Serial changes in granuloprival cerebellar cultures after transplantation with granule cells and glia: A timed ultrastructural study

Abstract

Granuloprival cerebellar cultures derived from neonatal mice were transplanted at nine days in vitro with granule cells and glia, and the changes induced in the host explants were examined daily with the electron microscope from one to nine days post-transplantation. Granule cells and astrocytes had migrated into the host cultures within 24 h, and astrocytic processes began to ensheath Purkinje cells and to interpose themselves between axon terminals and Purkinje cell somata, reducing the number of axosomatic synapses. Occasional degenerating Purkinje cells were present. At two days post-transplantation, synapse formation between parallel fibre terminals and Purkinje cell dendritic spines was initially evident, and Purkinje cells began to proliferate dendritic spines near astrocytic processes. Degenerating Purkinje cells were more frequently encountered. Myelin was first observed in host cultures at three days after transplantation, and astrocytes continued to ensheath Purkinje cells and reduce the population of axosomatic synapses, a process that began to stabilize at four days post-transplantation. At this time astrocytic ensheathment had extended to Purkinje cell dendrites and dendritic spine synapses. Proliferation of Purkinje cell dendritic spines accelerated, and occasional synapses with presumptive parallel fibre terminals were present among clusters of proliferated spines. At five days after transplantation, contours of Purkinje cells were rounded, and there was a decrease of somatic spines and of synapses with somatic spines. Purkinje cells were fully ensheathed by astrocytic processes by six days post-transplantation and had assumed a mature appearance. Homotypical parallel fibre-Purkinje cell dendritic spine synapses were predominant in more developed areas of cortical neuropil as heterotypical recurrent axon collateral-Purkinje cell dendritic spine synapses were reduced. Increasing synapse formation was evident among clusters of proliferated spines, which continued at seven days post-transplantation, as the spine clusters became less frequent. At eight days after transplantation, space between Purkinje cells had increased and the cortical neuropil resembled that of comparably aged control cultures. Occasional degenerating Purkinje cells were still evident at nine days post-transplantation, at which time residual clusters of proliferated unattached dendritic spines were scarce. The sequence of changes after transplantation was consistent with the specific roles of the transplanted elements. Astrocytes were involved with the regulation of synapse density, including reduction of some heterotypical synapses, and induced proliferation of Purkinje cell dendritic spines. Granule cell axons synapsed with Purkinje cell dendritic spines, further reducing heterotypical synapses and restoring cortical circuitry to a near control state. The loss of heterotypical synapses was associated with programmed cell death of excess Purkinje cells, reducing the Purkinje cell population to control levels.