005 cocaine F=17.794, β= 0.001 + 0.000, F=43.185, β= 0.001 + 0.000 , F=6.591, β=0.000 + 0.000, F=24.743, β=0.001 + 0.000, r2=0.178, p<0.0005 r2=0.345, p<0.0005 r2=0.074, p<0.01 r2=0.232, p<0.0005 control F=27.839, β= 0.146 + 0.028, F=47.311, β=0.221 + 0.032, F=24.915, β=0.162 + 0.032, F=28.807, β=0.175 + 0.033, 2 2 2 2 average r =0.128, p<0.0005 r =0.199, p<0.0005 r =0.116, p<0.0005 r =0.132, p<0.0005 amplitude at polydrug F=16.536, β= 0.170 + 0.042, F=34.280, β= 0.242 + 0.041, F=16.660, β= 0.178 + 0.044, F=25.336, β= 0.212 + 0.042, dom r2=0.118, p<0.0005 r2=0.217, p<0.0005 r2=0.118, p<0.0005 r2=0.170, p<0.0005 frequency cocaine F=9.224, β= 0.160 + 0.053, F=16.187, β= 0.229 + 0.057, F=12.897, β= 0.183 + 0.051, F=16.584, β=0.168, r2=0.168, r2=0.101, p<0.005 r2=0.165, p<0.0005 r2=0.136, p<0.001 p<0.0005
33 2.3.6.2. Perceived Behavioral Response Rating Differences (Summarized in Table 5):
Positive correlations between cry stimuli and perceived choice of behavioral action:
Control (p<0.001) and polydrug-exposed mothers (p<0.0005) showed a significant positive relationship between the average cry amplitude at the dominant frequency and their perceptual ratings of feed. They both also showed a significant positive relationship between the average amplitude at the peak fundamental frequency of the cry with their ratings of give pacifier (p<0.05). Control mothers also had a significant positive relationship between the average duration of cry stimuli and ratings to pick up baby (p<0.05). Both control (p<0.0005) and cocaine-exposed mothers (p<0.001) exhibited significant positive relationships between the average amplitude at dominant frequency of cries and their ratings of cuddle as well as between the average amplitude at the peak fundamental frequency and their choice to wait and see (Control, p<0.0005; Cocaine, p<0.01). Cocaine-exposed mothers exhibited a significant positive relationship between the average peak fundamental frequency of cries and their ratings for clean baby (p<0.01) not observed in the other two exposure groups. The polydrug-exposed mothers also showed a significant positive relationship between the average interval between expirations and ratings of give pacifier (p<0.01)
Negative correlations between cry stimuli characteristics and perceived behavioral actions:
Control and cocaine-exposed mothers presented with significant negative relationships between the average amplitude at the peak fundamental frequency of cries and ratings of feed (both: p<0.001) and between the average peak fundamental frequency of cries and their ratings to give pacifier (Control, p<0.05; Polydrug, p<0.005). Cocaine-exposed mothers did not show significant relationships between these measures. Controls mothers also had a negative relationship between the average duration of cry stimuli and their ratings to wait and see (p<0.01) neither of which was observed in the cocaine or polydrug-exposed maternal groups. Additionally, control (p<0.0005) and cocaine-exposed mothers (p<0.005) presented with negative relationships between the average amplitude at the dominant frequency of cries and between the average peak fundamental frequency with ratings of wait and see (Control, p<0.0005; Cocaine, p<0.001). Polydrug-exposed mothers did
34 Table 6: Linear regression results of perceived behavioral response for each maternal exposure group organized by specific acoustic features of stimuli and perceived behavioral response. Green highlighted areas indicate a difference between groups in relationship between acoustic stimuli and perceptual ratings
PICK UP CUDDLE FEED GIVE PACIFIER CLEAN WAIT AND SEE
control F=42.406, β=0.004 + 0.001, F=17.68, β=0.003 + 0.001, F=22.056, β=0.004 + 0.001, F=5.422, β= -0.002 + 0.001, F=1.812, β=0.001 + 0.001, F=39.533, β= -0.006 + r2=0.182, p<0.0005 r2=0.085, p<0.0005 r2=0.105, p<0.0005 r2=0.028, p<0.05 * r2=0.009, p=0.180 0.001, r2=0.173, p<0.0005 average peak polydrug F=19.477, β=0.004 + 0.001, F=4.925, β=0.002 + 0.001, F=25.593, β=0.006 + 0.001, F=9.26, β= -0.004 + 0.001, F=0.931, β=0.001 + 0.001, F=3.316, β= -0.002 + 0.001, fo r2=0.136, p<0.0005 r2=0.038, p<0.05 r2=0.171, p<0.0005 r2=0.071, p<0.005 *** r2=0.007, p=0.336 r2=0.026, p=0.071 cocaine F=11.753, β= 0.004 + 0.001, F=10.433, β= 0.003 + 0.001, F=8.124, β= 0.004 + 0.001, F=0.363, β= 0.001 + 0.001, F=7.059, β= 0.003 + 0.001, F=12.985, β= -0.005 + r2=0.125, p<0.001 r2=0.113, p<0.002 r2=0.091, p<0.01 r2=0.004, p=0.548 r2=0.079, p<0.01 ** 0.001, r2=0.138, p<0.001 control F=26.23, β= -0.078 + 0.015, F=7.910, β= -0.044 + 0.016, F=12.238, β= -0.071 + F=4.324, β= 0.048 + 0.023, F=2.003, β= -0.028 + 0.020, F=34.076, β= 0.125 + 0.021, r2=0.121, p<0.0005 r2=0.040, p<0.005 0.020, r2=0.061, p<0.001 r2=0.022, p<0.05 * r2=0.010, p=0.159 r2=0.153, p<0.0005 **** average dB at polydrug F=12.729, β= -0.073 + 0.02, F=4.546, β= -0.046 + 0.021, F=12.134, β= -0.092 + F=5.874, β= 0.075 + 0.031, F=1.230, β= -0.028 + 0.025, F=2.151, β= 0.037 + 0.025, peak Fo r2=0.093, p<0.001 r2=0.035, p<0.05 0.026, r2=0.089, p<0.001 r2=0.046, p<0.05 * r2=0.01, p=0.270 r2=0.017, p=0.145 cocaine F=4.590, β= -0.058 + 0.027, F=4.556, β= -0.05 + 0.023, F=2.889, β= -0.052 + 0.031, F=0.069, β= -0.007 + 0.028, F=3.650, β= -0.052 + 0.027, F=7.305, β= 0.09 + 0.033, r2=0.053, p<0.05 r2=0.053, p<0.05 r2=0.034, p=0.093 r2=0.001, p=0.793 r2=0.043, p=0.06 r2=0.083, p<0.008 ** control F=4.099, β= 0.433 + 0.214, F=1.163, β= 0.227 + 0.210, F=1.398, β= 0.327 + 0.277, F=1.587, β= -0.387 + 0.307, F=0.007, β= 0.022 + 0.263, F=6.872, β= -0.788 + 0.3, r2=0.021, p<0.05 * r2=0.006, p=0.282 r2=0.007, p=0.239 r2=0.008, p=0.209 r2=0.000, p=0.933 r2=0.035, p<0.01 ** average polydrug F=2.591, β= 0.449 + 0.279, F=0.904, β= 0.274 + 0.288, F=2.104, β= 0.528 + 0.364, F=2.255, β= -0.626 + 0.417, F=0.02, β= 0.048 + 0.337, F=0.014, β= -0.04 + 0.335, duration r2=0.02, p=0.110 r2=0.007, p=0.343 r2=0.017, p=0.149 r2=0.018, p=0.136 r2=0.000, p=0.887 r2=0.000, p=0.906 cocaine F=3.291, β= 0.650 + 0.359, F=2.768, β= 0.521 + 0.313, F=0.599, β= 0.316 + 0.409, F=1.837, β= 0.487 + 0.360, F=0.591, β= 0.280 + 0.365, F=2.936, β= -0.773 + 0.451, r2=0.039, p=0.073 r2=0.033, p=0.10 r2=0.007, p=0.441 r2=0.022, p=0.179 r2=0.007, p=0.444 r2=0.035, p=0.09 control F=36.976, β= -2.550 + F=27.165, β= -2.181 + F=20.802, β= -2.549 + F=0.969, β= 0.643 + 0.653, F=3.103, β= -0.974 + 0.553, F=33.474, β= 3.471 + 0.6, 0.419, r2=0.163, p<0.0005 0.418, r2=0.125, p<0.0005 0.559, r2=0.10, p<0.0005 r2=0.005, p=0.326 r2=0.016, p=0.08 r2=0.150, p<0.0005 average polydrug F=17.879, β= -2.365 + F=4.776, β= -1.315 + 0.602, F=30.122, β= -3.832 + F=6.772, β= 2.256 + 0.867, F=0.622, β= -0.563 + 0.713, F=5.733, β= 1.67 + 0.697, interval 0.559, r2=0.126, p<0.0005 r2=0.037, p<0.05 0.698, r2=0.195, p<0.0005 r2=0.053, p<0.01 ** r2=0.005, p=0.432 r2=0.045, p<0.05 cocaine F=9.703, β= -2.285 + 0.734, F=12.647, β= -2.237 + F=4.177, β= -1.748 + 0.855, F=0.012, β= -0.085 + 0.780, F=3.256, β= -1.375 + 0.762, F=7.74, β= 2.607 + 0.937, r2=0.106, p<0.003 0.629, r2=0.134, p<0.001 r2=0.049, p<0.05 r2=0.000, p=0.913 r2=0.038, p=0.075 r2=0.087, p<0.01 control F=51.567, β= 0.685 + 0.095, F=18.897, β=0.435 + 0.1, F=38.237, β=0.785 + 0.127, F=1.938, β= -0.213 + 0.153, F=8.560, β=0.375 + 0.128, F=52.514, β=-0.982 + 0.135, r2=0.213, p<0.0005 r2=0.090, p<0.0005 r2=0.169, p<0.0005 r2=0.010, p=0.166 r2=0.043, p<0.005 r2=0.217, p<0.0005 average polydrug F=21.688, β= 0.603 + 0.130, F=7.82, β= 0.390 + 0.140, F=35.524, β= 0.960 + 0.161, F=3.299, β= -0.376 + 0.207, F=4.036, β= 0.332 + 0.165, F=15.501, β= -0.624 + dysphonation r2=0.149, p<0.0005 r2=0.059, p<0.01 r2=0.223, p<0.0005 r2=0.026, p=0.07 r2=0.032, p<0.05 0.158, r2=0.112, p<0.0005 cocaine F=5.161, β= 0.401 + 0.177, F=5.744, β= 0.368 + 0.153, F=7.542, β= 0.544 + 0.198, F=2.496, β= -0.283 + 0.179, F=8.169, β= 0.497 + 0.174, F=16.975, β= -0.868 + r2=0.059, p<0.05 r2=0.065, p<0.05 r2=0.085, p<0.01 r2=0.03, p=0.118 r2=0.091, p<0.005 0.211, r2=0.173, p<0.0005 control F=0.000, β= -0.001 + 0.051, F=0.310, β= 0.028 + 0.05, F=0.054, β=0.015 + 0.065, F=0.745, β= 0.062 + 0.072, F=0.424, β=0.040 + 0.062, F=0.236, β= 0.035 + 0.072, r2=0.000, p=0.987 r2=0.002, p= 0.578 r2=0.000, p=0.816 r2=0.004, p=0.389 r2=0.002, p=0.516 r2=0.001, p=0.627 Number of polydrug F=0.031, β= -0.012 + 0.066, F=0.01, β= -0.007 + 0.068, F=0.017, β=0.011 + 0.086, F=0.310, β=0.055 + 0.099, F=0.051, β=0.018 + 0.079, F=0.425, β= -0.051 + 0.079, Expirations r2=0.000, p=0.860 r2=0.000, p=0.920 r2=0.000, p=0.898 r2=0.003, p=0.579 r2=0.000, p=0.821 r2=0.003, p=0.516 cocaine F=1.041, β= -0.087 + 0.085, F=0.433, β= -0.049 + 0.075, F=0.1, β= -0.031 + 0.097, F=2.14, β= -0.123 + 0.084, F=0.067, β= -0.022 + 0.086, F=1.001, β= 0.108 + 0.108, r2=0.013, p=0.311 r2=0.005, p=0.512 r2=0.001, p=0.752 r2=0.026, p=0.147 r2=0.001, p=0.797 r2=0.012, p=0.320 control F=55.028, β=0.001 + 0.000, F=19.013, β=0.000 + 0.000, F=33.541, β=0.001 + 0.000, F=5.498, β=0.000 + 0.000, F=3.845, β=0.000 + 0.000, F=52.563, β=-0.001 + 0.000, r2=0.225, p<0.0005 r2=0.091, p<0.0005 r2=0.151, p<0.0005 r2=0.028, p<0.05 * r2=0.02, p<0.05 * r2=0.218, p<0.0005 average polydrug F=24.079, β= 0.001 + 0.000, F=6.819, β=0.000 + 0.000, F=35.118, β= 0.001 + 0.000, F=7.697, β= 0.000 + 0.000, F=2.185, β= 0.000 + 0.000, F=7.622, β= 0.000 + 0.000, dominant r2=0.163, p<0.0005 r2=0.052, p<0.01 r2=0.221, p<0.0005 r2=0.059, p<0.01 ** r2=0.017, p=0.142 r2=0.058, p<0.01 frequency cocaine F=12.158, β= 0.000 + 0.000, F=10.313, β= 0.000 + 0.000, F=11.167, β=0.001 + 0.000, F=0.003, β= 0.000+0.000, F=10.325, β=0.000+0.000, F=21.488, β= -0.001 + r2=0.129, p<0.001 r2=0.112, p<0.005 r2=0.121, p<0.001 r2=0.000, p=0.958 r2=0.112, p<0.005 *** 0.000, r2=0.210, p<0.0005 control F=18.866, β=0.113 + 0.026, F=13.587, β=0.095 + 0.026, F=10.635, β=0.112 + 0.034, F=0.957, β= -0.038 + 0.039, F=0.860, β=0.031 + 0.033, F=19.688, β= -0.164 + r2=0.09, p<0.0005 r2=0.067, p<0.0005 **** r2=0.054, p<0.001 *** r2=0.005, p=0.329 r2=0.005, p=0.355 0.037, r2=0.094, p<0.0005 average amplitude at polydrug F=10.388, β= 0.111 + 0.034, F=2.872, β= 0.061 + 0.036, F=16.869, β= 0.179 + 0.044, F=4.909, β= -0.166 + 0.052, F=0.126, β= 0.015 + 0.043, F=2.356, β= -0.065 + 0.042, dom r2=0.077, p<0.005 r2=0.023, p=0.093 r2=0.120, p<0.0005 **** r2=0.039, p<0.05 * r2=0.001, p=0.723 r2=0.019, p=0.127 frequency cocaine F=10.838, β= 0.143 + 0.044, F=11.748, β= 0.129 + 0.038, F=2.856, β=0.086 + 0.051, F=1.234, β= 0.051 + 0.046, F=1.967, β= 0.064 + 0.046, F=8.351, β= -0.160 + 0.055, r2=0.117, p<0.001 r2=0.125, p<0.001 *** r2=0.034, p=0.095 r2= 0.015, p=0.270 r2= 0.023, p=0.165 r2=0.093, p<0.005 *** not show these same significant relationships but did have a negative relationship with the average amplitude at the dominant frequency of cries with their choice to give pacifier; neither of which was observed in the other maternal groups.
35 2.3.7. Maternal Hierarchical Patterning of Perceived Behavioral Responses to Cries
One-way ANOVAs assessing perceived behavioral category ratings (for each maternal group) and Tukey's HSD post hoc tests for comparisons between category ratings suggest nondrug exposed control mothers show a main effect of perceptual rating (F=71.458, p<0.0005, See Figure 8).
Specifically, control mothers said they were more likely to cuddle infants than clean, feed, give pacifier, or wait and see (p<0.0005 for each). Control mothers are likewise more likely to pick up infants than feed, clean, give pacifier, or wait and see (p<0.0005). Control mothers are more likely to clean than wait and see (p<0.0005) and also more likely to feed than give a pacifier (p<0.05) or wait and see (p<0.0005) and finally more likely to give pacifier than wait and see (p<0.0005).
A similar main effect of perceptual rating was observed in polydrug-exposed mothers
(F=66.126, p<0.0005). Specifically, polydrug-exposed mothers were more likely to cuddle than clean, wait and see, give pacifier (p<0.0005), or feed (p<0.05) infants emitting the cry sounds. They reported they would be more likely to pick up than clean (p<0.0005), wait and see (p<0.0005), give pacifier (p<0.005), or feed (p<0.001); more likely to feed than wait and see (p<0.0005), clean
(p<0.01), or give pacifier (p<0.005); and more likely to give pacifier or clean than wait and see
(p<0.0005).
Cocaine-exposed mothers also showed a main effect of perceptual ratings (F=27.607, p<0.0005), however, the same pattern of response as seen in control and polydrug-exposed mothers was not observed. Cocaine mothers were more likely to cuddle than clean (p<0.01) or wait and see
(p<0.0005) but not feed or give pacifier. They were also more likely to pick up than clean (p<0.05) or wait and see (p<0.0005); but not more likely to pick up than feed or give pacifier. Cocaine mothers did report they were more likely to clean, feed, give pacifier, and pick up than wait and see
(p<0.0005). No other differences were observed.
36
PICK UP **** **** CUDDLE * FEED CLEAN * ** **** GIVE PACIFIER 4.5 * **** *** WAIT AND SEE 4.0 ** *** **** 3.5 **** Figure 8. Hierarchy of Behavioral 3.0 Response Between Exposure Groups. **** 2.5 **** Control and Polydrug mothers showed a 2.0 clear hierarchy of responding to cries SCORE 1.5 with more “high bonding” behaviors (i.e. 1.0 cuddle and pick up) than “low bonding” 0.5 behaviors (i.e. clean and give pacifier). 0.0 Cocaine mothers did not show this same CONTROL POLYDRUG COCAINE response pattern. (*p<0.05, **p<0.01, MATERNAL EXPOSURE GROUP ***p<0.005, ****p<0.0005)
2.3.8. One month infant cry correlations with their mother’s maternal perception at three month postpartum
Individual subject ratings were averaged for each perceptual item (i.e. aroused, urgent, etc.) and the relationship between average maternal perception and the way a mother’s infant cried at one month following delivery was examined using linear regressions. A significant positive relationship was observed between mothers’ rating of how aversive cries sound and how long it took their own infants to emit the first cry when placed on the cold scale (r2=0.209, β =0.079 + 0.025, F=10.023, p<0.005). A positive relationship was also observed with maternal ratings of give pacifier and the average maximum amplitude of their own infant cries at one month (r2=0.071, β =0.06+0.029,
F=4.288, p<0.05). A trend was observed between ratings of sick and average dysphonation in cries
(r2=0.058, β = 0.245+0.132, F=3.455, p=0.068).
37 A. B. C.
Figure 9. Linear Regressions showing the relationship between the (A) infant’s latency to emit an expiration at time of cry elicitation and their mother’s maternal perception of cries as aversive (r2= 0.209, p<0.005), (B) infant’s average maximum amplitude across expirations and their mother’s maternal perception of likelihood of give a pacifier when listening to cries (r2= 0.071, p<0.05), and (C) infant’s average amount of dysphonation across expirations and their mother’s maternal perception of cries as sick sounding (r2= 0.058, p=0.068). (N= 17 control mothers, 14 polydrug‐exposed mothers, and 9 cocaine‐exposed mothers’ perceptual ratings with their own one month infant’s cry specifics).
