Establishment of primary cultures of rat olfactory bulb under serum-free conditions for studies of cellular injury

Abstract

Olfactory dysfunction has been implicated in various neurodegenerative diseases including Parkinson's and Alzheimer's disease but, despite intense interest in the neurobiology of the olfactory bulb (OB), studies of neurodegenerative mechanisms have not been attempted in primary OB cultures. This study was aimed at developing a primary OB culture under serum-free conditions in order to investigate injury and excitotoxicity in vitro. Olfactory bulbs from rat pups were rapidly trypsinised and mechanically dissociated and the resultant single cell suspension was centrifuged through a high bovine serum albumin concentration gradient to reduce cellular debris before being seeded in multi-well culture plates. Cells were plated in neurobasal medium containing 0.5 mM glutamine, 25 mM K⁺, 2% B27 and 10% fetal calf serum (FCS) for 24 h and, after 1 day in vitro (div1), were maintained without FCS. At div8, neurones exhibited extensive neuritic networks, were present as a monolayer and were mainly bipolar and immunopositive for γ-aminobutyric acid indicating that they were intrinsic OB neurones. At div8, neurones (positive for microtubule-associated protein-2, 73%) predominated over astrocytes (positive for glial fibrillary acidic protein, 27%). Cellular injury produced by staurosporine, hydrogen peroxide and kainate, when assessed by morphological and biochemical procedures, was shown to be concentration-dependent and significantly reduced the numbers of neurones and astrocytes. Further analyses of kainate-induced injury revealed the presence of TUNEL-positive cells (indicative of apoptosis) and increases in intracellular free calcium, both of which were antagonised by CNQX. Thus, the serum-free culture developed here is amenable to morphological and high throughput neurochemical analyses of mechanisms contributing to the injury of OB neurones in vitro.