Microglia are the immune cells of the brain and are known to respond to infectious and non-infectious insults to the nervous system. Recently, a new function has been demonstrated for microglia, active involvement in the maintenance of synapses, the connections between nerve cells, a function that has been likened to that of electricians. Using the transparent and genetically amenable brain of the zebrafish, this proposal explores this new function of microglia at the single cell level in the intact, behaving animal, through visualization of cellular components of the brain (neurons, glia, microglia, blood vessels, synapses), and through the genetic manipulation of synaptic density, and real time observation of microglia in the process.
Maintenance of synaptic integrity is of key importance for normal nervous system functioning. If synaptic connections are compromised, disease develops ranging from motor disturbances to dementia. The maintenance of synaptic integrity is only poorly understood while pruning of synapses during development and post-traumatic synaptic stripping are well-established processes in which microglial cells have a key role.
Microglial cells are generally referred to as the brain's defense and immune system. However, their functions in normal CNS have begun to attract considerable attention following the discovery that a compulsive behavioral trait is caused by loss of Hoxb8 activity in microglia. Thus, the interactions that take place between microglia and neurons are considerably more sophisticated than previously thought. We have proposed a new role for microglial cells in the maintenance of synaptic integrity analogous to electricians, and this proposition has gained significant support through findings by other groups. An electrician maintains and installs electrical equipment but is not involved in the actual circuitry.
What distinguishes synapses that are to be removed by microglia from those that are to stay? Microglia engaged in synaptic stripping following facial nerve axotomy in rodents strongly express complement receptors, and it has been shown subsequently that the classical complement cascade mediates CNS synapse elimination. In the past, synaptic connections had to be studied in complex mammalian systems where the analysis of effects caused by genetic manipulation is technically demanding and time-consuming. This has changed with the arrival of zebrafish as an experimental model.
We propose to use the zebrafish model to elucidate the spatial relationship between microglia and synapses and their dynamics in real time and to clarify the role of key molecules such as complement in microglia-synapse interactions. We will further test the hypothesis that behavioral changes ensue if synaptic density is altered following silencing or genetic ablation of the microglia, i.e. that microglia have an essential role in the maintenance of synaptic integrity.
THE SPECIFIC AIMS OF THIS PROJECT ARE
1. Establish a model for real-time observation of microglia-synapse interactions
2. Test the hypothesis that complement-activation represents a central mechanism for synapse removal by microglia
3. Genetically control synaptic density and analyze the microglial response
4. Silence microglia by over-expressing microRNA-124, ablate microglia using nitroreductase, and assay the effect on synapses and behavior
Evidence for an involvement of microglial cells in synaptic plasticity was originally obtained in the rat facial nerve axotomy model where microglial cell processes were observed to interpose between afferent axonal endings and the surface membrane of motor neurons after their peripheral axons had been cut. It was also in this model where the first evidence was obtained that microglial complement receptor 3 (CR3) and MHC class I antigens are involved in synaptic changes. Recently, it has been demonstrated that during normal brain development or preceding degeneration of adult retinal neurons, synapses are marked by the complement component C1q before they are removed.
The zebrafish has emerged as a powerful experimental tool for the examination of nervous system biology. The cellular complexity of the larval zebrafish CNS is three orders of magnitude lower than that of mammals, which facilitates experimental and microscopic analyses. Larvae are transparent and due to their small size and a rich genetic tool set, they represent a versatile model for in vivo studies of innate immune cells. These include macrophages and microglia.
The exact mechanisms underlying the interaction between microglia and synapses have remained essentially unknown. During development, neurons form far more synaptic connections than are maintained in the adult brain. Mice with a deficiency of the leukocyte-expressed fraktalkine receptor Cx3cr1 show a significant reduction in the number of microglia after birth and exhibit transient defects in synaptic connectivity and plasticity. Based on this, it has been suggested that complement-dependent synaptic pruning by microglia may be a common mechanism by which synaptic connections are physically modified during development. One study suggests that microglia participate in experience- dependent remodeling and elimination of synapses in the mature and normal adult brain. This lends support to the idea that microglia may influence neuronal circuit organization not only during postnatal development, but also throughout the animal’s lifespan.
The opportunity ID for this research opportunity is 1950