Acetylcholine induces neuritic outgrowth in rat primary olfactory bulb cultures

Abstract

The rat olfactory bulb is innervated by basal forebrain cholinergic neurons and is endowed with both nicotinic and muscarinic receptors. The development of this centrifugal cholinergic innervation occurs mainly in early postnatal stages. This developmental time-course and the demonstration that acetylcholine can modulate some aspects of neuronal proliferation, differentiation or death, suggests the possible involvement of cholinergic afferents in the morphogenesis and/or plasticity of the olfactory bulb. The purpose of the present work was to assess whether acetylcholine could modulate neuronal morphogenesis in the olfactory bulb. Toward this aim, we developed a primary culture model of rat olfactory bulbs. Three major cell types were identified on the basis of their morphological and immunocytochemical phenotype: neuronal-shaped cells expressing the neuronal markers neuron specific enolase, microtubule associated protein 2, neural cell adhesion molecule and β-tubulin III; glial-like cells immunoreactive for glial fibrillary acidic protein and flattened cells immunolabelled with antibodies against β-tubulin III and nestin, most likely neuronal precursors. After three to six days of treatment with 100-μM carbachol, a cholinergic agonist, significant increase in neuritic length was observed in cultured olfactory bulb neurons. The neurite outgrowth effect of carbachol was abolished by co-treatment with 1 μM α-bungarotoxin, an α7 subunit nicotinic receptor antagonist, but was not affected by the addition of 10 μM atropine, a general muscarinic antagonist. The effect of carbachol was also mimicked by the nicotinic agonists, nicotine (100 μM) and epibatidine (10 μM). This pharmacological profile suggested the involvement of nicotinic receptors of the α7-like subtype as confirmed using 125I-α-bungarotoxin receptor autoradiography. Taken together, these data argue for a role for nicotinic receptors in neuritic outgrowth in the rat olfactory bulb and provide a cellular support to the previously described effects of acetylcholine on olfactory bulb plasticity in vivo.