2.4. DISCUSSION
2.4.1. One Month Cold Scale Elicited Cries
We expected to find PCE coincided with variations in cry characteristics in infants at one- month of age. While PCE was related with differences in one-month old infant cries (elicited by placement on a cold weighing scale) we mainly observed trends for the prenatal cocaine-exposed infants to have a greater mean interval and maximum interval between expirations compared to control infants. After assessing if sex-specific changes may be contributing to variability in acoustic measures we observed that the increased interval between expirations in cries appears to be sex- specific in that cocaine-exposed female infants showed a significant increase in the interval between expirations but cocaine-exposed males did not (Figure 2) which was somewhat unexpected. Increased intervals between expirations is a component of the temporal rhythm of crying and is correlated with the rate of expirations and/or the number of expirations emitted over a unit of time (for more
38 information about infant cry analysis and acoustic descriptions see (Zeskind et al., 2011). While not quite significant, cocaine-exposed females did emit fewer expirations than other infants when placed on the cold scale. While many studies examining acoustic properties of cries have not directly examined the interval in between expirations, they have reported the number of expirations, with decreased expirations in newborns being a common finding following general prenatal insults such as environmental toxins and prenatal drug exposure (LaGasse et al., 2005). Data presented here suggest that PCE in females (but not males) may continue to exhibit depressed behavior at one month of age
(including decreased number of expirations and increased intervals of inspiration or breath holding between expirations). While this finding must be interpreted with care as only five cocaine-exposed female compared to eight male cries were analyzable at the time of this initial cohort comparison, the differences observed between minimum, mean, and maximum interval between expirations are striking compared to other infants and should be explored further with larger sample sizes which could then possibly allow numbers to reach statistical significance.
Sex differences in infant behavior are not surprising as behavioral differences have been observed between male and female infants (some as early as hours after delivery). Male newborns have been found to be less able to maintain eye contact (Hittelman and Dickes, 1979) and smile less compared to females (Korner, 1969). Male newborns engage in less self-comforting behaviors, such as sucking behavior and rhythmic body rocking (Brazelton et al., 1974;Feldman et al., 1980); yet when the mother-infant interaction is disturbed during still-face paradigms, girls appear to show more negative emotions than boys (Stoller and Field, 1982;Tronick et al., 1978). Sex-dependent differences in infant behavior could have a profound impact on the early care giving environment. One study found that mother-son dyads have higher synchrony of behavior scores, than mother-daughter dyads in a face-to-face still-face paradigm (Weinberg et al., 1999). That is to say that when mothers and infants are scored for emotional state during a social play task on a one to eight scale (one being maximum negative state, such as anger and hostility, and eight being maximum positive state, such as laughter and play) mother-son dyads show a higher synchrony in emotional state compared to
39 mother-daughter dyads. Mother-son dyads have also been found to share more joint attention, looking at one another or both looking at the same object, than mother-daughter dyads (Tronick and
Cohn, 1989). Interestingly, mothers who have used cocaine, have been shown to verbalize less during a still face test if the cocaine-exposed infant is a female (Lewis et al., 2009) further suggesting cocaine-exposed female infants might be exhibiting altered behavior (i.e. crying) contributing to environmental differences in their care taking environment.
Most studies examining cries have not alluded to sex differences, possibly because there are limited sex specific studies overall, but sex-specific differences in cries following a painful stimuli has been reported. Specifically, newborn boys have been found to have a shorter latency to cry and to have more cry cycles than girls following a heel-lance (Grunau and Craig, 1987). No previous study to date has reported sex-dependent changes in acoustic quality of infant cries following PCE but differential sex-dependent differences have been observed in other behaviors. For instance, male infants (12 to 36 months old) have been suggested to be more vulnerable to cocaine-exposure’s effects on height, motor development, and emotional regulation (Lewis et al., 2004), while deficits in language have been found to be more pronounced in female children following PCE (Beeghly et al.,
2006). Based on these studies, it may be that prenatal cocaine may have a differential impact on male and female infant crying behavior as early as at one month of age, and these variations could be differentially impacting the care taking environment.
In the present study, we found both male and female cocaine-exposed infants exhibited a greater minimum and mean amount of dysphonation in their expirations than control males and females but did not differ from controls in the maximum amount of dysphonation observed in expirations. This suggests that expirations emitted by cocaine-exposed infants have a greater degree of dysphonation, but infants from all three exposure-groups are capable of emitting expirations with the same high-degree of dysphonation. Dysphonation occurs when the vocal cords move irregularly generating turbulent noise, also referred to as “audible harshness” in the acoustic signal of cries (St
James-Roberts, 1999), during which the harmonic structure becomes obscure (Gustafson and Green,
40 1989). The resulting cry has been described as loud and raspy with the character of white noise
(Zeskind and Barr, 1997). To an extent it is a normal component of infant cries (Robb, 2003) and has been reported following both painful (Lester et al., 1992) and hunger (i.e. prefeeding) stimuli (St
James-Roberts, 1999). When assessing cry responses to invasive and non-invasive procedures no differences in quantity of dysphonation has been observed (Grunau et al., 1990) suggesting dysphonation to be a natural acoustic occurrence to discomfort. Dysphonation has also been suggested to be related more directly to growth retardation (Lester et al., 1991) following PCE; however, the present study found no differences between cocaine-exposed infants and controls in infant birth weight or in infant gestational age, weight, weight-for-length ratio of the ponderal index, and infant head circumference at the time of cry-elicitation (see Table 3) suggesting these confounding variables are not directly responsible for increased dysphonation (or increased intervals between expirations in cocaine-exposed females) observed at one month of age in this study.
Studies have previously suggested an association with PCE and increased dysphonation in newborn and one month old infant cries (Lester et al., 1991;Lester et al., 2002). Our findings further support that cocaine-exposed infants show increased dysphonation in their cries through one month of age, however findings in the present study are more generalizable as cries were elicited by a cold- scale used for weighing infants (not the typical painful heel stimulus used in both of these previous studies). Results in the present study therefore might be more representative of typical crying patterns observable in the infant’s home environment. Also since the present study analyzed all expirations infants emitted after placement on the cold scale, compared to previous studies assessing only the first one or up to first three expirations for spectral measures (Zeskind et al., 2011), the increased dysphonation reported here is more generalizable to infant crying compared to initial infant reaction to a pain stimulus. Cry studies have typically defined cries into two components: 1) cry reactivity or initial response- the first or first few expirations after a stimulus (i.e. pain) that represents how reactive the infant is to changes in the environment and 2) cry regulation or recovery- changes across all expirations representing the infant recovering from stimulus (LaGasse et al., 2005). Increased
41 dysphonation observed in this study could therefore indicate poor recovery following placement on the cold scale. While not shown, when comparing the first expiration infants emitted when placed on the cold scale we found no differences between groups in the quantity of dysphonation present. This further suggests that increased dysphonation observed in the present study potentially reflects poor regulation/ recovery in crying of infants exposed to cocaine.
It is important to note that effects we report here are very subtle and did not reach significance following an analysis adjusting for multiple comparisons and controlling for gestational age at the time of cry elicitation. More PCE infants are currently being recruited and larger sample sizes will help to clarify findings. It is likely that by controlling for multiple comparisons and infant gestational age at the time of cry elicitation (and with the relatively small sample size) we are masking the more subtle effects following PCE. Additionally, this present study only included full term, full birth weight, and healthy infants; thus we might be skewing the data by not including heavier cocaine-exposed infants. With these limitations in mind, results suggest that one month, healthy, full term infants exposed to cocaine in utero somewhat exhibit variations in acoustic characteristics in their cries. Understanding the effects these acoustic variations have on the infant’s environment and the mother-infant relationship is vital to begin understanding cocaine’s impact on infant developmental outcome.
2.4.2. Maternal Perception of Infant Cries
The present study compared cocaine-exposed mothers and controls on maternal perception of six unaltered, natural occurring, one-month old infant cries. We predicted that maternal cocaine- exposure would coincide with altered perception of infant cries at three month postpartum. Results support hypothesis in that cocaine-exposed mothers rated cries as less arousing and aversive and reported they would be more likely to give a pacifier and/or clean in response to cry stimuli. Results reported here are similar, though not identical, to previous findings in cocaine-using maternal populations. Cocaine-exposed mothers tested in the present study reported that they perceived cries as
42 less arousing and aversive sounding, but they did not report that cries sounded less urgent or sick sounding as previously described (Scheutze et al., 2003). Additionally, while cocaine-exposed mothers did report that they would be more likely to give a pacifier and clean, they did not report in this study that they would be more likely to wait and see or less likely to pick up or feed in response to cries as previously found (Scheutze et al., 2003).
Differential findings could have resulted from the types of acoustic stimuli employed in these two studies. Scheutze and colleagues’ stimuli was built from one single two-day-old female infant cry, which was elicited via a painful stimulus, and was then digitally altered to only increase the pitch of the cry by 100 Hz and 200 Hz respectively. When an infant’s arousal level changes (i.e. as via a painful stimulus) their cries not only change in pitch, but also in amplitude, duration, etc. The addition of these acoustic changes in six naturally occurring cries (see Table 1) compared to those used previously that only varied in fundamental frequency (Scheutze et al., 2003) most likely conveys differential perceptual salience which was detectable by our cocaine-exposed mothers. This could suggest that while cocaine-exposed mothers might be less sensitive to changes in one acoustic measure, when multiple changes occur they may be better able to detect these differences but are perhaps less sensitive to the arousing properties of the cries. Previously, it was found that when a cry was digitally altered to shorten or lengthen the expiration duration and paired with a decreased interval between expirations the cry stimuli was perceived as more sick sounding compared to the unaltered original cry, the original cry with altered duration alone, or the original cry with altered interval between expirations alone (Zeskind et al., 1992). This supports that stimuli used in this study, showing multiple acoustic differences, has a different saliency than cry stimuli used previously (that varies on one acoustic measure alone). Findings reported here are more generalizable and further suggest cocaine-exposed mothers perceive naturally occurring cries differently than control mothers.
Results observed in cocaine-exposed mothers are also similar to those previously seen in mothers diagnosed with depression (Schuetze and Zeskind, 2001). Cocaine-using mothers show an increased risk for depression, child abuse, and other demographic differences (Rounsaville et al.,
43 1991;Brooner et al., 1997). It is therefore possible that results reported here are more related to the indirect effects of cocaine, such as co-morbidity of depression. Studies have shown that differential demographic factors can have a profound impact on perception of infant cries, including culture
(Zeskind, 1983), parental age (Lester et al., 1989), the personality of the parent (Ziefman, 2003), and if the mother has other children already (Boukydis and Burgess, 1982;Zeskind, 1980). Neural responses to cries also appear to be modulated by time since infant delivery. Specifically, mothers show greater neuronal activation in neural circuits associated with parenting at two to four weeks postpartum compared to three to four months postpartum (Swain, 2008), suggested to be related to experience-dependent changes from mother-infant interactions (Landi et al., 2011). Neural responses to cries have also been shown to be related to breastfeeding (Kim et al., 2011). There were no differences between the cocaine-exposed and control maternal groups on measures of ethnicity, maternal age, maternal BMI, maternal age at cry perception testing, or percent of mothers with a male infant, suggesting none of these variables are responsible for cocaine associated changes in maternal perception. The cocaine-exposed maternal group did have a greater number of children already and breastfed their infants less than controls, however after controlling for these factors, findings remained significant suggesting at least in the present study these variables are not responsible for perceptual differences.
2.4.3. Synchrony of Arousal
We expected that as previous studies have suggested, cocaine-using mothers have a different synchrony of arousal to acoustic changes in cry stimuli as compared to controls (Scheutze et al.,
2003) which has been postulated as representing decreased sensitivity to variations in acoustic properties in infant cries. Findings reported here support the hypothesis that cocaine-exposed mothers may be differentially attending to the acoustic parameters of cries. We found that control and polydrug-exposed mothers (but not cocaine-exposed) showed significant positive relationships between the average duration of stimuli and ratings of urgency and between the average amount of
44 dysphonation in cry stimuli and ratings of sick. Conversely, we found that cocaine-exposed mothers
(but not control or polydrug-exposed) showed significant negative relationships between the number of expirations in acoustic stimuli and ratings of sick and aversive. These findings suggest that cocaine-exposed mothers may not only show a difference in synchrony of arousal to changes in acoustic stimuli but that they also maybe attending and placing higher perceptual value to other acoustic characteristics as compared to control mothers.
Cocaine and polydrug-exposed mothers both showed differential synchrony of perceived behavioral response with acoustic characteristics in stimuli compared to controls (for a full description see Table 4). Thus findings indicate that maternal drug-use in general can disrupt perception of behavioral responses to cry stimuli. More research is necessary to elucidate what is driving these changes and what interventions could be targeted in this population. Interpretation of these results is limited in that we did not assess maternal perception of one acoustic change at a time but rather each cry stimulus played varied on multiple acoustic measures. Perceptual differences could be contributed to mothers attending to one acoustic measure more than another, or it could be a combination of acoustic measures that mothers’ are synching their behavior with. Although findings are more naturalistic in the present study, future studies are needed to examine the saliency of multiple acoustic changes (compared to single) and impact on maternal perception.
2.4.4. Learned Helplessness Hypothesis of Cocaine-Exposure and Mother-Infant Interactions
We had hypothesized that we would find a disrupted hierarchy pattern of behavioral response in that cocaine-exposed mothers would be less likely to pick up and cuddle but more likely to give a pacifier and wait and see (opposite pattern than controls mothers). We found instead that cocaine- exposed mothers in our study reported they would be almost equally as likely to perform all behavioral responses (except wait and see). The multiple acoustic differences in our cry stimuli could be a factor in why we did not observe that cocaine-exposed mothers had a preference for non-active care giving behavioral responses (i.e. give pacifier and wait and see) over active care giving
45 behavioral responses (pick up and feed) as previously observed (Scheutze et al., 2003). In addition to the fact that our cocaine-exposed maternal population may have been a sample of cleaner subjects
(i.e. used less cocaine) than mothers included in the previous study. The present findings of a non- hierarchal behavioral response pattern could also represent a form of learned helplessness. It has been suggested that a mother’s success in interacting with her infant is related to her experience with her infant (i.e. learning that her behavioral response is effective at soothing and calming the infant) and that her success is a predictor of her attention and her ability to terminate infant crying (Donovan,
1981;Donovan et al., 1990). Donovan (1981) found that when mothers were pretrained that they can terminate a cry stimulus (in an experimental setting), they performed better on a later task at terminating a cry and showed cardiac deceleration not observed in mothers who had been pretrained that their actions did not reliably terminate the cry stimulus. Findings suggest that mothers with experience at being unsuccessful in terminating their infant cries, show differential processing of infant cues. The study further suggests that a mother’s experience with her own infant can have a profound effect on her perception and behavioral response to infant cries. It is therefore possible that the increased probability of a cocaine-exposed mother to not only cuddle and pick up her infant but also that she would just as likely give a pacifier or clean her infant could reflect in part, her failed success at soothing her infant at home and thus increase potential for less clear and decisive behavioral action.
With this in mind, we also hypothesized that we would see a correlation between the acoustic measures of a mother’s infant at one month (elicited on the cold-scale) and the mother’s perception/behavioral response to cry stimuli at three months postpartum. We found a significant positive relationship between mothers’ rating of how aversive cries sound at three month postpartum and the latency for their infant to emit an expiration when placed on the cold scale at one month of age (p<0.005). Mothers whose infants cried sooner when placed on the cold scale rated cries as less aversive. While we can only speculate, this could indicate that those mothers whose infants show a quick reactivity to stimulation (decreased threshold to begin crying) have more experience with
46 successfully soothing their infant when in crying state. Future studies exploring maternal perception and infant cries should add empirical evidence to support or refute this supposition by asking mothers to describe their experience with their own infant (i.e. describe your infant’s cry at home, does your infant cry a lot at home, do you feel you can easily soothe your infant, etc.). We also observed mothers were more likely to report they would give a pacifier in response to cries if their infant cried at a higher amplitude (average maximum amplitude, p<0.05) and were more likely to say cries sounded sick if their infant’s cries showed more dysphonation (p=0.068). Cocaine-exposed infants have been reported to have difficult temperaments (Alessandri et al., 1995) and have been characterized as showing higher reactivity and negative affect (Eiden et al., 2009a;Eiden et al.,
2009b;Lester et al., 1996). The experience these mothers have in interactions with their infant could therefore be different than comparison mothers and hence have subsequent effects on their own behavioral interactions with their infant. Future research aimed at exploring this relationship could hold promise for early intervention strategies in this population, including fostering a more nurturing mother-infant bond.
It’s important to note that other demographic differences between maternal groups could be playing a role in maternal confidence in their mothering ability. For instance, married first-time mothers have been found to feel more competent in their maternal role than single first-time mothers
(Copeland and Harbaugh, 2004). Single mothers may be vulnerable to more stress compared to married mothers due to reduced social support, financial hardships, and the physical burden of infant care (Mercer and Ferketich, 1995). This study did not have mothers rate their social support or financial hardship and so we are unable to say if these confounds are contributing to results reported.
Results do suggest maternal intervention may be promising. Prenatal and postpartum care incorporating assessments of maternal reports of self-confidence and social environment focused on providing individualized maternal support options or informative parenting classes could increase confidence in maternal ability and help to nurture the mother-infant bond (Kapp, 1998) and hence promote optimal infant development.
47 2.4.5. Secondary Analysis of Drug Exposure, Quantity, and Type
One limitation in human studies of drug-use is the large amount of variability between subjects that can often be contributed to high comorbidity for multiple drug use and large variability in drug exposure timing and quantity. To determine if quantity or timing of drug use during pregnancy is impacting infant cries and/or maternal perception we assessed the relationship between the number of times mothers used cocaine during their pregnancy and acoustic characteristics of infant cries. We found that the more mothers used cocaine in the first trimester, the lower the minimum amplitude at the peak fundamental frequency of their infant cries. However, we did not find any other relationships between cocaine use and infant crying. Since most of our cocaine-exposed infants were also exposed to nicotine we assessed the relationship between cigarette smoking during pregnancy with acoustic measures of infant cries and found several other significant relationships between the average number of cigarettes mothers smoked per week in the first trimester and acoustic measures. The more mothers smoked during the first trimester, the higher the minimum amplitude at the fundamental frequency. This is opposite to the relationship seen with cocaine-use in that the more cocaine mothers used, the lower the minimum amplitude at the fundamental frequency of their infant cries. This could suggest that cocaine alone is not driving this realtionship but that the quantity of cigarette smoking or combined cocaine and nicotine use might be underlying the relationship between cocaine use and minimum amplitude of infant cries. Unfortunately, we were not able to obtain
TLFBs for cigarette smoking from most of our cocaine-exposed mothers. We prioritized obtaining the
TLFB data for cocaine in these mothers and tried to obtain other TLFBs for other drugs used if time allowed (or if mothers were not becoming irritated or ready to terminate the testing). In most of our cocaine-exposed mothers this was not a possibility. We also found that the more mothers smoked in the first trimester, the higher the average amplitude at the fundamental frequency further supporting that cigarette smoking (i.e. nicotine), might be driving behavioral differences in amplitude at the fundamental frequency. Quantity of cigarette smoking in the first trimester was also positively related
48 to longer average intervals between cries and longer minimum intervals between cries, but negatively related to the average dominant frequency and the maximum dominant frequency. Results suggest that cigarette smoking during the first trimester may have a profound impact on crying behavior in the infant and potentially the care taking environment. While we found no relationships between quantity or timing of cocaine-exposure and maternal perception, we did find that the more mothers smoked cigarettes in the first trimester the more likely they were to report they would give a pacifier in response to cry stimuli at three months postpartum. More research is needed to explore the impact of cigarette smoking on infant behavior and meanings of these changes to the maternal environment.
We were unable to perform between subject comparisons as a factor of drug type, timing, and/or quantity because of the relatively small sample size and the high degree of variability between subjects in types of drugs used, drug use timing (first, second, third trimester, etc.), and quantity of use respectively. However, mothers who used three drugs during pregnancy reported they were less aroused by cries and thought cries sounded less urgent than mothers who used no drugs during pregnancy (regardless of type, timing, or quantity) and they were more likely to give a pacifier than those mothers that used no drugs during pregnancy (p=0.063). There is a high co-morbidity of drug abuse (most of our cocaine-exposed mothers smoked cigarettes and many used other drugs as well in the present study) and a high co-morbidity of psychiatric disorders in cocaine-using maternal populations (many of our cocaine-exposed mothers were also taking an antidepressant for depression as well). It is therefore important to consider the indirect effects of cocaine use on the mother-infant interaction in interpreting findings. For instance, maternal depression (Schuetze and Zeskind, 2001) and maternal psychopathology, i.e. paranoia, is also associated with altered care giving responses
(Scheutze et al., 2003). Substance use, including cocaine, is associated with a higher risk for co- morbid psychopathology, including major depression and anxiety disorders (Rounsaville et al.,
1991;Brooner et al., 1997). It is therefore likely that the more drugs an individual takes during pregnancy the greater the indirect effects of these drugs. These individuals most likely have different levels of environmental stress, potential lack of support from loved ones, lower socioeconomic status,
49 and increased risk of mental illness. It is therefore not surprising that mothers who used more drugs during pregnancy showed the greatest differences in maternal perception at three month postpartum.
