The goals of the proposed research are to understand the development of the opioid receptor and opioid systems. In order to simplify the multiplicity of influences exerted in the developing animal, the initial studies will be performed in a model cell line for differentiation, the PC12 rat pheochromocytoma cells. Subsequently, the information acquired in the cell line will be applied to the neonatal rat brain, specifically with respect to the development and differentiation of opioid systems in animals with drug-addicted mothers. Specifically: 1. In one subclone of PC12 cells, PC12h, low levels of opioid receptors markedly increase in response to nerve growth factor (NGF). The opioid receptor has been identified as delta, but no further characterization has been done. In this proposal, the receptor in PC12h cells will be further characterized with respect to its possible function, i.e., what are the consequences of opioid binding? 2. The possibility that NGF can induce opioid receptor expression in other subclones of PC12 cells will be explored in order to determine if PC12h cells are unique in this response to NGF or if NGF induction of opioid receptors is more universal. This relates to the question of the signals for gene expression in stem cells in the developing nervous system. 3. Once the response of receptors to opioids in PC12 cells has been established, the effect of opioids on differentiation will be examined. It is known that pre-natal morphine has numerous effects on development and differentiation. Thus, the following questions arise: Will opioids have any effect on NGF-induced differentiation in the cell line? Will opioids have a morphological or biochemical effect by themselves? 4. Recently, a novel molecule related to the neural cell adhesion molecule, N-CAM has been isolated which is related to opioid binding. The expression of this molecule, termed OB-CAM, will be examined in control and NGF treated cells, as it may be associated with the opioid receptors in PC12h cells. If it is found to be differentially expressed the molecule will be cloned from PC12h cells. 5. The level of G proteins, both mRNA and protein levels, will be examined in control and NGF treated PC12 cells as well as in opioid treated cells. These proteins are signalling molecules and probably have a role in the normal and drug-altered developing nervous system, which will be examined in the rat pup. 6. Are opioid peptides present in these cells? And if so, is their expression altered by NGF or opioid treatment? 7. In addition to examining the expression of specific molecules, such as OB-CAM and G proteins, it is a long term goal to clone molecules which are differentially expressed in control vs NGF treated PC12 cells. 8.
The final aims are to determine the role of NGF on opioid receptor development in neonatal rats as well as the effects of prenatal exposure to opioids on receptor development.
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| Abood, M E; Noel, M A; Farnsworth, J S et al. (1994) Molecular cloning and expression of a delta-opioid receptor from rat brain. J Neurosci Res 37:714-9 |
