Elsevier

Neurotoxicology and Teratology

Volume 84, March–April 2021, 106956
Neurotoxicology and Teratology

Full Length Article
Impact of preweaning stress on long-term neurobehavioral outcomes in Sprague-Dawley rats: Differential effects of barren cage rearing, pup isolation, and the combination

https://doi.org/10.1016/j.ntt.2021.106956Get rights and content

Highlights

  • Compare barren cage rearing to maternal separation (pup isolation) on behavior.

  • Barren cage rearing increased anxiety-like behavior in the elevated zero maze.

  • Isolation reduced open-field activity in males and increased it in females.

  • Isolation increased Cincinnati water maze (CWM) errors, barren cage reduced them.

  • Barren + isolation increased anxiety-like behavior and decreased CWM performance.

Abstract

Two developmental stressors were compared in preweaning rats exposed to either one stressor or both. Stressors were barren cage rearing or maternal separation (pup isolation). 40 gravid Sprague-Dawley CD/IGS rats were randomly assigned to two cage conditions: standard (Std) cage or barren cage (Bar), 20 litters/condition throughout gestation and lactation. After delivery, litters were randomly culled to 4 males and 4 females. The second stressor was maternal separation: Two male/female pairs per litter were isolated from their dam 4 h/day (Iso) and two pairs were not (Norm). Hence, there were 4 conditions: Std-Norm, Std-Iso, Bar-Norm, and Bar-Iso. One pair/litter/stress condition received the following: elevated zero-maze (EZM), open-field, swim channel, Cincinnati water maze, conditioned fear, and open-field with methamphetamine challenge. The second pair/litter/condition received the light-dark test, swim channel, Morris water maze, forced swim, and EZM with diazepam challenge. Barren rearing reduced EZM time-in-open, whereas isolation rearing reduced open-field activity in males and increased it in females. Effects on straight channel swimming were minor. In the Cincinnati water maze test of egocentric learning, isolation rearing increased errors whereas barren cage housing reduced errors in combination with normal rearing. Barren cage with maternal separation (pup isolation) increased Cincinnati water maze escape latency but not errors. Barren cage housing reduced hyperactivity in response to methamphetamine. Isolation rearing increased time in open in the EZM after diazepam challenge. Trends were seen in the Morris water maze. These suggested that barren cage and isolation rearing in combination reduced latency on acquisition on days 1 and 2 in males, whereas females had increased latency on days 2 and 3. Combined exposure to two developmental stressors did not induce additive or synergistic effects, however the data show that these stressors had long-term effects with some evidence that the combination of both caused effects when either stressor alone did not, but synergism was not observed.

Introduction

Neurodevelopmental cognitive disorders are the result of gene x environment interactions of which stress is a significant environmental factor. Stress during early childhood, such as poverty, neglect, and low socioeconomic status (SES), may alter neurodevelopment (Rebello et al., 2018). Children raised in very low SES households may experience psychosocial stress from neglect, family disruption, substandard housing, hunger, and violence (Evans, 2004; Taylor et al., 1997). Developmental exposure to such factors is associated with potentiated physiological stress. Elevated cortisol levels are found in children living in low SES households and in those in family environments characterized by conflict and neglect (Cohen et al., 2006; Evans and English, 2002; Evans and Kim, 2007; Lupien et al., 2001; Repetti et al., 2002). Chronic developmental stress is neurotoxic (van Bodegom et al., 2017) and induces dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis (Lupien et al., 2009; McEwen, 1998). Several days after birth in rats, the HPA axis goes through the stress hyporesponsive period (SHRP) that is characterized by a downregulated adrenal response and low basal glucocorticoid levels. The SHRP is hypothesized to be a neuroprotective mechanism that defends the developing brain from stress-induced excitotoxicity (De Kloet et al., 1988; Schmidt et al., 2002; Schmidt, 2019). However, some stressors overcome the buffering capacity of the SHRP and result in a maladaptive programming of HPA axis responsiveness and adverse functional outcomes (Levine, 2001; Mishra et al., 2019; Pihoker et al., 1993; van Bodegom et al., 2017).

