We also find that more non-PV neurons express m1 AChRs in MT than in V1. The implications of these data for ACh as a candidate mechanism that supports attentive states is discussed in the context of likely downstream targets for m1 AChR in various cell classes and in different species. Materials and Methods Histological preparation Three adult male macaque monkeys (two Macaca mulatta and one Macaca nemestrina) that had previously been used in unrelated electrophysiology Inhibitors,research,lifescience,medical recordings were used in this experiment. Tissue was obtained from the unrecorded

hemispheres. For further details of the standard protocols for the donor labs, see Oristaglio et al. (2006) and Nauhaus et al. (2012). All procedures Inhibitors,research,lifescience,medical were approved and performed in accordance with NIH and institutional guidelines for the care and use of animals. Animals were euthanized by intravenous injection of signaling pathway sodium pentobarbital (60 mg/kg). Following complete abolition of corneal and pedal reflexes, animals were transcardially

perfused with heparinized 0.01 mol/L phosphate-buffered saline (PBS, pH 7.4) followed by 4 L of chilled 4% paraformaldehyde (PFA) in 0.1 mol/L phosphate buffer (PB, pH 7.4). The fixative was run for at Inhibitors,research,lifescience,medical least 40 min. The brain was then removed and blocked as necessary to provide donor labs with tissue for their histological needs. The remaining tissue was post-fixed overnight at 4°C in 4% PFA. The following Inhibitors,research,lifescience,medical day, the brain was transferred to 30% sucrose in PBS as a cryoprotectant and stored at 4°C until it sank. Hemispheres to be sectioned were blocked in approximately the coronal plane at

the level of the lunate sulcus (with the whole lunate sulcus in the block) and at the anterior tip of the intraparietal sulcus. The tissue between these two blocking cuts was sectioned at a Inhibitors,research,lifescience,medical thickness of 50 μm on a freezing microtome. To provide reference sections for determining boundaries between cortical areas and cortical layers, two 1-in-6 series were set aside; one for Gallyas (Gallyas 1970) and the other for Nissl (cresyl violet) staining. The remaining sections were stored at 4°C in PBS with 0.05% sodium azide added. Source and characteristics of primary antibodies Please see Table ​Table11 for a summary of the antibodies used in this study. Table 1 Primary antibodies We detected m1 muscarinic ACh receptors (m1 AChRs) using a polyclonal antibody raised in rabbit against amino acids 227–353 of the intracellular loop i3 of the human many m1 AChR, obtained from Alomone Labs (Jerusalem, Israel, catalog #AMR-001, lot # AN-05). This region of the i3 loop has high sequence homology (99%) with the macaque m1 AChR. To detect parvalbumin (PV) we used a monoclonal antibody produced by hybridization of mouse myeloma cells with spleen cells from mice immunized with parvalbumin purified from carp muscles (Swant, Bellinzona, Switzerland, catalog #235, lot#10-11[F]).