Fig 1.
Images of olfactory bulb sections of adult Ai32/Ai32 mice stained with anti-EYFP, anti-NeuN, and anti-GFAP antibodies.
B and C show higher magnification images of dotted line (B) and solid line (C) boxes in A. B shows the glomerular cell layer, C shows mitral/granule cell layer. D shows a ‘negative’ region not showing leaky ChR2 expression (dotted line boxed region in B). n = 3 mice, scale bars = A-C 100 μm, D 25 μm.
Fig 2.
Images of cingulate cortex (A-C), hippocampus (D&E), and the medial forebrain bundle and islands of Calleja in the olfactory tubercle (F&G) sections in adult Ai32/Ai32 mice stained with anti-GFP, anti-NeuN, and anti-GFAP antibodies. B and C are higher magnification ChR2 expressing (B) and non-expressing (C) regions from A (solid line and dotted line boxes, respectively). E and G are higher magnification images of regions boxed in D and F respectively. n = 3 mice, scale bars = 100 μm.
Fig 3.
Images of cerebellar lobe (A&B) and vermis (C-E) sections of adult Ai32/Ai32 mice immunostained with anti-GFP, anti-NeuN and anti-GFAP antibodies. B shows a higher magnification view of region boxed in A. D and E show ChR2 expressing (D) and non-expressing (E) regions boxed in C (solid line and dotted line boxes, respectively). n = 3 mice, scale bars = 100 μm.
Fig 4.
Images of brainstem spinal trigeminal nucleus caudalis (A&B) and ventral pallidum (C-E) sections in adult Ai32/Ai32 mice stained with anti-GFP and anti-NeuN antibodies. B shows a higher magnification view of region boxed in A. D and E show ChR2 expressing (D) and non-expressing (E) regions boxed in C (solid line and dotted line boxes, respectively). n = 3 mice, scale bars = 100 μm.
Fig 5.
A. Images of cervical dorsal horn (DH) (A&B), thoracic DH (C), lumbar DH (D&E) and dorsal root ganglion (F&G) of adult Ai32/Ai32 mice stained with anti-GFP, anti-NeuN, and anti-GFAP antibodies. B shows a higher magnification view of ChR2 non-expressing region boxed in A. E and G show higher magnification views of regions boxed in D, F respectively. n = 3 mice, scale bars = 100 μm.
Fig 6.
Western blot analysis of ChR2 (H134R)-EYFP expression in Ai32 mice.
Olfactory bulb (OB) cell lysates were immunoprecipitated using a rabbit anti-GFP antibody and blotted with a chicken anti-GFP antibody. Lane 1: Positive control OmpCre; Ai32, lane 2,3,4: RosaChR2-EYFP/ChR2-EYFP homozygous Ai32 mice (n = 3 mice.), lane 5: Negative control CDI mouse. ChR2-EYFP = ~62 kDa.
Fig 7.
Leaky expression of ChR2(H134R)-EYFP in dorsal spinal cord neurons of four-week-old Ai32 mice is not sufficient to generate photocurrents.
(A) ChR2-EYFP expression in dorsal horn of lumbar spinal cord slice from 4 pw Ai32/Ai32 mice (scale bar 0.1 mm). (B) Whole cell recording from 12 of ChR2-EYFP expressing superficial dorsal horn (SDH) cells, light stimulation (0.1 ms or 1 s) did not induce any currents. (C) Representative trace of delayed firing in ChR2-EYFP expressing cell by 50 pA current injection. Table shows firing patterns of recorded cells: 5 cells showed delayed firing pattern (DFN), 2 cells showed phasic firing pattern (PFN), 2 cells showed initial bursting firing pattern (IBN).