This project aims to develop a novel system, Biolucida AT, which will greatly enhance the use of array tomography, the state-of-the-art method for investigating synaptic plasticity at the molecular level in human postmortem research and animal models of psychiatric, neurological and childhood developmental disorders. Synaptic transmission is essential for central nervous system (CNS) function, and its dysfunction is a known contributing factor in all major psychiatric, neurological and childhood developmental disorders. However, little is known about clinicopathological correlations of these disorders at the synapse level. Array tomography has overcome two major bottlenecks in investigating synaptic plasticity at the molecular level: the limited axial resolving power of confocal microscopy (rarely better than 700 nm, which is more than twice the synaptic active zone diameter of ~300 nm), and the need to characterize synapses with a number of antibodies (often 10 or more) that far exceeds the feasibility of conventional immunohistochemistry. Array tomography combines exceptional axial resolution with the possibility of characterizing synapses with a number of molecular markers by repeated rounds of immunofluorescent staining of the same ultrathin (70-200 nm) sections and repeated imaging, which is ideal for measuring numbers and densities of specific subtypes of synapses. However, despite the huge potential of array tomography, investigations of synaptic plasticity with this method have remained logistically very complex and in particular extremely time consuming. This is due to the fact that critical steps in an array tomography study are performed manually in the current protocols, using a combination of unspecialized commercial and free software. We have identified the key steps which can be automated - repeated imaging of the same sections after repeated rounds of immunofluorescent staining, managing thousands of multi-channel images, assembly of thousands of aligned 3D multi-channel image stacks from multiple adjacent sections, colocalization analysis and quantification. Biolucida AT will automate these critical steps in array tomography, thereby dramatically reducing logistic complexity and experimental time (by over 90%). By creating a new, specialized solution for array tomography, Biolucida AT will extend our Biolucida(R) technology developed during Phase II (SBIR Grant No. MH076188). We will develop Biolucida AT as a tested, validated, supported and fully documented system. The benefits of Biolucida AT for the neuroscience research, and society in general, will be (i) increasing the potential of performing many more array tomography studies than are currently done, thereby (ii) advancing the pace of research by obtaining valuable and currently unexplored information about synapses from human postmortem research and animal models of psychiatric, neurological and childhood developmental disorders, resulting in (iii) an improved basis for developing novel treatment strategies for complex CNS diseases.
Analysis of synaptic plasticity at the molecular level with the state-of-the-art method, array tomography, yields important information about nervous system function. This project creates an innovative system for automating and dramatically increasing the throughput of array tomography so neuroscience researchers can obtain more data about synapses, thereby advancing the pace of research by obtaining valuable, unexplored information about synapses from human postmortem research and animal models of psychiatric, neurological and childhood developmental disorders. This will result in an improved basis for developing novel treatment strategies for complex CNS diseases such as age-related cognitive decline, traumatic brain injury, neuropathic pain, autism, depression, and many other psychiatric, neurological and childhood developmental disorders.