Limitations of the GENSAT Egr1-EGFP transgenic mouse strain for neural circuit activity mapping.

Transgenic immediate-early gene reporter mouse strains are valuable tools for studying activity-dependent neural cell populations in vivo. However, routine characterization of the Gene Expression Nervous System Atlas (GENSAT) "Egr1-EGFP" reporter mouse strain produced results that were highly inconsistent with endogenous Egr1 expression. Activity-dependent EGFP expression was not observed, and EGFP protein did not co-localize with native Egr1 protein. This precautionary study outlines the limitations of the Egr1-EGFP transgenic line as a tool to study the activity-dependent expression of Egr1 and emphasizes the necessity of taking into account the potential loss of regulatory elements, stability determinants, or translational modulation in transgenic reporter strains.

A methodological pipeline for serial-section imaging and tissue realignment for whole-brain functional and connectivity assessment.

BACKGROUND: 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. NEW METHOD: 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. RESULTS: 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. COMPARISON WITH EXISTING METHODS: 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.). CONCLUSIONS: 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.