Elsevier

Neurobiology of Aging

Volume 23, Issue 3, May–June 2002, Pages 457-466
Neurobiology of Aging

Neurobiology of Aging
Comparison of maternal separation and early handling in terms of their neurobehavioral effects in aged rats12

https://doi.org/10.1016/S0197-4580(01)00320-7Get rights and content

Abstract

In the rat, relative to pup nonhandling (NH), early handling (EH) leads to old-adult offspring with a hyporesponsive HPA axis, superior spatial cognition, and greater hippocampal (HIPP) neuronal density. The present study compared the effects of EH and repeated maternal separation (MS), in the form of 6-hr separation on each of 4 days beginning at day 12, on spatial cognition, corticosterone (CORT) levels, and HIPP characteristics, in aged rats. Male Wistar rat pups were exposed to EH, MS, NH or our normal in-house husbandry (CON) and tested at 18–20 months. Relative to NH and CON, EH demonstrated superior spatial cognition, reduced CORT stress response, reduced CA-field volume and no change in HIPP neuronal number. MS demonstrated a trend to superior spatial cognition, an unaffected CORT stress response, reduced CA-field volume and no change in HIPP neuronal number. These findings are important in terms of the life-span mechanisms via which postnatal manipulations induce neurobehavioral effects, and the mechanisms via which CORT and HIPP structure relate to HIPP function.

Introduction

In a range of mammals, manipulations of the infant-mother relationship have been demonstrated to yield long-term changes in the neurobiology and behavior of the offspring. In the laboratory rat the most investigated postnatal manipulation procedure is early handling (EH), consisting of brief (3–15 min) daily separation of pups from the mother (and sometimes litter mates) on the first 7 to 21 postnatal days (PNDs) of life. Rats left completely undisturbed with the mother between birth and weaning, so called nonhandled (NH) subjects, normally serve as EH controls. EH as compared to NH has consistently been reported to produce robust effects on behavior in young adult rats, including decreased fear-related behavior in spontaneous and conditioned tasks [1], [28], [37], [38], [44] and increased selective attention [54]. Hypothalamic-pituitary-adrenal (HPA) effects have also been reported, with EH adults displaying attenuated corticotropin (ACTH) and corticosterone (CORT) stress responses relative to NH adults [2], [36], [38], [42], [44]. The reduced HPA stress response co-occurs in EH with increased hippocampal (HIPP) glucocorticoid receptor (GR) binding and GR mRNA expression, and it has been proposed that this altered EH HIPP GR phenotype mediates enhanced HPA CORT negative-feedback sensitivity [34], [36]. Taken together, the long-term EH-related changes in HPA function and behavior have led to the conclusion that EH reduces adulthood stress- and fear-related responsiveness to environmental challenge (e.g. [17]).

Rat EH, it has been reported, also leads to marked cognitive and neuroendocrine effects in old adulthood. Aged mammals generally, and non-aged human adults exhibiting one of several neuropsychiatric disorders, are characterized by the combination of memory impairment, elevated glucocorticoid (GC) levels, and HIPP neuronal atrophy [32]. Meaney and colleagues have described that aged EH rats do not demonstrate the age-related deficits in spatial learning and memory performance in the water maze that are typical of NH rats, and also do not demonstrate the age-related increase in basal and stress-related CORT levels that are typical of NH rats. Furthermore, this absence of elevated CORT and the attenuation of cognitive impairment are associated with maintenance of young-adult neuronal density and GR/GR mRNA densities in the CA1 and CA3 HIPP cell fields (males: [34]; females: [35]). These findings have been interpreted according to the GC cascade hypothesis, which proposes that elevated GC leads to HIPP neuronal loss and therefore GR loss, that HIPP GRs mediate GC negative feedback, and that their loss promotes further increase in GC levels [50]. This interpretation of the effects of EH on CORT and memory in old age is controversial: firstly, because recent studies have failed to demonstrate that elevated GC leads to HIPP neuronal loss [18], [26], [52]; secondly, because it has been suggested that increased hippocampal GRs reduce rather than increase HIPP GC negative feedback [10]. Nevertheless, the co-occurrence of reduced CORT levels and absence of cognitive decline in aged EH versus NH rats is clear-cut and the association could be causal.

