Functional N-methyl-D-aspartate receptors develop and persist in heterotopic cortical grafts and regulate expression of transcription factor KROX-24

Abstract

N-Methyl-D-aspartate (NMDA) receptors are a major subfamily of glutamate receptors and thought to play a pivotal role in developmental plasticity and synaptogenesis, neuronal migration and differentiation. NMDA receptors have also been implicated in neuronal degeneration, as glutamate binding to the receptor initiates rapid excitotoxic signal transduction. Molecular cloning of cDNAs has yielded different NMDA receptor subtypes with an essential NR1 subunit associated with various modulatory NR2 subunits. The NR1 gene is expressed at high levels in virtually all brain structures, but to a distinctly higher extent in cortex than in striatum. Here we report on the development, maintenance and function of glutamate receptors in intrastriatally located cortical grafts. Cortical primordia of rat fetuses (El4) were stereotactically grafted into the rostral striatum of adult recipient rats. Expression of NR1 mRNA was examined by in situ hybridization after post transplantation periods of 2, 6 and 12 months. Analysis of NR1 mRNA expression in grafts after a differentiation period of 2 months revealed equal levels compared to the intact neocortex of the host rats and that of rats with the same ontogenetic age. No downregulation of NR1 mRNA was seen 6 and 12 months after transplantation. To ensure normal function of NMDA receptors in grafts, we studied the effects of a blockade of receptor dependent gene expression, using Krox-24 as a reporter gene. In normal brain tissue, constitutive expression of KROX-24 protein is thought to be maintained by NMDA receptor mediated physiological synaptic activity and can be abolished by the non-competitive NMDA receptor antagonist MK-801. Immunostaining of KROX-24 protein was almost identical in grafts compared to the corresponding neocortex. This constitutive expression of KROX-24 could be abolished by treatment with MK-801. Thus, our data indicate normal development and long term persistence of glutamate receptors with intrinsic excitatory activity in transplants.