Long-term neurobehavioural impact of the postnatal environment in rats: manipulations, effects and mediating mechanisms

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Abstract

The major characteristics of the postnatal environment of the rat pup are its mother and littermates. The pup, which is poorly developed at birth, matures rapidly in this environment, and regulates the behaviour and physiology of the dam and littermates, as well as vice versa. The study of the impact of the rat's postnatal environment on its long-term neurobehavioural development is of fundamental importance. In fact, it is one of the major examples—at the interface of the biological, social and medical sciences—of animal models for the study of the interaction between the environment and the genome in both the acute and chronic regulation of the phenotype. Specific experimental manipulations of the rat postnatal environment have been demonstrated to exert robust and marked effects on neurobiological, physiological and behavioural phenotypes in adulthood. In the present review we present some of the major findings, including some original data, and discuss what these existing data can tell us about the long-term neurobehavioural effects of the postnatal environment in rats, the external and internal mechanisms that mediate these effects, and the most appropriate directions for future basic and applied research in this area.

Introduction

The importance of the environment in the regulation of brain, physiology and behaviour has long been recognised in the biological [1], social [2] and medical [3] sciences. Given that environmental regulation refers ultimately to modulation of the central nervous system's gene expression and proteomic activity [4], and given the marked recent advances at the genomic level resulting from the human and other species' genome projects [5], then we are now clearly at an important junction in the study of the causal relationships between physical and social environmental factors and neurobiological, physiological and behavioural phenotypes.

In mammals, including rodents, non-human primates and humans, the mother is the source of infant nutrition and a complex infant–mother relationship has evolved out of this nutritional need, such that the mother provides essential thermal, somatosensory, kinaesthetic, olfactory, visual and auditory stimulation, as well as maternal milk, for an extended period of postnatal development [6]. Hofer has been pioneering in describing how observable behavioural interactions of parent and offspring mediate non-observable events that have important and widespread homeostatic effects [7]. As such, the maternal environment constitutes one of the most significant environments that any mammal will encounter throughout its entire life span. In humans, there is a growing body of clinical and epidemiological evidence indicating that adverse postnatal environments associated with high levels of early life stress, for example, loss of a parent, parental neglect and/or abuse, or being cared for by a depressed parent, can impact on the long-term neurobiological and psychosocial development of offspring and markedly increase vulnerability to the development of psychiatric disorders [8], [9], [10], [11], [12], [13]. Therefore, as well as being of marked biological significance, the mammalian infant–mother relationship—in particular experimental manipulation thereof—is potentially also of high biomedical importance [3], [14], [15], [16], [17]. Animal models based on the impact of disrupted maternal care and leading to reliable and marked chronic effects on neurobiology, physiology and behaviour in adulthood, if identified, will be characterized by impressive levels of face and construct validity [3], [18].

In laboratory rats, the chronic effects of postnatal manipulation of the infant–mother relationship have been studied experimentally for nearly 50 years, a research field pioneered by the work of Seymour Levine [1], [19], [20], [21], [22], [23] and Victor Denenberg [24], [25]. Rat dams typically give birth to around 12 pups per litter, and these pups are poorly developed at birth (‘altricial’) and grow rapidly, with weaning taking place at 3–4 weeks. Maternal care occurs in bouts of retrieving, licking, and nursing, interspersed with periods when the dam is absent from the nest and litter [26]. Mother and littermates both constitute important components of the individual pup's environment and both can provide important sources of thermal, somatosensory, kinaesthetic, olfactory and auditory stimulation. As well as being stimulated by, each individual pup also stimulates, mother and littermates; an elegant example of the complexity of these three-way dyadic postnatal relationships is provided by the observation that arched back nursing (or kyphosis) and milk ejection by the dam are dependent on the combined suckling stimulus of several neonatal pups [26].

