title: A methodological pipeline for serial-section imaging and tissue realignment for whole-brain functional and connectivity assessment.
abstract: Understanding the neurobiological basis of cognition and behavior, and disruptions to these processes following injury and disease, requires a large-scale assessment of neural populations, and knowledge of their patterns of connectivity.
  
  
  
  We present an analysis platform for large-scale investigation of functional and neuroanatomical connectivity in rodents. Retrograde tracers were injected and in a subset of animals behavioral tests to drive immediate-early gene expression were administered. This approach allows users to perform whole-brain assessment of function and connection in a semi-automated quantitative manner. Brains were cut in the coronal plane, and an image of the block face was acquired. Wide-field fluorescent scans of whole sections were acquired and analyzed using Matlab software.
  
  
  
  The toolkit utilized open-source and custom platforms to accommodate a largely automated analysis pipeline in which neuronal boundaries are automatically segmented, the position of segmented neurons are co-registered with a corresponding image acquired during sectioning, and a 3-D representation of neural tracer (and other products) throughout the entire brain is generated.
  
  
  
  Current whole brain connectivity measures primarily target mice and use anterograde tracers. Our focus on segmented units of interest (e.g., NeuN labeled neurons) and restricting measures to these units produces a flexible platform for a variety of whole brain analyses (measuring activation, connectivity, markers of disease, etc.).
  
  
  
  This open-source toolkit allows an investigator to visualize and quantify whole brain data in 3-D, and additionally provides a framework that can be rapidly integrated with user-specific analyses and methodologies.
authors.affiliation: Canadian Centre for Behavioural Neuroscience, The University of Lethbridge, Lethbridge, AB, Canada. Electronic address: lilia.mesina@uleth.ca.
authors.forename: Lilia
authors.initials: L
authors.lastname: Mesina
authors.affiliation: Canadian Centre for Behavioural Neuroscience, The University of Lethbridge, Lethbridge, AB, Canada; Department of Neurobiology and Behavior, University of California, Irvine, CA, USA. Electronic address: awilber@uci.edu.
authors.forename: Aaron A
authors.initials: AA
authors.lastname: Wilber
authors.affiliation: Department of Psychology, The University of New Mexico, Albuquerque, NM, USA. Electronic address: bnjclark@unm.edu.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Clark
authors.affiliation: Canadian Centre for Behavioural Neuroscience, The University of Lethbridge, Lethbridge, AB, Canada.
authors.forename: Sutherland
authors.initials: S
authors.lastname: Dube
authors.affiliation: Canadian Centre for Behavioural Neuroscience, The University of Lethbridge, Lethbridge, AB, Canada.
authors.forename: Alexis J
authors.initials: AJ
authors.lastname: Demecha
authors.affiliation: Department of Neurobiology and Behavior, University of California, Irvine, CA, USA.
authors.forename: Craig E L
authors.initials: CE
authors.lastname: Stark
authors.affiliation: Canadian Centre for Behavioural Neuroscience, The University of Lethbridge, Lethbridge, AB, Canada; Department of Neurobiology and Behavior, University of California, Irvine, CA, USA.
authors.forename: Bruce L
authors.initials: BL
authors.lastname: McNaughton
mesh_terms: D000818:Animals; D001921:Brain / Q000166:cytology* / Q000378:metabolism*; D001931:Brain Mapping / Q000379:methods*; D005260:Female; D015870:Gene Expression; D017781:Genes, Immediate-Early / Q000502:physiology; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008297:Male; D008810:Mice, Inbred C57BL; D009434:Neural Pathways / Q000166:cytology / Q000378:metabolism; D056347:Neuroanatomical Tract-Tracing Techniques / Q000379:methods; D010363:Pattern Recognition, Automated / Q000379:methods; D011916:Rats, Inbred F344; D020318:Rats, Long-Evans; D012984:Software
keywords: Connectome; Functional networks; Immediate-early genes; Neural tracing; Neuroimaging; Tissue realignment