(A) Cre-dependent eNpHR(2.0)-YFP AAV.
(B–D) Injection of (A) into Crhr2α-eGFPCre LS
yielded eNpHR-YFP (green) in Cre+ cells (red).
(E) Recombination specificity and sensitivity; counts from areas containing
eNpHR-YFP+ cells:
eNpHR+/DAPI+ =
4.7±0.2%, Cre+/DAPI+
= 11.4±0.4%,
Cre+/eNpHR+=71.2±1.6%,
eNpHR+/Cre+=29.8±3.0%
(mean±s.e.m.). Total n = 8,216
DAPI+ cells from 2 mice.
(F–H) Recordings from
eNpHR-YFP+Crfr2α+
neurons. (G) Cell in response to the same current injections in absence
(top) or presence (bottom) of 593nm light.
(H) 593nm light significantly suppressed firing of eNpHR-YFP+
neurons (F1,108 = 28.69,
P<0.001).
(I,J) Surgical manipulations and behavioral testing.
(K) Photoinhibition of Crfr2α+
neurons only during IMS reduced anxiety in subsequent testing. hrGFP
(n=15) vs. eNpHR (n=17),
data presented Mann-Whitney U value, P value:
LDB, entries in light side (U = 64.0,
P<0.05), time in light side (U
= 62.5, P<0.05); OF, entries in center
(U = 69.0, P<0.05), time in
center (U = 64.5, P<0.05); NO,
entries in center (U = 73.5,
P<0.05), time in center (U = 34.0,
P<0.001).
(L) IMS mice that received photoinhibition of
Crfr2α+ neurons only during
behavioral testing showed reduced anxiety relative to controls. hrGFP
(n=11) vs. eNpHR (n=11):
LDB, entries in light side (U = 21.5,
P<0.01), time in light side (U
= 20.0, P<0.01); OF, entries in center
(U = 25.5, P=0.07), time
in center (U = 23.0, P<0.05); NO,
entries in center (U = 22.0,
P<0.05), time in center (U = 21.0,
P<0.05).
(M) Inhibiting Crfr2α+ neurons in the
absence of IMS does not alter anxiety. hrGFP(n=11) vs. eNpHR
(n=11): LDB, entries in light side
(11.5±1.9 vs. 11.7±2.3, P>0.9), time in
light side (218.7±44.6 vs. 206.4±45.2,
P>0.8); OF, entries in center (23.1±2.6 vs.
20.4±2.8, P>0.45), time in center (34.4±3.2
vs. 26.7±4.4, P>0.15); NO, entries in center
(43.0±6.3 vs. 39.0±3.9, P>0.6), time in
center (97.0±15.8 vs. 97.5±14.9,
P>0.95).
(N) Model for LS Crfr2α+ neuronal
valence based on optogenetic inhibition.
See also Figure S4.