New imaging tools are needed for brain-wide visualization of the neural activity underlying defined behaviors. Live imaging techniques such as fMRI and MEG measure activity across the brain, but these methods cannot resolve single cell activity. Moreover, in vivo electrophysiology and calcium imaging can reach cellular resolution, but only simultaneously survey a few brain regions at best. Capturing a brain-wide snapshot of recent neural activity at cellular resolution would provide an incredible tool for hypothesis generation and assay development for CNS drug discovery. To date, immunohistochemical imaging of the activity-dependent immediate-early gene (IEG) cFos is the only established platform that visualizes neural activity linked to a defined behavior at cellular resolution. To efficiently collect brain-wide snapshots of neuronal activity, two obstacles need to be overcome. First, since standard histological methods and modern imaging approaches (e.g., serial two-photon) require tissue slicing, these approaches to IEG imaging are time consuming and introduce anatomical distortions that prevent quantitative comparisons among brain regions. Second, since traditional IEGs like cFos are also regulated by paracrine signaling factors, the fidelity of IEG expression as a read-out of neuronal activity is compromised. To increase the throughput of whole-brain histological imaging, we will take advantage of recent developments in tissue clearing, fluorescence light sheet imaging and computational alignment and cell detection. For tissue clearing, we optimized the iDISCO tissue clearing method for whole brain immunostaining. For imaging, we have customized a commercial light sheet micrscope for large format imaging of iDISCO cleared tissue. With our adapted technology, we are imaging a whole mouse brain in less than three hours at higher spatial resolution than in previous reports. Using these methods, we have generated whole brain 3D images of genetically-encoded fluorophores. We will couple this technology to the use of a novel, high quality monoclonal antibody against Npas4, a unique IEG that is neuron-specific and exquisitely tuned to neuronal activity. Our automated quantification pipeline aligns samples to a common reference atlas and generates brain-wide cell counts of neuronal activity across >1,000 identified brain areas. This new imaging platform will overcome the time- and labor-intensive limitations of standard histology, and the use of Npas4 as a marker will provide the highest fidelity, brain-wide read-outs of neuronal activity to date.
Public Health Relevance Statement: Project Narrative To better establish the link between neural activity and behavior in health and disease states, new tools must be developed to capture brain wide patterns of activity at cellular resolution. By combining new advances in tissue clearing, whole brain light sheet imaging, computational alignment of brain anatomy and a new molecular marker for neural activity, our assay will provide 3D images of whole brain activity with unprecedented resolution and fidelity.
Project Terms: Anatomy; Anatomy Qualifier; Anatomical Sciences; Anatomic structures; Anatomic Structures and Systems; Anatomic Structure, System, or Substance; Anatomic Sites; Anatomic; Antibodies; Monoclonal Antibodies; mAbs; Clinical Treatment Moab; Antibody Affinity; antigen antibody affinity; Atlases; Behavior; Biological Assay; Biologic Assays; Bioassay; Assay; Brain; Encephalon; Brain Nervous System; Calcium; Cell Number; Cell Count; Cell Body; Cells; Central Nervous System Disorders; CNS disorder; CNS Diseases; Central Nervous System Diseases; Communities; Disorder; Disease; drug/agent; Pharmaceutic Preparations; Medication; Drugs; Pharmaceutical Preparations; electrophysiological; Neurophysiology / Electrophysiology; Electrophysiology; Electrophysiology (science); Equipment Design; Ethers; Fluorescence; Gene Expression; Health; Histology; Photoradiation; Light; Maps; Methods; Modernization; Murine; Mice Mammals; Mice; Mus; neuronal; Neurocyte; Neural Cell; Nerve Unit; Nerve Cells; Neurons; Neurosciences; Publishing; Survey Instrument; Surveys; Technology; Testing; Time; Body Tissues; Tissues; Generations; Imaging Technics; Imaging Procedures; Imaging Techniques; Measures; v-FOS FBJ Murine Osteosarcoma Viral Oncogene Homolog; c-fos Proto-Oncogenes; c-fos Gene; c fos; Protooncogene FOS; G0S7; FOS gene; Custom; Imagery; Visualization; Area; Phase; Histologic; Histologically; Immediate-Early Genes; Link; Evaluation; Individual; Measurement; Therapeutic; tool; Functional Magnetic Resonance Imaging; fMRI; Functional MRI; Hour; Pattern; Techniques; Paracrine Communication; Paracrine Signaling; Services; experience; paracrine; fluorophore; technology development; tech development; relating to nervous system; neural; novel; Reporting; Positioning Attribute; Position; Code; Coding System; Sampling; response; assay development; Three-Dimensional Imaging; 3D imaging; 3-D Imaging; drug discovery; 3D images; 3D image; 3-D image; 3-D Images; Three-Dimensional Image; Brain region; preventing; prevent; conditioned fear; fear conditioning; Detection; Imaging Device; Imaging Tool; Imaging Instrument; Recombinants; Reproducibility; Resolution; in vivo; Slice; Development; developmental; Behavioral; Image; imaging; cost; two-photon; 2-photon; novel strategies; novel strategy; novel approaches; new approaches; Consumption; Coupled; innovation; innovative; innovate; mouse model; murine model; activity marker; molecular marker; molecular biomarker; imaging platform; histological image; histologic image; NPAS4 gene; neuronal PAS domain protein 4; NPAS4; imaging approach; imaging based approach