The first postnatal manipulation model to be investigated in detail was the comparison between early handling and early non-handling (NH) [1], [19]. In these studies, early handling, or EH, was defined as the experimenter picking up the pup, removing it from the breeding cage and isolating it in a small compartment for several minutes, repeated across days between birth and weaning. Early NH was defined as the complete absence of handling (both experimental and husbandry related) in the breeding cage between birth and weaning [1]. As adults, EH rats were: more active, explored more, and defecated and urinated less in the open field; exhibited a lower plasma corticosterone (CORT) response to stressors including injection or placement in water; and demonstrated a more rapid maximal CORT response to electro-foot shock followed by a more rapid return to baseline titres [1], [19], [20], [21]. As well as providing landmark data on the importance of the postnatal environment for the long-term development of the emotional phenotype in the rat, Levine's studies also revealed a caveat that has continued to be a major theme, indeed a major problem, in the design and interpretation of rat postnatal manipulation studies conducted to this day. The issue concerns the nature of the control group in the EH–NH model. Given that EH constitutes a physical manipulation and NH a non-manipulation, then it is a logical step to regard the former condition as the experimental group and the latter condition as the control group. However, another interpretation is that rat pups require a minimal amount of stress and/or stimulation in order to develop into adult rats that exhibit a behavioural profile typical for laboratory rats, and that NH is below this minimum and therefore constitutes an experimental group. Levine referred to this caveat in an early review of his own EH–NH emotionality work ([1], p. 81). A clear problem with measures of emotionality is that it is very difficult to define what is normal or typical for a laboratory rat, of any strain, maturing in the constant laboratory environment. Studies based on the actual absence of an otherwise ubiquitous cognitive phenomenon, namely latent inhibition (LI), have subsequently confirmed that NH does indeed yield adults exhibiting abnormal behaviour, and is therefore in itself an experimental treatment. LI consists of retardation in the classical conditioning of a neutral stimulus (e.g. tone, light) to an unconditioned stimulus (e.g. electro-foot shock) as a consequence of its prior non-reinforced preexposure [27]. It is a ubiquitous behavioural phenomenon, interpreted as being highly adaptive as it allows an organism to ignore irrelevant information in its environment. In humans disrupted LI is a common and important feature of the behaviour of schizophrenic patients [27]. In male rats, NH leads to the absence of LI, supporting both the importance of the EH–NH model to biomedicine and the validity of the interpretation that NH constitutes the manipulation in this model ([28], [29], [30]; see also Ref. [31]). In the last 15 or so years, as reviewed below, a large number of laboratories have applied the EH–NH model to ask questions concerning the importance of the postnatal environment for long-term neurobehavioural development, in the contexts of both emotionality and cognition. The model has also been used in an attempt to explain the mediating mechanisms via which postnatal manipulation can lead to alterations in the adulthood phenotype at the neurobiological, physiological and behavioural levels.

In parallel to this recent increased interest in the EH–NH model, other manipulations (e.g. maternal separation (MS), early deprivation (ED)) of the infant–mother–litter relationship have been developed and their effects studied. The findings, again at the neurobiological, physiological and behavioural levels, are reviewed below. Here, it is important to point out that this research area has become very complex, both conceptually and methodologically. Firstly, there are many forms of manipulation that are currently in use and it would seem to be essential to recognise that a large number of potentially very important variables are being manipulated differently in the many laboratories active in this area. A recent review from our laboratory has addressed this very issue [16]. Second, there is clearly a need for a framework that facilitates recognition of these variables and that provides clear and unambiguous nomenclature for the different forms of postnatal manipulation. Recently [32] we proposed the following: MS should be used to describe separation of the intact litter from the dam for one or more hours per day across several postnatal days [33], [34], [35], [36], and single MS to describe separation of the intact litter from the dam for a single 24 h period [37], [38], [39], [40], [41], [42]. Infant or early combined with either isolation or deprivation should be used to describe separation of the pup from the dam and the litter for one or more hours per day across several postnatal days [43], [44], [45]. Our preference is for ED, because this indicates similarity to EH and thereby emphasises the important and reciprocal relationship between these two manipulations: the ‘patent form’ of EH constitutes separation of the pup from the litter and the dam [1], as does ED; EH does not constitute deprivation in that the isolation period is shorter than species-typical periods between successive bouts of maternal care, whereas ED clearly does. A third important point with regard to the current complexity of postnatal manipulation research in the rat concerns the nature of the control group. The fragility of NH as a control treatment has already been highlighted above, and recently rats that as pups have experienced the human interventions inherent to cage cleaning have been used as controls. Animal facility rearing (AFR) is the title given to this group, and AFR, as reviewed below, has already been applied to study specific long-term effects of EH, MS, ED and, indeed, NH [15], [44], [45], [46], [47]. In a recent review, Levine has highlighted that AFR is also not without its problems, given that, ‘Variations in animal husbandry are as numerous as there are animal facilities.’ ([31], p. 540).

