Ventral Mesencephalic Grafts in the Neostriatum of the Weaver Mutant Mouse: Structural Molecule and Receptor Studies

Abstract

Mesencephalic cell suspensions were prepared from E12 wild-type (+/+) mouse embryos and stereotaxically implanted into the dorsal neostriatum of weaver mutant mice (wv/wv), which have a genetic mesostriatal dopamine (DA) deficiency. Survival of DA neurons in the grafts was documented by tyrosine hydroxylase (TH) immunocytochemistry. Axon growth was monitored by immunocytochemistry using a battery of antibody markers, and the cellular localization of structural protein and receptor RNA transcripts was studied by in situ hybridization histochemistry using [32P]oigo-nucleotide probes. The cellw suspension grafts exhibited strong immunoreactivity for neural cell adhesion molecule (N-CAM), growth-associated phosphoprotein GAP-43, micro-tubule-associated protein 2 (MAP2), β-amyloid protein precursor (βAPP), and phosphorylated neurofilament epitopes (clone SMI-31); intermediate-to-high levels of immunoreactivity were seen for synaptophysin. High levels of hybridization were found in the grafts for the RNA transcripts of GAP-43, MAP2, and isoforms βAPP₆₉₅, βAPP₇₁₄ and βAPP₇₅₁ of the βAPP. No hybridization signal was detected in the grafts for DA D₂ or neurotensin receptor mRNAs, both of which are normally expressed by nigral DA neurons. DA receptor autoradiography using the D₂/D₃ agonist [3H]CV 205-502 as a ligand showed no binding in the transplants, indicating an apparent abnormality of grafted cells; neurotensin binding sites, labeled with [125I]neurotensin, were visualized in the suspensions, indicating the possibility that receptors could be present but that RNA message levels might be too low to allow detection. These findings offer a molecular correlate of axonal, dendritic and structural protein expression by transplanted mesencephalic neurons; further, they suggest that specific functional properties of grafted nigral cells are maintained after transplantation, while other aspects of their cellular biology may be compromised.