Research reportRestraint as a stressor in mice:: Against the dopaminergic neurotoxicity of d-MDMA, low body weight mitigates restraint-induced hypothermia and consequent neuroprotection☆
Introduction
In research projects aimed at understanding the impact stress can have on various body systems and disease models, the perceived ease of application, wide involvement of higher and lower brain centers and activation of both the HPA-axis (release of CRH, ACTH and glucocorticoids) as well as the sympathetic nervous system make restraint a widely used manipulation [8], [15], [23]. In previous work we chose to use restraint to examine how alterations in homeostasis (i.e., stress) would affect the dopaminergic striatal neurotoxicity induced by the amphetamines just as others have shown that a variety of stressful manipulations (e.g, electric shock, alterations in caging, etc.) including restraint can alter the general lethality and toxicity of these agents as well as their activating and sensitizing properties [16], [30], [2], [3], [29], [6], [7].
Amphetamine, as well as its derivatives d-methamphetamine, d-methylenedioxyamphetamine (d-MDA) and d-methylenedioxymethamphetamine (d-MDMA), are striatal neurotoxicants as evidenced by marked increases in glial fibrillary acidic protein (GFAP), a biochemical measure of injury induced gliosis, as well as the presence of argyrophilia and long-term decrements in dopamine (DA), its metabolites and tyrosine hydroxylase (TH) protein. The decrements in DA-associated parameters indicate dopaminergic projections to the striatum appear to be the likely target [16], [21].
The actions of the amphetamines have long been linked to their thermoregulatory actions and at neurotoxic dosages elevated core body temperatures are observed. The degree of dopamine depletion and other indices of neural damage can be blocked or reduced by manipulations that will lower body temperature during the course of amphetamine administration. These observations confirm that body temperature plays some as yet undetermined role in their neurotoxic capabilities [16]. Restraint of the mouse results in body temperature reductions through factors that are not yet fully elucidated but include reduced motor activity [26]. In previous studies, restraint-induced hypothermia during the course of d-MDMA administration resulted in nearly complete protection from the dopamine-depleting effects of the drug [16].
In recent work examining the contribution of mouse strain, we included C57Bl6/J mice as a comparison strain in each experiment as this strain has been used extensively in our work documenting the d-MDMA neurotoxicity and the neuroprotection of restraint. Over the course of these experiments, we noted a wide range in the degree to which restraint could protect against the striatal dopamine depletions induced by a body weight-adjusted dose of d-MDMA, despite the same neurotoxic regimen, a consistent application of the restraint procedure and the utilization of the same restraint devices from our previous work. Mouse age and weight were the only two factors varied among the set of ten experiments, though held consistent within a given experiment. We hypothesized that one or both of these variables makes a marked contribution to the effects observed when utilizing restraint. The following report describes an in-depth analysis of the ways in which each affects restraint-induced body temperature reduction and consequent neuroprotection. Mouse weight appears to be the dominant factor in the neuroprotective effects induced by restraint of the C57Bl/6 female mouse against a body-weight adjusted neurotoxic dose of d-MDMA.
Section snippets
Materials
The following drugs and chemicals were kindly provided by or obtained from the sources indicated: high-performance liquid chromatography (HPLC) standards (Sigma Chemical, St. Louis, MO, USA); d-MDMA (Research Technology Branch, National Institute on Drug Abuse, Rockville, MD, USA). Reagents used for HPLC were of HPLC grade (ESA, Chelmsford, MA, USA).
Animals
All procedures were carried out according to protocols approved by the institutional Animal Care and Use Committee and in accordance with the NRC
Effects of d-MDMA, restraint and the combination thereof, on striatal dopamine in C57Bl6/J mice
d-MDMA administered to unrestrained female C57Bl6/J mice causes a reproducible decline in striatal dopamine. Over a series of ten individual experiments, with an accumulated sample size of 61 animals that received d-MDMA 20 mg/kg of mouse body weight, s.c. every 2 h for a total of four injections, the average dopamine decrement was 82%. That is, d-MDMA treated unrestrained mice exhibited striatal dopamine concentrations that were 18% of controls (saline, unrestrained) Table 1. The dopamine
Discussion
Our data replicate our previous work and show that restraint of the C57Bl/6J female mouse in 35-ml centrifuge tubes will reduce body temperature relative to baseline temperature and, provided sufficient reduction is achieved, will protect against the striatal dopamine depletion induced by the neurotoxic amphetamine d-MDMA [16], [17]. Our data also demonstrate that both the temperature reduction and hence the neuroprotection are markedly dependent on body weight despite the use of a body-weight
Acknowledgements
The authors gratefully acknowledge F.X. Ma, M. Graziani, M.A. Hammer, B. Billig and C. Felton for their expert technical assistance.
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This work was supported by the U.S. Department of Health and Human Services/Centers for Disease Control/National Institute for Occupational Safety and Health.