Three days after the tracer injections, mice were deeply anesthetized with an overdose of Ketamine/Xylazine and perfused through the heart with phosphate buffered saline, followed by 1% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB, pH 7.4). The cortex was immediately separated from the rest of the brain, flattened or completely unfolded, placed white matter down on a filter paper laying on top of a thin strip of sponge and covered with a glass slide (25x75x1 mm). The assembly was postfixed in a petri dish filled with 4% PFA and stored overnight at 4°C. After postfixation the tissue was cryoprotected in 30% sucrose and cut on a cryostat or freezing microtome in the tangential plane at 50 μm.
To identify the injected area as well as the targets of anterogradely BDA labeled projections we visualized in every case the regional myeloarchitecture and the callosal connections. Previously, we have used these landmarks as reference to locate and/or directly identify the visuotopically organized areas V1, LM, P, LI, POR, AL, RL, A, PM and AM (Wang and Burkhalter, 2007 (link)). Both histological patterns were imaged in wet mounted sections with a CCD camera (CoolSnap EZ). Sections through layer 4 were imaged under a dissecting microscope (Wild M5), equipped with dark field optics. Under these conditions, heavily myelinated areas appeared lighter than the background (Fig. 1c). Sections through layer 2/3 were imaged under a fluorescence microscope (Nikon Eclipse 80i) equipped with UV optics. This illumination revealed blue retrogradely bisbenzimide labeled callosal projection neurons (Fig. 1a). To parcel the rest of cortex in which myeloarchitecture and callosal patterns showed less structure, we stained complete sets of sections of each BDA injected hemisphere with an antibody against type 2 muscarinic acetylcholine receptor (m2AChR, MAB367, Millipore), visualized the expression with Alexa 647 labeled secondary antibody (A21247, Invitrogen) and imaged the sections under IR fluorescence (Wang et al., 2011 (link)). To further validate the m2AChR-based parcellation scheme, we stained alternate series of tangential sections from three flatmounted hemispheres, which were not injected with BDA, for cytochrome oxidase (CO) histochemistry and Nissl substance. BDA labeled projections were visualized by incubating sections in avidin, biotinylated HRP (Vectastain ABC Elite) and intensifying the diaminobenzidine reaction product with AgNO3 and HAuCl2 (Jiang et al, 1993 (link)). The reacted sections were dehydrated, cleared and cover slipped with DPX. The intensified, BDA labeled sections were imaged under dark field illumination.
Digital overlays of BDA labeled projections with images of the myeloarchitecture, callosal connections, m2AChR and CO staining patterns were used for assigning terminal clusters to cortical areas. Superimpositions with m2AChR immunofluorescence were performed by matching blood vessels within the same case. Alignments with CO patterns were done across cases, by first overlying the intensely labeled areas V1 and S1 with corresponding myeloarchitectonic regions of another brain, and then matching the BDA labeled projections to the CO stained template.