Against the above background, the overall aim here is to review the evidence for the long-term impact of specific postnatal manipulations of the pup–mother–litter relationships on emotional and cognitive responses to specific environmental challenges in adult rats. The following specific themes are addressed: long-term neurobehavioural effects of early handling; long-term neurobehavioural effects of MS; long-term neurobehavioural effects of ED; identifying the appropriate control group and, mediating mechanisms. The long-term effects that are reviewed are neurobiological, physiological and behavioural.

Section snippets

Long-term neurobehavioural effects of early handling

As stated in Section 1, the initial studies with the EH–NH model were aimed at comparing effects on anxiety-like behaviour and hormonal stress responses, and demonstrated that EH was associated with increased open-field exploration, reduced open-field defecation and urination, suggesting reduced activation of the sympathetic branch of the autonomic nervous system (ANS), and a more rapid and less prolonged plasma CORT response to a range of stressors, suggesting altered activity of the

Long-term neurobehavioural effects of maternal separation

There are many instances in the original research reports and reviews of the Meaney–Plotsky group where it is stated that MS leads to effects opposite to those of EH. For example, ‘In the rat and mouse, postnatal handling decreases the magnitude of behavioral and endocrine responses to stress in adulthood. In contrast, longer periods (i.e. 3–6 h) of daily separation from the mother increase behavioral and endocrine responses to stress’. ([67], p. 129); ‘As adults, animals exposed to repeated MS

Long-term neurobehavioural effects of early deprivation

ED, involving deprivation of not only the mother but also the littermates for an extended period of time, and this repeated across days, would appear to constitute a more severe postnatal manipulation than MS, and might be expected to constitute a postnatal stressor. In fact clear evidence that this latter assumption is indeed the case is provided by the study of McCormick and colleagues, which demonstrates that ED for 60 min per day on PNDs 2–8, potentiates the CORT response to ED on PND 9

Identifying the appropriate control group

Clearly, throughout the study of the long-term neurobehavioural impact of postnatal manipulations in rats, NH has predominated as the control group. Equally clearly, this approach has shortcomings, as described above, and yet, some 40 or so years after the pioneering EH–NH stuides there are many experiments where NH is still being used. Our laboratory has also been ‘guilty’ of this practice, and in our self-defence we would argue (as of course others are equally entitled to do) that this was at

Mediating mechanisms

In our above consideration of what constitutes the most appropriate control group for specific postnatal manipulations, we of course also began referring to the mechanisms that mediate the effects of these manipulations. Just as the Meaney–Plotsky group has held a high profile in the study of the effects of postnatal manipulations, so has the Meaney group held a similar profile in theories of the mechanisms mediating these effects. Specifically, this group has proposed that, at least as far as

Conclusions

Our goals with this review have been (i) to provide a timely analysis of the current evidence for the chronic effects and mediating mechanisms of specific experimental postnatal manipulations on neurobiological, physiological and behavioural phenotypes in adulthood in laboratory rats, and (ii) to provide insights into important future directions in this area of research. In attempting to achieve these goals we have focussed on the research of some of the most eminent and influential research

Acknowledgements

We sincerely thank Daniela Rüedi-Bettschen for her important contribution to the studies from our laboratory described in this review. These studies were supported by the Research Commission of the Swiss Federal Institute of Technology Zurich (grant TH-24./99-4).

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