VTA-projecting cerebellar neurons mediate stress-dependent depression-like behavior

Although cerebellar alterations have been implicated in mental depression, the exact contribution of the cerebellum to depressive symptoms remains to be elucidated. Here, we demonstrated the crucial role of cerebellar neurons projecting to the ventral tegmental area (VTA) in the chronic stress-induced development of depression-like behavior. The combination of adeno-associated virus-based circuit mapping and electrophysiological recording identified network connections from crus I to the VTA via the dentate nucleus (DN) of the deep cerebellar nuclei. Chronic chemogenetic activation of inhibitory Purkinje cells in crus I suppressed c-Fos expression in the DN and depression-like behavior, which were triggered by chronic stress application. Furthermore, specific inhibition of neurons in the DN that project to the VTA prevented stressed mice from showing depression-like behavior, whereas specific activation of these neurons alone triggered depression-like behavior that was comparable with the one triggered by chronic stress application. Our results indicate that the VTA-projecting cerebellar neurons proactively regulate depression-like behavior, raising the possibility that cerebellum may be an effective target for the prevention of depressive disorders.

whereas specific activation of these neurons alone triggered depression-like behavior that 28 was comparable with the one triggered by chronic stress application. Our results indicate 29 that the VTA-projecting cerebellar neurons proactively regulate depression-like behavior, 30 raising the possibility that cerebellum may be an effective target for the prevention of 31 depressive disorders. 32

Introduction 41
Whereas the cerebellum has traditionally been considered to be important solely for motor 42 coordination and learning, it became apparent that the cerebellum is also involved in higher 43 cognitive functions (Rochefort et  The cerebellum interacts with many brain areas through both direct and indirect 63 synaptic connections, and one of the brain areas receiving direct inputs from the deep 64 cerebellar nuclei (DCN), which is the major cerebellar output, is the ventral tegmental area application. The c-Fos expression in the RS group was significantly enhanced compared 274 with the CTR group even by 3 days of RS application, and was maintained at elevated level 275 after 7-or 10-day RS application ( Figure 4H; 3, 7, or 10 days: p < 0.001). Thus, the DCN 276 neurons in the DN were already activated at the early stage of chronic stress, suggesting 277 that the DCN neuron activity may encode the stressful situations.  The increase in activity was also confirmed in vivo by immunohistochemical analysis of c-296 Fos, which showed that intraperitoneal administration of CNO into mice expressing Gq in 297 PCs resulted in an increased c-Fos level in Gq-expressing PCs in crus I ( Figure 5C). In 298 addition, when we used this chemogenetic tool for enhancing crus I PC activity by the daily 299 injection of CNO for the 2 weeks prior to RS application ( Figure 5D), c-Fos expression in the 300 DN was reduced compared with when only RS was applied ( Figure 5E). Quantified data 301 showed that c-Fos-positive cells in the DN after chronically enhancing the activity of crus I 302 PCs during RS were significantly decreased and the level was equivalent to the control 303 ( Figure 5F; RS -Gq-CNO: t(11) = -4.25, p < 0.001; CTR -Gq-CNO: t(11) = -0.43, p = 0.67). 304 Thus, our chemogenetic manipulation of PCs appeared to be an appropriate experimental 305 system for testing the involvement of the cerebellum in the development of depressive 306