This supports the idea that the observed effects are not solely caused by the direct effects of cocaine- exposure, but also by indirect effects through the maternal environment. Hopefully a larger sample size will allow for a more direct assessment of cocaine’s direct biological and behavioral effects.
2.4.6. Limitations/Considerations of Outcomes
The present study used maternal self-report of drug use (timing and quantity of drug exposure) during pregnancy. Zuckerman previously found that a large number of women who deny cocaine use by self-report will test positive on urine analysis and that positive urine analysis is associated with heavier users (Zuckerman et al., 1989). Corwin and colleagues also previously reported that cocaine effects on infant birth weight and infant cry analysis differences was twice as large in infants whose mothers urine tested positive for cocaine compared to infants only identified by self-report (Corwin et al., 1992). This suggests that there may be a dose-response relationship in cocaine-exposure and infant behavioral outcome; however, we might not have accessed enough highly-exposed mother/infant dyads to make these comparisons. Mothers in the cocaine-exposed maternal group were recruited from a residential treatment program and hence were not using cocaine at the time of testing and probably use less cocaine than cocaine abusing populations that are not in residential programs. Additionally, while studies have suggested that TLFB data is not as reliable as other biological methods, especially for heavy users (Griffith et al., 2009), recent studies show strong correlations between TLFB cannabis reports and blood plasma THC, suggesting that when assessing use greater than 20 days in the past, TLFB data may be more reliable (Hjorthoj et al., 2011a;Hjorthoj et al., 2011b). Since many of our cocaine-exposed mothers were light to moderate users (many only reported use in the first trimester it is likely that TLFB data obtained is as, if not more, reliable than blood or urine testing.
50 It is very important to note that while certain acoustic measures of infant cries have been shown to correlate with later developmental outcome (Huntington et al., 1990;Lester, 1987), not all infants showing a particular cry feature will develop poorly. The interaction of cries with the care giving environment should be considered as an equally important, if not more important, underlying factor in infant developmental outcome. For instance, a recent pilot study found that fundamental frequency and number of expirations in cries emitted by infants at one month, correlated with later cognitive scores at three months of age, however patterns of maternal synchrony of arousal were a better predictor of infant cognitive outcome and minimized variability observed when the cry itself was used to correlate with outcome (Cox et al., 2010). Studies suggest the relationship between an infant considered at increased risk for non-optimal development and a synchronous care taking environment or a non-synchronous care taking environment is an important factor in infant outcome.
Infants who score higher on the Brazelton scale (as being more alert and responsive) also coincide with having mothers who are more responsive and sensitive (Osofsky, 1976) further supporting how critical the maternal environment is on infant behavioral outcome.
2.4.7. Summary
In summary, we found an association between PCE and variations in neonatal cries at one month of age following delivery. We also found that cocaine-exposed mothers exhibited differential maternal perception of infant cries that vary on multiple acoustic features at three months postpartum.
All mothers tested had full term, full birth weight, and healthy infants at delivery and hence results could suggest that even low cocaine-exposure can alter maternal and infant behavior. Future studies including heavier doses of cocaine-exposure in infants could help clarify cocaine’s relationship with behavioral outcomes. Findings reported here are more generalizable to what cocaine-exposed mothers might hear at home, as cries were elicited with a non-painful stimulus at one month and cries picked for the auditory stimulus was left unaltered for playback. Data suggest that cocaine-exposed mothers may be experiencing a form of learned helplessness and prior experience with their infant’s
51 crying behavior may be having an impact on their perception and behavioral response to cries. Future studies should further elucidate cocaine’s direct and indirect effects on the mother-infant relationship to better clarify infant outcome following cocaine-exposure. Human clinical studies are inherently limited in the number of confounding variables that can have a direct and indirect effect on behavior and which cannot be controlled for. Strong preclinical animal models are helpful to elucidate cocaine’s effects on infant behavior, and infant behavioral impact on the maternal environment (see
Chapter 3), as well as the neurobiology underlying behavioral changes following drug-exposure (see
Chapter 4).
52 CHAPTER 3. RODENT PCE: EFFECTS ON RODENT PUP VOCALIZATIONS AND RODENT DAM PREFERENCE BEHAVIOR
3.1. INTRODUCTION
Parallel to studies on human cocaine abuse, animal models indicate gestational cocaine- exposure alters maternal behavior with the greatest deficits occurring during the early postpartum period (PPDs 1-5) (Johns et al., 2005;Johns et al., 1994;Vernotica et al., 1999;Kinsley et al.,
1994;Vernotica et al., 1996). Cross-fostering studies suggest cocaine-induced deficits in maternal behavior could be in part a consequence of altered behavior of offspring following PCE (Johns et al.,
2005). It is now widely accepted that somatosensory input, thermal, and motor cues from offspring are important in onset and retention of maternal behavior (Rosenblatt, 1975;Stern and Johnson,
1990;Lonstein and Stern, 1997;Morgan et al., 1992). Additionally, when rodent offspring are isolated outside of the nest, away from their rodent mother (referred to as dam) and litter mates, they emit
USVs. While these neonatal USVs are inaudible to the human ear they communicate to the dam parallel to the human infants’ cry communicating discomfort or need to their environment. These neonatal vocalizations are now widely accepted as an important early offspring cue facilitating early interactions between dam and rodent offspring (referred to as pups) (Farrell and Alberts,
2002b;Noirot, 1972;Brunelli et al., 1994), and because of their highly translational value to human cries, many studies have begun characterizing rodent USVs (Brudzynski et al., 1999;Zeskind et al.,
2011) related to their importance in the early rearing environment (Stern, 1997).
Sustained high-rate vocalizations emitted by pups are the most effective for eliciting retrieval from dams (Farrell and Alberts, 2002a;Deviterne et al., 1990;Zimmerberg et al., 2003a;Fu et al.,
53 2007) although several studies suggest vocalizations provide directionality for retrieval while olfactory cues provide the motivation for retrieval (Smotherman et al., 1974;Stern, 1997;Brunelli et al., 1994). Studies also suggest that frequency modulation (Burgdorf et al., 2008;Wohr et al., 2008b) and amplitude of USVs (Wohr et al., 2008a) may be important acoustic cues for maternal response.
However, few studies examine other acoustic parameters of USVs and the impact that subtle changes in these acoustic parameters may have on the environment. USVs are an important stimulus for consumption of pup excretions during maternal anogential licking (Brouette-Lahlou et al., 1992) and have been associated with increased nest-building activities (Noirot, 1972) indicating important roles for USVs in other maternal behaviors following retrieval.
In addition to USVs serving a communication role with the neonatal environment, animal models suggest vocalizations may be a sensitive behavioral marker for central nervous system damage following prenatal insult and teratogen exposure. Most studies have reported decreased vocalizations following prenatal insult, including prenatal exposure to pesticides (Venerosi et al.,
2009), alcohol (Tattoli et al., 2001;Kehoe and Shoemaker, 1991), cannabinoids (Antonelli et al.,
2005), 3,4-Methylenedioxymethamphetamine (MDMA) (Winslow and Insel, 1990), malnutrition
(Tonkiss and Galler, 2007), and cocaine (Hahn et al., 2000). Prenatal cocaine has also been found to be associated with increased fundamental frequency of vocalizations and decreased rate of vocalizations (Hahn et al., 2000;McMurray, 2011). Most studies examining USV differences following prenatal drug exposure have focused solely on the rate of USV emissions, warranting the need for extending USV analysis to other acoustic measures, and even fewer studies have explored how variations in acoustic characteristics of vocalizations might affect the maternal environment.
Since PCE is associated with increased maternal neglect (Johns et al., 2005) understanding how variations in pup USVs impact the maternal environment could help to further elucidate the mechanism(s) underlying rodent maternal neglect.
54 It is now accepted that maternal behavior is not dependent solely on the dam or the pup but more likely dependent on the interaction between the two (Fleming and Walsh, 1994;Stern and
Johnson, 1990). Studies have found that virgin rodents will display signs of maternal responsiveness to rodent pups following either blood transfusion from newly parturient mothers (Terkel and
Rosenblatt, 1972) or administration of progesterone and/or estrogen (Bridges, 1984;Stern and
McDonald, 1989). These studies support theories that initial maternal behavior onset is largely associated with changes in the maternal endocrine system (Numan, 1994;Numan and Insel,
2003;Keverne, 1988;Rosenblatt, 1990). Cocaine use has been shown to decrease oxytocin, a neuropeptide strongly linked to parturition and maternal behavior onset (Johns et al., 2010;McMurray et al., 2008;Strathearn and Mayes, 2010), and decrease progesterone synthesis during pregnancy
(Ahluwalia et al., 2010). Studies also suggest acute estrogen replacement alters cocaine-induced changes in behavior in rodents (Niyomchai et al., 2006) suggesting cocaine’s behavioral effects may be in part more directly related to cocaine-induced changes in estrogen levels. Progesterone and estrogen administration has also been shown to enhance maternal responsiveness to vocalizing pups in virgin rodents (Farrell and Alberts, 2002a) further suggesting gestational cocaine-exposure may affect how rodent dams respond to changes in vocalizations at least in part though cocaine’s direct effects on the neuroendocrine system during the early postpartum period.
Recently our lab found that PCE changes in whole litter (four pups) vocalizations were not associated with differential maternal retrieval in a specialized retrieval apparatus (Williams, 2011) although individual pup cry characteristics for these litters were not discerned. Studies have found that when littermates are isolated together sensory cues attenuate USV production (Hofer and Shair,
1987); while this effect may be age and experience dependent it suggests isolated pups might have a differential impact on maternal response behavior dependent on variations in vocalizations. To our knowledge, no study has focused on individual pup vocalizations following PCE in regard to the impact variations in vocalizations may have on early postpartum maternal behavior in cocaine- exposed and control dams. The present study examined differences in maternal preference behavior
55 of rodent dams (cocaine-exposed and controls) by observing the amount of time spent with a neonatal pup when given the choice between a cocaine-exposed or an untreated male pup. It was also determined if dam preference behavior was correlated with variations in pup USVs on each respective test day to elucidate to what extent a dam’s preference-like behavior is influenced by variations in
USV characteristics such as call rate, fundamental frequency, and amplitude and if correlations were dam experience-dependent.
3.2. METHODS
3.2.1. Animals
Following a one-week habituation period, virgin female (200-240 grams) Sprague-Dawley rats (Charles River, Raleigh, NC) were placed with males on a breeding rack until a sperm plug was found, which was designated as gestation day (GD) zero. Subjects were randomly assigned to one of three treatment or control groups or one of two pup-provider groups and singly housed and maintained on a reversed 12:12 reverse light cycle (lights off at 0900 hours) for seven days. They were then transferred to a room with a regular light cycle (lights on at 0700 hours) for the remainder of the experiment, a procedure that generally results in the majority of dams delivering their litters during daylight hours (Mayer and Rosenblatt, 1998). All procedures were conducted under federal and institutional animal care and use committee guidelines for humane treatment of laboratory subjects.
3.2.2. Treatment
Treatment groups included: chronic cocaine (CC), and two control groups: chronic saline
(CS) and untreated (UN) dams. CC and CS dams received subcutaneous (SC) injections on alternating flanks of 15 mg/kg cocaine HCL (dose calculated as the free base, Sigma Chemical
Company, St. Louis, MO) dissolved in 0.9% normal saline (total volume 2 ml/kg), or the same volume of normal saline (0.9%), respectively. Injections were delivered twice daily (at
56 approximately 0800 and 1600 hours) throughout gestation beginning on GD 1 and continuing until the day before delivery (GD 20) with the CS dams serving as controls for injection stress and early food consumption rates. UN dams were weighed and handled daily, but received no drug treatment.
CC and UN dams had free access to water and food (rat chow), while CS-treated dams were yoke-fed over the first week to match maximum consumption rates of CC dams to control for the anorectic effects of cocaine, as previously described (Johns et al., 1994). A second group of dams were bred simultaneously and treated as the three treatment groups described above but served as pup-providers for the test dams. Pup-providers either received no treatment (UN dam), other than weighing and handling, or they were treated with cocaine as described above for CC-treated dams. The pup providers reared their natural culled litters (ten pups, six males, four females) for behavioral testing.
Each test dam had two pup-providers assigned to her for postpartum testing so each dam would be tested with unfamiliar pups other than her own. No pup from a pup-provider litter was used twice during preference testing.
3.2.3. Maternal Preference Testing
3.2.3.1. Preference Apparatus: As depicted on Figure 10 the maternal preference apparatus consisted of a rectangular Plexiglas box (80 cm x 22 cm x 30 cm). On either side of the apparatus were partial dividing walls that were 32.5 cm from both left and right ends of the box, thus dividing the box into a left, right, and center (no choice) chamber. Attached to the center of the box was a start box (20 cm x 15 cm x 15 cm) where the dam was placed immediately prior to the start of her test session. The start box had a removable door which was raised by a wire permitting entry into the center chamber of the testing box and promptly closed after dam entry into the apparatus. In each side chamber there was a mesh cage attached to the wall for placement of the stimulus pup. Above each mesh cage, microphones were secured for recording rodent pup ultrasonic vocalizations during preference testing. USVs were recorded with model 4939 microphones connected to (model 2670) preamplifiers, run through a 2-channel model 4939 power amplifier (all Brüel and Kjær), which is
57 connected to a laptop computer through a National Instruments instrumentation recorder (779193-01-
DAQPad-6015) sampling at a rate of 200kS/s (200,000 samples per second). The microphones have a
A. B.
Figure 10. Preference Box Apparatus. (A) Schematic Diagram originally proposed. (B) Image of Apparatus During Testing. Microphones were moved to above the rodent pup cages to maximize acoustic localization. flat frequency response across the anticipated frequency range. Software capable of recording USVs,
Adobe Audition v1.5, begins acquisition of USVs at the session start and terminates as appropriate.
Sessions were stored on a laptop hard drive and backed-up to DVD for later analysis. The temporal mode of rat pup USVs were reduced (“slowed down”) by an appropriate factor to reduce the ultra- high frequencies to the frequency range required for spectral analysis by the Multi-Speech Laboratory
(MSL; KayPENTAX, Inc.) software program.
3.2.3.2. Preference Testing Procedure: On GDs 16 and 17 all dams were shuttled from the animal housing to the testing room to habituate to travel and testing room. On GD 19 each test dam
(not pup-providers) were habituated to the preference apparatus for 15 minutes. On the day of delivery after all pups were born, cleaned, and had a visible milk band (designated as PPD one) a triad consisting of a test dam with her litter, and two pup provider litters (one CC and one UN litter that were born on the previous day before the test litter) were brought to the test room. A 10 minute rest period was allowed following travel. After this period, the PPD 1 test dam was separated from her pups and placed in isolation for 20 minutes. Pups from the test dam litter were placed on a heating
58 pad together and were culled during this isolation period. At this same time, a CC and UN male pup from a CC and UN pup-provider litter respectively was removed from the pup-provider litter and isolated on a heat pad in two separate secondary containers. During the first ten minutes of isolation, the test dam was placed in the start box and allowed ten minutes of exploration of the box which was videotaped and subsequently analyzed for baseline measures. The dam was then returned to her isolated cage for the remaining 10 minutes of the 20 min period. Dams were in a separate room and unable to here pup cries during their isolation period. At the end of the dam’s 20 minute isolation period the isolated CC and UN male pup-provider pups were placed and secured on opposite sides of the testing apparatus in the wire mesh cage. Side of pup placement varied between test dams and test days. Pup vocalizations were recorded for one minute prior to dam entry into the testing apparatus.
Dams were placed in the start box and given five minutes to leave the start box after which the door was closed behind them. Preference behavior was videotaped for ten minutes and stimulus pup vocalizations continued to be recorded for later analysis and correlation with preference. Ten minute preference tapes and USVs were subsequently scored and analyzed. After preference testing the test dam was returned to her home cage along with her culled natural litter. Both the CC and UN male pups from pup-provider litters were marked with a sharpie so as not to be reused and returned to the pup-provider litter. Test dam and litter weight (before and after culling) were recorded, along with the weight of the two stimulus pups from pup-provider litters prior to testing. Additionally, the temperature of each pup was recorded using a laser thermometer held a half inch from the lower back of the pup for four seconds. The testing triad was returned to the animal colony room until the next testing day.
On PPDs three and five the same test dams, litters and unused provider pups were shuttled back to the testing room for preference testing as described above. On PPDs three and five dams did not undergo baseline testing during their 20 min isolation. Procedures for CC and UN male pup providers were the same and placement side for pup treatment group was alternated on PNDs 1, 3, and 5. Dam preference behavior and pup vocalizations were recorded as before for later analysis.
59 3.2.3.3. Maternal Preference Behavioral Scoring: Video recordings of maternal preference testing trials (baseline, PPDs 1, 3, and 5) were scored by two independent observers (blind to stimulus pup side) for frequency, duration and latency of behaviors displayed by dams. All behavioral scoring was reconciled to inter and intra-reliability set at 95-100% concurrence for frequency and latency and
80% or better for duration of behaviors. The following behaviors were scored and were the focus of preference analysis: in center (begins when dam leaves the start chamber and enters the center chamber, i.e. all four feet cross the threshold), in chamber left or right (scored differentially when dam enters one of the chamber sides where the pup cage is (chamber left or right was later defined as
CC or UN pup side), touch/sniff pup stimulus cage in left or right chamber (scored when dam touches or sniffs either pup cage on the left or right side, respectively). Scored behavioral data was analyzed for frequency, duration, and latency of the scored preference behaviors using Generalized
Estimating Equations (GEE).
Interval Until Duration Next USV
Frequency and Amplitude at Maximum Frequency
Frequency and Amplitude at Maximum Amplitude Frequency
Frequency and Amplitude at Minimum Frequency
Time
Figure 11. USV analysis schemiatic showing temporal and spectral acoustic measures assessed for each individual vocalization. The mean was subsequently calculated from individual USV characteristics.
60 3.2.4. Rodent Pup Ultrasonic Vocalization Analysis
The acoustic characteristics of rat pup USVs were determined from sound spectrographic analyses using previously established methods in analyses of rodent and human infant distress vocalizations (Zeskind et al., 2011). Analysis of acoustic characteristics was conducted with the aid of the MSL (KayPENTAX, Inc.) computer program by experienced researchers blind to treatment group membership and experimental conditions. The software program provides a wide range of digitally-based acoustic analyses and visual displays, including (1) sound spectrograms of user- selected segments of the vocal signal, (2) frequency by amplitude power spectra resulting from Fast
Fourier Transforms (FFT) of the acoustic signal at user-selected points in the spectrogram, (3) displays of frequency and amplitude at cursor placement in all acoustic analysis procedures, including power spectra and (4) time intervals between user-juxtaposed cursors. Software routines also allow summation, means and summary statistics of measures of fundamental frequency (basic pitch in Hz), amplitude (dB) and durations of voiced components (sec) across user-selected points of time.
Temporal measures including the number of USVs emitted, mean duration, and mean interval between USVs were collected along with spectral measures including the mean frequency and amplitude at the a) maximum amplitude, b) minimum frequency, and c) the maximum frequency within a USV in the first minute (before the dam had entered the chamber) and the second minute
(one minute following the dam entering the testing chamber). Additionally, the standard deviation of the fundamental frequency and amplitude of USVs were calculated.
3.2.5. Statistical Analysis
3.2.5.1. Gestational, Litter, and Pup Stimulus Comparisons: Gestational and postpartum weight gain of test and pup-provider dams were compared with a one-way Analysis of Variance
(ANOVA) for treatment group. Litters were assessed for weight at birth, total number of offspring, percent of offspring being males, culled litter weight, and culled litter weight gain (PNDs 1-5) with a one-way ANOVA for litter group. To assess that stimulus used for all test dams were the same a two-
61 way ANOVA (for stimulus pup group by test dam group) were ran for pup weight, temperature of pup at the start of testing, and pup temperature change during testing on each test day.
