PAC1- and VPAC2 receptors in light regulated behavior and physiology: Studies in single and double mutant mice - Fig 4

<p>Distribution of PACAP (green) and PAC1 receptor (red) immunoreactivity (A-C), and VIP (red) and VPAC2 (green) immunoreactivity in the mouse wild type SCN (D-F). PACAP immunoreactive fibers from the eye are found in the retinorecipient part of the SCN in close apposition to the PAC1 receptor immunoreactivity (A). 47 digital sections (Z- stack) were used to make the 3D reconstruction seen in B-C. Imaris co-localization module (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188166#sec002" target="_blank">material and methods</a>) was used to determine close appositions (indicated blue in B-C) most likely representing synaptic appositions. C represents the frame indicated in B. VIP immunoreactivity is found in nerve cell bodies in the ventral SCN (red) with nerve fiber projections in–and outside the SCN, many reaching the dorsal part of the SCN where the VPAC2 receptors (green) are present (D). In 3D reconstruction (Z-stack of 90 images) as indicated with frame in D of the ventral SCN, close appositions most likely representing synaptic appositions between VIP immunoreactive nerve fibers and VPAC2 receptor are indicated (blue in E). Arrows in E and F indicate VIP cells also expressing the VPAC2 receptor. PAC1 and VPAC2 receptor are expressed in separate neurons of the mouse SCN (G-L). The VPAC2 receptor (green) is widely distributed in the SCN with slightly higher occurence in the shell region (G). The PAC1 receptor (red) is also expressed in the SCN with a slightly higher occurrence in the retino-recipient part (H). In I both receptors are stained on the same section. High magnification and 3D analysis on a Z-stack consisting of 37sections in the mid-ventral SCN (marked with * in I) are shown in J-L. In K-L examples of a SCN neuron expressing the PAC1 receptor is indicated by a, a neuron expressing both the PAC1 and the VPAC2 receptor is indicated by b, and one neuron expressing the VPAC2 receptor is indicated with c. 3v; third ventricle, oc; optic chiasm. Scale bars: A; 50 μm, B and E; 20 μm, D; 100 μm, C;7 μm, F; 5 μm, G-I; 50 μm, J-L; 5 μm.