Associations between respiratory sinus arrhythmia reactivity and internalizing and externalizing symptoms are emotion specific.

Internalizing and externalizing disorders are often, though inconsistently in studies of young children, associated with low baseline levels of respiratory sinus arrhythmia (RSA). RSA is thus considered to reflect the capacity for flexible and regulated affective reactivity and a general propensity for psychopathology. However, studies assessing RSA reactivity to emotional challenges tend to report more consistent associations with internalizing than with externalizing disorders, although it is unclear whether this is a function of the type of emotion challenges used. In the present study, we examined whether baseline RSA was associated with internalizing and/or externalizing severity in a sample of 273 young children (ages 5-6) with elevated symptoms of psychopathology. Following motivation-based models of emotion, we also tested whether RSA reactivity during withdrawal-based (fear, sadness) and approach-based (happiness, anger) emotion inductions was differentially associated with internalizing and externalizing symptoms, respectively. Baseline RSA was not associated with externalizing or internalizing symptom severity. However, RSA reactivity to specific emotional challenges was associated differentially with each symptom domain. As expected, internalizing symptom severity was associated with greater RSA withdrawal (increased arousal) during fearful and sad film segments. Conversely, externalizing symptom severity was related to blunted RSA withdrawal during a happy film segment. The use of theoretically derived stimuli may be important in characterizing the nature of the deficits in emotion processing that differentiate the internalizing and externalizing domains of psychopathology.

Aggression as an equifinal outcome of distinct neurocognitive and neuroaffective processes.

Early onset aggression precipitates a cascade of risk factors, increasing the probability of a range of externalizing and internalizing psychopathological outcomes. Unfortunately, decades of research on the etiological contributions to the manifestation of aggression have failed to yield identification of any risk factors determined to be either necessary or sufficient, likely attributable to etiological heterogeneity within the construct of aggression. Differential pathways of etiological risk are not easily discerned at the behavioral or self-report level, particularly in young children, requiring multilevel analysis of risk pathways. This study focuses on three domains of risk to examine the heterogeneity in 207 urban kindergarten children with high levels of aggression: cognitive processing, socioemotional competence and emotion processing, and family context. The results indicate that 90% of children in the high aggression group could be characterized as either low in verbal ability or high in physiological arousal (resting skin conductance). Children characterized as low verbal, high arousal, or both differed in social and emotional competence, physiological reactivity to emotion, and aspects of family-based contextual risk. The implications of this etiologic heterogeneity of aggression are discussed in terms of assessment and treatment.

Focus on methodology: salivary bioscience and research on adolescence: an integrated perspective.

The characterization of the salivary proteome and advances in biotechnology create an opportunity for developmental scientists to measure multi-level components of biological systems in oral fluids and identify relationships with developmental processes and behavioral and social forces. The implications for developmental science are profound because from a single oral fluid specimen, information can be obtained about a broad array of biological systems and the genetic polymorphisms related to their function. The purpose of this review is to provide a conceptual and tactical roadmap for investigators interested in integrating these measurement tools into research on adolescent health and development.

Assessing genetic polymorphisms using DNA extracted from cells present in saliva samples.

BACKGROUND: Technical advances following the Human Genome Project revealed that high-quality and -quantity DNA may be obtained from whole saliva samples. However, usability of previously collected samples and the effects of environmental conditions on the samples during collection have not been assessed in detail. In five studies we document the effects of sample volume, handling and storage conditions, type of collection device, and oral sampling location, on quantity, quality, and genetic assessment of DNA extracted from cells present in saliva. METHODS: Saliva samples were collected from ten adults in each study. Saliva volumes from .10-1.0 ml, different saliva collection devices, sampling locations in the mouth, room temperature storage, and multiple freeze-thaw cycles were tested. One representative single nucleotide polymorphism (SNP) in the catechol-0-methyltransferase gene (COMT rs4680) and one representative variable number of tandem repeats (VNTR) in the serotonin transporter gene (5-HTTLPR: serotonin transporter linked polymorphic region) were selected for genetic analyses. RESULTS: The smallest tested whole saliva volume of .10 ml yielded, on average, 1.43 ± .77 µg DNA and gave accurate genotype calls in both genetic analyses. The usage of collection devices reduced the amount of DNA extracted from the saliva filtrates compared to the whole saliva sample, as 54-92% of the DNA was retained on the device. An "adhered cell" extraction enabled recovery of this DNA and provided good quality and quantity DNA. The DNA from both the saliva filtrates and the adhered cell recovery provided accurate genotype calls. The effects of storage at room temperature (up to 5 days), repeated freeze-thaw cycles (up to 6 cycles), and oral sampling location on DNA extraction and on genetic analysis from saliva were negligible. CONCLUSIONS: Whole saliva samples with volumes of at least .10 ml were sufficient to extract good quality and quantity DNA. Using 10 ng of DNA per genotyping reaction, the obtained samples can be used for more than one hundred candidate gene assays. When saliva is collected with an absorbent device, most of the nucleic acid content remains in the device, therefore it is advisable to collect the device separately for later genetic analyses.