Models of early life stress in rodents such as barren cage (also referred to as limited nesting) stress and maternal separation/pup isolation cause HPA axis dysregulation and long-term behavioral alterations (Bolton et al., 2017; Gilles et al., 1996; Lajud et al., 2012; Tractenberg et al., 2016). Barren cage rearing, as a model of environmental impoverishment, was used to induce chronic stress during development in numerous studies (Brunson et al., 2005; Gilles et al., 1996; Ivy et al., 2008; Rice et al., 2008; Sprowles et al., 2018; Vorhees et al., 2014; Walker et al., 2017). Barren cage rearing caused impaired learning and memory (Brunson et al., 2005; Molet et al., 2016b; Sprowles et al., 2018), emotion-related behavioral changes (Dalle Molle et al., 2012; Molet et al., 2016a; Sprowles et al., 2018), alterations in basal corticosterone levels (Arp et al., 2016; Avishai-Eliner et al., 2001; Rice et al., 2008), and abnormal hippocampal volume and structure (Molet et al., 2016b). The maternal separation stress paradigm, as a model of neglect wherein pups are separated from the dam, is one of the most frequently used neonatal stress protocols (van Bodegom et al., 2017). Maternal separation stress causes learning and memory deficits (Aisa et al., 2007; Grassi-Oliveira et al., 2016), increased anxiety-like behavior (Aisa et al., 2007; Gracia-Rubio et al., 2016), increased depressive-like behavior (Aisa et al., 2007; Kundakovic et al., 2013), HPA axis hyper-reactivity (van Bodegom et al., 2017), epigenetic changes, and altered expression of hippocampal and striatal molecular markers (Tractenberg et al., 2016). We chose maternal separation without pup warming.

Exposure to multiple early stressors may pose greater neurodevelopmental effects compared with one stressor alone. The purpose of this study was to assess whether simultaneous exposure to two stressors interact to adversely affect cognition and behavior more than either one separately. We used barren cage rearing and maternal separation/pup isolation. Both models of developmental stress cause long-term effects (Bolton et al., 2017; Gilles et al., 1996; Ivy et al., 2008; Rice et al., 2008; Tractenberg et al., 2016; van Bodegom et al., 2017). Rats were reared in barren or standard cages and/or were subjected to daily periods of isolation from their dam or remained with their dam prior to weaning, a period of brain development analogous to late gestation to early childhood in humans (Clancy et al., 2001; Clancy et al., 2007a; Clancy et al., 2007b). The hypothesis was that developmental exposure to a combination of stressors would produce greater cognitive and anxiety-related changes compared with either stressor alone.

Section snippets

Animals

Male and nulliparous female (175–200 g when received) Sprague-Dawley CD/IGS rats (strain 001) were obtained from Charles River Laboratories, Raleigh, NC and acclimated for not less than one week before breeding. Rats were maintained in an AAALAC International accredited vivarium on a 14:10 h light-dark cycle (lights on at 600 h) with controlled temperature (19 ± 1 °C) and humidity (50 ± 10%). Rats were provided with ad lib NIH-07 diet (LabDiet, Richmond, IN) and reverse osmosis, UV filtered

Growth

Rats were weighed prior to weaning and as adults. Repeated measures ANOVA on body weight showed effects of cage (F(1,38.7) = 29.29, p < 0.0001), isolation (F(1,272) = 423.30, p < 0.0001), cage x day (F(5,1183) = 82.15, p < 0.0001), isolation x day (F(5,1183) = 145.57, p < 0.0001), and cage x isolation x day (F(5,1183) = 3.76, p < 0.005). The data are shown in Fig. 1A,B. All experimental groups differed significantly from Std-Norm starting on P11; differences increased thereafter through weaning

Discussion

Table 1 summarizes the primary effects of the developmental stress conditions and the combination of both together. While there were several main effects of barren cage and maternal separation, most of the effects were interactions of the two. The interactions, however, did not support the hypothesis that these two stressors would interact synergistically to cause greater effects than either one alone. Instead, the interactions were specific to particular combinations and were seen where

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Supported by NIH grant R01 ES015689 and training grant T32 ES007051.

