Figure 3: Photostimulation of PVN CRH^ChR2 neurons triggers behaviours in the absence of stress.

(a) Construct of Cre-dependent AAV-DIO-ChR2-eYFP virus. (b) Schematic maps show the injection of virus into the PVN of CRH-Cre/tdTomato mice (left) and the implantation site of the light ferrule (right). (c) Confocal image shows expression of ChR2-eYFP (green) and tdTomato (red) in the PVN. (d) Optical stimulation in current clamp (top) and voltage clamp (bottom) shows delivery of blue light reliably controls PVN CRH neurons. (e) Blood samples taken before and 15 min after the onset of optical stimulation show increase in CORT levels specifically in CRH^ChR2 mice (CRH^eYFP: 0.528 μg dl⁻¹ increase, n=6; versus CRH^ChR2: 5.327 μg dl⁻¹ increase, n=5; P=0.0316; t-test). (f) Detailed analysis shows the pattern of eight different behaviours observed in CRH^eYFP (left) and CRH^ChR2 (right) animals during 5 min of optical stimulation in an observational chamber to which mice were previously habituated to in the absence of stress. Each row represents one animal. (g) Histograms show percentage of animals grooming in each group during optical stimulation. (h) Cumulative graph illustrates relative extent grooming. (i) Optical stimulation of PVN CRH neurons increased grooming time (CRH^eYFP: 6.8±1.9 s, n=12; versus CRH^ChR2: 112.6±13.6 s, n=11; P<0.0001; t-test). (j) Histograms show percentage of animals rearing in each group during optical stimulation. (k) Cumulative graph illustrates relative rearing. (l) Rearing time as a fraction of non-grooming behaviours is decreased by photostimulation of CRH neurons (CRH^eYFP: 9.9±1.5%, n=12; versus CRH^ChR2: 5.4±1.4%, n=11; P=0.0396; t-test). (m) Histograms show percentage of animals walking during the optical stimulation. (n) Cumulative graph illustrates relative walking. (o) Fractional walking time is unaltered by optical stimulation (CRH^eYFP: 28.8±3.4%, n=12; versus CRH^ChR2: 28.7±3.8%, n=11; P=0.9784; t-test). Scale bars: c, 50 μm; d, Top: 200 pA and 500 ms, Bottom: 20 mV and 200 ms; (g,h,j,k,m,n), 20%; NS, not significant, *P<0.05; ****P<0.0001; Error bars±s.e.m.