symptoms. 307
In the behavioral experiments of this study, in which we analyzed the effects of 308 chemogenetic manipulation on RS-dependent depression-like behaviors, AAVs expressing 309 chemogenetic molecules were stereotaxically injected 3 weeks before the RS procedures, 310 and CNO or saline was intraperitoneally administered 30 minutes before the RS application 311 every day during the 2-week RS period ( Figure 5D), unless stated otherwise. To enable the 312 comparison within a series of behavioral experiments even in the presence of mild variability 313 between different series, we included the CTR and the RS groups in all series of 314 concurrently performed behavioral experiments, and compared groups of saline or CNO 315 administration to the CTR and the RS groups. When saline (RS-Sal) or CNO (RS-CNO) was 316 administered to the mice that were subjected to RS without stereotaxic AAV injection, the 317 effects of RS were not altered, as seen by the significantly longer immobile times than the 318 CTR group, but the similar immobile times to the RS group, in both the TST and FST (  and that both tests rely on immobility to evaluate same dimension of depression, i.e., despair 373 or hopelessness. Two further analyses were thus conducted. Firstly, we examined the 374 correlation of individual data to see the inter-relationship in different combination among 375 three behavioural tests. There was a significant correlation between TST and FST, but not 376 between OFT and TST or FST (Table 1;    Approximately 2 weeks after AAV injection for neuronal tracing analysis, 5-week-old mice 699 were anesthetized with isoflurane and perfused transcardially with 4% paraformaldehyde 700 (PFA) in 0.1 M sodium phosphate buffer (pH 7.4). To confirm successful AAV injection in 701 mice subjected to behavior analysis, or to observe c-Fos expression, 10 to 12-week-old mice 702 were also similarly perfused after all behavior tests or right after 2 weeks of RS, respectively. 703 To deliver restraint stress, 8 to 9-week-old of PCP2-Cre or C57BL/6J mice were each placed 800 in a well-ventilated 50 mL conical tube with holes along the side and at the tip, where the 801 nose is positioned. Depending on the size of the mouse, a 3.5 to 7-cm-long tube (15 mL) 802 was inserted into the 50 mL tube to fill the leftover space after the mouse is placed into the 803 50 mL tube. The 50 mL tube was closed with a cap that had a hole for letting the tail out. 804 Mice were unable to move both forward and backward in the tube. Mice were subjected to 805 2 hours of this RS every day for 2 weeks, which is the protocol of RS in this study. The main symptoms of depression include feelings of worthlessness and helplessness. To 851 evaluate depression, two behavioral tests, i.e., the TST and FST, which are widely applied 852 in depression research were primarily used. After AAV injections at P35, the mice were left 853 for another 3 to 4 weeks to ensure full expression of the constructs, and then the handling 854 process (5 minutes for 3 days) was started when the mice were 8 to 9-weeks old. After the 855 2 weeks of RS and/or CNO administration, three behavioral tests were performed in the next 856 3 consecutive days, i.e., the OFT for testing general locomotor activity, and the TST and 857 FST for assessing depression-like behaviors. In separate sets of behavioral experiments, 858 depression-like behaviors were assessed by the SST and NSFT, which are tests for a lack 859 of self-care and hyponeophagia or anhedonia, respectively, performed in the next 2 860 consecutive days after the last day of RS or chemogenetic manipulation. In addition, to test 861 whether chemogenetic activation of VTA-projecting DCN neurons could result in persistent 862 depression-like behaviors, the OFT, TST, and FST were performed a week after the SST 863 and NSFT. The order of behavioral tests was OFT, TST, and FST, or SST and NSFT. 864 Although we cannot deny a possibility that the order of test may affect behavioral 865 consequences, such as relatively smaller effects of RS in TST than FST, depression-like 866 behaviors were always confirmed by including the CTR group and the RS group in one 867 series of concurrently processed behavioral experiments. The results of the behavioral tests 868 were analyzed using Ethovision software (Noldus) with an immobility threshold of 5% for the 869 results of the TST and FST. 870 871

Tail suspension test (TST) 872
Each mouse was suspended with a 20-cm-long tape from a rod, which was horizontally 873 placed 50 cm above the floor. A 3-cm-long 15 mL conical tube was placed through the tail 874 to prevent the mice from climbing back. The TST lasted for 6 minutes and video recordings 875 of the last 4 minutes were used for the analysis. Immobility was considered as a mouse 876 being completely motionless while being hung. 877 878

Forced swimming test (FST) 879
Each mouse was put into an acrylic cylinder (30-cm high and 15 cm in diameter) filled with 880 water at 22 to 24 °C. The depth of the water was up to 15 cm of the cylinder. Trials were 881 video-recorded for a total of 6 minutes, and the last 4 minutes of the recording were used 882 for analysis. Immobility was considered as remaining motionless except for movements that 883 were necessary for the mice to float and to keep their balance or keep their head/nose above 884 the water.

Sucrose splash test (SST) 896
The SST was performed in the home cage with wooden bedding. A mouse was placed in 897 the cage, and sprayed with approximately 200 μl sucrose solutions (10%) directly onto its 898 back. Grooming behavior was then recorded for 5 min, and total duration of grooming was 899 measured. Touching, scrubbing and licking their fur was considered as the grooming 900 behaviors. 901

Novelty suppressed feeding test (NSFT) 903
The NSFT was performed by scoring the latency to feed, when a food-deprived mouse is 904 introduced to an unfamiliar environment in the white plexiglass container (50 cm ´ 50 cm ´ 905 35 cm) with the floor covered by wooden bedding. All food was removed 24 hours prior the 906 test in the home cage. A single sweet food pellet (soaked in 50% sucrose) was placed in 907 the center of the container that was brightly illuminated (500 lux). Once a mouse was placed 908 in the test container, the latency to feed was measured during 10 min. Mice that exceeded 909 10 minutes without eating the pellet were excluded from the data analysis. After the test, the 910 mouse was immediately returned into its home cage and further recorded for 5 min to 911 measure latency to feed in the home cage as a sign of control feeding drive, which was not 912 significantly different between groups ( Figure 6G, Figure7-figure supplement 1H).  unpaired Student's t-test was used for two-group comparisons, and one-way ANOVA 921 followed by the Fisher's least significant difference (LSD) post hoc test was conducted to 922 compare significant differences between more than two groups. We described summary 923 statistics (t-tests) in Results, and listed details of statistical information, including exact p 924 values and the statistical tests used, in Source data 1. To explore the possibility that 925 individual variability in the TST and FST would be in part due to the degree of depression, 926 or due to the degree of abnormal locomotion, correlation between TST and FST, TST and 927 OFT, or FST and OFT was examined using Pearson's correlation coefficient (Table 1)