Maternal separation disrupts dendritic morphology of neurons in prefrontal cortex, hippocampus, and nucleus accumbens in male rat offspring
Introduction
Maternally deprived children have higher incidence to develop mood-related disorders, poor impulse control, and psychoses that persist throughout life (Mullen et al., 1996, Heim and Nemeroff, 2002, Bebbington et al., 2004, Freyd et al., 2005). Meanwhile, good parent–infant bonding has been shown to reduce such tendencies (Swain et al., 2007). Maternal manipulations during the early postnatal period may induce prolonged effects on the response to stress in rat offspring (for review see Meaney, 2001). In maternal separation (MS) and the substituted human handling (HH) paradigms, pups are separated from their mothers several times during a critical postnatal period. In HH paradigm, visual, tactile, auditory, and olfactory communication is possible between the siblings, whereas these sensory inputs are not available to rats subjected to maternal separation (D’Amato et al., 2005, Pascual and Zamora-León, 2007). Additionally, the effects of separation are time-length dependent (Lehmann and Feldon, 2000, Pryce and Feldon, 2003, Ladd et al., 2004). For example, MS for 3 h from postnatal day (PND) 2 to 12 leads to a heightened adrenocorticotropic hormone release after air-puff startle, and a decrease in the expression of the mRNA of glucocorticoid receptor in the hippocampus and frontal cortex (Plotsky and Meaney, 1993, Ladd et al., 2004). Moreover, MS in rats produces a stress hyper-reactivity, anxious behavior in the elevated plus-maze, anhedonia, increased ethanol consumption, and hyper-reactivity of the hypothalamic–pituitary–adrenal (HPA) axis in response to stress (Plotsky and Meaney, 1993, Francis and Meaney, 1999, Caldji et al., 2000, Newport et al., 2002, Plotsky et al., 2005, Kikusui and Mori, 2009, Rivarola and Suárez, 2009). These behavioral and endocrine dysfunctions persist throughout adulthood (Daniels et al., 2004, Macri and Würbel, 2006).
Recent data suggest that MS, performed prior to the stress hyporesponsive period (SHRP) of the HPA axis (PND1–3), induces a reduction in the dendritic spine density of the pyramidal neurons from layer 3 of prefrontal cortex (PFC) (Bock et al., 2005); whereas MS carried out during the SHRP (PND5–7) does not cause any changes in the dendritic morphology of the aforementioned region. In addition, the SHRP is characterized by low basal levels of stress hormones and a relative non-responsiveness to external stressors (Rosenfeld et al., 1992; Levine, 2002), and was proposed to protect the juvenile brain against the deteriorating effects of high levels of stress hormones (Meaney et al., 1991).
The hippocampus and the amygdala are interconnected with the medial PFC, and send the major excitatory projections to the nucleus accumbens (NAcc) (Heidbreder and Groenewegen, 2003). The PFC and hippocampus are involved in the regulation of HPA function (Sullivan and Gratton, 2002, Herman and Mueller, 2006), and dysfunction of the HPA axis has been associated with depression-related disorders (Holsboer, 2000). More importantly, long-term stress or repeated Corticosterone administration have been associated with altered dendritic arborization and spinogenesis of neurons of the corticolimbic circuit. Interestingly, rats with neonatal ventral hippocampus lesions exhibit locomotor hyperresponsiveness to stress (Flores et al., 1996, Silva-Gómez et al., 2003) with morphological changes such as atrophy of pyramidal neurons of the prefrontal cortex (PFC) and medium spiny neurons of the NAcc (Flores et al., 2005, Alquicer et al., 2008). Interestingly, a recent report using a maternal separation paradigm suggests that proteins related to oxidative stress regulation are changed in the CA1 ventral hippocampus (Marais et al., 2009). Therefore, these studies suggest an involvement of the CA1 ventral hippocampus region in stress. However, the effect of MS on the pyramidal cells of the ventral hippocampus and the medium spiny neurons of the NAcc remains elusive, since both structures, together with the PFC pyramidal neurons, play a critical role in emotional as well as learning and memory processes.
Therefore, the present study was conducted to examine the potential role of different maternal care conditions (MS, HH or undisturbed animal care) on the dendritic morphology of the PFC, CA1 ventral hippocampal pyramidal cells, and medium spiny neurons of the NAcc at two critical ages of development, pre-pubertal (PND35) and post-pubertal (PND60) age, using the Golgi-Cox staining method.
Section snippets
Experimental subjects
Pregnant Sprague–Dawley rats were obtained at gestational day 14–17 from our facilities (University of Puebla). The day of birth was designated as PND0. After birth, litters were culled to 12 pups (six males and six females) and housed together with their mother in standard rat cages (56 cm × 34 cm × 25 cm), in a room with controlled temperature (22 ± 2 °C), and a 12 h light–dark cycle (lights on at 7:00 am). Fresh drinking bottled water and food pellets (Harlan Teklad 2018S, Madison, Wisconsin) were
Locomotor activity
The effect of MS and HH on spontaneous locomotor activity in a novel environment is illustrated in Fig. 1. All animals (MC, MS, HH), initially showed enhanced locomotion, reflecting an active exploratory behavior in a novel environment. The locomotor activity then gradually declined in 40–60 min to a stable level (Fig. 1A and B). A two-way ANOVA analysis of the total locomotor activity data for the entire 120 min revealed that locomotion was significantly affected by age (F1, 54 = 24.23, P < 0.01)
Discussion
Disturbances in maternal care have been associated with mood-related disorders (for review see Champagne, 2008). However, the neurobiology of disrupted maternal care is not fully understood. In this study, we characterize three maternal care conditions that induce changes at two critical ages of neurodevelopment in key brain regions associated with mood-related disorders and in locomotor activity in novel environment. Before discussing the main findings of the present study, certain aspects of
Acknowledgements
This study was supported by grants from VIEP-BUAP (No. FLAG-SAL10-G) and CONACYT (No. 102327). We are grateful to Dr. Carlos Escamilla for his help and suggestions related to animal care. The authors wish to thanks Julio Cesar Morales-Medina for helpful comments and suggestions. EM and EHT are students of the BUAP. GF acknowledges the National Research System of Mexico for membership. Thanks to Mira Thakur and Stephanie Newton for editing the English language text.
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