A different type of postnatal manipulation, namely maternal separation (MS), constitutes either a single 24-hr period or repeated 3–6-hr periods of separation of litters or individual pups from the dam [22]. There are several reports that MS schedules yield long-term effects in young adulthood opposite to those of EH, including increased fear-related behavior [41], [56], increased HPA stress response [48], [53], and disruption of attentional processes ([11], [12]; although see [25]). This apparent dichotomy between EH and MS is intuitively attractive, because the effects of EH are proposed to be mediated via effects on maternal care: EH increases overall levels of certain maternal behaviors, namely licking and arched-back nursing [30], [43], and receipt of high levels of licking and arched-back nursing have been reported to lead to reduced fearfulness and attenuated HPA stress responsiveness [7], [30]. If it is increased maternal care which leads to the neurobehavioral effects of EH then it would be parsimonious to expect MS to induce the opposite long-term effects to those of EH. However, in several studies from different laboratories such opposing effects of EH and MS have not been forthcoming. Firstly, there are several reports that MS is without effect on fearfulness and stress responsiveness relative to NH [8], [42], despite the different profiles of maternal care that these two groups receive (see [43]). Second, several laboratories have reported that MS schedules yield EH-like effects; for example, a reduced CORT response to restraint, relative to NH adults, was observed in adults that had experienced a daily 4.5-hr MS on PNDs 1–21 [39], and a recent study from our laboratory yielded the same effect using a 4-hr MS over the same developmental period [44]. We also recently reported on a MS schedule that yielded behavioral effects in the same direction as those yielded by EH: MS was carried out for 6 hr on each of four PNDs, namely PND 12, 14, 16 and 18. As adults, attentional function of subjects was assessed via latent inhibition (LI) of aversive classical conditioning; LI was increased in MS subjects [25], as it is in EH subjects [13], [54]. Therefore a range of MS schedules lead to effects in the same direction as those induced by EH.

In the present study we directly compared the neurobehavioral effects of EH and the PND12–18 repeated MS schedule described above, in aged rats. Specifically, we conducted a direct comparison in 18–20-month-old Wistar rats of the effects of EH and MS on spatial learning and memory in the Morris water maze, stress-related CORT response to restraint, and HIPP volume and neuronal number. Since behavioural, endocrine and HIPP neuronal measures of aging have been shown to be “protected” in EH subjects, we aimed to test whether the opposite effects, i.e. increased aging-related cognitive deficit, increased CORT stress reactivity, and reduced HIPP size, could be observed following a repeated MS paradigm. The majority of EH studies have used NH as the control group whereas some MS studies have used control animals subjected to normal animal husbandry, including regular pre-weaning human contact inherent to activities such as cage cleaning. In the present study we therefore included both NH and in-house postnatal husbandry control groups. Furthermore, it has been proposed that NH leads to high fearfulness and stress responsiveness relative to EH, rather than vice versa [5], [14], [27], [44]; the inclusion of both control groups allowed for analysis of whether NH also constitutes a “manipulation” in terms of neurobehavioral aging.

Section snippets

Subjects

Experiments were performed on adult male Wistar rats bred in the in-house animal facility. Animals were housed under reversed cycle lighting (lights on 19:00–07:00 hours) in a temperature (21 ± 1°C) and humidity (55 ± 5%) controlled environment. Food (Nafag 9431, Eberle Nafag AG, Gossau, Switzerland) and water were available ad libitum in the home cage. Four experimental groups were generated, with all subjects unrelated within and between groups, that is, only one subject per litter was

Acquisition

The 3 × 6 ANOVA of latency to locate the platform revealed a significant main effect of day (F = 17.01, df = 5/125, p < 0.001) indicating reduced escape latencies over days of acquisition. There was a significant main effect of treatment (F = 5.68, df = 2/25, p < 0.01), reflecting the intertreatment differences depicted in Fig. 1, and no interaction between treatment and day (F < 1, p > 0.84). Post-hoc analysis using Fisher’s protected LSD revealed a significant difference between EH and CONH (

Discussion

This study aimed to test the hypothesis that repeated MS at the developmental stage of PNDs 12–18 would have opposite effects on aging-sensitive neurobehavioral parameters in aged rats in comparison to those reported for EH. Therefore, we compared the effects of EH and repeated MS on cognitive performance, CORT reactivity, and HIPP parameters in 18–20-month-old male Wistar rats, relative to each other and to their respective control groups. In line with the report in [34], in aged EH subjects

Acknowledgements

The research was supported by a grant from the Swiss Federal Institute of Technology, Zurich. Special thanks are due to Elisabeth Weber and Corinne Späte for technical assistance in the neuroanatomy laboratory, animal services for animal husbandry and care, and Bonnie Strehler for her help with the preparation of the manuscript.

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  • Cited by (0)

    1

    Present address: Max Planck Institute for Evolutionary Anthropology, Inselstr. 22, D-04103 Leipzig, Germany.

    2

    Present address: Janssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse Belgium.

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