Elsevier

Behavioural Brain Research

Volume 197, Issue 2, 11 February 2009, Pages 292-300
Behavioural Brain Research

Research report
Repeated stress in combination with pyridostigmine: Part II: Changes in cerebral gene expression

https://doi.org/10.1016/j.bbr.2008.08.032Get rights and content

Abstract

Organophosphates (OP) represent a potential threat in terrorism or during military conflicts. Due to its faculty to protect cholinesterase (ChE) activity against irreversible inactivation by OP, pyridostigmine bromide (PB) was used as a prophylaxis treatment during the first Persian Gulf War. To explain dysfunctions reported by Gulf War Veterans (GWV), it was suggested a potentiation of the operational stress effects by PB given to soldiers. Our companion paper (see part 1 in the same journal issue) describes that PB treatment administered in repeated stress conditions results in long-term perturbations of learning and social behaviour. The present paper examines, in adult male Wistar rats, consequences of the association of repeated stress and PB treatment on gene expression in hypothalamus and hippocampus. PB treatment (1.5 mg/kg/day) was orally administered 30 min before each stress session to inhibit 40% of blood ChE as recommended by NATO. 10 days of stress alone induce a decrease in hypothalamic Il-1α expression. Treatment with PB alone increases mineralocorticoid receptor expression in hypothalamus which means that PB may thus modify stress perception by animals. Stressed-PB animals showed increase in hippocampal expression of BDNF, TrkB and CamKIIα, three genes implicated in memory development. As a supplement to previous studies showing behavioural and biochemical effects of the association of stress with PB, our data reveal that behavioural effects of this association may be linked with genomic changes in hippocampus. Mechanisms underlying these modifications and their link with memory disturbances reported by GWV remain to be further determined.

Introduction

Organophosphates (OP) are the most frequently used insecticides in the world. These compounds irreversibly inactivate cholinesterase (ChE) activity which is essential to nerve function. Moreover, OP, like soman or sarin, represent a potential threat in terrorism or during military conflicts. Due to its faculty to protect ChE from inactivation by OP [45], pyridostigmine bromide (PB) was proposed as a pretreatment against OP poisoning. Indeed, Dirnhuber and collaborators [21] demonstrated that administration of PB (0.2 mg/kg iv) prevents soman-induced death in primates. PB is widely used in the treatment of myasthenia gravis (Mestinon®, 360 mg/day) but was also employed as a pretreatment during the first Persian Gulf War (PGW) (PB 30 mg per os, three times a day) [41], [84]. Since their return from the first PGW, some Gulf War veterans (GWV) have reported various symptoms designated as “Gulf War Illness” [50], [62]. Among these symptoms, GWV reported memory disturbances, aggressiveness, fatigue, anxiety and musculoskeletal disorders. Recently, epidemiological studies were conducted to evaluate prevalence of symptoms in GWV. These studies demonstrated in particular hypothalamo–pituitary–adrenal (HPA) axis disorder [30] and increased risk for development of chronic widespread pain and fatigue [24]. In combination with stressor environment, PB was suspected of leading to side effects in the central nervous system (CNS). In this view, our companion paper [49] presents evidence that PB pretreatment associated with chronic stress procedure induces learning dysfunctions (see part 1). In addition a lot of experimental works demonstrated that PB treatment could result in an amplification of stress effects in rodents [25], [40], [80], [79]. However, similar studies failed to find such effects [48], [36], [39], [75]. Moreover epidemiological studies [42], [77] also suggest that the association of PB with other factors such as stress could explain some symptoms reported by GWV. As an anticholinesterase drug, PB induces an important cholinergic tone as a result of accumulation of ACh at synapses [74]. Thus, submitted to a stress situation, soldiers treated per os with PB may differently react than non-treated veterans. This hypothesis is sustained by findings from Taysse and co-workers [79] who demonstrated that in mice, peripheral administration of PB associated with stress procedure leads to functional changes in neurons and may affect CNS controlled functions.

The aim of our work was to determine in two rat brain areas the effects on gene expression of (i) a stress procedure, (ii) a repeated PB pretreatment and (iii) the association of PB pretreatment with repeated stress. The cerebral areas studied were hippocampus, a structure particularly sensitive to stress [61], which plays important role in learning and memory [29], [43], [47] and hypothalamus because of its relationships with the HPA axis, a neuroendocrine system involved in the stress response [82], [18]. Genes known to be either (i) affected by stress, (ii) implicated in learning and memory functions or (iii) linked to the activation of the HPA axis were selected. Thus, expression of heat shock protein 70 (Hsp70) [38], [54] and inhibitory-κB (IκB) [58], [11] the natural inhibitor of nuclear factor kappa B (NF-κB) were assessed as stress markers. The expression of brain-derived neurotrophic factor (BDNF), of its intracellular kinase-activating receptor, tropomyosin-related kinase B (TrkB) [46] and of calcium/calmodulin-protein kinase II alpha (CamKIIα) [88] was also measured because of their implication in learning and memory processes. Expression of interleukin-1 (Il-1) was assessed because of its contribution to the regulation of memory processes within hippocampus [4], [20] and its implication in the activation of HPA axis [89], [34]. Effects of stress and PB treatment were also evaluated on the expression of both glucocorticoid receptors (GR) and mineralocorticoid receptors (MR) as major effectors of stress response during HPA axis activation.

Section snippets

Chemicals

Pyridostigmine bromide (PB: 3-dimethylamino carbonyloxy-N-methylpiridinium bromide) was purchased from Sigma Chemical (Saint-Quentin Fallavier, France). PB was dissolved in water and freshly prepared on the day of its administration. All other chemicals used in this study were obtained as specified.

Subjects

Animals used in the present work were 4-month-old male Wistar rats (Harlan, Gannat, France). The animals were housed four per cage on a 12 h dark/light cycle. They were given food and water ad libitum

PCA test

Fig. 1 presents the number of trials per PCA session for each day and each subgroup of animals. There was a significant effect of both subgroups (F(3, 28) = 45.89, p  0.001) and day (F(9, 252) = 6.083, p  0.001) on the number of trials and a significant subgroups × day interaction (F(27, 252) = 2.291, p  0.001). A day-by-day analysis was therefore performed. Newman–Keuls analysis showed that stressed animals presented a significant decrease in the number of trials per session for each delay (p  0.001,

Discussion

To our knowledge, no study has been performed to analyse the effects of a combination of stress and PB pretreatment on gene expression in rats. The present study evidences that: (i) repeated PCA test decreases Il-1α expression, (ii) PB treatment, by increasing MR expression, modifies MR/GR balance in hypothalamus, suggesting a different stress response in treated animals and (iii) association of stress with PB treatment activates expression of genes implicated in learning and memory in

Acknowledgements

We are indebted to Mr D. Coulon for his help in technical assistance. We thank technical staff of the medical analyses lab in CRSSA for ChE determination, particularly Mrs. J. Denis and Mrs. V. Leroux. We are very grateful to Dr G. Lallement and Dr P. Filliat for their helpful suggestions in the redaction of this manuscript.

This work was supported by grants from the French Service de Santé des Armées (fellowship to Dr L. Barbier) and the Délégation Générale pour l’Armement (contract 05co017-05).

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