References (108)

  • A. Chocyk et al.

    Maternal separation affects the number, proliferation and apoptosis of glia cells in the substantia nigra and ventral tegmental area of juvenile rats

    Neuroscience

    (2011)
  • A. Chocyk et al.

    The impact of maternal separation on the number of tyrosine hydroxylase-expressing midbrain neurons during different stages of ontogenesis

    Neuroscience

    (2011)
  • B. Clancy et al.

    Translating developmental time across mammalian species

    Neuroscience

    (2001)
  • B. Clancy et al.

    Extrapolating brain development from experimental species to humans

    NeuroToxicology

    (2007)
  • E.R. De Kloet et al.

    Stress, glucocorticoids and development

  • D.S. Fareri et al.

    Effects of early life stress on amygdala and striatal development

    Dev. Cognit. Neurosci.

    (2016)
  • E.E. Gilles et al.

    Abnormal corticosterone regulation in an immature rat model of continuous chronic stress

    Pediatr. Neurol.

    (1996)
  • M.S. Golub et al.

    Statistical modeling with litter as a random effect in mixed models to manage “intralitter likeness”

    Neurotoxicol. Teratol.

    (2020)
  • I. Gracia-Rubio et al.

    Maternal separation induces neuroinflammation and long-lasting emotional alterations in mice

    Prog. Neuro-Psychopharmacol. Biol. Psychiatry

    (2016)
  • R. Grassi-Oliveira et al.

    Cognitive impairment effects of early life stress in adolescents can be predicted with early biomarkers: impacts of sex, experience, and cytokines

    Psychoneuroendocrinology

    (2016)
  • A. Guadagno et al.

    Morphological and functional changes in the preweaning basolateral amygdala induced by early chronic stress associate with anxiety and fear behavior in adult male, but not female rats

    Prog. Neuro-Psychopharmacol. Biol. Psychiatry

    (2018)
  • F.S. Hall et al.

    Isolation rearing in rats: pre- and postsynaptic changes in striatal dopaminergic systems

    Pharmacol. Biochem. Behav.

    (1998)
  • K. Hashimoto et al.

    Critical role of brain-derived neurotrophic factor in mood disorders

    Brain Res. Rev.

    (2004)
  • H.J. Hulshof et al.

    Maternal separation decreases adult hippocampal cell proliferation and impairs cognitive performance but has little effect on stress sensitivity and anxiety in adult Wistar rats

    Behav. Brain Res.

    (2011)
  • A.S. Ivy et al.

    Dysfunctional nurturing behavior in rat dams with limited access to nesting material: a clinically relevant model for early-life stress

    Neuroscience

    (2008)
  • A. Kapoor et al.

    The effects of prenatal stress on learning in adult offspring is dependent on the timing of the stressor

    Behav. Brain Res.

    (2009)
  • E.D. Knuth et al.

    Long-term behavioral consequences of brief, repeated neonatal isolation

    Brain Res.

    (2007)
  • T.A. Kosten et al.

    Chronic neonatal isolation stress enhances cocaine-induced increases in ventral striatal dopamine levels in rat pups

    Dev. Brain Res.

    (2003)
  • T.A. Kosten et al.

    Memory impairments and hippocampal modifications in adult rats with neonatal isolation stress experience

    Neurobiol. Learn. Mem.

    (2007)
  • M.-C. Lai et al.

    Neonatal isolation enhances anxiety-like behavior following early-life seizure in rats

    Pediatr. Neonatol.

    (2008)
  • N. Lajud et al.

    Periodic maternal separation decreases hippocampal neurogenesis without affecting basal corticosterone during the stress hyporesponsive period, but alters HPA axis and coping behavior in adulthood

    Psychoneuroendocrinology

    (2012)
  • S. Levine

    Primary social relationships influence the development of the hypothalamic–pituitary–adrenal axis in the rat

    Physiol. Behav.

    (2001)
  • J. Maniam et al.