3.2.5.2. Rodent Dam Preference Behavior: Rodent preference behavior on each test day was adjusted for baseline measures as some dams showed a clear preference for a side at the time of baseline testing. Baseline adjustments were made by subtracting the duration (or frequency/latency) of time dams spent engaging in a behavior on a particular side during baseline testing from the duration of time dams spent engaging in a behavior on a particular side during testing. Rodent dam adjusted behavior was analyzed on each test day separately, with a two-way ANOVA (dam group by pup exposure group associated side) for the duration of time dams spent on the CC or UN pup associated side (but not including touch/sniff behavior) and for the duration of time dams spent on the
CC or UN pup associated side in touch/sniff behavior. The frequency of behavior and latency until dams touch/sniffed a pup associated cage, the latency to enter a pup associated side, the latency to leave the start box, and the duration and frequency of dams to enter the center compartment was also assessed with a two-way ANOVA. Following a significant dam group or interaction effect Tukey
HSD post hoc tests were used to make between group comparisons.
3.2.5.3. Rodent Offspring Stimulus- Vocalizations: Rodent offspring USVs which served as a part of the stimulus to elicit preference behavior were assessed for differences in acoustic measures on each respective test day with a two-way ANOVA for offspring exposure group (CC vs. UN pup- provider) and minute of testing (first minute- before dam entered the chamber; second minute- first minute dam was in the chamber). The first and second minutes were analyzed separately to determine if rodent pups differed in their vocalizations once the dam entered the chamber. Significant interaction effects were followed with Tukey HSD post hoc tests to make between group/minute comparisons.
3.2.5.4. Maternal Preference Behavior Relationship with Pup Vocalizations: Pearson
Correlations were used to assess whether maternal preference behavior was related to offspring
62 vocalizations during testing. We examined the relationship between total time spent on a pup side
(adjusted for baseline preference) and mean vocalization measures from the pup on that respective side during the second minute of testing (first minute dam was in the test chamber) for each dam treatment group. Correlations between the duration of time dams spent in T/S on a pup side (adjusted for baseline preference) and mean vocalization measures during the second minute from the pup on that respective side for each dam treatment group were run as this was the first minute of vocalizations dams heard when entering the testing apparatus.
3.3. RESULTS
3.3.1. Gestational and Litter Comparisons
There were no significant treatment group differences in test dam gestational or postpartum weight gain (ANOVA F=2.607, p=0.088). However, we did see a main effect in gestational weight gain among pup-provider dams (ANOVA F=5.690, p<0.05) where CC pup providers gained less weight (136.37 +/- 4.062 grams) during gestation than did UN (149.0 +/- 3.96) pup providers (Tukey
HSD, p<0.05) but there was no difference between pup-providers in postpartum weight gain. There was no difference among test or pup-provider dams in the number of pups in their litter, weight of unculled litters, gender ratio, or weight gain of culled litters during the postnatal testing period.
3.3.2. Pup Stimulus Comparisons
There were no differences in pup stimulus weight, temperature before testing, or temperature change during testing on any PPD of testing between UN and CC stimulus pups used for any test dam group.
63 3.3.3. Rodent Dam Preference Behavior
3.3.3.1. PPD 1: A significant dam treatment group by pup side interaction was observed on
PPD 1 in the duration of time dams spent on a pup associated side (F=4.210, p<0.05, See Figure 12).
Specifically, CC dams spent more time on the UN pup associated side than the CC pup associated side (p<0.05). CC dams also spent less time on the CC pup side than CS (p<0.05) and UN (p<0.05) dams spent on the CC pup side. UN and CS dams did not differ in the amount of time they spent on the UN or CC pup side. There was no difference in the duration, frequency, or latency until dams touched/sniffed a pup associated cage. There was no difference in the latency to enter a particular pup associated side or the latency to leave the start box. There were no group differences in the duration of time dams spent in the center compartment or the frequency that they entered into the center from the left or the right side.
3.3.3.2. PPD 3: There were no statistically significant group differences observed in any pup preference behaviors on PPD 3.
3.3.3.3. PPD 5: A significant dam treatment group by pup side interaction was observed on
PPD 5 on the duration of time dams spent on a pup associated side (F=3.615, p<0.05). Specifically,
CC dams spent more time on the UN pup side than did CS (p<0.012) and UN dams (p<0.05). There was also a main dam group effect on duration (F=3.228, p<0.05) and frequency (F=3.235, p<0.05) of time spent touching/sniffing pup cages. Specifically, CC dams touched/sniffed both UN and CC pup cages less frequently (Tukey, p<0.05) and for a shorter duration than did UN and CS dams (Tukey, p<0.05). There were no differences in the latency to touch/sniff pup cages, to enter a pup associated side or to leave the start box and no other group differences for PPD 5 measures.
64 PPD 1 A. UN
150 CS 100 CC 50 Figure 12. Rodent dam 0 preference behavior for an -50 -100 UN or CC male offspring -150 during early postpartum * -200 UN CC UN CC testing . Graphs depict total time dams spent on a DURATION ON DURATION T/S SECONDS (CHANGE BASELINE) FROM SECONDS respective pup‐associated PPD 3 side (but not in T/S B. behavior) and total time 150 dams spent in T/S on a 100 respective pup side. (A) On 50 PPD 1 CC dams spent less 0 time on the CC‐pup -50 associated side on PPD 1 -100 compared to other test -150 -200 dams . B) On PPD 3 no UN CC UN CC significant differences were DURATION ON DURATION T/S SECONDS (CHANGE FROM BASELINE) SECONDS found. (C) On PPD 5 CC test dams spent more time on C. PPD 5 the UN‐pup associated side compared to other test 150 100 dams. CC dams T/S less on both regardless of pup‐ 50 * * * 0 associated side on PPD -50 * 5.(*p<0.05). -100 -150
-200 UN CC UN CC DURATION ON DURATION T/S SECONDS (CHANGE FROM BASELINE) SECONDS
65 3.3.4. Rodent Offspring Vocalizing Behavior
3.3.4.1 PPD 1 Stimulus Pup Vocalization Comparison: As seen in Figure 13, CC offspring emitted fewer USVs (F=7.022, p<0.01), USVs with a higher minimum frequency (F=3.916, p<0.05), and USVs with a smaller standard deviation in the fundamental frequency (F=3.812, p<0.05) in both the first and second minute of PPD 1 testing compared to UN offspring. CC offspring also had a greater interval between USVs in the second minute of testing (F=4.989, p<0.05) compared UN offspring (Tukey HSD, p<0.01), and in comparison to both CC (Tukey HSD, p<0.005) and UN offspring (Tukey HSD, p<0.001) during the first minute. Both CC and UN offspring also emitted
USVs with a great standard deviation of amplitude in the second minute compared to the first
(F=6.614, p<0.01).
UN A.PPD 1 Number of B. PPD 1 Interval B/W CC USVs Emitted USVs 35 12.5 30 10.0 25 ** 20 7.5 *** *** ** 15 ** 5.0 10 2.5 Number Observed 5
0 0.0 1ST 2ND Interval BetweenUSVs (sec) 1ST 2ND
C. PPD 1 Minimum Frequency D. PPD 1 Standard Deviation at the Fundamental of Amplitude *
50000 0.65 0.60 * * 0.55 0.50 45000 0.45 0.40 0.35 0.30 40000 0.25 0.20 0.15 0.10 at Fundamental Freq. 0.05 35000 0.00 1ST 2ND 1ST 2ND Standard Deviation of Amplitude MinimumFrequency at Fundamental (Hz) Figure 13. PPD 1 Stimulus Pup Vocalization Comparison. (A) Total number of USVs emitted , (B) Interval Between USVs, (C) Minimum frequency at the fundamental frequency, (D) Standard Ddeviation in the amplitude of USVs . CC stimulus pups emitted fewer USVs, with longer intervals in between them (in the second minute only), and USVs with a higher minimum frequency. (*p<0.05, **p<0.01, ***p<0.005).
66
3.3.4.2. PPD 3 Stimulus Pup Vocalization Comparison: There were no differences between
CC or UN stimulus pup vocalizations during the first or the second minute. We did observe that both
CC and UN offspring vocalized less (F=9.639, p<0.005), had a greater interval between USVs
(F=8.214, p<0.005), a higher peak fundamental frequency (F=4.821, p<0.01), a higher max peak frequency (F=5.970, p<0.05), and a greater standard deviation of the fundamental frequency
(F=4.952, p<0.05) in the second minute (when dam entered the testing apparatus) than the first minute on PPD 3.
3.3.4.3. PPD 5 Stimulus Pup Vocalization Comparison: There were no differences between
CC or UN stimulus pup vocalizations during the first or the second minute. We did observe that both
CC and UN offspring vocalized less in the second minute (when the dam entered the testing apparatus) than during the first minute (F=4.501, p<0.05).
3.3.5. Dam Preference Correlations with Stimulus Offspring Vocalizing Behavior
3.3.5.1. PPD 1 Preference Behavior Correlated to Pup Vocalizations: When assessing the relationship between dam preference behavior and USVs recorded during the second minute of testing (first minute dam was in the testing chamber) we found a positive correlation with CC dams spending more time on a side where stimulus pup emitted a greater number of USVs on that respective side (χ=4.043, p<0.05, See Figure 14), however CS and UN dams did not show a significant relationship between these measures. Conversely, we found that UN dams spent less time in T/S behavior on a side where the offspring was emitting USVs at higher frequencies (See Figure
14). Specifically, UN dams spent less time in T/S on the side where the pup was emitting USVs at a higher frequency at the maximum amplitude (χ=9.139, p<0.005), minimum fundamental frequency
(χ=10.602, p<0.001), and maximum fundamental frequency of USVs (χ=8.681, p<0.005). These relationships were not observed in either the CS or the CC group.
67
A. UNTREATED CHRONIC SALINE CHRONIC COCAINE 60 80 80
70 70 50 60 60 40 50 50
30 40 40
30 30 20 Respective Side Respective Side Respective Side Respective 20 20 10 10 10 Number of USVs of USVs Number Emitted a on of USVs Number Emitted a on of USVs Number Emitted a on
0 0 0 -500 -400 -300 -200 -100 0 100 200 300 -500 -400 -300 -200 -100 0 100 200 300 -400 -300 -200 -100 0 100 Duration (sec) Dams Spent Duration (sec) Dams Spent Duration (sec) Dams Spent ON a Respective Side ON a Respective Side ON a Respective Side
B. UNTREATED CHRONIC SALINE CHRONIC COCAINE 60,000 60,000 60,000
50,000 50,000 50,000
40,000 40,000 40,000
30,000 30,000 30,000 Fundamental Fundamental Fundamental
Minimum Frequency at at Frequency Minimum 20,000 at Frequency Minimum 20,000 at Frequency Minimum 20,000
10,000 10,000 10,000 -100 0 100 200 300 400 -100 0 100 200 300 -100 0 100 200 300 Duration (sec) Dams Spent Duration (sec) Dams Spent Duration (sec) Dams Spent in T/S on a Respective Side in T/S on a Respective Side in T/S on a Respective Side Figure 14. PPD 1 Correlations between dam preference behavior and stimulus pup vocalizing behavior. (A) Total number of USVs emitted on a respective side correlated with duration dams spent ON that side (but not in T/S). CC dams showed a significant positive relationship not observed in other two controls. (B) Minimum frequency at the fundamental of USVs emitted by pups on a respective side correlated with duration dams spent in T/S behavior on that respective side. UN dams showed a significant negative relationship not observed in other chronic Saline or chronic cocaine dams.
3.3.5.2. PPD 3 Preference Behavior Correlated to Pup Vocalizations: When assessing the relationship between dam preference behavior and USVs recorded during the second minute of testing (first minute dam was in the testing chamber) we did not find any significant correlations between stimulus pup vocalizations and duration of time spent on a respective side (but not touch/sniffing). We did see a significant inverse relationship for UN dams (but not CC or CS) to spend more time in T/S behavior on a side where the stimulus pups were emitting USVs with a shorter average duration (F=4.328, p<0.05).
3.3.5.3. PPD 5 Preference Behavior Correlated to Pup Vocalizations: When assessing the relationship between dam preference behavior and USVs recorded during the second minute of
68 testing (when dam was first in the testing chamber) we did not find any significant correlations between stimulus pup vocalizations and dam preference behavior (See Figure 15).
UNTREATED CHRONIC SALINE CHRONIC COCAINE A. 150 150 150
100 100 100
50 50 50 Respective Side Respective Side Respective Side Number of USVs Emitted on a a on Emitted USVs of Number a on Emitted USVs of Number a on Emitted USVs of Number
0 0 -400 -300 -200 -100 0 100 200 300 400 0 -300 -200 -100 0 100 200 -400 -300 -200 -100 0 100 200 Duration (sec) Dams Spent Duration (sec) Dams Spent Duration (sec) Dams Spent ON a Respective Side ON a Respective Side ON a Respective Side
B. UNTREATED CHRONIC SALINE CHRONIC COCAINE 60,000 60,000 60,000
50,000 50,000 50,000 Fundamental Fundamental Fundamental 40,000 40,000 40,000 Minimum Frequency at at Frequency Minimum at Frequency Minimum at Frequency Minimum
30,000 30,000 30,000 -100 0 100 200 -100 0 100 200 -50 0 50 100 150 Duration (sec) Dams Spent Duration (sec) Dams Spent Duration (sec) Dams Spent in T/S on a Respective Side in T/S on a Respective Side in T/S on a Respective Side Figure 15. PPD 5 Correlations between dam preference behavior and stimulus pup vocalizing behavior. (A) Total number of USVs emitted on a respective side correlated with duration dams spent ON that side (but not in T/S). (B) Minimum frequency at the fundamental of USVs emitted by pups on a respective side correlated with duration dams spent in T/S behavior on that respective side. No significant correlations were observed on PPD 5.
3.4. DISCUSSION
Newborn rodent pups are highly salient stimuli to postpartum dams, especially during the earlier postpartum period (Mattson et al., 2001). Findings reported here further support that early neonatal pups are highly salient stimuli to postpartum dams, especially early postpartum as all dams tested here decreased the amount of time they spent on a side (not touch-sniffing pup cages) and increased the amount of time they spent engaged in touching and sniffing the pup-containing cage on
69 PPD one compared to baseline testing (when no pups were present). This shift in behavior decreased over the postpartum testing period and was not as dramatic on PPD five compared to PPD one. While this finding could be in part associated with decreased novelty of the testing apparatus, pilot studies
(not shown) found that if PPD five dams were tested again with PND one pups their T/S behavior resembled what is reported here on PPD one testing, suggesting that younger neonatal pups have a different saliency than older rodent pups. Previous studies suggest that as pups age their changing physical characteristics (i.e. size and functional capability) may be less salient to rodent dams
(Reisbick et al., 1975;Bridges et al., 1972;Stern and Mackinnon, 1978). Decreased T/S behavior across postpartum test days therefore most likely reflects a decrease in the saliency of pup stimuli used (age matched to be 18-24 hours older than postpartum age at testing) or changes in the maternal endocrine system across the postpartum period. Additionally, pilot studies in lab found that virgin dams do not show the same increase in T/S behavior when tested in the apparatus with neonatal pups suggesting findings are dam and pup dependent.
It has previously been shown that cocaine administration can interfere with preference behavior for neonatal pups in a dual-choice conditioned place preference apparatus when given the choice of a pup or cocaine-associated side (Seip and Morrell, 2007). In this study we found that gestational cocaine-exposure interferes with preference of pups when given a choice between a CC- exposed or UN male pup on PPD 1. CC dams spent more time on the UN pup side compared to the
CC pup side, but did not differ in the amount of time they T/S both pup cages. We had hypothesized that all dams would spend less time on the CC pup side, based on previous work showing that all dams (regardless of treatment history) show differential maternal behavior towards CC-exposed pups
(Johns et al., 2005). However, both UN and CS dams did not differ in the duration of time they spent on the CC or UN pup side. Differences between this study (where the dams can see, hear, smell but not touch the vocalizing pup) and the previous study (where dams can see, hear, smell, and touch vocalizing pups) could be underlying differential findings. UN and CS dams did not alter their preference behavior of UN and CC pups but if given the option to nurse, lick, and rest in the nest with
70 the pups their behavior might resemble that previously reported. Rodent offspring emit thermal, olfactory, somatosensory, and auditory (i.e. USVs) cues which impact the maternal environment.
Somatosensory input has been shown to be an important pup cue in nursing behavior (Stern and
Lonstein, 1996) and differences in somatosensory behavior between CC and UN offspring could in part drive differences in maternal behavior following retrieval. Additionally, we previously found CC offspring show increased body temperature compared to UN offspring when isolated on PND one
(McMurray, 2011), a behavioral cue which dams may use as a stimulus and hence dams may be more likely to rest out of the nest as observed in previous cross fostering studies when the dam has contact with pups. In the present study (while we did not assess olfactory cues (i.e. urine), or motor behavior between CC and UN pups) we found that CC pups do vocalize less than UN pups during testing on
PPD one, but do not differ in body temperature, although our temperature instruments may have been less robust than those reported in the prior study (McMurray, 2011).
Previous studies have shown that high-rates of vocalizations emitted by pups are the most effective for eliciting retrieval from dams (Brunelli et al., 1994;Farrell and Alberts, 2002a;Deviterne et al., 1990;Zimmerberg et al., 2003a;Fu et al., 2007) but few studies have explored preference-like behavior of rodent dams to whole pup-stimuli (not their own biological pup), but where dams are unable to touch/ retrieve pups. Based on the present work it can be suggested that differences in the rates of vocalizations were not salient enough to directly alter dam preference behavior as we did not observe any correlations between stimulus pup vocalizations with control UN or CS dam preference behavior. Interestingly, CC dams’ preference behavior was found to positively correlate with number of USVs pups emitted. Specifically, we observed that CC dams spent more time on a pup side where the pup emitted more USVs. This could suggest that CC dams are differentially ranking cues in their environment or differentially attending to multiple environmental cues ultimately reflecting a failure of the CC dam to react normally in the maternal environment.
It was previously shown in this animal model that CC dams show avoidance-like behavior toward CC-pup urine on PPD one (not observed in UN dams) but not on PPDs three or five
71 (Williams, 2011). It is therefore plausible that CC dams are attending to multiple cues in the early postpartum period that they find aversive, specifically olfactory and auditory (urine and USVs). As mentioned above, rodent dams in this study were exposed to complex, multimodal stimuli: they could hear stimuli, but could also see and smell stimuli. It is possible that the pups that emitted fewer USVs could also have the greatest differences in urine olfactory cues based on level of cocaine-exposure as cocaine and it’s metabolites have been detected in the urine of PCE pups to at least postnatal day three (pilot work). Preference of CC dams for an UN pup side over a CC pup side could therefore be in part due to olfactory differences between offspring stimuli. Previous studies have found that olfactory cues from offspring play an important role in maternal behavior (Levy et al., 2004), but studies suggest that accurate maternal approach/response requires both vocalization and olfactory stimuli (Smotherman et al., 1974;Farrell and Alberts, 2002b). In light of these studies and previous findings that CC dams find CC-exposed urine aversive (but not UN dams) it is very likely that olfactory cues are also playing a role in CC dam preference for the UN pup side on PPD one. Future studies aimed at elucidating the differential impact of olfactory cues and USVs, including own pup versus other pup stimuli, in this model would prove useful in understanding the mechanism(s) underlying maternal response.
We also observed that CC male stimulus pups vocalized at a higher minimum fundamental frequency and showed decreased standard deviation of the fundamental frequency compared to UN male stimulus pups. While UN dams did not significantly differ in their preference behavior for one pup side over another we did observe that UN dams spent less time on a pup side where USVs being emitted had a higher pitch. This correlation was not observed in either the CC or CS dam suggesting gestational stress (CS) may also alter preference or perception of pup cues.
