Resolving neuronal circuits of emotional memory with confocal microscopy

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We request funds to address a critical gap in the research infrastructure at the University of Toronto Scarborough (UTSC) with the purchase of a laser scanning confocal fluorescence microscope for imaging mammalian tissue. Together our labs consist of 27 HQP that share a goal to understand how defined neuronal circuits give rise to emotional behaviour. At UTSC there is no core laser scanning confocal microscope available to our labs. Laser scanning confocal microscopes are located at other University of Toronto sites, but these locations charge $36 per hour and are over 30 km from the UTSC campus. Together these factors generate an unsustainable situation. If funds are not received our research programs will be severely impeded because either (1) we must travel long distances and pay exorbitant fees for offsite microscopes of (2) regress our research hypotheses to a level that is out of step with modern behavioural neuroscience and current publishing standards. The applicants require a confocal microscope because interpreting their experimental results hinges upon generating fine spatial resolution images of neuronal tissue that can disambiguate multiple fluorescent protein targets. The applicants share a conventional widefield fluorescence microscope that is unable to provide focused single cell resolution due to background fluorescence that arises from tissue outside of the field of view. This issue is compounded when imaging multiple fluorescent proteins in tissue that contains multiple cell layers and complex neuronal morphology. A laser scanning confocal microscope is the only method that can dissect neuronal circuits at this level because it optically sections neural tissue in the axial (depth) plane to create unambiguous, high-resolution, neuronal images. Optical sectioning technology is essential to correct the current misalignment between the specificity of our sophisticated neuronal manipulation and recording techniques, and our outdated current microscopy infrastructure. Reception of RTI funds will correct this disparity and allow us to perform pioneering research. Without a laser scanning confocal microscope on site, the breadth and impact of our research scope is restricted and diminished because we cannot validate transgenic, viral, and neural recording strategies at the levels currently required for publication in high-impact neuroscience journals. Instead, reception of a confocal microscope will (1) allow our research programs to discover activity-dependent, genetically-defined, and pathway-specific neuronal circuits that generate emotional behaviour, (2) modernize UTSC's research infrastructure to the standard of most Canadian neuroscience programs, (3) fill a technological gap that provides HQP with training and research opportunities using a fundamental microscopy method in the life sciences.