Figure 7. Manipulation of PHb neuronal function alters affective behavior.

(A)c-Fos^CreERT/+ mice injected with an AAV5/DIO-hM3D-mCherry in the PHb, received a light pulse and 4-OH-Tam. Injection sites were evaluated for mCherry, and c-Fos staining was used to confirm CNO- neuronal activation. (B-C) Chronic activation of light-responsive PHb neurons leads to behavioral alterations. n=10–16. (D) The SPT was evaluated in c-Fos^CreERT/+ and control injected-mice that received CNO twice a day (see STAR Methods). c-Fos^CreERT/+ injected-mice displayed a significant reduced preference for sucrose, vs. control group. n=5–7. (E) vmPFC-projecting PHb cells were chronically activated in mice that were bilaterally injected in the vmPFC with a retrogradely-transported AAV/Cre (J-K), and an AAV5/DIO-hM3D-mCherry in PHb. (F-G) CNO treatment in DREADD-injected mice caused a significant increased in the immobility time in the TST (F), and FST (G), vs. control mice. n=12–15. (H-I) CNO treatment in DREADD-injected mice had no effect on cognitive functions. For the NOR test (H), the statistical analysis vs. 50% (dotted lines) was: Control: p<0.05; DREADD: p<0.05; by one sample t-test. Using the MWM test, we found no significant deficits during the test (I) trial. n=12–14. (J) AAV5/Cre-GFP was bilaterally injected into the PHb of mice that express tetanus toxin (tetX) in a Cre-dependent manner. GFP expression confirms injection site that included the PHb and was largely restricted to immediately adjoining thalamic nuclei. (K-M) Suppressing PHb synaptic output eliminated any statistical difference in the TST (K), FST (L), and SPT (M) between mice housed under the T24 or T7 cycles. For the SPT, the statistical analysis compared to 50% (dotted lines, M) was: T24 p<0.001; T7 p<0.001; by one sample t-test). n=14–15. Data are mean±SEM. */> 0.05; ***p<0.001, by Student’s / test. Hb: habenular complex. Scale bars: 200μm. See also Figures S6 and S7.