Elsevier

Neuroscience

Volume 165, Issue 4, 17 February 2010, Pages 1353-1369
Neuroscience

Neuropharmacology
Research Paper
Lithium prevents excitotoxic cell death of motoneurons in organotypic slice cultures of spinal cord

https://doi.org/10.1016/j.neuroscience.2009.11.034Get rights and content

Abstract

Several studies have reported the neuroprotective effects of lithium (Li) suggesting its potential in the treatment of neurological disorders, among of them amyotrophic lateral sclerosis (ALS). Although the cause of motoneuron (MN) death in ALS remains unknown, there is evidence that glutamate-mediated excitotoxicity plays an important role. In the present study we used an organotypic culture system of chick embryo spinal cord to explore the presumptive neuroprotective effects of Li against kainate-induced excitotoxic MN death. We found that chronic treatment with Li prevented excitotoxic MN loss in a dose dependent manner and that this effect was mediated by the inhibition of glycogen synthase kinase-3β (GSK-3β) signaling pathway. This neuroprotective effect of Li was potentiated by a combined treatment with riluzole. Nevertheless, MNs rescued by Li displayed structural changes including accumulation of neurofilaments, disruption of the rough endoplasmic reticulum and free ribosome loss, and accumulation of large dense core vesicles and autophagic vacuoles. Accompanying these changes there was an increase in immunostaining for (a) phosphorylated neurofilaments, (b) calcitonin gene-related peptide (CGRP) and (c) the autophagic marker LC3. Chronic Li treatment also resulted in a reduction in the excitotoxin-induced rise in intracellular Ca2+ in MNs. In contrast to the neuroprotection against excitotoxicity, Li was not able to prevent normal programmed (apoptotic) MN death in the chick embryo when chronically administered in ovo. In conclusion, these results show that although Li is able to prevent excitotoxic MN death by targeting GSK-3β, this neuroprotective effect is associated with conspicuous cytopathological changes.

Section snippets

Embryos and in vivo pharmacological experiments

Fertilized chicken eggs (Hy-line white) were purchased from Avigan Terralta (Vinallop, Tarragona, Catalonia, Spain) and incubated in the laboratory at 37.5 °C and 60% relative humidity. At the time of sampling, embryos were staged according to the Hamburger and Hamilton (1951) stage series. All experimental protocols used were previously approved by the Committee for Animal Care and Use of the University of Lleida.

Some embryos on embryonic day (E) 10 were treated with increasing volumes (50–600

Chronic treatment with lithium does not prevent programmed cell death of developing MNs

To investigate whether Li is able to prevent naturally occurring, caspase-dependent, apoptosis of MNs that takes place during normal avian development (Li et al., 1998, Ayala et al., 1999), Li was administered in ovo from E6 to E10, the main period of developmental cell death of lumbar MNs in the chick. This process leads to about 50% reduction of the initially differentiated MN pool (Burek and Oppenheim, 1999). Embryos were treated with different volumes (50–600 μl) of 0.15 M LiCl solution.

Discussion

In addition to the well known beneficial effects of Li for the treatment of maniac-depressive disorders, this agent has also been reported to have neuroprotective actions in distinct in vivo and in vitro paradigms in which neuronal death has been induced by excitotoxic stimuli or potassium/serum deprivation (see for review Chuang et al., 2002). In the present study, excitotoxic cell death of mature MNs was induced in an organotypic culture system of chick embryo spinal cord by exposure to KA,

Acknowledgments

We thank Imma Montoliu for technical assistance, Dr. R.W. Oppenheim for editing and comments on an earlier version of the manuscript, and Drs. Y. Uchiyama and M. Shibata for providing the anti-LC3 antibody. This work was supported by grants from the Ministerio de Ciencia y Tecnología financed jointly with FEDER (SAF2006-00619; SAF2005-01535) and the Fundació La Marató de TV3 (063030).

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