Fredrick J. Seil

Astrocytes play a role in regulation of synaptic density

Exposure of neonatal cerebellar explants to cytosine arabinoside destroys granule cells and arrests surviving glia in an early stage of maturation. Purkinje cells lack astroglial ensheathment and are hyperinnervated by sprouted Purkinje cell recurrent axon collateral terminals. Such granuloprival cultures were transplanted with optic nerve in order to supply mature glial cells. It was observed that not only were Purkinje cells almost completely ensheathed by astroglia, but there was a greater than 60% reduction in the number of somatic synapses compared to the non-transplanted granuloprival cultures. This astroglial ensheathment, which may be neuronally directed, could be the physical element provoking the reduction in the number of synapses.

Morphological correlates of altered neuronal activity in organotypic cerebellar cultures chronically exposed to anti-GABA agents

Organotypic cerebellar cultures derived from newborn mice were chronically exposed to medium containing picrotoxin or bicuculline from explanation until they were recorded from extracellularly or fixed for morphological studies. Cultures exposed to anti-GABA agents for 13-18 days in vitro had decreased spontaneous cortical discharge rates when compared with sister control cultures and prolonged inhibitory responses to cortical stimulation. Electron microscopic examination of exposed cultures after 14-16 days in vitro revealed a hyperinnervation of Purkinje cell somata by inhibitory terminals predominantly of basket cell origin. The sprouted terminals penetrated otherwise intact Purkinje cell astrocytic sheaths. These changes represent a departure from the usual developmental pattern, a departure induced by exposure to anti-GABA agents that increased neuronal activity early in the development of the cerebellar circuitry in vitro. The precise signals that initiated the changes are unknown, but the altered development is most likely in response to increased Purkinje cell activity.

Induction of dendritic spine proliferation by an astrocyte secreted factor.

A number of neuron-supporting functions have been ascribed to astrocytes. In this study we found that a proliferation of Purkinje cell dendritic spines, a target site for presynaptic axon terminals, was induced in cytosine arabinoside-treated cerebellar cultures by exposure to astrocyte-conditioned medium. This result suggests that astrocytes may instigate the elaboration of postsynaptic neuronal elements prior to the appearance of axons. This may be an important mechanism in neural development, postdevelopmental neuroplastic change, or regeneration.

BDNF and NT-4, but not NT-3, promote development of inhibitory synapses in the absence of neuronal activity

Development of the full complement of inhibitory synapses in cerebellar cultures requires the presence of neuronal activity. The neurotrophins, BDNF, NT-3 and NT-4, were applied to cerebellar explants during activity blockade. Control numbers of inhibitory Purkinje cell axosomatic synapses developed in the presence of the TrkB receptor ligands, BDNF and NT-4, but not the TrkC receptor ligand, NT-3. The results suggest that BDNF and NT-4 have a role in the promotion of activity-dependent inhibitory synaptogenesis.

Cytosine arabinoside effects in mouse cerebellar cultures in the presence of astrocytes

Organotypic cerebellar cultures derived from neonatal mice were exposed to recent preparations of cytosine arabinoside that destroyed oligodendrocytes and drastically reduced granule cells, but did not reduce the astrocyte population. The cultures were analysed by light and electron microscopy, and by extracellular electrophysiological recording. Purkinje cells survived in greater numbers than in untreated explants and sprouted excess recurrent axon collaterals that formed heterotypical synapses with Purkinje cell dendritic spines. These changes were similar to those found in earlier studies with a cytosine arabinoside preparation that did reduce the astrocyte population, in addition to destroying oligodendrocytes and granule cells. Results with recent cytosine arabinoside preparations that differed from those obtained previously included astrocytic ensheathment of Purkinje cells and apposition of many unattached dendritic spines, encasement of heterotypical synapses by astroglial processes, a loss of Purkinje cell somatic spines, and a lack of somatic hyperinnervation of Purkinje cells by sprouted recurrent axon collateral terminals. All of these differences were attributed to the presence of adequate numbers of competent astrocytes. Heterotypical synapses formed by sprouted recurrent axon collateral terminals and Purkinje cell dendritic spines were functional, as indicated by cortical inhibition in response to antidromic Purkinje cell activation in the absence of somatic hyperinnervation. These results give further definition to the role of astrocytes in cerebellar development and plasticity.

Transplanted astrocytes reduce synaptic density in the neuropil of cerebellar cultures

Cytosine arabinoside-treated neonatal mouse cerebellar cultures, devoid of granule cells and mature glia, demonstrate heterologous synapses between sprouted Purkinje cell recurrent axon collaterals and dendritic spines in the neuropil. Such cultures were transplanted with optic nerve as a source of glia, and the effect on neuropil synapses was investigated. There was a significant reduction in the number of synapses in the neuropil and an increase in the number of free dendritic spines. Many of these spines occurred in clusters, unapposed by glial processes. The effect on the synapse density was not due to a comparable increase in the area occupied by the added astrocytes or an increase in nerve terminal diameter. The results suggest that astrocytes alter the density of neuropil synapses and may also induce the sprouting of dendritic spines.

