Mature astrocytes transform into transitional radial glia within adult mouse neocortex that supports directed migration of transplanted immature neurons.

Neuronal migration is an essential step in normal mammalian neocortical development, and the expression of defined cellular and molecular signals within the developing cortical microenvironment is likely crucial to this process. Therapy via transplanted or manipulated endogenous precursors for diseases which involve neuronal loss may depend critically on whether newly incorporated cells can actively migrate to repopulate areas of neuronal loss within the adult brain. Previous studies demonstrated that embryonic neurons and multipotent precursors transplanted into the neocortex of adult mice undergoing targeted apoptosis of pyramidal neurons migrate long distances into neuron-deficient regions, undergo directed differentiation, accept afferent synaptic input, and make appropriate long-distance projections. The experiments presented here: (1) use time-lapse digital confocal imaging of neuronal migration in living slice cultures to assess cellular mechanisms utilized by immature neurons during such long distance migration, and (2) identify changes within the host cortical astroglial population that may contribute to this migration. Prelabeled embryonic day 17 mouse neocortical neurons were transplanted into adult mouse primary somatosensory cortex undergoing targeted apoptotic degeneration of callosal projection neurons. Four to 7 days following transplantation, living slice cultures containing the region of transplanted cells were prepared and observed. Sequential time-lapse images were recorded using a video-based digital confocal microscope. Transplanted cells displayed bipolar morphologies characteristic of migrating neuroblasts and moved in a saltatory manner with mean rates of up to 14 microm/h. To investigate whether a permissive glial phenotype may provide a potential substrate for this directed form of neuronal migration, slice cultures were immunostained with the RC2 monoclonal antibody, which identifies radial glia that act as a substrate for neuronal migration during corticogenesis. RC2 does not label mature stellate astrocytes, which express glial fibrillary acidic protein (GFAP). RC2 expression was observed in glial cells closely apposed to migrating donor neurons within the slice cultures. The timing and specificity of RC2 expression was examined immunocytochemically at various times following transplantation. RC2 immunostaining within regions of neuronal degeneration was transient, with peak staining between 3 and 7 days following transplantation. Strongly RC2-immunoreactive cells that did not express GFAP were found within these regions, but not in distant cortical regions or within control brains. RC2-positive cells were identified in recipient transgenic mice which express beta-galactosidase under a glial specific promoter. Coexpression of RC2 and beta-galactosidase identified these cells as host astroglia. These results demonstrate that adult cortical astrocytes retain the capacity to reexpress an earlier developmental phenotype that may partially underlie the observed active migration of transplanted neurons and neural precursors. Further understanding of these processes could allow directed migration of transplanted or endogenous precursors toward therapeutic cellular repopulation and complex circuit reconstruction in neocortex and other CNS regions.

Embryonic neurons transplanted to regions of targeted photolytic cell death in adult mouse somatosensory cortex re-form specific callosal projections.

In the neocortex, the effectiveness of potential transplantation therapy for diseases involving neuronal loss may depend upon whether donor neurons can reestablish the precise long-distance projections that form the basis of sensory, motor, and cognitive function. During corticogenesis, the formation of these connections is affected by tropic factors, extracellular matrix, structural pathways, and developmental cell death. Previous studies demonstrated that embryonic neurons and multipotent neural precursors transplanted into neocortex or mice undergoing photolytically induced, synchronous, apoptotic neuronal degeneration selectively migrate into these regions, where they differentiate into pyramidal neurons and accept afferent synaptic input. The experiments presented here assess whether embryonic neurons transplanted into regions of somatosensory cortex undergoing targeted cell death differentiate further and develop long-distance axons and whether this outgrowth is target specific. Neocortical neurons from Gestational Day 17 mouse embryos were dissociated, prelabeled with fluorescent nanospheres and a lipophilic dye (DiI or PKH), and transplanted into adult mouse primary somatosensory cortex (S1) undergoing apoptotic degeneration of callosal projection neurons. Donor neurons selectively migrated into and differentiated within regions of targeted neuronal death in lamina II/III over a 2-week period, in agreement with our prior studies. To detect possible projections made by donor neurons 2, 4, 6, 8, or 10 weeks following transplantation, the retrogradely transported dye fluorogold (FG) was stereotaxically injected into contralateral S1, ipsilateral secondary somatosensory cortex (S2), or ipsilateral thalamus. Ten weeks following transplantation, 21 +/- 5% of the labeled donor neurons were labeled by FG injections into contralateral S1, demonstrating that donor neurons sent projections to the distant area, the original target of host neurons undergoing photolytically induced cell death. No donor neurons were labeled with FG injections into ipsilateral S2 or thalamus, nearby targets of other subpopulations of neurons in S1. These data indicate that in the adult neocortex: (1) transplanted immature neurons are capable of extending long-distance projections between hemispheres through the mature white matter of the corpus callosum and (2) these projections are formed with specificity to replace projections by neurons undergoing synchronous degeneration. These experiments provide an experimental system with which to test factors affecting such outgrowth and connectivity. Taken together, these results suggest that the reconstruction and repair of cortical circuitry responsible for sensory, motor, or cognitive function may be possible in the mature neocortex, if donor neurons or precursor cells are provided with the correct combination of local and distant signals within an appropriately permissive host environment.