Salivary cortisol mediates effects of poverty and parenting on executive functions in early childhood.

In a predominantly low-income population-based longitudinal sample of 1,292 children followed from birth, higher level of salivary cortisol assessed at ages 7, 15, and 24 months was uniquely associated with lower executive function ability and to a lesser extent IQ at age 3 years. Measures of positive and negative aspects of parenting and household risk were also uniquely related to both executive functions and IQ. The effect of positive parenting on executive functions was partially mediated through cortisol. Typical or resting level of cortisol was increased in African American relative to White participants. In combination with positive and negative parenting and household risk, cortisol mediated effects of income-to-need, maternal education, and African American ethnicity on child cognitive ability.

Differences in saliva collection location and disparities in baseline and diurnal rhythms of alpha-amylase: a preliminary note of caution.

Identified in the early 1980s as a surrogate marker of the sympathetic nervous system component of the stress response, there has been renewed interest in measuring salivary alpha-amylase (sAA) to test biosocial models of stress vulnerability. This brief report presents studies that document that oral fluids from the parotid and submandibular gland areas had higher sAA values than did whole saliva specimens, and sAA values in whole saliva were higher than levels measured in oral fluids from the sublingual gland area. sAA in oral fluids from the parotid and submandibular gland areas showed the highest and more pronounced diurnal variation than levels in whole saliva, and sAA in sublingual saliva showed the lowest and shallowest diurnal variation. When this source of inherent variability in sAA activity levels is not controlled for by collecting oral fluids consistently from specific gland areas, the detection of individual differences, associations between sAA and "behavioral" variables, and intra-individual change in sAA levels may be compromised. Awareness, and management, of this ubiquitous source of measurement error in sAA are essential to ensure the success of future research on the correlates and concomitants of sAA levels, stress-related reactivity and recovery, and diurnal variation.

Maternal and child contributions to cortisol response to emotional arousal in young children from low-income, rural communities.

Relations of maternal and child characteristics to child cortisol reactivity to and recovery from emotional arousal were examined prospectively at approximately 7 months of age (infancy) and then again at approximately 15 months of age (toddlerhood). The sample was diverse and population based (N = 1,292 mother-infant dyads) and included families from predominantly low-income, rural communities. Maternal behavior, family income-to-need ratio and social advantage, and child temperament, attention, and mental development were assessed, and children's saliva was sampled before and after standardized procedures designed to elicit emotional arousal. Maternal engagement in infancy was associated with greater cortisol reactivity at the infancy assessment and with reduced overall cortisol level at the toddler assessment. Also at the toddler assessment, child attention, mental development, and temperamental distress to novelty were associated with increased cortisol reactivity and regulation, whereas temperamental distress to limitations and African American ethnicity were associated with reduced cortisol reactivity. Findings are consistent with prior work linking early caregiving to the development of the hypothalamic-pituitary-adrenal axis stress response system and with a conceptual model in which developing temperament is characterized by the interplay of emotional reactivity and the emergence of the ability to effortfully regulate this reactivity using attention.

Salivary alpha-amylase and cortisol in toddlers: differential relations to affective behavior.

