We used a model system in which dopaminergic (DA) neurons from embryonic rat mesencephalon undergo spontaneous and selective degeneration as they develop in culture. Here, we show that DA cell loss can be prevented efficiently by low concentrations of the Na ϩ channel agonist veratridine. The survival promoting effect of veratridine was reproduced by, but independent of, glial cell line-derived neurotrophic factor. Neuroprotection by veratridine was exquisitely specific to DA neurons, short-lived after withdrawal, and abolished by tetrodotoxin, indicating that activation of voltage-gated Na ϩ channels was crucially involved. Calcium measurements showed that veratridine-induced Na ϩ influx was necessary to maintain intracellular Ca 2ϩ within a neuroprotective range through the stimulation of low-voltage activated T-type calcium channels, a mechanism that was distinct from that elicited by high K ϩ -evoked depolarization. Interestingly, increasing neuronal excitability by treatment with apamin, an inhibitor of Ca 2ϩ -activated K ϩ channels, or with ouabain, a blocker of the Na ϩ /K ϩ -ATPase pump, was also neuroprotective by a mechanism involving T-type calcium channel activation. These results support the idea that mesencephalic DA neurons depend primarily on excitatory input for their survival during development.

We have developed a model system in which rat basal forebrain cholinergic neurons degenerate progressively when maintained in culture conditions that make them susceptible to low-level oxidative stress. In this study, we showed that cholinergic neurons identified by acetylcholinesterase cytochemistry or choline acetyl transferase immunocytochemistry are rescued efficiently by the neurotransmitter noradrenaline (NA). The effect of NA required neither adrenoceptor activation nor intracellular accumulation. NA operated via a mechanism that precluded activation of a cell death pathway in which reactive oxygen species (ROS) and proapoptotic caspases were crucially involved. It is noteworthy that NA remained protective even when applied late in the degenerative process but before intracellular ROS began to increase. The high efficacy of iron chelators and catalase in preventing the death of cholinergic neurons in this model suggested that NA neutralized the effects of hydroxyl radicals produced through a Fenton-type reaction. Pyrocatechol [the diphenolic moiety of NA] was sufficient in itself to prevent ROS production and cholinergic cell demise, indicating that the catechol structure was instrumental for the neuroprotective function of NA. Therefore, the noncatecholic neurotransmitter GABA failed to prevent neurodegeneration. Nerve growth factor and brain derived neurotrophic factor, two trophic peptides for septal cholinergic neurons, did not afford protection by themselves and did not improve neuroprotection provided by NA. However, in the presence of NA, they both retained their efficacy to stimulate cholinergic parameters. These data indicate that NA-based therapeutic strategies may be of interest in such neurodegenerative conditions as Alzheimer's disease, where progressive cholinergic deficits occur.

We evaluated the neuroprotective potential of tachykinin peptides using a model system in which mesencephalic dopaminergic (DA) neurons die spontaneously and selectively as they mature. The three native tachykinins, substance P (SP), neurokinin (NK) A, and NKB afforded substantial protection against DA cell demise. The selective NK 1 receptor antagonist [D-Pro9,[spiro-␥-lactam] Leu10,Trp11]substance P (GR71251) was sufficient in itself to suppress the effect of SP, whereas a cotreatment with GR71251 and the NK 3 receptor antagonist (R)-N-[␣-(methoxycarbonyl)benzyl]-2-phenylquinoline-4-carboxamide (SB218795) was required to prevent the effects of both NKA and NKB. Consistent with these results, D-Ala-[LPro9,Me-Leu8]substance P(7-11) (GR73632), a selective agonist of NK 1 receptors and [pro7]-NKB, a selective agonist of NK 3 receptors, conferred protection to DA neurons, whereas (Lys3, Gly8-R-␥-lactam-Leu9)neurokinin A(3-10) (GR64349), which activates specifically NK 2 receptors, did not. DA neurons rescued by tachykinins accumulated [ 3 H]DA efficiently, which suggests that they were also totally functional. Neuroprotection by tachykinins was highly selective for DA neurons, rapidly reversed upon treatment withdrawal, and reproduced by but independent of glial cell line-derived neurotrophic factor. Survival promotion by tachykinins was abolished by blocking voltage-gated Na ϩ channels with tetrodotoxin or N-type voltagegated Ca 2ϩ channels with -conotoxin-MVIIA, which indicates that an increase in neuronal excitability was crucially involved in this effect. Together, these data further support the notion that the survival of mesencephalic DA neurons during development depends largely on excitatory inputs, which may be provided in part by tachykinins.

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