    A diet high in fat and sugar reverses anxiety-like behaviour induced by limited nesting in male rats: impacts on hippocampal markers

    Psychoneuroendocrinology

    (2016)
  • E.M. Marco et al.

    Maternal deprivation effects on brain plasticity and recognition memory in adolescent male and female rats

    Neuropharmacology

    (2013)
  • E. Martisova et al.

    Long lasting effects of early-life stress on glutamatergic/GABAergic circuitry in the rat hippocampus

    Neuropharmacology

    (2012)
  • K. Matthews et al.

    Early experience as a determinant of adult behavioural responses to reward: the effects of repeated maternal separation in the rat

    Neurosci. Biobehav. Rev.

    (2003)
  • C.M. McCormick et al.

    Neonatal isolation alters stress hormone and mesolimbic dopamine release in juvenile rats

    Pharmacol. Biochem. Behav.

    (2002)
  • F. Modir et al.

    Prenatal stress decreases spatial learning and memory retrieval of the adult male offspring of rats

    Physiol. Behav.

    (2014)
  • A. Muhammad et al.

    Maternal separation altered behavior and neuronal spine density without influencing amphetamine sensitization

    Behav. Brain Res.

    (2011)
  • C. Pihoker et al.

    Maternal separation in neonatal rats elicits activation of the hypothalamic-pituitary-adrenocortical axis: a putative role for corticotropin-releasing factor

    Psychoneuroendocrinology

    (1993)
  • R.D. Porsolt et al.

    Behavioural despair in rats: a new model sensitive to antidepressant treatments

    Eur. J. Pharmacol.

    (1978)
  • R.D. Porsolt et al.

    Immobility induced by forced swimming in rats: effects of agents which modify central catecholamine and serotonin activity

    Eur. J. Pharmacol.

    (1979)
  • V. Ryu et al.

    Post-weaning isolation promotes food intake and body weight gain in rats that experienced neonatal maternal separation

    Brain Res.

    (2009)
  • E.C. Sarro et al.

    Unpredictable neonatal stress enhances adult anxiety and alters amygdala gene expression related to serotonin and GABA

    Neuroscience

    (2014)
  • M.V. Schmidt

    Stress-hyporesponsive period

  • M. Schmidt et al.

    The HPA system during the postnatal development of CD1 mice and the effects of maternal deprivation

    Dev. Brain Res.

    (2002)
  • S.Y. Shin et al.

    Adolescent mice show anxiety- and aggressive-like behavior and the reduction of long-term potentiation in mossy fiber-CA3 synapses after neonatal maternal separation

    Neuroscience

    (2016)
  • V.C. Sousa et al.

    Maternal separation impairs long term-potentiation in CA1-CA3 synapses and hippocampal-dependent memory in old rats

    Neurobiol. Aging

    (2014)
  • J.L.N. Sprowles et al.

    Developmental manganese, lead, and barren cage exposure have adverse long-term neurocognitive, behavioral and monoamine effects in Sprague-Dawley rats

    Neurotoxicol. Teratol.

    (2018)
  • S.G. Tractenberg et al.

    An overview of maternal separation effects on behavioural outcomes in mice: evidence from a four-stage methodological systematic review

    Neurosci. Biobehav. Rev.

    (2016)
  • Cited by (3)

    • Effects of Early Life Adversities upon Memory Processes and Cognition in Rodent Models

      2022, Neuroscience
      Citation Excerpt :

      It is important to consider that distinct regions of the PFC are involved in attentional shifting and reversal learning, and these regions and processes could be differently affected by early stress. On the other hand, a few studies investigating the effects of ELS reported no impairment in spatial reversal learning memory, investigated in the Barnes maze test (Daun et al., 2020), MWM test (Sprowles et al., 2021), or in the left–right discrimination test (Martín-Sánchez et al., 2021). Although seemingly conflicting, these results are noteworthy because those cognitive alterations may be age-dependent.

    • Determinants of Addiction: Neurobiological, Behavioral, Cognitive, and Sociocultural Factors

      2022, Determinants of Addiction: Neurobiological, Behavioral, Cognitive, and Sociocultural Factors
    View full text