Supplementary Figure 1: Hcrt-IRES-Cre validation and Hcrt-ChR2-LH RTPT behavioral and pharmacological analyses. | Nature Neuroscience

Supplementary Figure 1: Hcrt-IRES-Cre validation and Hcrt-ChR2-LH RTPT behavioral and pharmacological analyses.

From: Parallel circuits from the bed nuclei of stria terminalis to the lateral hypothalamus drive opposing emotional states

Supplementary Figure 1

(a) Schematic of targeting vector for Hcrt-IRES-Cre knockin mouse line. (b) Knockin strategy for Hcrt-IRES-Cre. (c) Representative images showing co-expression of AAV-DIO-eYFP viral labeling (green) and Hcrt (top) or melanin-concentrating hormone (MCH; bottom) immunostaining (red) in LH of Hcrt-Cre mice, replicated independently with similar results in four mice. (d) Quantification of specificity and efficiency of Hcrt-LH infection with Cre-inducible viruses in Hcrt-Cre mice, n=4. (e) Raw Ca2+ activity traces from in vivo fiber photometry recordings of Hcrt-LH neurons in Hcrt-Cre mice injected with AAV-DIO-GCaMP6f virus. Traces show 60s baseline (left of dashed line), and 60s of Ca2+ activity data following exposure to salient odorant stimuli (right of dashed line) (f) Mean (± S.E.M.) fluorescent Ca2+ activity traces from in vivo fiber photometry recordings of Hcrt-LH neurons in control mice injected with AAV-DIO-GFP virus during baseline (left of dashed line), and following exposure to salient odorant stimuli (right of dashed line). Inset; mean (± S.E.M.) fluorescence levels during the 60s stimuli exposure phase (n=2 mice; 1-2 stimulus trials per subject). (g) Number of entries into the stimulation side for Hcrt-LH-ChR2 and control mice tested in the RTPT (n=7 eYFP, n=10 ChR2-eYFP, two-way RM-ANOVA; ChR2 x stimulation interaction: F2,30 = 1.37, p = 0.27). Centre and error bars are mean ± S.E.M. (h) Mean time spent per entry to the stimulation side for Hcrt-LH-ChR2 and eYFP control mice tested in the RTPT (n=7 eYFP, n=10 ChR2-eYFP mice, two-way RM-ANOVA; ChR2 x stimulation interaction: F2,30 = 2.28, p = 0.12, Bonferroni post-hoc comparison **p < 0.005 vs. Hcrt-LH-eYFP). Centre and error bars are mean ± S.E.M. (i) Hcrt-LH-ChR2 real-time place avoidance is significantly blocked by pharmacological treatment with double Hcrt-R antagonist MK-6096 (5mg/kg i.p., n=10 mice, two-tailed paired t-test; t9= 1.11, p = 0.297, n.s.). Centre and error bars are mean ± S.E.M. (j) Significant Hcrt-LH-ChR2 real-time place avoidance following pharmacological treatment with KOR antagonist norBNI (10 mg/kg i.p., n=6 mice, two-tailed paired t-test; t5 = 4.33, p = 0.0075, **p < 0.01 vs. No Light control). Centre and error bars are mean ± S.E.M. Scalebars: all 100 μm.

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