Selective breeding for infant rat separation-induced ultrasonic vocalizations: developmental precursors of passive and active coping styles

Abstract

Human depression and anxiety disorders show inherited biases across generations, as do antisocial disorders characterized by aggression. Each condition is preceded in children by behavioral inhibition or aggressive behavior, respectively, and both are characterized by separation anxiety disorders. In affected families, adults and children exhibit different forms of altered autonomic nervous system regulation and hypothalamic-pituitary-adrenal activity in response to stress. Because it is difficult to determine mechanisms accounting for these associations, animal studies are useful for studying the fundamental relationships between biological and behavioral traits. Pharmacologic and behavioral studies suggest that infant rat ultrasonic vocalizations (USV) are a measure of an early anxiety-like state related to separation anxiety. However, it was not known whether or not early ultrasound emissions in infant rats are markers for genetic risk for anxiety states later in life. To address these questions, we selectively bred two lines of rats based on high and low rates of USV to isolation at postnatal (P) 10 days of age. To our knowledge, ours is the only laboratory that has ever selectively bred on the basis of an infantile trait related to anxiety. The High and Low USV lines show two distinct sets of patterns of behavior, physiology and neurochemistry from infancy through adulthood. As adults High line rats demonstrate "anxious"/"depressed" phenotypes in behavior and autonomic nervous system (ANS) regulation to standard laboratory tests. In Lows, on the other hand, behavior and autonomic regulation are consistent with an "aggressive" phenotype. The High and Low USV lines are the first genetic animal models implicating long-term associations of contrasting "coping styles" with early attachment responses. They thus present a potentially powerful model for examining gene-environment interactions in the development of life-long affective regulation.

Figure 1

Mean (±SEM) number of USV in P10 pups for 2 minutes (y-axis) over 20 generations (x-axis). Low and High USV lines diverged significantly in rates of USV from the Randomly-bred control line in the S1 and S3 generations respectively. Data based on litter means. USV was transduced by a bat detector (UltraSound Advice, London) set on broadband mode (10 – 100 kHz), and therefore the frequencies of USVs selected for in the lines were theoretically anywhere within this range. From Brunelli, 2005.

Figure 2

A. Mean (±SEM) birth weights (grams) High, Low and Random lines, across the 14^th through 21^st generations: Line F(2,1196) = 44.634, p = 0.000; Generation: F(6, 1196) = 6.277, p = 0.000; Generation *Line: (12,1196) = 2.146, p = 0.012). Data based on litter means (unpublished data). B. Mean (±SEM) birth weights averaged over 22^nd – 28^th selected generations, showing stable differences between Low and High/Random litters Line (F(2,49)= 7.175, p = 0.002). Data based on litter means (unpublished data).

Figure 3

A. Mean (±SEM) DA and DOPAC levels in accessory cingulate cortex (ACC), measured as picograms per milligram of tissue (n=12 pups All ps < .01 per line). Low line DOPAC significantly greater than High and Random lines). From Brunelli & Kehoe, 2005. P10 pups from 6 High, 6 Low and 6 Random line litters were rapidly decapitated and the brains quickly removed, dissected and frozen in dry ice after: 1) 10-min isolation at ambient temperature; or 2) removal directly from the litter pile in the home cage. Two pups per litter per group were used for each brain sample. Dissected brains were frozen at -80°C until monoamines were extracted and measured by means of high performance liquid chromatography. B. DOPAC/DA ratio in ACC). All ps < .01. Low line significantly greater than High and Random lines. From Brunelli & Kehoe, 2005. C. Mean (±SEM) DA, DOPAC and HVA levels in periaqueductal gray (PAG), measured as picograms per milligram of tissue. All ps < .01. High and Low line HVA significantly greater than Random. From Brunelli & Kehoe, 2005.

Figure 4

A. Mean (±SEM) levels of 5-HT (picograms per milligram of tissue) and 5-HT turnover measured as levels of 5-HIAA (picograms per milligram of tissue) in the bed nucleus of the stria terminalis (BNST) All ps < .01. High and Low line levels of 5-HT and 5-HIAA significantly greater than Random line. From Brunelli & Kehoe, 2005. B. Mean (±SEM) levels of 5-HT (picograms per milligram of tissue) and 5-HT turnover in striatum (caudate + putamen) and nucleus accumbens (NA). High line levels of 5-HIAA significantly greater than Low and Random lines.

Figure 5

A. Mean (±SEM)number of pins in 10 minutes of play during three successive play sessions in High, Low and Random juvenile sibling pairs. Low line significantly less than Random line. Male and female pairs are shown separately for each line. Frequencies shown are for sibling pairs, which were scored as one unit. From Brunelli, et al., 2006. B. Mean (±SEM) number of 55 kHz vocalizations emitted by High, Low and Random sibling pairs in 10 minutes during three successive play sessions. Low line significantly less than Random line. From Brunelli, et al., 2006. C. Mean (±SEM) number of nape contacts in 10 minutes of play during three successive play sessions (one per day over three days), in High, Low and Random line sibling pairs. Low and High lines significantly less than Random line. From Brunelli, et al., 2006.

Figure 6

Mean (±SEM) latency to emerge from a cylinder into an open field by males High and Low USV lines. From Zimmerberg, et al., 2005.

Figure 7

A. (left)Mean (±SEM) duration immobile (floating) in the Porsolt Swim by males in the High, Low and Random lines in the 15^th generation (S15); and B. (right) in High and Low line males in the 19^th (S19) generation of selection. From Shair et al., 2000; and Zimmerberg, et al., 2005.

Figure 8

Mean (±SEM) concentrations in ng/g tissue of allopregnanolone (3-,5-THP) in amygdala/hippocampal tissue of males (A), and proestrus females and diestrus females (B) from selectively bred High and Low USV lines (significant main effects of line and hormone status, n's = 13–17 subjects per bar). From Zimmerberg, et al., 2005.

Figure 9

Mean (±SEM) heart rates (HRs, in beats per minute [bpm]) of Postnatal Day (P) 18 control pups in the High-USV, Low-USV, and Random-USV lines, from Baseline in the home cage (Home Cage, left) through Isolation in a novel cage (Novel Cage, middle), to Recovery back in the home cage (Home Cage, right). From Brunelli, et al., 2002.

Figure 10

A. Mean (±SEM) adult male heart rates over 30-min of restraint at 10-min intervals. Significant Line effect (p=.013), which post hocs demonstrate are due to Low > Random line heart rates. Significant Line × Epoch effect (p = .05) due to Low > Random/High line heart rates at the 30 minute epoch. From Shair et al., 2000. B.) Mean (±SEM) adult male r-MSSD (± standard error), calculated as root mean square of successive beat-to-beat variability (an index of high frequency variabilty) [Murphy, et al., 1991; 99]) over 30-min of restraint at 10-min intervals. Significant Line × Epoch effect (p = .05) due to Low < Random/High line at the 30 minute epoch. From Shair et al., 2000.