Findings in the animal study are similar to those reported in humans exposed to cocaine during pregnancy (Chapter 2) as human cocaine-exposed mothers (but not control comparison mothers) showed a significant relationship between number of expirations in cry stimuli they listened to and ratings of how sick and aversive cry stimuli sounded. Additionally, human control comparison
72 mothers evidenced correlations with the fundamental frequency of stimuli and perceived behavioral response to cry stimuli that was not observed in cocaine-exposed mothers (Chapter 2). These results support our hypotheses of translational mechanisms for human and animal cocaine effects on maternal response. Results suggest cocaine-exposure may be affecting the way mothers either perceive or attend to acoustic characteristics of cries resulting in cocaine-exposed mothers attending and/or placing higher perceptual value to different acoustic parameters as compared to control mothers. Cocaine’s effects following gestational treatment in the animal model employed here extend and hopefully inform findings from human studies (Chapter 2) suggesting that cocaine alone
(not paired with other drugs) is capable of altering maternal perception/preference. Results also suggest that other confounding variables observed in our human studies (differential environments, maternal age, parity, breastfeeding, etc.) may not be driving these behavioral differences as much as previously thought given our animal model controlled for these confounds. Of course we realize that generalizations to human studies from animal studies are tenuous and based on many differences but in the sense that there are similarities, future studies may be directed towards further comparative experiments.
While we found no differences in dam preference-like behavior on PPD three we found on
PPD five CC-exposed dams spent more time on the UN-pup side compared to UN and CS dams and touched /sniffed pup cages less (regardless of which pup associated side they were on) compared to
UN and CS dams. However, preference behavior on PPD five did not correlate with pup stimulus vocalizations on that respective day. Together with previous studies (Williams, 2011), data suggests that preference behavior observed on this day is not directly associated with vocalizations or olfactory differences between stimulus pups. Future studies are needed to elucidate the mechanism(s) underlying this later preference behavior following cocaine-exposure including impact of continued experience with litters.
It has previously been shown that a human mother’s maternal behavior is in part dependent on her experience with her own infant (Donovan, 1981). It’s been shown that a rodent dam’s
73 maternal behavior is also somewhat experience-dependent. Rodent dams exposed to a predator odor on PPD one, but not PPD three, show altered maternal behavior through PPD five (Mashoodh et al.,
2009), suggesting early environmental differences impact rodent maternal behavior and that maternal vulnerability to environmental changes is timing dependent. It is therefore possible that early postnatal olfactory (Williams, 2011) and vocalization differences reported here and in other studies
(Hahn et al., 2000;McMurray, 2011) following PCE could be having a direct impact on later maternal/ preference behavior and hence maternal care pups receive by PPD five. We can only speculate if this may be a factor, as we did not test vocalizations of each test dams’ own litter to correlate with her preference behavior on respective test days. Additionally, the stimulus pups used in this study were born 24 hours before test mothers delivered so their ages were actually slightly older than the dam’s own biological pups, which could have been a factor in preference-like behavioral differences. However we do not feel this is the case as pilot studies found no differences between PND one and PND two rodent pup vocalizations or dam preference behavior towards PND one or two pups respectively. Cross fostering studies aimed at examining pup behavior and the impact these changes have on the later maternal environment would prove useful.
We did not observe differences in vocalizations between CC and UN stimulus pups on PPDs three or five. Generally speaking, it is therefore not surprising that we did not observe any significant relationships between stimulus pup vocalizations and dam preference-like behavior on PPDs three or five if indeed USVs are a salient stimulus for preference. We did observe a significant negative relationship for UN dams to spend more time in T/S behavior on a side where the stimulus pup was emitting USVs with a shorter average duration on PPD three. This suggests that USV differences can still impact preference-like behavior later in the postpartum period and perhaps if we manipulated
USV properties, we would have observed greater differences in dam preference-like behavior. Future studies manipulating auditory stimuli (i.e. USV playback in the absence of pups) that dams are exposed to are now underway to elucidate the role USVs have in the early postpartum environment
(alone and in combination with olfactory cues).
74 We assessed differences in USV behavior in the first minute of vocalization recording
(before the dam entered the testing apparatus) and the second minute of vocalization recording (first minute after the dam entered the testing apparatus). Previous studies have suggested that older pups will stop vocalizing, called contact-quieting, when they are reunited with their dam (Shair et al.,
2009). Interestingly on PPDs three and five all pups emitted fewer USVs in the second minute compared to the first suggesting pups were perhaps on some level influenced by the dam’s presence, however based on the pups early age we would not expect this. While pups may not have been able to hear the dam if she called out, it is possible they are sensitive to olfactory and vibratory cues we were not aware of. Secondary analyses correlating dam position in the apparatus with timing of reduced vocalizations may give us more insight into these questions. Future studies should explore contact-quieting and maternal potential of USVs (isolation of pup, followed by reunion with the dam, and then re-isolation of pup) to further phenotype cocaine-exposed pups’ behavior with the maternal environment.
The overall findings of this study are in accord with established literature and indicates that cocaine’s effects on the mother-infant relationship is likely synergistic in that it influences both mother and offspring independently and concertedly. The importance of continued research in this field is highlighted by its direct application in translational value for informing mother-infant studies of cocaine abuse in humans. Continued research has the possibility to unlock clues into the mechanisms by which mother-infant communication fails and whether such failure is driven primarily by altered changes in the maternal sensory and perceptual systems, by altered infant cues, or through interactions between the mother and infant.
75 CHAPTER 4. DELAYED DEVELOPMENTAL CHANGES IN NEONATAL VOCALIZATIONS CORRELATES WITH VARIATIONS IN VENTRAL MEDIAL HYPOTHALAMUS AND CENTRAL AMYGDALA DEVELOPMENT IN THE RODENT NEONATE: EFFECTS OF PRENATAL COCAINE
4.1. INTRODUCTION
Variations in the acoustic parameters of neonatal crying have been consistently shown to reflect the integrity of central nervous system development in the human infant. Infants with such prenatal and perinatal insults as brain damage, prenatal malnutrition, and prenatal drug exposure, have been differentiated by a wide range of measures of infant crying, including a higher fundamental frequency (F0), longer latency to cry, and shorter overall durations of crying (Zeskind and Lester,
1978;LaGasse et al., 2005;Zeskind and Lester, 2001). Recent translational analyses suggest that comparable measures of distress vocalizations can be found across several mammalian species, including the USVs of rat pups (Zeskind et al., 2011). While physioacoustic models of infant crying have described the role of coordinated activity among brainstem, midbrain and limbic systems in the production of variations in human infant cry characteristics (Newman, 2007;Lester, 1984;Golub and
Corwin, 1985;Porges and Lewis, 2010), a paucity of research has explored the neurobiological bases of variations in vocalizations.
Animal models are often used to control for external variables in the human population and mirror clinical studies which show human infant cries are age- and experience-dependent (Blass and
Camp, 2003;Scheiner et al., 2002;St James-Roberts and Plewis, 1996;St James-Roberts and Halil,
1991). Neonatal offspring in isolation emit infant 40 kHz distress calls, whereas older rodents emit 22
76 kHz calls in aversive situations (Blumberg and Alberts, 1991;Brunelli et al., 2006). Vocalizations also differ as a result of the postnatal environment. For instance, litter size has been shown to affect rodent infant vocalizations (Hofer et al., 1993), suggested to be related to altered maternal care individual pups receive in a small litter versus a large litter. It has also been shown that juvenile stress alters adult rodent vocalizations, including potentiation of 22 kHz USVs (Yee et al., 2012).
Studies of adult rodents indicate that specific brain regions involved in particular attributes of vocalization control, elicitation, and complexity and are also experience-dependent (Herschkowitz et al., 1997;Chan et al., 2011;Gonzalez-Lima and Frysztak, 1991;Dujardin and Jurgens, 2005); however the neurobiology underlying vocalizations during early development, in a high-risk rodent model, has not been explored. Preclinical animal studies support the hypothesis that maturation of specific brain regions involved in adult vocalizing behavior, i.e. the amygdala (Lee and Kim, 2004;Mallo et al.,
2009), ventral medial hypothalamus (VMH) (Borszcz, 2006), and periaqueductal gray (PAG)
(Jurgens, 1994;Chen et al., 2009;Li et al., 1990;Calizo and Flanagan-Cato, 2002) are associated with typical developmental changes in offspring behavior, including emergence of fear-like behavior
(Moriceau et al., 2004;Wiedenmayer, 2009) and behavioral sex-differences (Lee et al., 2006).
However whether differential maturation of these regions contribute to variations in neonatal vocalizations in an animal model of high–risk infants, such as those prenatally exposed to drugs, is unknown.
The present study investigated whether PCE is associated with normal developmental changes in USVs in male and female rat offspring at three different age groups corresponding to neonate (designated as PND 1), infant (PND 14), and early juvenile (PND 21) periods. Neuronal maturation rates in the dorsal (dPAG) and ventral PAG (vPAG) on PND 1 (before emotional control of USVs start), and additionally in the central (CeA) and basal lateral (BLA) amygdala and also the
VMH on PNDs 14 and 21 (to correlate with emergence of emotional-dependent USVs) were measured and correlated with vocalizations. We hypothesized that delayed PAG maturation would be
77 associated with decreased number of USVs on PND 1 while delayed CeA and VMH maturation would coincide with increased number of USVs and a lower percentage of 22 kHz USVs on PND 21 in cocaine-exposed offspring. Additionally, we assessed development in the nucleus accumbens
(NAc) (Brudzynski et al., 2011;Burgdorf et al., 2001;Burgdorf et al., 2007), a region associated with
55 kHz positive affect vocalizations, to control for reward circuitry/ motivational influence in vocalization developmental differences.
4.2. METHODS
4.2.1. Animals
Following a one-week habituation period, virgin female (200-240 grams) Sprague-Dawley rats (Charles River, Raleigh, NC) were placed with males on a breeding rack until a sperm plug was found, which was designated as gestation day (GD) zero. Subjects were randomly assigned to one of three treatment or control groups and singly housed and maintained on a reversed 12:12 reverse light cycle (lights off at 0900 hours) for seven days. They were then transferred to a room with a regular light cycle (lights on at 0700 hours) for the remainder of the experiment, a procedure that generally results in the majority of dams delivering their litters during daylight hours (Mayer and Rosenblatt,
1998). All procedures were conducted under federal and institutional animal care and use committee guidelines for humane treatment of laboratory subjects.
4.2.2. Treatment
Treatment groups included: chronic cocaine (CC), and two control groups, chronic saline
(CS), and untreated (UN) dams. CC and CS dams received subcutaneous (SC) injections on alternating flanks of 15 mg/kg cocaine HCL (dose calculated as the free base, Sigma Chemical
Company, St. Louis, MO) dissolved in 0.9% normal saline (total volume 2 ml/kg), or the same volume of normal saline (0.9%), respectively. Injections were delivered twice daily (at approximately
0800 and 1600 hours) throughout gestation beginning on GD 1 and continuing until the day before
78 delivery (GD 1-20) with the CS dams serving as controls for injection and nutritional stress. UN dams were weighed and handled daily, but received no drug treatment. CC and UN dams had free access to water and food (rat chow), while CS-treated dams were yoke-fed over the first week to match maximum consumption rates of CC dams to control for the anorectic effects of cocaine, as previously described (Johns et al., 2005;Johns et al., 1994). To investigate neuronal postnatal development in offspring, pregnant dams from all three treatment groups received an injection of
Bromodeoxyuridine (BrdU) (10mg/kg) between 0800 and 0900 hours, before any other injections or handling, for three consecutive days (GDs 13-15). A small cohort of animals were tested in a pilot study to examine the effects of BrdU on neonatal vocalizations, maternal behavior, and weight gain in all three treatment groups and findings suggested no BrdU-related differences at the dose employed here (data not shown). While BrdU has been found to have adverse effects on development when administered gestationally (Kuwagata et al., 2001), the dose we employed is lower than that previously found to have no effect on cellular kinetics (Lewandowski et al., 2003) and therefore caused no problems in the present study.
4.2.3. USV Testing Procedures
Offspring were left undisturbed with their biological dams for three hours following delivery
(designated as PND 1) and then brought to the test room and allowed to habituate to the room for 15 minutes. One male and female offspring were removed from the litter, weighed, and placed together in a plastic holding cage on top of a heating pad for five minutes. At the end of five minutes, skin temperature on the rear flank of each offspring was recorded with a laser thermometer (Fischer
Scientific, Model 15-077-966). The male and female offspring from each litter were simultaneously placed onto two separate individual cold scales in two Med Associates sound attenuated boxes, each with a Med Associates Ultrasonic Vocalization Detector (model number ANL-937-1) attached to a unidirectional microphone and powered by SG-501 power supply. Med Associate USV detectors scan ultrasonic frequencies every 30 milliseconds and record the amplitude of sound at each
79 frequency between 20kHz and 100kHz. Detectors are connected to a laptop computer and data acquired and analyzed through ANL-937-1 MED USV Application Software (SOF-937-1) and the
“MED-USV.xls” macro for Microsoft excel. USV testing lasted for 5 minutes, and after testing, offspring skin temperature was again recorded. The temperature of the cold scale was measured before and after testing to control for confounding environmental temperature differences using a laser thermometer. Following testing, both offspring had their temperature assessed and were sacrificed and their brains collected for immunohistochemical analysis. The offspring’s biological dam and littermates were returned to the animal facility and left undisturbed until the next assigned test days. On PNDs 14 and 21, litters were brought to the testing room and the same PND 1 testing procedure was carried out on another male and female offspring pair.
4.2.4. USV Analysis
Following vocalization testing with ANL-937-1 MED USV Application Software (SOF-937-
1), data were loaded and analyzed with the “MED-USV.xls” macro for Microsoft excel. Data output included the amplitude of sound at each frequency between 20 and 100kHz for every instance that sound exceeded the manually set threshold level of 25dB. Data output was examined by generated 3-
D area graphs, where the x-axis was frequency (kHz), y-axis was amplitude (dB), and z-axis was time
(sec). Data was then manually categorized as a USV or other broad spectrum sound (i.e. from offspring movement in the box). To ensure proper categorization, a subset of rodent offspring were tested for USVs simultaneously with the Med Associates Detection software and with a model 4939 microphone connected to (model 2670) preamplifiers, running through a 2-channel model 4939 power amplifier (all Brüel and Kjær), and connected to a computer through a National Instruments instrumentation recorder (779193-01-DAQPad-6015) sampling at a rate of 200kS/s (200,000 samples per second). Individual USVs that occurred within 30 milliseconds of one another were considered as part of the same call. All of the USVs occurring during the five minute test or up to the first 60 USVs were categorized and subsequently analyzed. Acoustic features assessed for each animal included the
80 duration (average, and total) of USVs, the average interval of time between USVs, the latency to emit the first USV, latency to a USV with an observable harmonic, total number of USVs emitted during the test (up to 60), and the percentage of these USVs with at least one harmonic. Based on their peak fundamental frequency (F0, the highest point at which F0 occurred within each USV), USVs were categorized either occurring at 22 (20-29kHz), 40 (30-46kHz), 55 (47-59kHz), or greater than 60kHz.
Percentage of calls emitted at each frequency band was calculated for each pup. If an animal did not emit a USV, its data was only used in the analysis of total number of USVs emitted during the test.
Additionally, to assess if differences in amplitude of calling exists amongst groups the loudest USV emitted was identified and the peak F0 and the amplitude at the peak F0 were recorded for each offspring.
4.2.5. Tissue Fixation and Sectioning
All PND 1 tissue was paraffin-embedded following a previously established protocol (Dunty,
Jr. et al., 2001). Based on the tissue consistency of the rodent brain on PND 1, we found that paraffin embedding was the best choice for maintaining tissue integrity through the length of the study.
Immediately following vocalization testing, brains were removed and drop-fixed in 4% paraformaldehyde in 0.1M phosphate-buffered saline (PBS) for 48 hours followed by two overnight
(O/N) rinses in PBS. Specimens were dehydrated through a graded series of alcohols, cleared with two washes of toluene, and finally embedded in molten paraffin. The embedded specimens were serially sectioned at 10um and immediately mounted to slides and stored at room temperature until immunohistochemistry was performed. Conversely, immediately following USV testing on PND 14 and 21 offspring were deeply anesthetized with pentobarbital (60 mg/kg, 10 ml/kg, i.p.) and perfused transcardially with 0.1M PBS followed by 4% paraformaldehyde in PBS. The brains were removed from the skull and placed in the same fixative solution for at least 24 hours before being washed with
PBS and sliced on a Leica VT 1000S vibrating microtome into 50 um sections. The free-floating sections were stored in cryoprotectant at -20C until immunohistochemistry was performed.
81 4.2.6. Immunofluorescence
Fluorescent immunohistochemistry was adopted from previously described methods
(Stevenson et al., 2009;Nixon and Crews, 2004). The colocalization of BrdU with neuronal-specific nuclear protein (NeuN) or glial fibrillary acidic protein (GFAP) was used to characterize the expression of surviving neural precursor cells that were labeled during BrdU injections on the mornings of GDs 13-15. Immunofluorescent assays were conducted as follows: free-floating tissue was washed in PBS, and then treated with 0.6% hydrogen peroxide to block endogenous peroxidase activity. Next, tissue was incubated in 50% formamide/2X SSC for thirty minutes at room temperature, then in 2N HCl for one hour at 37ºC. After neutralizing in 0.1M boric acid, tissue was rinsed in PBS, and incubated in a blocking solution (3% goat serum, 0.2% Triton-X, 0.1M PBS) for one hour. Tissue was then incubated in a solution containing primary antibodies (1: 400 rat anti-
BrdU, Accurate, Westbury, NY, 1:1000 mouse anti-NeuN, Santa Cruz, CA, 1:2000 rabbit anti-GFAP,
Dako, Glostrup, Denmark). Antibody dilutions were chosen based on dilution curves and antibody specificity verified in all immunohistochemical assays. The following day, sections were rinsed in
PBS, and then incubated in the dark with fluorescent-coupled secondary antibodies appropriate for each primary antibody (Invitrogen, CA) for one hour. Tissue was then rinsed in PBS, mounted onto slides and cover-slipped with Fluoromount-GTM (Southern Biotech, Birmingham, AL), taking care to protect the sections from the light.
Sections selected for analysis from the PND 1 were deparaffinized with xylene, rehydrated through a graded alcohol series, and quenched using 5% H2O2 in methanol. Steam antigen retrieval was performed using a citrate buffer (Antigen Retrieval Citra, BioGenex, San Ramon, CA). DNA was denatured with 2N HCl for one hour at 37C, neutralized with 0.1 M Borate Buffer, and then incubated with blocking solution (3% goat serum, 0.2% Triton-X, 0.1M PBS) before incubation in primary antibodies. Primary antibodies consisted of mouse anti- NeuN (Santa Cruz, CA, 1:1000) and rat anti-
BrdU (Accurate Chemical and Scientific Corp., NY, dilution 1:400). The following day, slides were
82 rinsed in PBS, and then incubated in the dark with fluorescent-coupled secondary antibodies appropriate for each primary antibody (Invitrogen, CA). Slides were rinsed in PBS and cover-slipped using Fluoromount-GTM (Southern Biotech, Birmingham, AL). PND 1 tissue was not stained for glial fibrillary acidic protein (GFAP) after a pilot expression assay showed no GFAP presence in PND 1
PAG tissue. Protein expression at PND 1 was only assessed in the PAG since studies assert that vocalizations at this neonatal age are not emotionally based, but controlled by brain stem mechanisms
(Middlemis-Brown et al., 2005).
4.2.7. Visualization and Analysis of Immunofluorescence
Triple-label (PND 14, 21) or double-label (PND 1) immunofluorescence was visualized with a Leica SP2000 confocal scanning microscope with a 40 x oil objective. Anatomy hallmarks for each region were employed to ensure consistent imaging during microscopy. Co-localization was confirmed by confocal microscopy optimized for the analysis of tissue sections. For each subject, images were acquired from the left and right hemisphere from a minimum of four tissue sections for each brain region. Image analysis was adapted from previously described methods (Byun et al.,
2006). In brief, optimal thresholds for BrdU, NeuN, and BrdU/NeuN co-localization was identified for each age group and kept constant for all subjects. Total number of nuclei stained was assessed using the plug-in for nucleus detection in Image J for microscopy. Total number of GFAP and
BrdU/GFAP co-localization was counted manually, and required a full stained body and at least one process to be considered positive for staining.
