Article Figures & Data
Figures
- Figure 1.
Changes in AHA neuronal activity during footshock-cue conditioning. A, Strategy for expression of jGCaMP7s with GRIN lens implantation for imaging calcium-dependent signals in the AHA. B, Representative image of GRIN lens placement (white dotted lines) and viral microinjection of the AHA. Scale bar, 200 µm. C, Experimental timeline depicting the shock-paired conditioning session on Day 1 and testing session on Day 2 with two trials for each session. D, Representative Z-scored traces from individual neurons during a Day 1 session. For each pair of black dotted lines, the first line indicates the time of tone onset, and the second line indicates the time of shock delivery. E, Heat maps showing changes in AHA neuronal activity for each neuron on Day 1 as well as mean response to the tone on Day 2. The three dotted lines indicate the onset of the tone (line 1), shock onset (line 2), and shock offset (line 3). Note that no shock was delivered on Day 2. F, Comparison of calcium sensor signal intensity during shock and tone deliveries on Day 1 showed significant increases (n = 121 neurons) during shock 1 and shock 2 delivery compared with the prior stage, a significantly higher response to tone 2 compared with tone 1, and a significantly lower intensity level during shock 2 compared with shock 1. No effects were detected during tone 1 presentation on Day 1. G, There was a significant correlation on Day 1 between average Z-scored fluorescence intensity (x-axis) from jGCaMP7s in AHA neurons with locomotor speed (y-axis, n = 8 mice). Black dots indicate data before the shock, purple dots indicate data during the shock, and green dots indicate data in the 5 s following the shock. H, Freezing analysis indicates significantly increased freezing on Day 2 compared with Day 1 (n = 9 mice). I, Peri-event analysis showing Z-scored AHA activity 3 s before (“Pre”) and the 10 s duration of the tone (n = 90 neurons, data shown as mean ± 1 SEM). J, Area under curve analysis (AUC) indicates significantly higher levels of Z-scored AHA activity during tone delivery on Day 2 compared with Day 1 (n = 90 neurons). K, Significantly increased Z-scored mean intensity of AHA neuronal response was observed following tone delivery on Day 2 (n = 90 neurons). L, Peri-event analysis centered around the onset of grooming at time point zero (n = 90 neurons, data shown as mean ± 1 SEM). M, Z-scored quantification of intensity changes showed significantly decreased activity in AHA neurons (n = 90 cells) during bouts of grooming compared with the pregrooming period on Day 2. Statistical tests are detailed in Extended Data Table 1-1.
- Figure 2.
Photoactivation of the VMH→AHA pathway increases jumping and grooming behaviors. A, Viral injection strategy to determine selectivity of AAV/Camk2a-ChR2-YFP for glutamatergic neurons. B, Expression of AAV/Camk2a-ChR2-YFP does not colocalize with AAV/FLEX-tdTomato in the VMH of VgatCre mice. Scale bars: 200 µm (left), 50 µm (right). White box represents approximate area of zoomed image of the dorsomedial VMH (right). C, Colocalization of AAV/Camk2a-ChR2-YFP and AAV/FLEX-tdTomato in the VMH of Vglut2Cre mice. Scale bars: 200 µm (left), 50 µm (right). White box represents approximate area of zoomed image of the dorsomedial VMH (right). D, Viral microinjection strategy for the expression of AAV/Camk2a-ChR2-YFP or YFP control fluorophore in VMH neurons with an optical fiber implanted above the VMH axonal projections in the AHA. ChR2-expressing mice, n = 7; fluorophore control mice, n = 11. E, Representative image depicting YFP fluorophore control expression in the VMH (left) and the immunostained (GFP-488) axon field with optical fiber tract (dotted white outline) in the AHA (right). Scale bar, 500 μm. F, Representative image depicting ChR2-YFP expression in the VMH (left) and the immunostained (GFP-488) axon field with optical fiber tract (dotted white outline) in the AHA (right). Scale bar, 500 μm. G, There was significantly greater fluorescence intensity in the VMH compared with other hypothalamic regions. H, Diagram depicting order of optogenetic behavioral experiments. I, No significant stimulation × transgene effect was detected for locomotor behavior in the OFT. J, A significant stimulation × transgene interaction was detected for jumping behavior marked by higher levels of jumping in ChR2-expressing mice. K, No stimulation × transgene effects were detected for time spent immobile. L, A significant stimulation × transgene effect was observed for time spent grooming marked by a higher amount of grooming in ChR2-expressing mice compared with fluorophore controls. M, A significant stimulation x transgene effect was observed for rearing behavior marked by reduced rearing in ChR2-expressing mice compared with fluorophore controls. N, No effects on time spent in the center zone were detected. Statistical tests are detailed in Extended Data Table 1-1.
- Figure 3.
Photoactivation of the VMH→AHA pathway drives avoidance and OFF zone repetitive grooming behavior. A, Mean heat maps for groups during RTPP showed that ChR2 photoactivation causes mice to spend more time in the Laser-OFF zone. B, Time spent in the laser-paired ON zone was significantly reduced in ChR2-expressing mice compared with fluorophore controls. C, The average duration in the Laser-ON zone was significantly reduced in ChR2-expressing mice compared with fluorophore controls. D, There were no significant differences detected in the number of Laser-ON zone entries between groups. E, Time immobile was significantly increased in ChR2 compared with fluorophore controls. F, Time spent grooming in the Laser-OFF zone showed ChR2-expressing mice exhibit significantly more grooming behavior than fluorophore controls, even when normalized for time in the OFF zone. G, A significant suppression of rearing behavior was observed in the ChR2-expressing mice compared with fluorophore controls in the Laser-ON and Laser-OFF zone. H, VMH→AHA photostimulation resulted in a significant suppression of digging behavior in the Laser-ON and Laser-OFF zone. Statistical tests are detailed in Extended Data Table 1-1.
- Figure 4.
Repeated intermittent photoactivation of the VMH→AHA pathway promotes lasting grooming behavior. VMH→AHA stimulation increased the percentage of time spent grooming postsession compared with fluorophore controls (p < 0.05 each day). Multiple comparisons reveal that grooming is greater in ChR2-expressing mice on Day 2 after the session (post) compared with behavior during the session. Statistical tests are detailed in Extended Data Table 1-1.
- Figure 5.
Photoactivation of the VMH→AHA pathway increases neuronal activity in the amygdala and ventral striatum. FDG-PET overlaid on structural MRI shows that VMH→AHA stimulation significantly increases FDG uptake compared with controls (p < 0.05, unpaired t test; n = 6 ChR2 and 10 YFP) in a region encompassing the lateral/ventrolateral striatum (left) and amygdala (right). Statistical tests are detailed in Extended Data Table 1-1.
Extended Data
Extended Data
Download Extended Data, ZIP file.
Table 1-1
Statistical analyses. Download Table 1-1, DOCX file.
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