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. Author manuscript; available in PMC: 2022 May 1.
Published in final edited form as: Addict Biol. 2020 Aug 20;26(3):e12961. doi: 10.1111/adb.12961

Figure 3.

Figure 3.

Silencing the IC-vBNST pathway is anxiogenic without affecting locomotor activity. A) C57/Bl6 mice were infected with the inhibitory opsin, Arch in the IC cell bodies and had an optical fiber implant in the vBNST for terminal silencing. Nissl stain of the Arch injection site (GI: granular insula; DI: dysgranular insula; AIP: agranular insula). B) 40x magnification of IC cell bodies expressing Arch. C) Nissl stain of Arch expression in IC terminals and optical fiber site within the vBNST (ACP: anterior commissure posterior limb; mvBNST: medial ventral bed nucleus of the strial terminalis; lvBNST: lateral ventral bed nucleus of the stria terminalis). D) 40x magnification of IC(CaMKIIa::Arch) terminals in the vBNST. E) Mice underwent an open field assay with continuous photoinhibition staggered off and on for 3 minute bins over 21 minutes (7 bins). Heat maps from both a IC(CaMKIIa::eYFP) (left) and an IC(CaMKIIa::Arch) (Right) expressing subject during the open field assay. The white box in the center represents the “center zone”. F) We then measured the total distance traveled in the open field assay when the laser was either OFF or ON. We found no effect of IC terminal inhibition on locomotor activity. G) We then examined changes in time spent in the center of the open field as a measure for anxiety. We found that Arch inhibition significantly reduced the time spent in the center in a within-groups comparison. H) Mice expressing either IC(CaMKIIa::eYFP) or IC(CaMKIIa::Arch) underwent a Real Time Place Preference assay. Heat maps from both a IC(CaMKIIa::eYFP) (Top) and a IC(CaMKIIa::Arch) (Bottom) expressing subject during RTPP. I) We found that optical inhibition of Arch-expressing IC terminals in the vBNST caused aversion for the stimulationpaired chamber. J) Measured time spent in stimulus chamber vs. non-stimulus chamber to examine aversion for the photoinhibition over time. Mice begin showing significant aversion for the stimulation chamber by the last 5min bin. (*p≤0.05, **p≤0.01, ***p≤0.001, ****p<0.0001)