4.2.8. Statistical Analysis
A one-way ANOVA for treatment group was used to examine the effects of PCE on gestational and postnatal litter data. Fisher LSD post hoc tests were conducted to find the differences between the three treatment groups. A 2 (sex) by 3 (treatment group) ANOVA was conducted for measures of offspring weight and temperature for each respective test day. Because groups differed
83 in the number of offspring emitting vocalizations on each test day Kruskal-Wallis tests (for continuous results) or Fisher’s exact test (for binary questions such as if offspring emitted a vocalization or not) were used to assess treatment group differences. Conover-Inman Test for all pairwise comparisons were conducted to evaluate which treatment groups were different from each other. Wilcoxon signed rank test (paired for continuous results) or McNemar’s test (for binary results) were also used for evaluating sex differences by day and treatment group.
A 2 (sex) by 3 (treatment group) ANOVA was conducted for each brain regions at each age group on measures of total NeuN-positive labeled cells, BrdU-positive labeled cells, GFAP-positive labeled cells, and the percentage of BrdU-positive cells that co-labeled as a NeuN or GFAP-positive neuron or astrocyte respectively. Following overall significance Fisher LSD post-hoc tests were conducted to find the differences between the three treatment groups. Pearson product-moment correlations were employed to examine the relationship between rodent vocalizations and development of different brain regions. These measures include the total number of NeuN-positive cells, total number of GFAP-positive astrocytes, and the percentage of BrdU-positive cells that co- labeled as a mature neuron or astrocyte at each respective age. Only those animals that produced a
USV were included in the correlations involving latency and number of USVs.
4.3. RESULTS
4.3.1. Gestational/ Litter Data
Results of the one-way ANOVA for treatment group showed a significant main effect in gestational weight gain of dams (F=4.158, p<0.05), specifically CC dams (n=11) gained less weight across the gestational period than did UN dams (Fisher, p<0.01). There were no differences between
CS dams (n=10) and UN dams (n=11) on this measure. CC dams also gained more weight between
PPDs 1-21 (ANOVA, F=4.305, p<0.05) than did UN (Fisher HSD, p<0.01) but not CS dams. By
PND 21, there were no significant differences in weight between treatment groups. There were no group differences in litter size or sex ratio, litter birth weight, culled litter weight, or the culled litter
84 weight gain from birth to PND 21. Group offspring weights on PND 1, 14, or 21 were not significantly different. Results of the one way ANOVA showed a significant treatment group effect
(F=3.087, p<0.05) on offspring temperature on PND 1 before vocalization testing, such that CC offspring were warmer than CS offspring (Fisher, p<0.05), and had a trend for being warmer than UN offspring (Fisher, p<0.09) with no differences following testing cessation. There were no group differences in temperature measures at any measurement time on either PND 14 or 21 and the plate temperatures were the same for all tests.
4.3.2. Characterization of Vocalizations
Since USVs were manually categorized as a vocalization or ‘other’ noise a subset of rodent offspring were tested for USVs simultaneously with the Med Associates Detection software and with a model 4939 microphone connected to (model 2670) preamplifiers, running through a 2-channel model 4939 power amplifier (all Brüel and Kjær), and connected to a computer through a National
Instruments instrumentation recorder (779193-01-DAQPad-6015) sampling at a rate of 200kS/s
(200,000 samples per second). Spectrograms of vocalizations recorded simultaneously from both the
Figure 16: Characterization of Vocalizations with MedAssociates Equipment. (A) Spectrogram recording from an untreated male pup, (B) graphed fast fourier transform (FFT) at the time point represented by the line in A, (C) 3‐D area graph created with MedAssociates Microsoft excel macros at the same time point depicted by the line in A and depicted in B showing proper categorization of a USV. # symbol represents the fundament frequency.
85 model 4939 microphones and the med associate detector equipment were created and FFTs of USVs were created every 30 msec and compared to 3-D area graphs from Med Associate data output (which detects sound every 30 msec) at every time point a USV was occurring to ensure proper categorization of USVs with med associate equipment (See Figure 16).
4.3.3. Neonatal Rats
4.3.3.1. PND 1 Vocalizations: CC exposure decreased vocalizations in both PND 1 male and female offspring in a sex and temperature dependent manner. Treatment effects were seen in PND 1 females in the latency to emit the first USV (Z=7.415; p<0.05). As indicated by Conover-Inman
Tests, CC females had a longer latency to emit the first USV than did CS (p<0.01) and UN females
(p<0.05). Based on high variability of the data, a linear mixed model was used to evaluate sex and treatment group effects adjusted for starting temperature and weight of offspring. We found that variability in the total number of USVs emitted could be explained in part by temperature of the offspring. A higher body temperature at time of testing was directly related to a higher total number of USVs (p<0.05), while offspring weight did not show this association (p=0.8232). Using this adjustment, there was a significant main effect of group for total number of USVs emitted (p<0.05), with both male and female CC offspring vocalizing less overall compared to CS (p<0.01) and UN offspring (p=0.06 (non-significant), Figure 18A) with no significant difference in the total duration of vocalizing (Figure 18B). CC offspring that were measured as warmer than controls at the start of testing vocalized as much as controls, while CC offspring with the same temperature as controls vocalized less. Interestingly, while change in body temperature during testing was not different between groups, there was a positive correlation between temperature at the start of testing and body temperature change during testing (χ2=59.951, p<0.0005); specifically, those offspring that were warmer at the start of testing lost more heat during testing, supporting previous studies showing neonatal offspring’s inability to thermoregulate (Blake, 2002;Blumberg and Sokoloff, 1998). Starting temperature and weight were not associated with any other acoustic measures.
86 There was a treatment effect for PND 1 males on the percentage of calls that had at least one observable harmonic (Z=7.633; p<0.05, Figure 18C) and on the latency to emit a USV with at least one observable harmonic (Z=5.852; p<0.05). PND 1 CC males produced a smaller percentage of calls with a harmonic compared to UN (Conover Inman, p<0.005) and CS (Conover Inman, p=0.059) males; and a longer latency to emit a USV containing a harmonic than both UN and CS male offspring (Conover Inman, p<0.05).
Amygdala VMH PAG
Figure 17. Schematic of regions imaged on PNDs 14 and 21. Left column shows schematic of CeA (pink square) and BLA (blue square) sections imaged. Middle column shows schematic of VMH (green squares) sections imaged. Right column shows schematic of dPAG (orange) and vPAG (brown) sections imaged. Atlas images obtained from Paxinos G. and Watson C. The rat brain in stereotaxic coordinates: compact third edition (1997) Academic Press.
4.3.3.2. PND 1 IHC Cell Counts: Sections selected for analysis from the PND 1 age group contained the PAG region of interest (coronal plate 19-20 in embryonic day 22 rodent offspring as cited in Atlas of Prenatal Rat Brain Development (Altman and Bayer, 1995)). There were no group effects associated with dPAG developmental changes on PND 1 (See Figures 18D-K). There was however a main effect in the percentage of BrdU cells that co-labeled as a NeuN-positive neuron in the vPAG on PND 1 (ANOVA, F=3.808, p<0.05, See Figures 18G-I,J,K), showing that CS offspring had a significantly greater percentage of BrdU cells that co-labeled as a NeuN-positive neuron in the
87 vPAG compared to UN (Fisher LSD, p<0.01) but not CC offspring. There were no other developmental differences observed in the PAG on PND 1.
ABPND 1 Total Number PNDTOTAL 1 Total DURATION Duration P1 C PND 1 Percent Harmonic J 400 PND 1 Total NeuN 0.55 *** 20 1.4 * ** 1.3 0.50 UN 1.2 0.45 15 1.1 0.40 300 CS * ** 1.0 0.9 0.35 CC 0.8 0.30 Observed 10 * 0.7 0.25 0.6 200
0.20 Observed
0.5
5 Seconds 0.4 0.15
0.3 Percentage 0.10 0.2 100 0.1 0.05 0 0.0 0.00 Number MALES FEMALES MALES FEMALES MALES FEMALES
Number 0 PND 1 Untreated PND 1 Chronic Saline PND 1 Chronic Cocaine MALES FEMALES MALES FEMALES dPAG vPAG DE F K PND 1 % BrdU co‐labeling 0.20 with NeuN **
0.15 ** dPAG 0.10
Percentage 0.05
0.00 MALES FEMALES MALES FEMALES GH I dPAG vPAG L PND 1 vPAG Development: 2.0 Duration of USVs (sec)
1.5 vPAG 1.0 Duration
0.5
Total 0.0 0.0 0.1 0.2 0.3 % BrdU co‐labeling with NeuN in vPAG Figure 18: PND 1 vocalizing behavior and PAG development in neonatal rat male and female offspring following CC, CS, or no prenatal exposure. (A) Total number of USVs emitted. CC exposed offspring emitted fewer USVs compared to controls. (B) Total duration of USVs. No significant differences were observed in total duration of USVs. (C) Percentage of USVs emitted with an observable harmonic. CC males showed a decrease in the percentage of calls emitted with an observable harmonic. (D‐F) Representative Images of the dPAG at 40X magnification from UN, CS, and CC‐exposed male offspring respectively. (G‐I) Representative images of the vPAG at 40X magnification from UN, CS, and CC‐exposed male offspring respectively. (J) Quantitative depiction of neuronal density in the dPAG and vPAG. No significant differences were observed in neuronal density. (K) Quantitative depiction of percentage of BrdU‐positive cells co‐labeling as a mature neuron. CS offspring had a greater percentage of BrdU‐positive cells co‐labeling as a mature neuron in the vPAG on PND 1. (L) Correlation between total duration of USVs on PND 1 and the percentage of BrdU‐positive cells that co‐label as a mature neuron in the vPAG on PND 1 showing no significant relationship. (***p<0.001, **p<0.01, * p<0.05; N= 10‐11 animals per treatment group and sex analyzed for vocalizations; 8‐10 animals per treatment group and sex analyzed for the NAc core, CeA, VMH, and vPAG).
88 4.3.3.3. PND 1 Vocalizations and Regional Development Correlations: There were no significant correlations between the dPAG or the vPAG in total number of NeuN-positive cells or percentage of BrdU-positive cells that co-label as a NeuN-positive neuron with variation in rodent offspring vocalizations on PND 1 (See Figure 18L).
4.3.4. Infant rats
4.3.4.1. PND 14 Vocalizations: There were no significant treatment or sex group differences in vocalizations of offspring on PND 14 (See Figure 19 A-C).
4.3.4.2. PND 14 IHC Cell Counts: For PND 14 tissue, free-floating sections were picked from each animal for the CeA and BLA (bregma -2.12 to -2.80mm), VMH (bregma -2.30 to -3.60), and the dPAG and vPAG (bregma -6.72 to -7.80) (See Figure 17 for Schematic Representation). To control for our hypothesis that differences in vocalizations observed will be fear circuitry and not reward/motivation circuitry based sections were also picked for the NAc core and shell (bregma 1.70 to 1.20mm). CC and CS exposure was differentially associated with sex-dependent developmental differences in the NAc core, CeA and vPAG but not in the NAc shell, BLA, VMH, or dPAG.
4.3.4.2.1. NeuN-positive Cells: As seen in Figure 19D-F,M there was a group by sex interaction (ANOVA, F=5.387, p<0.01) in the total number of NeuN-positive neurons in the CeA on
PND 14. CC females had less NeuN-positive cells compared to UN females (Fisher LSD, p<0.01) and CC males (Fisher LSD, p<0.05). CS males also had significantly fewer NeuN-positive cells compared to CC males (Fisher LSD, p<0.05, with a strong trend (p=0.062) for fewer than UN males), and both CS (Fisher LSD, p<0.05), and UN females (Fisher LSD, p<0.005). There was a trend for an interaction effect for total NeuN+ neurons (ANOVA, F=2.938, p=0.067) in the NAc core.
Specifically, CC females have less NeuN-positive neurons compared to UN females and CC males
(Fisher LSD, p<0.05).
4.3.4.2.2. BrdU-positive Cells: There was a treatment group by sex interaction (ANOVA,
F=3.964, p<0.05) in the total number of BrdU-positive cells in the CeA. CC males had more BrdU-
89 A BC PND 14 Total Number PND 14 Total Duration PND 14 Percent P PND 14 TotalNeuN NeuN UN NUMB USVS P14 TOT DUR P14 PERC HARM P14 CS Harmonic UN 175 CC 60 6.5 0.75 CS 6.0 50 5.5 CC ***** 5.0 150 40 4.5 0.50 ** 4.0 3.5 * ** Observed 30 125 3.0 2.5 20 2.0 0.25
Seconds 1.5 100 10 1.0 Percentage Observed
0.5 0 0.0 0.00 MALES FEMALES MALES FEMALES MALES FEMALES 75 Number PND 14 Untreated PND 14 Chronic Saline PND 14 Chronic Cocaine 50
Number 25 DE F 0 MALES FEMALES MALES FEMALES MALES FEMALES MALES FEMALES NAC CORE CEA VMH vPAG Q UN PND 14 % BrdU co‐ CS Nac Core CC ** labeling% with NeuN NeuN 0.4 ** * GH I * 0.3 **
Percentage 0.2 CeA 0.1
0.0 MALES FEMALES MALES FEMALES MALES FEMALES MALES FEMALES CEA VMH vPAG JKL NAC CORE
R PND 14 VMH Development: S PND 14 VMH Development: VMH UN Duration of USVs UN Amplitude of USVs 11 CS 80 CS 10 CC Call CC 9 70 8 60 (sec) 7 50 6
Loudest 40
MN O 5 4 30 of
3 20 Duration 2 10 1 0
Total 0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
vPAG 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Amplitude % BrdU co‐labeling % BrdU co‐labeling with NeuN in VMH with NeuN in VMH
Figure 19: PND 14 vocalizing behavior and NAc core, CeA, VMH, and vPAG development in infant rat male and female offspring following CC, CS, or no prenatal exposure. (A) Total number of USVs emitted. (B) Total duration of USVs. (C) Percentage of USVs emitted with an observable harmonic. No significant differences were observed amongst treatment groups or sex in USV measures on PND 14. (D‐F) Representative Images of the NAc core at 40X magnification from UN, CS, and CC‐exposed female offspring respectively. (G‐I) Representative images of the CeA at 40X magnification from UN, CS, and CC‐exposed female offspring respectively. (J‐L) Representative images of the VMH at 40X magnification from UN, CS, and CC‐exposed female offspring respectively. (M‐O) Representative images of the vPAG at 40X magnification from UN, CS, and CC‐exposed female offspring respectively. (P) Quantitative depiction of neuronal density in the NAc core, CeA, VMH, and vPAG. CC female offspring showed a decrease in neuronal density in the CeA and NAc core compared to UN female and CC male offspring but did not significantly differ from controls in the VMH or dPAG. (Q) Quantitative depiction of percentage of BrdU‐positive cells co‐labeling as a mature neuron. CC male offspring showed a greater percentage of BrdU‐positive cells co‐labeled with NeuN in the Nac core than UN males and CC females. Conversely CC females showed less co‐labeling than UN females. CC male offspring also had a greater percentage of BrdU‐positive cells co‐labeling with NeuN in the CeA on PND 14 compared to CS male and CC female offspring but did not differ from controls in the VMH or vPAG. (R) Correlation between total duration of USVs on PND 14 and the percentage of BrdU‐positive cells that co‐label as a mature neuron in the VMH on PND 14 showing no significant relationship. (S) Correlation between amplitude of the loudest call on PND 14 and the percentage of BrdU‐positive cells that co‐label as a mature neuron in the VMH on PND 14 showing a significant inverse relationship. (***p<0.001, **p<0.01, * p<0.05; N= 10‐11 animals per treatment group and sex analyzed for vocalizations; 8‐10 animals per treatment group and sex analyzed for the NAc core, CeA, VMH, and vPAG). positive cells in the CeA than UN males (Fisher, p<0.01), and both CC and UN females (Fisher, p<0.05). CS females also had more BrdU-positive cells in the CeA than UN (Fisher, p<0.01) and CC
90 females (Fisher, p<0.05), and both CS (Fisher, p=0.058 non-significant), and UN males (Fisher, p<0.01).
4.3.4.2.3. BrdU positive cells co-labeled as NeuN-positive Neurons: There was a group by sex interaction (ANOVA, F=3.945, p<0.05, see Figure 19N) for the percentage of BrdU-positive cells that co-labeled as a NeuN-positive neuron in the CeA. CC males had a larger percentage of BrdU cells that co-labeled as a NeuN-positive neuron compared to CC females (Fisher, p<0.05) and CS males (Fisher, p<0.05). There were no other significant differences in the CeA. Additionally there was a group by sex interaction (ANOVA, F=6.103, p<0.01) in the percentage of BrdU positive cells that co-label as a NeuN positive neuron in the NAc core. Specifically UN females showed more
BrdU colabeling with NeuN compared to UN males (p=0.055), suggesting a sex effect. CC females had a smaller percentage of cells that colabel as a NeuN+ neuron compared to CC males (p<0.01) and
UN females (p<0.05) while CC males had more compared to UN males (p<0.01) suggesting cocaine- exposure reverses the pattern of naturally occurring sex differences in the NAc core.
4.3.4.2.4. BrdU-positive Cells Co-labeled as GFAP-positive Astrocytes: There was also a main effect of treatment group for the percentage of BrdU-positive cells that co-labeled as a GFAP- positive astrocyte in the vPAG (ANOVA, F=4.739, p<0.01). CC (Fisher, p<0.05) and CS (Fisher, p<0.01) offspring (males and females) had a smaller percentage of BrdU-positive cells that co-labeled as a GFAP astrocyte compared to UN offspring (data not shown). No other developmental effects were observed in the vPAG or in any other region on PND 14.
4.3.4.3. PND 14 Vocalizations and Regional Development Correlations: By PND 14, some acoustic measures of vocalizations began to correlate with VMH developmental differences (See
Figure 19-O,P). A significant inverse relationship was observed for the percentage of BrdU cells that co-label as a mature neuron in the VMH and the amplitude of the loudest call produced (χ2=8.188, p<0.005). Specifically, a greater percentage of BrdU cells that co-labeled as a neuron in the VMH correlated with a lower amplitude of the loudest call. There were no other relationships between PND
14 rodent offspring vocalizations, and total number of NeuN positive cells, GFAP positive cells, or
91 percent of BrdU positive cells that co-label as a NeuN positive neuron or a GFAP positive astrocyte in any other brain regions.
4.3.5. Juvenile rats
4.3.5.1. PND 21 Vocalizations: CC and CS exposure were associated with vocalization differences on PND 21 in a sex-dependent manner.
4.3.5.1.1. Males: There were no significant treatment effects in the total number of USVs on
PND 21 (Figure 20A), but there was a main effect (Kruskal-Wallis) of treatment group for males in the total duration of USVs (Z=6.157; p<0.05, Figure 20B), the minimum interval between USVs observed (Z=7.528; p<0.05), and the amplitude of the loudest call measured (Z=9.268; p<0.01). CC and CS males vocalized longer (total duration-Conover Inman, p<0.05); cried at higher amplitudes at the loudest call emitted (Conover Inman, CC p<0.05, CS p<0.005) and had a smaller minimum interval between USVs (Conover Inman, CC p<0.05, CS p<0.01) than did UN males on PND 21.
There were no differences in the percentage of USVs that had a harmonic (See Figure 20C) or other vocalization measures between male offspring.
4.3.5.1.2. Females: There was a main effect of treatment group in the percentage of USVs emitted that fell into the 22 kHz range (Z=9.720; p<0.01, See Figure 21B) in female offspring. CS
(Conover Inman, p<0.005) and CC females (Conover Inman, p<0.05) emitted fewer 22kHz USVs compared to UN females on PND 21.
4.3.5.1.3. Within Group Sex Effects: CC and CS exposure was associated with USV sex differences on PND 21 that were not observed in UN offspring. Wilcoxon signed rank tests indicated that CC males (n=10) emitted a greater total number of USVs (p<0.05, Figure 20A), a longer total duration of USVs (p<0.05, Figure 20B), and had a greater percentage of USVs that fell into the 55
kHz range compared to CC females (n=10, p<0.05, Figure 21A). CS males (n=10) had a shorter average interval between calls (p<0.05) and a greater amplitude of the loudest call emitted compared to CS females (n=10, p<0.05). The only within group sex effect observed for UN offspring
92 was that UN males (n=11) had a shorter latency to emit the first USV compared to UN females
(n=11, p<0.05), with a strong trend observed for UN males to emit more calls that fell into the 55kHz range than UN females (p=0.06),
4.3.5.2. PND 21 IHC Cell Counts: For PND 21 tissue, free-floating sections were picked from each animal for the CeA and BLA (bregma -2.12 to -2.80mm), VMH (bregma -2.30 to -3.60), and the dPAG and vPAG (bregma -6.72 to -7.80) (See Figure 2). To control for our hypothesis that differences in vocalizations observed will be fear circuitry and not reward/motivation circuitry based sections were also picked for the NAc core and shell (bregma 1.70 to 1.20mm). Developmental differences previously observed in the CeA and NAc core on PND 14 normalized in CC and CS offspring by PND 21 (See Figures 20D-F), however, sex-dependent differences began to emerge in the VMH (See Figures 5G-I) and vPAG (See Figures 20J-L) in CC and CS offspring.
