Dividing neuronal precursor cells in the adult rat SVZ give rise to progeny which migrate rostrally into the OB to become the granule and periglomerular interneurons. The ORNs of the peripheral olfactory epithelium synapse upon the periglomerular neurons, as well as the mitral/tufted neurons of the OB. If the olfactory epithelium ablated postnatally, ORNs die and there is also a loss of granule neurons in the OB, suggesting that there may be a relationship between peripheral olfactory input and survival of SVZ-generated neurons in the OB. However, it is not known if re-innervation of the OB by ORNs is associated with a compensatory replacement of olfactory bulb interneurons. Methods for culturing SVZ cells from adult rats have been established, and the dependence of these neurons upon extrinsic factors for their survival in vitro has been demonstrated in the laboratory in which the Investigator is a postdoctoral fellow. In addition, an antibody marker directed against the Hu protein, which is useful for labeling SVZ-generated neurons, has also been characterized at least partially. The first specific aim will test the hypothesis that signals from ORNs regulate survival of OB interneurons generated by the SVZ. Cultures of neonatal rat olfactory epithelium will be prepared according to published methods, and plated onto monolayers of astrocytes harvested from adult SVZ explant outgrowths. Explants of adult SVZ, labeled in vivo with 3H- thymidine, will be cultured in the presence or absence of the olfactory epithelium cultures in a nutrient rich medium. In the second set of experiments, SVZ cultured alone or in co-culture with OE cells will be tested in the presence or absence of 20 ng/ml BDNF. Cultures will be fixed at 1, 4, and 8 weeks in vitro, immunostained for the neuron specific marker Hu and the ORN-specific marker Olfactory Marker Protein (OMP), and then processed for autoradiography to identify 3H-thymidine- labeled SVZ-derived neurons. Neurons that are double-labeled with anti- Hu and 3H-thymidine will be counted, as will OMP+ neurons for each unique combination of treatment condition and time point. The second specific aim will test the hypothesis that regeneration of ORNs and their reinnervation of the olfactory epithelium of adult rats according to published methods. Reinnervation of the OB is known to occur from 4-6 weeks following MBG treatment. Following MBG treatment, rats will be divided into 2 groups. Group 1 will be injected with 3H- thymidine 1 day prior to sacrifice at 1, 4, and 8 weeks following MBG exposure, and Group 2 will be injected at 1, 4, and 8 weeks following MGB exposure, but then sacrificed one month later. Brain tissue will be fixed and sectioned, immunostained for Hu antigen, and processed for autoradiography. Sections through both OB and SVZ will be analyzed. The average number of HU+ olfactory bulb neurons and the proportion of these which are labeled with 3H-thymidine will be determined at each time point after MBG exposure, in both injection paradigms. In SVZ sections, the total number of SVZ cells and the proportion of these which are 3H- thymidine+/Hu+ will be similarly assessed. Comparisons of the numbers of double-labeled cells in each condition will permit confirmation of previous reports showing the loss of olfactory bulb neurons following loss of ORN innervation of the bulb, and determination of whether proliferation and/or survival of SVZ-generated interneurons can be up- regulated following ORN recovery.
| Kirschenbaum, B; Doetsch, F; Lois, C et al. (1999) Adult subventricular zone neuronal precursors continue to proliferate and migrate in the absence of the olfactory bulb. J Neurosci 19:2171-80 |