This study applies a minimally invasive and multi-system measurement approach (using salivary analytes) to examine associations between the psychobiology of the stress response and affective behavior in toddlers. Eighty-seven 2-year-olds (48 females) participated in laboratory tasks designed to elicit emotions and behavior ranging from pleasure/approach to fear/withdrawal. Saliva samples were collected pretask and immediately posttask, and assayed for markers of sympathetic nervous system (alpha-amylase or sAA) and hypothalamic-pituitary-adrenal axis (cortisol) activity. Individual differences in sAA were positively associated with approach behavior and positive affect; whereas, cortisol was positively associated with negative affect and withdrawal behavior. The findings suggest that individual differences in sAA may covary specifically with positive affect and approach behaviors or the predominant emotional state across a series of tasks. The results are discussed with respect to advancing biosocial models of the concomitants and correlates of young children's affective behaviors.

Salivary flow and alpha-amylase: collection technique, duration, and oral fluid type.

There has been renewed interest in salivary alpha-amylase (sAA), a surrogate marker of autonomic/sympathetic activity, in biosocial research on stress vulnerability, reactivity, and recovery. This study explored the impact of saliva flow rate on sAA measurement by examining the influence of (1) the technique used to collect oral fluid-synthetic swab, cotton pledget, hydrocellulose microsponge, or passive drool; (2) collection point duration--the length of time the technique is employed (1-5min); and (3) oral fluid type--whole unstimulated saliva (not absorbed by any material) or oral fluid sampled from areas near the parotid, submandibular, or sublingual salivary glands. sAA activity (U/mL) was the highest in oral fluid collected from the parotid and submandibular gland areas. The volume (mL) of oral fluid collected increased, and the activity of sAA (U/mL) decreased, as collection point duration lengthened. The magnitude of these effects varied according to collection technique and oral fluid type. Across all conditions, there were positive correlations (range .70-.88) between sAA activity (U/mL) and sAA output (U/min). Management of these potential sources of measurement error will be essential to ensuring the success of future research on the correlates and concomitants of sAA activity, stress-related reactivity and recovery, and diurnal variation.

Medication effects on salivary cortisol: tactics and strategy to minimize impact in behavioral and developmental science.

The non-invasive measurement of cortisol in saliva has enabled behavioral scientists to explore the correlates and concomitants of the interaction between the activity of the hypothalamic-pituitary-adrenal (HPA) axis, intrinsic factors, and social forces as they occur naturally in everyday life. The widespread integration of salivary cortisol into behavioral science has also revealed that omnipresent features of everyday life such as, over-the-counter and prescription medications, have the capacity to influence measurement validity. We identify several pathways by which pharmacologic agents could influence salivary cortisol, including (a) direct agonistic and antagonistic effects on the HPA axis, (b) indirect effects on physiological systems networked with the HPA axis, (c) moderation or mediation effects on cortisol secretion via pharmacologically induced change in subjective experience, (d) iatrogenic effects on the availability or composition of saliva, or the diffusion of serum constituents into oral fluid, and (e) cross-reactivities with antibodies used to detect cortisol by immunoassay. Specific medications with the capacity to influence salivary cortisol via these pathways are documented in an effort to procedurally and statistically minimize this potential source of error variance in the next generation of studies.

Individual differences in salivary cortisol and alpha-amylase in mothers and their infants: relation to tobacco smoke exposure.

Tobacco smoke exposure affects the activity of both the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS). Statistics reveal 41 million children in the U.S. are regularly exposed to tobacco smoke, but we know little about the effects of environmental tobacco smoke exposure on HPA and SNS activity in early childhood. This study assayed cotinine (a metabolite of nicotine), cortisol, and alpha-amylase (sAA) in the saliva of mother-infant dyads from 197 low income and ethnically diverse families. The dyads were identified as tobacco smoke exposed (N = 82) or nonexposed (N = 115) based on maternal self-reports of smoking and salivary cotinine levels greater or less than 10 ng/ml. As expected, higher rates of maternal smoking behavior were associated with higher levels of cotinine in mothers' and their infants' saliva. On average, smoking mothers' salivary cotinine levels were 281 times higher compared to their nonsmoking counterparts, and 23 times higher compared to their own infant's salivary cotinine levels. Infants of smoking mothers had salivary cotinine levels that were four times higher than infants with nonsmoking mothers. Mothers who smoked had higher salivary cortisol levels and lower sAA activity compared to nonsmoking mothers. There were no associations between maternal smoking behavior, infant's salivary cotinine levels, or tobacco exposure group, and cortisol or sAA measured in infant's saliva. The findings are discussed in relation to the influence of smoking tobacco on the validity of salivary biomarkers of stress.