In VitroGeneration of Adult Rat Olfactory Sensory Neurons and Regulation of Maturation by Coculture with CNS Tissues

Abstract

Olfactory sensory neurons (OSNs) are continually generated throughout life. Although previous studies have examined neurogenesis in olfactory cell cultures derived from embryonic or newborn rodents, we demonstrate neurogenesis in cell cultures derived from adult rat tissues. Dissociated cells taken from adult rat nasal mucosal tissues (ANM cells) were plated onto a feeder layer of newborn rat cortical glia (astrocytes) in serum-free conditions. Immature OSNs (stained for neuron-specific tubulin, NST) increased in number between 1 and 5 d in vitro (DIV) and in mass thereafter. Mature OSN (stained for olfactory marker protein, OMP) numbers decreased between 1 and 5 DIV, then increased over 5 DIV values by 12 and 15 DIV. Pulse labeling with [3H]thymidine confirmed in vitro neurogenesis. To determine whether the target cells for OSNs, olfactory bulb (OB) neurons, provide trophic support, dissociated newborn rat OB cells were cocultured with ANM cells on glia. This resulted in greater numbers of OMP-positive (OMP+) neurons after 9 DIV than ANM-alone cultures. This neurotrophic effect was not OB specific. Addition of newborn rat cerebellar and embryonic rat ventral mesencephalic cells to ANM cells also increased OMP+ neurons, whereas addition of newborn rat cortical cells or controls (purified glia or fibroblasts) did not. Changes in numbers of dopaminergic neurons (stained for tyrosine hydroxylase), present in OB and VM cultures, did not correlate with OMP+ neuronal increases. Thus, cultures of adult rat OSNs demonstrate neurogenesis, and trophic/maturation support is variably provided by CNS neurons (and not glia).