Early locomotor and social effects in vasopressin deficient neonatal rats

Abstract

This study investigated behavioral differences between neonatal rats with vasopressin AVP deficiency (di/di) and those with little (+/di) or no deficiency (+/+) using a number of open field metrics. Infant rats (pups) at days 7 and 10 postpartum were videotaped in individual and group trials in a temperature-controlled arena for 12 min. Pups were tracked every 5 s for tip-of-the-nose and base-of-the-tail coordinate positions. These positional data were transformed, using computer algorithms, into measures of activity, change in orientation, distance moved, cells occupied, wall contact, number of corners visited, and degree of aggregation. Analysis of these data revealed some phenotypic differences among genotypes for 7-day-old pups in individual trials, but there was no clear pattern of genotypic differences. By day 10, however, there were dramatic differences. AVP deficient pups differed significantly from both +/+ and +/di pups on most individual and group metrics. As measured by effect size, group effects were especially large. The behavioral results were consistent with previous findings in adult di/di rats and indicated that 10-day-old di/di pups were hyperactive and had social deficits. AVP deficient pups also appeared to have accelerated locomotor development and to exhibit stereotypic behavior. These results suggest that Brattleboro, di/di, pups are a potentially important animal model for investigating the role of AVP in early locomoter and social development. These behavioral metrics are generalizable to infants of other rodent species and thus may allow early behavioral phenotyping and the assessment of behavioral deficits.

Introduction

The neuropeptide vasopressin (AVP) affects social behavior by modulating aggression, pair bonding, and social recognition [20], [39], [34]. Disregulation of AVP may play a role in several mental disorders such as autism spectrum disorder (ASD) [15], [40], [65], [36], [21], schizophrenia [31], [17], [16], and effective disorders [20], [58]. For example, ASD may be associated with vasopressin resistance due to hyporesponsiveness of AVP V1a receptors [15], [29]. Knockout mice lacking V1a receptors have deficits in social recognition resembling aspects of ASD [65], [10], [11]. AVP deficient Brattleboro rats (di/di), exhibit abnormalities in emotional reactivity [63], [64], memory and attention [37], [41], and social recognition [30]. Indeed, AVP was first implicated in behavioral deficits in studies that found avoidance learning and memory deficits in di/di rats [61].

Little, however, is known about the affects of AVP deficiency on neonatal behavioral and brain development. Fifteen-day-old di/di pups exhibit reduced social attachment as measured by odor preference and approach latency learning [46]. By day 10, di/di pups have an impaired adrenocorticotropin stress response to separation from their mother [66]. These results indicate that AVP deficiency is behaviorally detectable early in life and that these early developmental effects could be related to abnormalities previously detected in adult di/di rats [63], [64], [37], [41], [30]. The main aim of this study was to detect whether AVP deficiency in neonatal di/di rats has behaviorally detectable effects on locomotor and social behavior.

The Brattleboro, di/di, rat strain was discovered in the laboratory of Henry A. Schroeder in Brattleboro, VT, USA in 1964 who cloned the normal rat AVP gene and then identified the mutation responsible for AVP deficiency in these rats [60]. It turned out to be a single base deletion at nucleotide 1552 in a conserved region of exon B resulting in a frame shift mutation that produces an altered amino acid sequence [54]. Immunohistochemical and in situ hybridization studies showed that the vasopressin gene is transcribed and translated in di/di rats, but with an impaired response to physiological stimulation [42], [43], [54]. Brattleboro rats develop diabetes insipidus at least by the time of weaning with indications of AVP deficiency even earlier in development [26].

Open field behavior has long been used as a method for assessing behavioral responses in rodents and has been used to assess behavioral deficits in di/di rats [63], [64]. Neonatal rats that are 10-days old or less, can crawl about on a surface with characteristic patterns of movement that are age specific [28]. Even though they are incapable of adult thermoregulation and thus quickly cool down even at room temperature [3], [4], cooling can be controlled by regulating ambient and surface temperature [7], [48]. Thus, when placed on the flat surface of an arena in a temperature-controlled chamber, pups exhibit sustained activity, locomotion, and aggregation dynamics [48], [49], [50].

The development of computational models of neonatal rat locomotion and aggregation required the specification of metrics that extract precise information on the dynamics of locomotion and aggregation in open field contexts [48], [50], [53], [52]. These metrics aim to maximize (within practical constraints) information available in terms of activity, distance moved, changes in orientation, contact with other pups and walls, and location in the arena; thereby allowing the detection of even subtle behavioral changes induced by AVP deficiency. Thus, another aim of this research was to determine whether these metrics could be used to detect phenotypic differences among genotypes and, if so, which of these metrics are best at detecting phenotypic differences as measured by effect size [25].

The interpretation of these metrics is important for relating neonatal behavior to adult behavior and for discovering possible analogies to human disorders. Early work showed that di/di rats exhibit more open field activity than +/+ rats and that their increased activity was not dependent on sex or weight [63], [64]. Increased activity of di/di pups could therefore be an indicator of early effects of AVP deficiency in neonatal rats. Social deficits are found in adult di/di rats [30]. Group trials, in which an entire litter of pups is observed for activity and aggregation, provide the opportunity to measure potential social deficits. The ability to aggregate into contact groups in an arena should be an indicator of early social deficits, since previous research showed that pup aggregation could be explained by computational models that represent pup preferences for other pups and contact-dependent activity (i.e., coupled activity) [48], [50]. Indeed, the developmental emergence of coupled activity in group contexts is an indicator of the emergence of social behavior by day 10 [50]. Thus, analysis of open field behavior in two contexts – individual and group – should reveal a number of behavioral indicators of AVP deficiency relatable to adult behavior.

In rats, the central pattern generator (CPG) in the spinal cord (for leg locomotion) begins to operate at birth (see Fig. 1) allowing infant rats to crawl in their nest and huddle [23], [24]. Up to about day 10, crawling locomotion continues with the development of spinal neural networks and descending brain connections proceeding cephalocaudally. Since early locomotor development proceeds cephalocaudally, the kinematics of crawling change at each day of development during the immature stage [28]. Of particular interest during the immature stage are day 7, when the corticospinal tract begins to develop [23], [24], and day 10, which is the end of the immature stage and when coupled activity in groups can be detected [50]. It is at these days of development that we should see the most dramatic changes in the kinematics of locomotion during the immature stage. If AVP affects the development of the neonatal brain and spinal chord, then we should detect genotypic effects, especially those related to social behavior, by day 10.

The present study investigated the open field performance of ++, di/di, and +/di pups in individual and group trials at postnatal days 7 and 10. Based on previous findings, I hypothesized that di/di pups at day 10, would exhibit greater locomotor activity (and related metrics) and more social deficits in aggregation than +/+ and +/di pups. In addition, the metrics used were evaluated for their power to detect effects as measured by effect size [25].