4.3.5.2.1. NeuN-positive Cells: As seen in Figures 20 M,N, there was a significant treatment group by sex interaction in total number of NeuN-positive neurons in the VMH on PND 21
(ANOVA, F=3.120, p<0.05) but not the percentage of BrdU cells that co-labeled with NeuN. CC males had a greater density of mature neurons in the VMH on PND 21 than CC females, CS females, and UN males (Fisher, p<0.05). CS males also had more NeuN-positive neurons in the VMH than
CS females (Fisher, p<0.05).
4.3.5.2.2. BrdU-positive Cells: There was a treatment group by sex interaction effect in the total number of BrdU-positive cells in the VMH on PND 21 (ANOVA, F=3.289, p<0.05). Fisher
LSD post hoc comparisons indicated CS males had more BrdU cells compared to CS (p<0.005), CC
(p<0.005) and UN females (p<0.05) as well as UN males (Fisher, p<0.01). There was also a significant treatment group by sex interaction in the number of BrdU cells in the vPAG (ANOVA,
F=3.539, p<0.05). UN males had more BrdU-positive cells than UN females (Fisher, p<0.05) in the vPAG. There were no other significant differences on developmental measures on PND 21 in any other brain regions assessed.
93 UN A C UN PND 21 Total Number B PND 21 Total Duration PND 21 Percent P CS CS PND 21 Total NeuN CC 2.5 Harmonic NeuN 40 0.225 CC 0.200 2.0 120 0.175 30 0.150 110 1.5 0.125 100
Observed 20 0.100 * 1.0 * * 90 0.075 Seconds 10 80
Percentage 0.050 0.5 Observed 0.025 70 0 0.0 0.000 60 * MALES FEMALES MALES FEMALES Number MALES FEMALES 50 * 40
PND 21 Untreated PND 21 Chronic Saline PND 21 Chronic Cocaine Number 30 20 DE F 10 0 MALES FEMALES MALES FEMALES MALES FEMALES MALES FEMALES NAC CORE CEA VMH vPAG
PND 21 % BrdU co‐ Nac Core Q UN labeling% NeuN with NeuN CS CC 0.65 0.60 0.55 GH I 0.50 0.45 0.40 0.35 0.30 0.25
CeA 0.20 Percentage 0.15 0.10 0.05 0.00 MALES FEMALES MALES FEMALES MALES FEMALES MALES FEMALES JK L NAC CORE CEA VMH vPAG
R S VMH PND 21 VMH Development: PND 21 VMH Development: Duration of USVs UN Amplitude of USVs UN
6 Call CS 65 CS 60 5 CC 55 CC
(sec) 50
4 45 40 Loudest 35 3 MN O 30 of 2 25
Duration 20 15 1 10 5
Total 0 0 vPAG 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80
NeuN Amplitude Total NeuN in VMH Total NeuNNeuN in VMH
Figure 20: PND 21 vocalizing behavior and NAc core, CeA, VMH, and vPAG development in infant rat male and female offspring following CC, CS, or no prenatal exposure. (A) Total number of USVs emitted. CC males emitted more USVs than CC females. (B) Total duration of USVs. CC and CS males showed a longer total duration of USVs compared to UN males. CC males also had a greater total duration than CC females. (C) Percentage of USVs emitted with an observable harmonic. No significant differences were observed in the percentage of calls with an observable harmonic. (D‐F) Representative Images of the NAc core at 40X magnification from UN, CS, and CC‐exposed male offspring respectively. (G‐I) Representative images of the CeA at 40X magnification from UN, CS, and CC‐exposed male offspring respectively. (J‐L) Representative images of the VMH at 40X magnification from UN, CS, and CC‐ exposed male offspring respectively. (M‐O) Representative images of the vPAG at 40X magnification from UN, CS, and CC‐exposed male offspring respectively. (P) Quantitative depiction of neuronal density in the NAc core, CeA, VMH, and vPAG. CC male offspring showed an increase in neuronal density in the VMH compared to UN male and CC and CS female offspring. CS males showed an increase in neuronal density in the VMH compared to CS females. No significant differences were observed in the NAc core, CeA, or vPAG. (Q) Quantitative depiction of percentage of BrdU‐positive cells co‐labeling as a mature neuron. No significant differences were observed in the NAc core, CeA, VMH, or vPAG. (R) Correlation between total duration of USVs on PND 21 and the percentage of BrdU‐positive cells that co‐label as a mature neuron in the VMH on PND 21 showing a significant positive relationship. (S) Correlation between amplitude of the loudest call on PND 21 and the percentage of BrdU‐positive cells that co‐label as a mature neuron in the VMH on PND 21 showing a significant positive relationship. (***p<0.001, **p<0.01, * p<0.05; N= 10‐ 11 animals per treatment group and sex analyzed for vocalizations; 8‐10 animals per treatment group and sex analyzed for the NAc core, CeA, VMH, and vPAG).
4.3.5.3. PND 21 Vocalizations and Regional Development Correlations: Correlations between vocalization acoustic measures and specific brain region developmental differences, specifically the VMH and CeA, began to emerge by PND 21. The total number of NeuN-positive
94 neurons in the VMH correlated positively with the total duration of USVs (χ2=11.322, p<0.001,
Figure 20O) and the amplitude of the loudest USV (χ2=13.933, p<0.0005, figure 20P). Interestingly, the total number of NeuN-positive neurons in the VMH (χ2=4.260, p<0.05, Figure 21C) and in the
CeA (χ2=3.866, p<0.05, Figure 21D) were inversely related to the percentage of calls that fell into the
22kHz USV range.
The percentage of BrdU cells that were NeuN-positive neurons in the VMH were positively related to the amplitude of the loudest call (χ2=5.189, p<0.05) and the percentage of BrdU cells that co-labeled as NeuN-positive neurons in the VMH was also inversely related to the percentage of calls that were categorized as a 22kHz USV (χ2=3.762, p<0.05). To ensure correlations were not a false positive based on a large number of offspring not emitting any vocalizations in the 22kHz range and potentially skewing the data correlations were re-ran for only those offspring that emitted at least one
22kHz USV. Correlations between the number of 22kHz USVs offspring emitted and either the
VMH or CeA developmental measures became more significant (See insert Figure 21 C,D).
95 110 A MALES C 100 90 80 % 22 110 70 60 22 110 100 50 % 40 40 100 30 20 90 10 0 90 % 55 Percent kHz 80 0 10 20 30 40 50 60 70 80
80 kHz PND 21 Total NeuN in VMH % >60 70 70
22 60 60 UN 50 T # 50 40 40 CS 30 30 CC 20 20 Percent 10 10 0 0 UN CS CC UN CS CC UN CS CC 0 10 20 30 40 50 60 70 80 PND 1 PND 14 PND 21 PND 21 Total NeuN in VMH B D FEMALES 110 % 22 100 90 110 80
110 % 40 70
22 60 100 50 100 40 % 55 30 90 20 90 10 0 % >60 Percent 0 kHz 25 50 75 100 125 150 175
80 kHz 80 70 70 PND 21 Total NeuN in CeA 22 60 60 50 50 UN 40 40 30 CS 20 30 CC Percent 10 * 20 0 10 UN CS CC UN CS CC UN CS CC 0 PND 1 PND 14 PND 21 0 25 50 75 100 125 150 175 200 PND 21 Total NeuN in CeA Figure 21: PND 1‐21 Developmental Changes in frequency categories of USVs and relationship between 22kHz USVs and VMH and CeA development. (A) Percentage of vocalizations emitted that fell into the 22, 40, 55, or greater than 60kHz range for male offspring at each age of testing. No significant differences were observed between treatment groups at any age. (B) Percentage of vocalizations emitted that fell into the 22, 40, 55, or greater than 60kHz range for female offspring at each age of testing. CC and CS female offspring emitted less 22kHz USVs compared to UN females. CC and UN females also emitted less 55kHz USVs compared to CC (#=p<0.05 sex effect) and UN males (T=trend sex effect). (C) Correlation between percentage of USVs that were in the 22kHz range on PND 21 and the total number of NeuN‐positive mature neurons in the VMH on PND 21 showing a significant inverse relationship. Graph insert is same correlation but with those offspring that emitted zero 22kHz USVs emitted showing an increase in significance of correlation. (D) Correlation between percentage of USVs that were in the 22kHz range on PND 21 and the total number of NeuN‐positive mature neurons in the CeA on PND 21 showing a significant inverse relationship. Graph insert is same correlation but with those offspring that emitted zero 22kHz USVs emitted showing an increase in significance of correlation. (*p<0.05; N= 10‐11 animal per treatment group and sex analyzed for vocalizations on PND 1, 14, and 21; 8‐10 animals per treatment group and sex analyzed CeA and VMH correlations with percentage of 22kHz USVs).
96 4.4. DISCUSSION
We predicted that USVs at different ages would be differentially disrupted following PCE and that differences would correlate with variations in development of specific brain regions. As hypothesized, cocaine-induced changes in neonatal vocalizations were sex and age-dependent. On
PND 1, CC decreased the number of vocalizations in both sexes and CC males had a smaller percentage of calls with at least one observable harmonic than did UN males at this age. Following a painful experience, human infants emit cries with fewer harmonics which are then perceived to be more urgent (Porter et al., 1986). Maternal substance abuse is correlated with neglect in humans
(Leventhal et al., 1997) and rodents (Nelson et al., 1998;Johns et al., 1994) perhaps partially mediated through variation in infant cues (Johns et al., 2005). Vocalization characteristics, including harmonics, could potentially be an important cue impacting human and rodent early care. For instance, if human male infants prenatally exposed to cocaine emit expirations that have acoustic differences in harmonics this could influence maternal perception (i.e. feeling of urgency) and hence parental response toward the infant. No study to date has explored the impact of harmonics on early dam-pup interactions in a rodent model. Since gestational cocaine-exposure has been found to alter maternal behavior (with the greatest deficits occurring around PPD one) future studies should target early translational behavioral differences in CC pups and their impact on the maternal environment to continue elucidating the mechanisms underlying neglect.
The PAG is a region critical for triggering vocalizations (Jurgens, 2009;Larson and Kistler,
1984) in response to limbic emotional (Adamec et al., 2003;Zhao et al., 2009) and somatosensory input (Mayer, 1984;Sandkuhler et al., 1991). We hypothesized that variation in PAG development would therefore impact vocalization production, even during the neonatal period when vocalizations are speculated to be an acoustic by-product of thermoregulation (Blumberg and Alberts, 1990).
Vocalization changes in CC offspring did not however correlate with altered PAG development on
PND 1. Future studies targeting later proliferation times (i.e. GDs 16-17) might help to clarify
97 findings as proliferation in the PAG occurs between GDs 13-17 in a ventral to dorsal gradient
(Altman and Bayer, 1981). Since neonatal vocalizations are thought to be produced via a ‘brainstem model’ in that regions rostral to the midbrain are not required for vocalization production during early infant development (Newman, 2007), perhaps other regional changes in the brainstem such as the nucleus ambiguous (Wetzel et al., 1980), should be explored in CC offspring.
PND 14 was found to be a peak vocalizing period for all offspring, as evidence in more vocalizations that were longer and had shorter intervals between individual USVs than at other ages.
PND 14 in rodents is thought to be roughly equivalent in age to one month old human infants who have a peak crying period around six to eight weeks (Barr et al., 1996;Prescott, 1975) followed by a decrease in vocalizations (Barr et al., 1996;Prescott, 1975). Rodent infants show a similar developmental shift to human infants, first producing high rates of isolation-induced USVs (Hofer et al., 1998;Hofer and Shair, 1980;Shair et al., 2003) followed by signs of fear-like behavior (i.e. freezing and decreasing USV production) when in an aversive environment (i.e. predator odor or isolation) (Kabitzke and Wiedenmayer, 2011). Our age comparisons support this developmental progression in rodent infants with both CC and CS-exposure in males prolonging higher rates of vocalizing. While human PCE has been suggested to decrease neonatal crying (Corwin et al., 1992) similar to what we observed on PND 1, it has also been found to increase excessive crying and irritability (Keller, Jr. and Snyder-Keller, 2000;Eyler et al., 1998). In general, “excessing crying” has been referred to as a common symptom following substance exposure (Galanter and Kleber, 2008).
Since prenatal stress has also been shown to result in excessive crying (van der Wal et al., 2007) with a peak period of fussiness occurring between three and six months of age (Wurmser et al., 2006), similar findings in both CC and CS males could suggest effects at this age may reflect a common or similar early developmental stress effect. As not all differences were identical for CC and CS-exposed males, PCE specific changes are likely resulting from more direct effects of drug exposure. The sex specific effects suggest that males may be more sensitive to these manipulations which support previous reports. Sex-dependent developmental differences in rats have been reported for a number of
98 measures following PCE (Johns et al., 2002;Lewis et al., 2009;Hamilton et al., 2011;Dow-Edwards,
2010) and prenatal stress (Bale, 2011) with males suggested to be more vulnerable to prenatal complications, including stress (Gerardin et al., 2011). These findings are particularly interesting in light of the many neurodevelopmental disorders (many with increased prevalence in males) that are associated with altered early communication in humans. For instance, parents of children with autism spectrum disorder often report that they cannot understand why their child is crying in the first year.
These parents describe their child’s cries as unexpected and loud (Esposito and Venuti, 2010).
Altered crying behavior in males could also have a detrimental impact on the maternal environment, especially in populations more vulnerable to neglect-like behavior such as those abusing cocaine, further strengthening the need for more research exploring the implications of sex-dependent changes in vocalizations.
We hypothesized that developmental delays in fear-related circuitry would correlate with altered vocalizations on PND 21 in CC offspring, including increased rates of vocalizations and delayed onset of 22 kHz USVs. As the limbic system matures in the developing human infant the first displays of fear-like behavior begin to emerge (Herschkowitz et al., 1997). Previous animal studies indicate that fear-like behavior is associated with plasticity of the VMH (Pagani and Rosen,
2009) and amygdala (Collins, 2011;Takahashi et al., 2007). In the present study CC and CS female
(significant) and male (non-significant) offspring had a reduction in the percentage of USVs that fell into the 22 kHz range on PND 21 suggesting that CC and CS exposure may alter emergence of USV expression associated with fear-like behavior in both sexes though this effect was stronger in females in the present study.
On PND 21 CC and CS males had a greater density of VMH neurons compared to controls.
Increased neuronal density (and larger percentage of BrdU cells co-labeled as a neuron) was found to positively correlate with the total duration of vocalizations suggesting dysregulated VMH development (previously suggested as playing a role in infant crying (Joseph, 1996)), may possibly have contributed to the altered crying in CC and CS males. Greater neuron density in the VMH and
99 CeA was negatively correlated with the percentage of 22 kHz USVs emitted, suggesting a role for
CeA and VMH developmental differences with delayed onset of fear-like behavior which should be further explored. It is important to note that data appears to be bi-modal for many acoustic measures.
Fear circuitry has recently been shown to be modulated by the medial prefrontal cortex (Chan et al.,
2011) and hence other regions could be playing a role in vocalization production and control as offspring appear to fall into two categories: those that vocalize and those that don’t suggesting other mechanisms of inhibition are occurring.
We were surprised that CC-exposure in males was associated with greater neuron density in the VMH on PND 21 as we hypothesized that they would have delayed neuronal maturation. PCE has previously been shown to decrease proliferation but not effect cell survival (Lee et al., 2008), increase apoptosis in the fetal brain (Xiao and Zhang, 2008), delay postnatal astroglial maturation
(Clarke et al., 1996), disrupt migration, and/or enhance differentiation in offspring (Lee et al., 2011).
Few studies have examined later periods in development for maturational changes in the brain. Based on these results, increased differentiation could actually be a mechanism to explore, however, it is important to note that cell death continues to occur in the brain through adolescence and is also sexually dimorphic (Nunez et al., 2001;Nunez et al., 2002) thus future studies are needed to clarify these findings.
CC and CS males vocalized at higher amplitudes on PND 21 compared to UN males and regional differences in the VMH were related to amplitude of the loudest call emitted during testing.
Stimulation of the VMH has been previously associated with increased USV amplitude parallel to pain-induced increases in USV amplitude (Borszcz, 2006). It was interesting that the correlation of
VMH with amplitude was age-dependent. PND 14 offspring showed a negative inverse relationship between amplitude and the percentage of BrdU cells that co-labeled as a neuron (Figure 19P) while
PND 21 offspring showed a positive relationship between amplitude and the density of VMH neurons
(Figure 20P). Defensive behavior and what is perceived as a threatening stimulus changes in an age- dependent manner during early development (Wiedenmayer, 2009). Differential age-specific
100 relationship between VMH development and amplitude of calling could represent a developmental change in VMH function or changing sensitivity to the isolation/ cold scale stimulus used for USV elicitation in this study.
Recent findings suggest changes in the amplitude of vocalizations are associated with early differences in the rearing environment (i.e. maternal care), and might be functionally relevant as a behavioral marker of early environmental differences (Wohr et al., 2008a). With this in mind, it is important to note that a limitation of this study is that we did not differentiate between the consequences of prenatal cocaine-induced changes in offspring vocalizations with that of differences in the postnatal environment. Previous studies have found that early environmental experience alters vocalizations in rodent pups. Specifically, pups experiencing five days of brief maternal separation/deprivation (an animal model of maternal neglect) from PND two through six vocalized less and show subtle changes in the sonographic structure of USVs than control pups when re-isolated from their dam and litter on PNDs seven and twelve (Zimmerberg et al., 2003b;Zimmerberg et al.,
2003a). Maternal deprivation has been shown to decrease neurogenesis in the hippocampus (Fabricius et al., 2008) and to effect neural proliferation differentially in male and female offspring. These gender differences are speculated to coincide with behavioral differences in adulthood and imply that early life stress establishes sex differences in neural plasticity, contributing to alterations in the HPA axis (Oomen et al., 2009). Prenatal stress also differentially affects neural proliferation in male and female offspring (Weinstock, 2007;Vaido et al., 2000;Mandyam et al., 2008). These studies and our findings here might suggest differences in vocalizations and differences observed in the VMH on
PND 21 might be primarily related to environmental-induced differences. It is interesting that only males and not females showed an increase in both amplitude and total duration of USVs on PND 21 which coincided with increased neuronal density in the VMH. Future studies employing cross fostering paradigms should focus on determining the role of the postpartum environment as an independent mediator of these effects.
101 As hypothesized, we did not observe any correlations between offspring NAc development and variations in vocalizations. Based on the testing paradigm employed in the present study
(eliciting vocalizations via cold isolation) we hypothesized that developmental differences in fear- associated regions would correlate with differences in USVs and not reward-associated regions. The differences observed in the NAc core of CC offspring are interesting and could impact infant social behavior, including vocalizations, in positive/ rewarding testing paradigms (i.e. play behavior or social interactions with dam). Just like rodent dams find pups highly salient the presence of the dam is thought to also be very rewarding to the pup. As early as PND 11, pups undergo “contact quieting”, in which they attenuate USV production, following reunion with dam. Contact quieting was found to be dopamine-dependent, involve the NAc, and dam-specific as reunion with littermates is was not salient enough to attenuate USVs (Shair et al., 2009). Additionally, re-isolation following dam reunion leads to maternal potentiation, a greater number of calls observed than what was previous observed in isolation before dam reunion, however re-isolation following litter mate reunion does not (Hofer et al., 1998;Hofer et al., 1994;Muller et al., 2009). These studies and studies showing anxiolytic drugs can reduce isolation-induced USVs (Hamed et al., 2009;Winslow and Insel, 1991) underlies theories that dam-reunion attenuated USVs decreases the stress of the pup through rewarding salience of the dam. Developmental differences observed in the NAc core of CC offspring could have profound impact on dopamine signaling and hence the rewarding value of the dam to the offspring. Human studies have suggested that cocaine-exposed mothers have a more difficult time soothing their infant (Eiden et al., 2009a;Eiden RD et al., 2011); which could be a consequence of altered infant NAc development. Most studies examining USVs using an isolation testing paradigm.
