5,7-Dihydroxitryptamine toxicity to serotonergic neurons in serum free raphe cultures.
5,7-Dihydroxytryptamine (5,7-DHT), is an experimentally widely used selective serotonergic neurotoxin, though the mechanisms of toxicity remain to be fully elucidated. In the present study, we evaluated 5,7-dihydroxitryptamine (5,7-DHT) induced serotonergic neurotoxicity in foetal raphe serum free cultures from the rat. For this purpose, a model of foetal raphe serum free neuronal cultures from the rat was established, containing about 16% serotonergic neurons and studied up to 3 months. Two weeks old raphe cultures were exposed to the serotonergic neurotoxin 5,7-DHT (concentration range 10-100 microM) for 72 h, after which the medium was replaced and neurotoxicity was evaluated by immunocitochemistry 1 week later. Lactate dehydrogenase release into the medium, 72 h after exposure to 5,7-DHT, showed a concentration-dependent neurotoxicity. To access morphologically the serotonergic toxicity tryptophan hydroxylase (TPH) was used as a specific marker of these neurons. Immunocitochemistry using TPH antisera showed a concentration-dependent serotonergic neurotoxicity induced by 5,7-DHT. Serotonergic neurons showed the typical pattern of "pruning" accompanied by axon terminals and dendrites loss, which were either partial or total. The axotomy induced by the neurotoxin was morphologically characteristic of retrograde axonal degeneration. Fluoxetine (0.1 microM) pre-treatment reduced 5,7-DHT-induced serotonergic neurotoxicity. These results indicate that the mechanism by which 5,7-DHT-induces serotonergic neurotoxicity is, at least partially, dependent on the toxin uptake by the serotonin transporter. Finally, we have established a robust model of primary raphe neuronal culture to evaluate serotonergic neurons development and the mechanisms of toxicity involving this neuronal population.
REQUIMTE (Rede de Química e Tecnologia), Toxicology Department, Faculty of Pharmacy, University of Porto, Rua Aníbal Cunha 164, 4099-030 Porto, Portugal. joaoc@ufp.pt <joaoc@ufp.pt>
João Paulo
JP
Capela
Marion
M
Lautenschlager
Ulrich
U
Dirnagl
Maria Lourdes
ML
Bastos
Félix
F
Carvalho
Andreas
A
Meisel
D015116:5,7-Dihydroxytryptamine / Q000633:toxicity*; D000818:Animals; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004305:Dose-Response Relationship, Drug; D005473:Fluoxetine / Q000494:pharmacology; D018817:N-Methyl-3,4-methylenedioxyamphetamine / Q000633:toxicity; D009410:Nerve Degeneration; D009412:Nerve Fibers / Q000187:drug effects; D011903:Raphe Nuclei / Q000187:drug effects* / Q000473:pathology; D051381:Rats
D016895:Culture Media, Serum-Free; D005473:Fluoxetine; D015116:5,7-Dihydroxytryptamine; D018817:N-Methyl-3,4-methylenedioxyamphetamine