Dopaminergic neurotoxins require excitotoxic stimulation in organotypic cultures

• Published in: Neurobiology of disease Vol. 20; no. 3; pp. 639 - 645
• Main Authors: Kress, Geraldine J., Reynolds, Ian J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2005; Elsevier
• Subjects: 1-Methyl-4-phenylpyridinium - pharmacology; Animals; Animals, Newborn; Brain - metabolism; Brain - physiopathology; Cell Death - drug effects; Cell Death - physiology; Cell Nucleus - drug effects; Cell Nucleus - pathology; Cerebral Cortex - metabolism; Cerebral Cortex - physiopathology; Corpus Striatum - metabolism; Corpus Striatum - physiopathology; Dopamine - metabolism; Drug Synergism; Excitotoxicity; Glutamic Acid - metabolism; N-Methylaspartate - pharmacology; Nerve Degeneration - chemically induced; Nerve Degeneration - metabolism; Nerve Degeneration - physiopathology; Neurons - drug effects; Neurons - metabolism; Neurons - pathology; Neurotoxins - metabolism; Neurotoxins - pharmacology; NMDA receptors; Organ Culture Techniques; Organotypic culture; Oxidopamine - pharmacology; Parkinson Disease - metabolism; Parkinson Disease - physiopathology; Parkinson's disease; Rats; Rats, Sprague-Dawley; Rotenone - pharmacology; Substantia nigra; Substantia Nigra - metabolism; Substantia Nigra - physiopathology; Tyrosine 3-Monooxygenase - metabolism; Tyrosine hydroxylase; Tyrosine hydroxylase; Parkinson's disease; NMDA receptors; Excitotoxicity; Substantia nigra; Organotypic culture
• ISSN: 0969-9961; 1095-953X
• DOI: 10.1016/j.nbd.2005.04.019

We have investigated the properties of the dopaminergic neurotoxins 6-hydroxydopamine, 1-methyl-4-phenylpyridinium and rotenone using an organotypic culture that included slices of substantia nigra, striatum and cortex maintained for about 20 days in vitro. At this age, the organotypic culture contains dopaminergic neurons, visualized using tyrosine hydroxylase (TH) immunohistochemistry, that project into the striatal slice and extend up to 1 mm into the cortical slice. Using TH immunohistochemistry to assess survival of dopaminergic neurons, we found that the three dopaminergic toxins alone were not selectively neurotoxic. However, the addition of a low concentration of N-methyl-d-aspartate together with each individual toxin resulted in profound injury to the dopaminergic neurons, reflected by the loss of cell bodies and the fragmentation of processes. The combined toxicity was completely blocked by MK801. To assess the specificity of the injury, we measured the diameter of cell nuclei in the organotypic culture stained with Hoechst 33342 because the nucleus shrinks when neurons are injured. These measurements showed that the combined toxin treatment selectively injured only the TH immunoreactive cells. Thus, in a model culture system where dopaminergic neurons innervate appropriate targets, excitotoxicity appears to be essential for the manifestation of the toxic actions of 6-hydroxydopamine, 1-methyl-4-phenylpyridinium and rotenone.