Synaptic activity and the formation and maintenance of neuronal circuits

One of the most fundamental features of neurons is their polarized organization with two types of neurites extending from the cell body, axons and dendrites that are both functionally and morphologically distinct. During development, both axons and dendrites possess highly dynamic and actin-rich growth cones and filopodia extending from their shafts, which are subsequently replace by fundamentally stable axonal varicosities and dendritic spines. Together they form the basic elements of mature synapses. To mimic in vivo neuronal development, I have used organotypic cultures of brain tissue from transgenic mice expressing either green fluorescent protein (GFP) bearing a surface membrane localization signal or actin-GFP in combination with live cell imaging system. This approach provided me with high-resolution images of developing neurons’ fine structure in organized tissue. Co-cultures of fluorescent and non-fluorescent hippocampal slices enabled me then to examine simultaneously dendrite differentiation in the fluorescent slice and to track the fate of fluorescent axons growing into the non-fluorescent slice. Together this granted me a powerful tool to study neuronal network formation and developmental maturation of axons and dendrites. Co-cultures of embryonic tissue showed a sustained cross-innervation of axonal projections. Over time neurons in these co-cultures formed a dense axonal network with numerous axonal varicosities along their shaft. This axonal plexus remained present beyond 2 months in vitro. Dendrites in these embryonic co-cultures subsequently switched from producing labile filopodia to fundamentally stable dendritic spines. These mature dendritic spines had morphologies similar to those reported from studies of adult brain. Both axons and dendrites exhibited a successive focalisation of actin-based dynamics to the site of the synaptic junction. The observed changes in shape of mature axonal varicosities and dendritic spines together with the rapidly extension and retraction of actin-rich protrusions from the top of varicosities and spine heads suggest a retained capacity for experience-dependent fine-tuning e.g. during either periods of learning and memory or during brain damage resulting in an altered connectivity for both pre- and postsynaptic compartments in the mature mammalian central nervous system. The observed morphological dynamics suggest a high degree of preservation of morphological plasticity at the synapse in mature neuronal networks. Co-cultures of postnatal brain slices showed intensive invasion of axonal projections during the first two weeks in culture, followed by dramatic axonal regression and resulting in a near complete absence of cross-innervating axons after 1 month in vitro. In contrast, dendrite development in each of these postnatal cultures was fundamentally normal and occurred similar to that observed in embryonic co-cultures. I then co-cultured embryonic and postnatal slices to investigate whether the difference in capacity to cross-innervate between postnatal co-cultures and embryonic co-cultures were the result of tissue maturation. We found that the postnatal slice degenerated so that after 1 month in culture it had almost disappeared whereas the neighbouring embryonic slice had matured without noticeable problems. Staining these co-cultures of embryonic and postnatal slices showed a massive invasion of microglial cells into the dying postnatal slice. The difference between embryonic and postnatal neurons in their capacity to maintain cross-innervating synaptic connection suggests the existence of a developmental switch resulting in the inability of sustained afferent cross-innervation between postnatal brain slices. At the same time, in heterochronic co-cultures it causes miscommunication between postnatal and embryonic cells leading to profound degeneration of postnatal tissue. The thick layer of microglia surrounding postnatal tissue suggests their involvement in neuronal degeneration similar to that observed in axotomy-induced neuronal death and various neurodegenerative conditions such as Alzheimer’s disease. The earlier suggested preservation of morphological plasticity at the synapse in mature neuronal networks was illustrated by cooling mature hippocampal slices, either acutely cut brain slices or organotypic cultures, to room temperature. Dendritic spines are highly sensitive to reduced temperature with rapid loss of actin-based motility followed by disappearance of the entire spine structure within 12 hours. However, rewarming these cooled slices to 37˚C resulted in the rapid extension of filopodia from the surface of dendrites and re-establishment of dendritic spines within several of hours. These data underline the high degree of plasticity retained by neuronal connections in the mature CNS and suggest a link between dendritic spine structure and global brain function