ABSTRACT
Nitric oxide has been investigated widely both during neurodevelopment and in neurological diseases. However, whilst it has been established that nitric oxide-producing enzymes of nitric oxide synthase family are expressed in cerebellar Purkinje neurons, the effects of nitric oxide on the viability and morphology of these neurons remain unknown. Here, we have demonstrated that the activity of neuronal nitric oxide synthase, but not the inducible or endothelial forms of this enzyme, is required to support the survival of a proportion of cerebellar Purkinje neurons in vitro. We discovered that donation of high concentrations of exogenous nitric oxide reduces Purkinje neuron survival in culture and that peroxynitrite is also toxic to these cells. Finally, we demonstrated that exogenous nitric oxide and peroxynitrite reduce both the magnitude and the complexity of the neurite arbour extended by cerebellar Purkinje neurons. Taken together, these findings reveal that whilst a low level of endogenous nitric oxide, released by the activity of neuronal nitric oxide synthase, is beneficial to cerebellar Purkinje neurons in vitro, high levels of exogenous nitric oxide and peroxynitrite are detrimental to both the survival of these neurons and to their ability to extend processes and form functional neural networks.
Subject(s)
Neurites/drug effects , Nitric Oxide/toxicity , Purkinje Cells/drug effects , Animals , Benzoates/toxicity , Cell Count , Cells, Cultured/drug effects , Cells, Cultured/enzymology , Cells, Cultured/ultrastructure , Cerebellar Cortex/cytology , Cerebellar Cortex/embryology , Citrulline/analogs & derivatives , Citrulline/toxicity , Female , Imidazoles/toxicity , Isothiuronium/analogs & derivatives , Isothiuronium/toxicity , Mice , Mice, Inbred C57BL , Nerve Degeneration , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/physiology , Neurites/ultrastructure , Nitric Oxide/pharmacology , Nitric Oxide Donors/toxicity , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/physiology , Nitroso Compounds/toxicity , Peroxynitrous Acid/toxicity , Pregnancy , Purkinje Cells/enzymology , Purkinje Cells/ultrastructure , Reactive Nitrogen Species/toxicity , Thiourea/analogs & derivatives , Thiourea/toxicityABSTRACT
Nitric oxide is a diffusible messenger that plays a multitude of roles within the nervous system including modulation of cell viability. However, its role in regulating neuronal survival during a defined period of neurodevelopment has never been investigated. We discovered that expression of the messenger RNA for both neuronal and endothelial nitric oxide synthase increased in the early postnatal period in the cerebellum in vivo, whilst the expression of inducible nitric oxide synthase remained constant throughout this time in development. Whilst scavenging of nitric oxide was deleterious to the survival of early postnatal cerebellar granule neurons in vitro, this effect was lost in cultures derived at increasing postnatal ages. Conversely, sensitivity to exogenous nitric oxide increased with advancing postnatal age. Thus, we have shown that as postnatal development proceeds, cerebellar granule cells alter their in vitro survival responses to both nitric oxide inhibition and donation, revealing that the nitric oxide's effects on developing neurons vary with the stage of development studied. These findings have important consequences for our understanding of the role of nitric oxide during neuronal development.