In light of the present findings, other testing paradigms (i.e. contact quieting, social play) should be explored as differences observed in these paradigms may be more related to mother-infant interactions.
102 4.5. CONCLUSIONS
In conclusion, PCE disrupted offspring vocalizations in an age-dependent fashion with neonatal offspring exhibiting decreased rates of vocalizations and juvenile offspring displaying increased overall rates of vocalizations. Males appear to be more sensitive to cocaine and stress- induced changes in vocalizations as juveniles than do females. Data also suggests developmental differences in the VMH and CeA are associated with variations in vocalizations supporting previous reports that the neonatal cry is both a social and biological signal. Additional research is needed to elucidate how infant behavior and neural development interact with the infant’s environment, to influence long-term developmental outcomes. Understanding the neural basis of behavioral changes following prenatal insults, commonly found to be correlated with neurodevelopmental disorders has great promise for discovering early biomarkers for these disorders.
103 CHAPTER 5. GENERAL DISCUSSION
5.1. OVERALL CONCLUSIONS, LIMITATIONS, AND FUTURE DIRECTIONS
The main goals of the studies reported here were to assess if PCE coincided with variations in infant vocalizations (human cries and rodent USVs respectively) and how these changes might be translationally impacting early maternal response (human perception and rodent preference-like behavior respectively). We found that in human and rodent neonates PCE coincides with variations in neonatal vocalizations. Our human infants showed subtle trends of having more dysphonation in their cry expirations and greater intervals between expirations following PCE (Chapter two). While results must be interpreted with care, as only healthy, full term infants were included in this study, they suggest that even low levels of PCE may induce variations in infant behavior and hence mother- infant interactions. Low levels of cocaine-exposure has also been found recently to impact maternal care in an animal model (Morrell et al., 2011) further supporting findings reported here. However, secondary analyses of cocaine and nicotine TLFB data suggest that at the low levels of cocaine- exposure (which most of our cocaine-exposed mother-infant dyads were exposed to) infant behavioral results are possibly more related to nicotine-exposure. Few studies have explored nicotine-exposure on infant development and warrants more focus. However, our animal model further supports the human study in that PCE also altered rodent neonatal vocalizations (in the absence of nicotine). On
PND one, rodent pups prenatally exposed to cocaine emitted fewer USVs (Chapters three and four)
USVs with a smaller standard deviation of the fundamental frequency (Chapter three), and USVs with fewer observable harmonics (Chapter four). Interestingly, the greatest differences on PND one in
104 vocalizations were found in males prenatally exposed to cocaine. This is interesting in light of our human study which suggested that cocaine-exposed females show the greatest differences in cry expirations at one month of age. Future studies should aim to clarify human findings by increasing the number of infants assessed and extending cry analysis to later developmental ages. Differential sex-dependent changes in rodent USV production were observed depending on the age of pups at testing. While cocaine-exposed males emitted fewer USVs on PND one, they did not differ in their vocalizations on PND 14, and showed an increase in USVs on PND 21. In light of these age- dependent changes cry analysis in human newborn infants and infants at six months of age might resemble results reported here in the rodent pups on PNDs one and 21 respectively. More work is needed to understand how PCE affects infant vocalizations.
Our rodent vocalization studies also suggest earlier variation in vocalizations following PCE may be more directly related to cocaine as PND one testing occurred hours after birth. Recent work in our lab has shown cocaine is still present in pup urine on PND one (McMurray, 2011), suggesting cocaine could be directly depressing pup vocalizations at this respective age. Results also suggest that differences following PCE may normalize in the early postpartum period (as cocaine is slowly cleared from the pup’s system) as we did not see any difference in vocalizations between CC and UN male pups on PPDs three and five (Chapter 3). Later differences in vocalizations observed on PND 21
(Chapter 4) are therefore thought to be more related to variations in the postnatal environment.
However, since increased vocalizations observed on PND 21 were found to be sex-dependent (only males showed this difference) and increased vocalizations were observed in both CC and CS male offspring more research is needed to understand the mechanism(s) behind this behavioral phenotype.
It is possible that increased vocalizations on PND 21 are more directly related to prenatal stress or environmental differences associated with prenatal stress; however, if this is the case then data suggests that males are more sensitive to this stress-induced environmental challenge.
We also found that maternal cocaine-exposure in our human study coincided with altered perception of infant cries at three months postpartum. In general, cocaine-exposed mothers reported
105 cry stimuli sounded less arousing and aversive than comparison mothers. Cocaine-exposed mothers also reported that they would be more likely to give a pacifier and clean infants producing the cry sounds than comparison mothers. Interestingly, cocaine-exposed mothers reported that they would be very likely to perform all behaviors (except wait and see) in response to infant cries. This is a different pattern of response from comparison mothers who showed a hierarchy in behavioral response for being more likely to perform high mother-infant bonding behaviors (pickup and cuddle) compared to low mother-infant bonding behaviors (clean and give a pacifier). We think this may be in part a model of learned helplessness and that maternal experience with her infant for the first three months following delivery differentially impacted her behavioral responses to infant cries.
Significant correlations between infant crying behavior and maternal perception of cry stimuli corroborated this hypothesis in that infants with a longer latency until they emitted their first expiration (less reactive to cry) correlated with mothers finding cry stimuli more aversive.
Our rodent model of gestational cocaine-exposure supported human findings and suggests cocaine-exposure can alter maternal preference behavior. CC dams showed a preference for the UN pup-associated side on PPD one. This preference behavior correlated with USV differences emitted by stimulus pups and since UN and CS dams did not show this same preference behavior results are thought to be an interaction between cocaine’s direct effects on the maternal brain and cocaine’s direct effects on infant behavior. Observed PPD five decreases in touch/sniff behavior of CC dams
(regardless of which pup-associated side CC dams were on) we hypothesize might be a consequence of cocaine-exposed dams’ interactions with their litters and environment over the first five postpartum days. Future studies should more closely examine the synergistic relationship between infant behavior and the maternal environment. An important next step is to understand how behavior of one shapes the other and trying to tease apart the chicken or the egg dilemma. In human and rodents, it has been observed that maternal interaction with an infant affects later infant vocalizing behavior. In humans, differences in cry behavior at the end of the first year of life are strongly associated with maternal responsiveness during earlier months of the infant’s life (Bell and Ainsworth, 1972). In rodent pups,
106 maternal-infant interactions (i.e. maternal separation/deprivation earlier in life) correlate with later differences in offspring vocalizations (Zimmerberg et al., 2003b;Zimmerberg et al., 2003a).
Additionally, maternal behavior has been shown to differ as a consequence of differential postpartum environments in humans (Donovan, 1981;Donovan et al., 1990) and rodents (Mashoodh et al., 2009).
The studies presented here are not able to address if differences in infant behavior or maternal behavior are a consequence of cocaine-exposure’s direct effects on the infant or maternal brain or the synergistic interaction between the mother and infant. Studies aimed at examining maternal perception and infant crying at birth, and then longitudinally throughout the postpartum period could help establish our knowledge on how these behaviors shape one another, the maternal-infant environment, and infant developmental outcome.
Since many cocaine-exposed children experience adverse maternal care environments, behavioral or biological disorders may be exacerbated in these children (Eiden et al., 1999;Smith,
1992;Zuckerman and Brown, 1993). Likewise, children who have experienced significant parental neglect or abuse are more likely to have personality disorders (Bernstein et al., 1998;Johnson et al.,
1999), difficulty with social relationships (Delaney-Black et al., 1998), and to abuse drugs themselves in later life (Jantzen et al., 1998). Mother-infant interactions and the care giving environment have significant influences on infant regulation, which in turn alters subsequent maternal care received by the infant. Understanding the dynamic interplay between biological vulnerabilities of the infant and the mother and how postnatal factors, i.e. mother-infant interactions, shape developmental outcome of each (Mayes, 1996;Harvey and Kosofsky, 1998;Zuckerman and Brown, 1993) is an important area of research that warrants more focus.
The work presented here also suggests that variations in CeA and VMH neuronal developmental correlate with variations in offspring vocalizations at later developmental ages. While these neuronal differences could be in part a consequence of prenatal exposure to cocaine or stress we speculate that they are most likely a product (at least to some degree) of the early maternal
107 environment. Maternal deprivation, an animal model of maternal neglect, has been shown to decrease neurogenesis in the hippocampus (Fabricius et al., 2008) and to effect neural proliferation differentially in male and female offspring. Maternal deprivation also increases neuronal and glial apoptosis (Zhang et al., 2002) and decrease the expression of brain-derived neurotrophic factor
(Roceri et al., 2002), a neurotrophin involved in neuronal survival and differentiation (Lewin and
Barde, 1996). Gender differences observed following maternal deprivation are speculated to coincide with behavioral differences in adulthood and imply that early life stress establishes sex differences in neural plasticity, contributing to alterations in the hypothalamic-pituitary-adrenal axis (Oomen et al.,
2009). Prenatal stress also differentially affects male and female offspring neural proliferation
(Weinstock, 2007;Vaido et al., 2000;Mandyam et al., 2008). Based on these previous findings future studies using a cross-fostering paradigm would prove useful in teasing apart environmental contributions to infant behavior (USVs) and correlating neuronal development (increased neuronal density in the VMH and CeA). Furthermore, preclinical animal work is needed to advance our understanding of the neurobiology underlying altered vocalizations in a high-risk animal model.
Studies that temporarily inactivate, stimulate, or pharmacologically manipulate specific brain regions, such as the VMH and CeA, would provide more evidence of causation compared to the studies reported here that were correlational. However, neurobiological findings are very interesting and should be explored further. Understanding the neurobiology underlying complex behaviors early in typical and atypical development has great promise for early intervention and treatment.
Comparisons between human and rodent infant results need to be done with care as our rodent model experienced heavy cocaine-exposure throughout the gestational period while most of our human infants experienced a lighter cocaine-exposure level in comparison. Additionally, our human infants were not solely exposed to cocaine, with many also exposed to nicotine and/or other drugs. Future animal models need to explore the impact of multiple drugs on infant development as this paradigm is most likely more translational to human studies. An animal model that employs drug self-administration and a multiple drug administration would be more translational to human studies.
108 Human mothers who used cocaine during pregnancy are also exposed to cocaine before pregnancy.
Additionally, the stress associated with willingly taking cocaine in our human maternal group is undoubtedly different compared to our rodent maternal group in which we administered cocaine subcutaneously (only during gestation with no prior cocaine experience). Future animal models that more accurately match human conditions are needed for results to be quickly and safely translated into clinical practice.
It is important to note that there is not a specific cry phenotype that can predict detrimental outcome. While changes in cry acoustics has been shown to correlate with differential developmental outcome (Huntington et al., 1990;Lester, 1987), it is widely accepted that developmental outcome is a mixture of infant vulnerability and environmental factors (Lester, 1979). An infant that doesn’t cry often (potentially because of a depressed nervous system following PCE) but still has an environment of high maternal responsiveness has a better predicted developmental outcome than infants whose mothers do not show the same responsiveness. Additionally, infants whose cries are high-pitched, previously suggested to negatively correlate with developmental outcome (Lester, 1987), and who experience a maternal environment in which the mother shows a synchrony in her behavior to her infant’s behavioral signals (i.e. as the cry increases in pitch the feeling of arousal increases in the mother prompting her behavioral responsiveness) have been shown in preliminary studies to resemble typical developing infants compared to infants whose mothers do not show the same synchrony in behavior to changes in cry pitch (Cox et al., 2010). Additionally, maternal demographics (including maternal socioeconomic status and maternal age) has been shown to correlate with infant developmental outcome (Huntington et al., 1990) further suggesting one must be careful when thinking of causation in developmental outcome in a group of infants considered at risk. The studies presented in this thesis further support an extensive amount of literature showing just how complex development is and how the environment (i.e. cocaine and/or maternal neglect) can alter the developmental trajectory in offspring. More research is needed to continue growing in our understanding of how prenatal and postnatal environments interact to produce developmental
109 outcomes. Finding sensitive windows of developmental plasticity and implementing intervention strategies is needed to improve long term outcome in vulnerable populations.
Estrogen Stress Response/HPA reactivity Progesterone Childhood experience A Simplified Story: Oxytocin Drug Use/ Withdrawal Prolactin Physiological State/Mood The Complex Neuroendocrine Disorders Changes Mother‐ Infant Relationship Parity Maternal Current Infant Mothering Maternal Learned Helplessness Model Behavior experience Caretaking environment Social Poverty Support Maternal Responsiveness Health care Single/Married Genetic Education Drug exposure/ SES Variation Infant Withdrawal experience Infant experience Nutrition Childhood experience Drug Use Stress Response Prenatal Nutrition environment Maternal stress Infant Behavior Drug exposure Known to be directly Hypoxia (or indirectly) Epigenetic Genetic Variation effected by cocaine Prematurity CNS integrity Birthweight Maternal/Paternal use or exposure Prenatal Insult Nutrition Genetic disorders Nurturing Environment Figure 22. Schematic showing the complexity of the mother‐infant relationship. Several factors (social and hormonal) can affect maternal and infant behavior which in turns affects the other symbiotically. Gestational/prenatal cocaine‐exposure has been shown to alter each of these factors which can in turn contribute to variations in maternal care and infant behavior and ultimately infant long‐term infant outcome.
5.2. CONCEPTUAL MODEL FOR FUTURE WORK
The work presented here further supports how complex the mother-infant relationship is.
One cannot think of the impact of differential maternal behavioral without thinking about the effect and contributions of infant behavior (and vice versa). Many confounding factors impact maternal and infant behavior (See Figure 22). Most (if not all) of these confounding factors can be directly or indirectly affected by cocaine use or exposure. This makes interpreting cocaine’s effects on
110 behavioral outcome in mothers and infants very challenging. In light of these challenges preclinical animal models are not only useful based on their similarity with humans but strongly needed to elucidate the mechanisms underlying altered mother-infant interactions and altered developmental outcome in the infant. However care needs to be taken when directly comparing between species because of intrinsic differences between animal models in a laboratory setting compared to a human mother’s environment (See Figure 23). Data presented in this thesis support previous studies in that gestational/prenatal cocaine exposure directly and indirectly (through continued interactions between the mother and infant) impacts maternal and infant neurobehavior, altering the HPA axis, and long- term outcome in the offspring (See Figure 24). These changes in offspring behavior can lead to drug abuse later in life and ultimately intergenerational transfer of poor maternal behavior in the offspring.
As Figure 24 depicts these long-term behavioral changes following cocaine-exposure are a result of the vast array of cocaine-induced changes in neuroendocrinology, altered monoamine dynamics, and
HPA reactivity in both mother and infant. As the mother-infant relationship continues to grow during the postpartum days following birth we theorize that environmental influences play the primary role in long-term neurobehavioral outcome. These changes result in increased likelihood for mood and anxiety disorders, relapse or continued drug-use in mothers, and/or development of drug problems in developing offspring. We therefore feel a critical window for intervention and behavioral therapies could be during this transition from Figure 23. Value of Translational Studies cocaine’s indirect effect on behavior Human Mother/Infant Rodent Mother/ Infant
(through cocaine’s direct impact on Hormonal changes in MB Infant Developmental Progression neuroendocrinology, etc.) to environment- Infant cues: cries, olfactory Cognitive/reward circuitry contribute to relationship ‐Synchrony of Behavior ‐Contact quieting/maternal induced changes in behavior (See Figure ‐Own vs. other potentiation ‐Own vs. other 25). Early educational and support Long‐term outcome ‐Emotional, Social, Cognitive ‐Emotional, Social, Cognitive ‐Choice of Drug Use ‐Forced Drug Exposure interventions to alter the environmental ‐Home environment ‐Laboratory Environment SES, abuse single vs. group house drug/psychopathology comorbidity handling conditions associated with adverse genetic variation inbred strains
111 Altered Brain Development
Behavioral Dysregulated Prenatal/ Poor Responses to Stress Gestational Offspring Maternal Hormone Cocaine Abuse Outcome Environment Response Social Behavior Emotional Behavior ‐Aggression ‐Mood/Anxiety ‐Impaired MB Poor Maternal Cognition Stress Response Behavior Drug Abuse
Gestational Period Postpartum Period Figure 24. Prenatal and gestational cocaine‐exposure results in behavioral and brain changes in both the mother and the infant. These changes, in turn, affect each other and programs stress response/reactivity as offspring ages which ultimately can result in poor offspring outcome including disrupted social and emotional behavior, cognition, stress response, and drug use as an adult.
developmental outcome could hold the greatest promise for successful intervention. More translational research is needed to understand how behavioral changes in the mother, infant, and environment drives long-term outcome and when/what interventions should be applied.
5.3. GENERALIZABILITY OF FINDINGS
The findings presented in this thesis can be applied across multiple fields of study outside of the prenatal cocaine model. Infants prenatally exposed to cocaine are considered to be high-risk for developing many of the behavioral abnormalities commonly seen in other neurodevelopmental disorders. Many neurodevelopmental/genetic disorders (such as Autism Spectrum Disorder, Williams
112 syndrome, and Downs syndrome) are associated with altered language development. These neurodevelopmental disorders are also associated with altered social behavior. Few studies have explored early communication (i.e. infant vocalizations) in these populations and the impact these changes might be having on the infant’s environment and hence subsequent development. As discussed previously infant studies in neurodevelopmental disorders are rare but warranted to understand how particular phenotypes develop (Jarvinen-Pasley et al., 2008). Altered neonatal
LEGEND Cocaine direct impact on maternal brain Vulnerable window when Environmental direct impact on maternal brain intervention and therapy could Cocaine direct impact on fetal CNS target preventing non‐optimal developmental outcome Environmental direct impact on infant brain
OT, Estrogen, Progesterone HPA reactivity
Depressed/Excitatory CNS reactivity HPA reactivity
Target Interventions Neurobehavior
to
Importance
Relevant Birth (PPD/ PND age) Weaning
Figure 25. Schematic of cocaine and environment’s relevant importance to neurobehavior. Immediately prior and following birth cocaine’s direct effects on the maternal brain (through neuroendocrinology changes) and fetal brain (through CNS reactivity) are primarily responsible for initial behavioral profiles in mother and infant. As the postpartum period progresses environment plays a vital role in long‐term outcome of both the mother and infant. This behavioral shift to environmental‐driven behavior we theorize is a critical time in behavioral plasticity when interventions might hold the most promise.
113 communication could be interfering with the infant’s environment and more directly related to the atypical development of other behavioral phenotypes, i.e. attention problems commonly observed at older ages. Longitudinal studies are a necessity to begin to understand how phenotypes develop and influence others behaviors across a lifespan.
Additionally, many neurodevelopmental disorders are now being linked to alterations in adult neurogenesis. Adult neurogenesis has been found to be altered in animal models of schizophrenia, addiction, epilepsy, and mood disorders, and therapies used to treat these disorders normalize alterations in neurogenesis (Chen et al., 2000;Malberg et al., 2000;Abrous et al., 2005;Pittenger and
Duman, 2008). Most of our current therapies employ treating symptoms of a disorder versus treating the cause. It is not known if neurogenesis or other neuronal developmental deficits occur as a by- product of these disorders or as a causative or contributing mechanism, although it is clear that adult neurogenesis alterations are an important contributor to these complex disorders (for a review see
(Eisch et al., 2008)). The PCE animal model of high-risk infant development employed in the studies presented here further suggests that altered developmental outcome is a consequence of complex interactions between infant vulnerability (genetic disorders, prenatal complications, including stress and drug exposure, etc.) and the postnatal environment (i.e. maternal education, socioeconomic class, maternal neglect, etc.). Parallel human and animal models, multidisciplinary collaborations, longitudinal examination of neuronal development, subsequent behavioral manifestation of these changes, and the role of the environment are all critical for the design and implementation of new therapeutic strategies and interventions in these populations.
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