Limbic system activation is affected by prenatal predator exposure and postnatal environmental enrichment and further moderated by dam and sex

Highlights

• A novel prenatal stressor increased dam glucocorticoids and decreased pup birth weight.

• Pup body weight remained significantly decreased at two weeks of age.

• Postnatal environmental enrichment restored body weight and had effects on seizure.

• Early life manipulations exerted effects on limbic system activity.

• Sex- and dam-dependent effects for seizure and FosB immunoreactivity.

Abstract

Epilepsy is a relatively common and chronic neurological condition, affecting 1–2% of the population. However, understanding of the underlying pathophysiology remains incomplete. To identify potential factors in the early environment that may increase the risk for experiencing seizures, maternal stress and environmental enrichment (EE) were utilized. Pregnant Long-Evans rats were exposed to an ethologically relevant predator stress (PS) and maternal glucocorticoid (GC) response was assessed across the exposure period. At birth, litters were divided into standard care (SC) and EE groups until postnatal day 14 (PD14) when a model of febrile convulsions was used to determine seizure susceptibility of the various groups. Pup brains were then processed for immunohistochemical detection of FosB from several structures in the limbic system as a measure of neuronal activation. Maternal PS-induced GC levels were elevated early in the exposure period, and pup birth weights, in both sexes, were lower in litters from dams exposed to PS. Seizure scores at PD14 were highly individualized and litter dependent, suggesting a dam-dependent and variable effect of controlled pre- and postnatal environmental factors. Further, analysis of FosB-immunoreactive (-ir) patterns revealed an activity dependent distribution, reflecting individual seizure susceptibility. EE had a varying effect on FosB-ir that was dependent on region. In the hippocampus FosB-ir levels were greater in the EE groups while extra-hippocampal regions showed lower levels of FosB-ir. Our results support the concept that pre- and postnatal environmental influences affect fetal programming and neurodevelopment of processes that could underlie seizure susceptibility, but that the magnitude of these effects appears to be dam- or litter-dependent.

Introduction

Epilepsy is a relatively common neurological disorder, which affects people of all ages [1], with one of the highest incidence of new onset seizures occurring within the first few years of life [2]. The etiology underlying the development of epilepsy during early life can be broadly grouped as symptomatic (hypoxic–ischemic encephalopathy, tumour, trauma), genetic (channelopathies, neurodevelopmental diseases), and cryptogenic (etiology unknown) [3], [4]. In cryptogenic epilepsies the lack of diagnostic yield (approximately 50–60% of all people with epilepsy have no identified etiology) despite considerable investigation suggests a problem at the molecular or ultra-structural levels [3]. To gain a better understanding of the factors leading to the development of epilepsy, an appreciation of the pathophysiology underlying an increased susceptibility for having seizures is required. One potential approach, which is consistent with the high incidence of first seizures occurring during early life, is to examine factors affecting neurodevelopment and brain organization.

Studies continue to demonstrate an association between an adverse maternal environment and a risk of developing disorders of brain function such as schizophrenia [5], [6], [7], anxiety [8], [9], [10], and attention deficit disorders [11], [12], [13]. Although less conspicuous than a teratogen, maternal stress is common and has diverse and long-lasting effects on neurodevelopmental of offspring in a variety of species [9]. Further, the high rate of co-morbid association between epilepsy and neuropsychiatric, as well as neurodevelopmental disorders [14], [15], [16], which also have no identified diagnostic etiology, warrants further investigation in terms of a link between prenatal stress, neurodevelopment and seizure susceptibility.

Previous studies have demonstrated that repeated restraint stress during the last week of gestation lowers the after-discharge threshold and increases the kindling rates in postnatal day (PD) 14 male and female pups, and in adult males [17]. In addition, maternal prenatal stress increases the severity and the risk of mortality, particularly in male pups, in pilocarpine-induced seizures, [18], and the severity of evoked seizures in a model of febrile convulsions [19]. It is important to note, however, that the effects of prenatal stress on neurodevelopmental disorders is highly mutable, and can be influenced by a number of factors such as maternal genotype [20], [21], offspring sex [22], [23], exposure timing [24], and the intensity of the stressor [25].

Not surprisingly, the type of stressor is extremely important and significant differences have been documented in the neural and behavioural responses to physical and psychosocial stressors [26]. This is particularly notable in prenatal stress paradigms [27]. Most prior studies [17], [18], [19] have used physical stressors such as restraint but it can be argued that psychosocial stressors are more representative of ‘natural’ stress exposure, and perhaps also a better model of typical stressors experienced by pregnant women. Predator exposure represents an excellent example of a psychosocial stressor that has been used successfully to increase anxiety behaviour in animal models using tests such as social interaction, elevated plus maze, or the hole board (reviewed in [28]). In the present study, we examined whether exposure of pregnant Long-Evans rat dams to a predator threat (live cat exposure), modified the vulnerability to, and characteristics of, induced convulsions in the offspring. We also determined whether prenatal stress modifies the pattern of seizure-induced neuronal activation, measured by the immediate early gene FosB, specifically within the limbic system and the HPA-axis. FosB has been used extensively to measure neuronal activation following seizure and large effects have been demonstrated in the hippocampus, hypothalamus, and amygdala at time-points ranging from 18 h to 36 h following seizure [29], [30], [31].

In addition to determining the risk factors that may increase seizure susceptibility, there is significant clinical interest in identifying factors that may ameliorate these deleterious influences. One of the more commonly recognized influences on neurodevelopment is environmental enrichment (EE) [32], [33]. In adult rats, EE has been shown to prevent or delay the development of seizures [34], [35], [36], as well as prevent the development of depressive symptoms after seizures early in life [37]. Most research to date in this area has focused on prevention in normal adults, or on recovery from initial status epilepticus in normal adults (reviewed in [38]), while fewer studies have focused on an earlier prevention/intervention. However, EE is known to have a number of effects on hippocampal neurogenesis, including recovering the capacity for long-term potentiation following prenatal stress [39] and recovering deficits induced by prenatal stress [40] in early life. Many of these factors are also involved in the development of seizures, which suggests that there may be a role for EE before the need for recovery. In this study, we investigated the role of EE from birth in reducing/preventing vulnerability to initial febrile convulsions, both as a stand-alone treatment and as a potential ‘rescue’ measure to prevent changes induced by prenatal stress.