Enhanced Purkinje cell survival in granuloprival cerebellar cultures

The number of large cortical neurons that survived in cerebellar cultures in which granule cells had been destroyed by exposure to cytosine arabinoside was 3-4 times the number in normal cultures. Transplantation of granuloprival cerebellar cultures with granule cells and glia resulted in a reduction of the large cortical neuron population (predominantly Purkinje cells) to normal, while the number of such neurons remained elevated after transplantation with glia alone. These results indicated that granule cells were critical for the reduction of large cortical neurons. The rescue of large cortical neurons in granuloprival cultures was attributed to an expanded target field for Purkinje cell axon collateral projections.

The extracellular matrix molecule, laminin, induces purkinje cell dendritic spine proliferation in granule cell depleted cerebellar cultures.

Granule cells and glia were eliminated or reduced in organotypic cerebellar cultures exposed to cytosine arabinoside. Transplantation of such granuloprival cultures with glia or exposure to astrocyte conditioned medium in the absence of parallel fibers (granule cell axons) resulted in proliferation of Purkinje cell dendritic spines. The aim of the present study was to identify specific astrocyte secreted factors that induced dendritic spine proliferation. Known astrocyte secreted, neurite promoting factors were screened by application to granuloprival cultures and assayed for dendritic spine proliferation by electron microscopy. An extracellular matrix molecule, laminin, evoked sprouting of Purkinje cell dendritic spines. Dendritic spine proliferation was not associated with known neurite promoting parts of the laminin molecule, as two laminin-derived peptides with identified neurite promoting domains did not induce dendritic spine sprouting. The purpose of laminin-induced dendritic spine proliferation may be to elaborate postsynaptic membrane, thereby increasing the target area for arriving axon terminals during development or regeneration, both of which have been associated with the presence of laminin secreting astrocytes.

NEURAL PLASTICITY IN CEREBELLAR CULTURES

Cerebellar granule cells and oligodendrocytes are destroyed and astrocytes are functionally compromised by exposure of organotypic cerebellar cultures derived from newborn mice to cytosine arabinoside for the first 5 days in vitro. Consequently, myelin does not form and Purkinje cells survive in increased numbers, but without astrocytic ensheathment. In the absence of glial sheaths, Purkinje cells have altered membrane properties and reduced input resistance. Their inhibitory recurrent axon collaterals sprout enormously and hyperinnervate the unensheathed somata of other Purkinje cells and form heterotypical synapses with Purkinje cell dendritic spines normally occupied by homotypical excitatory parallel fiber (granule cell axon) terminals. This reorganization of the cortical circuitry, in which recurrent axon collaterals are the dominant inhibitory elements, allows retention of some inhibition in the absence of parallel fiber excitation of the inhibitory interneurons. In the absence of neuronal activity, the full complement of inhibitory synapses is not developed and the cultures exhibit sustained cortical hyperactivity after recovery from the blockade. If granule cells and glia are replaced, a second round of reorganization ensues, in the direction of restoration of the normal cortical circuitry. The cultures are myelinated and the number of recurrent axon collaterals is reduced. Astrocytes ensheath Purkinje cell somata and strip excess axosomatic synapses, as well as eliminate some of the heterotypical synapses in the cortical neuropil. Parallel fibers synapse with already present Purkinje cell dendritic spines and with newly proliferated spines, the latter induced by an astrocyte secreted factor. As homotypical synapses develop and heterotypical synapses decline, Purkinje cells undergo apoptosis and their population is reduced to control levels. With the restoration of parallel fiber excitation, recurrent axon collaterals are no longer the dominant cortical inhibitory elements. If neuronal activity is blocked as the granule cells and glia are replaced, there is incomplete formation of inhibitory synapses, and cortical discharges are hyperactive after recovery from activity blockade.

Demyelinating and myelination-inhibiting factors induced by chloroform-methanol insoluble proteins of myelin.

A host of proteins was seen when the chloroform-methanol insoluble protein (CMIP) fraction of bovine brain myelin was transferred from polyacrylamide gels to cellulose nitrate sheets. Inoculation of rabbits with the CMIP fraction generated a number of antibodies which were demonstrated by the immunoblot technique. These antisera against CMIP contained antibodies which induced demyelination and inhibited myelin formation in central nervous system cultures. The demyelinating factor was specific for centrally myelinated fibers, and did not demyelinate peripherally myelinated axons.