Inclusion Criteria

The MBA project uses mice purchased from Jackson Labs (Catalog# 664): (1) Strain = C57BL/6; (2) Sex = Male; (3) Age= 53±4 days; (4) Weight= 18.8 – 26.4 grams and (5) Quarantine Period = 7-14 days.

Animal Care All experiments and procedures were discussed and approved by the CSHL Institutional Animal Care and Use Committee, and conform to all federal regulations, including the NIH Guidelines for the Care and Use of Laboratory Animals. Part of our motivation in this project is to generate a community-wide digital database of neuronal circuitry in mouse. Ultimately, therefore, this should result in fewer animals being needed in other experimental protocols, throughout the US (and internationally).

Injection Coordinates

The target injection coordinates for the connectivity dataset were based on the Allen Reference Atlas (ARA). A total of 250 targets were placed in the left-hemisphere of the brain. These injections were volumetrically divided across the structures in the ARA. Regions defined in the atlas that occupied less than 0.4% of the left-hemisphere were subsumed into larger structures (of which they are subdivisions according to the ARA). For each resulting structure, a sphere packing schema was assumed to ensure uniformity of injection spacing, and one injection site is targeted at the center of each sphere. In order to separate the regions, the injection sites are constrained not to be too close to the boundary of the targeted region. For neocortex, we placed injections in a columnar line (normal to the cortical surface).

Classical Neuronal Tracers

Cholera Toxin subunit B (CTB) [4] is used for retrograde tracing and biotinylated dextran amine (BDA) [5] is used for anterograde tracing. CTB (1%) is prepared from a stock solution (List Biological Laboratories) and BDA (10%) is used in the 10,000 MW form (Invitrogen) to ensure primarily anterograde tracing [3].

Viral Neuronal Tracers

Both rabies and adeno-associated (AAV)[6] viral tracers are used as part of the connectivity project. The AAV viral tracers are obtained from the Virus Core Facility at the University of Pennsylvania, in two principle forms: (1) AAV2/1.CB7.CI.EGFP.WPRE.RBG, which expresses green fluorescent protein (EGFP) and (2) AAV2/1.CAG.tdTomato.WPRE.SV40, which expresses red fluorescent protein (tdtomato). For cortical injections, both colors are used, per animal, to target different cortical layers. Modified rabies viruses are used for retrograde tracing (RV-4GFP(B19G)) as well as for anterograde tracing (RV-1E5nB6SR(VSVG)). These viruses were produced by Ian Wickersham (MIT) [7, 8]