CCK signaling is critical for LTP induction in the BLA.
As emotionally charged memory is associated with neuronal activity or neuroplasticity in the amygdala (Cahill, Babinsky et al. 1995), we first examined the dependency of CCK signaling in LTP induction in the BLA.
We adopted a brain-slice electrophysiological system with multiple slices and microelectrode arrays (fig.1 a) to compare the LTP induction in the BLA between the CCK-KO mice and its wild-type control. CCK-KO mice showed a deficit in TBS induced LTP in the BLA, as compared to their wild-type controls (two-way RM ANOVA, significant interaction, F [1,20] = 19.7, p = 2.5E-4 < 0.001; pairwise comparison, post hoc Bonferroni test; first 10 min before vs. last 10 min after the TBS in CCK-KO mice, 100.5 ± 0.3% vs. 112.4 ± 2.8%, P > 0.05; first 10 min before vs. last 10 min after the TBS in wild-type mice, 100.4 ± 0.5% vs. 161.4 ± 8.9%, P = 1.0E-7 < 0.001, fig.1 d).
It is known that LFS does not induce LTP in the field-potential recording setting. LFS induces long-term depression (LTD) when both the number of stimulation pulses (typically 900 pulses) and the current of stimulation are large (Collingridge, Peineau et al. 2010). As previously reported, we concluded that HFS induced the release of CCK from the presynaptic terminals, leading to LTP in the neocortex and amygdala (Chen, Li et al. 2019, Feng, Su et al. 2021). We then hypothesize that the application of the CCKBR agonist (CCK4) induces LTP in the BLA even under the LFS paradigm.
Application of CCK4 with LFS (100 pulses) induced LTP in the BLA, whereas application of the vehicle did not (two-way RM ANOVA, significant interaction, F [1,18] = 6.6, p = 0.02 < 0.05; pairwise comparison, post hoc Bonferroni test; with vehicle, first 10 min before vs. last 10 min after LFS, 99.4 ± 0.5% vs. 108.2 ± 4.6%, P > 0.05 ; with CCK4, first 10 min before vs. last 10 min after LFS, 99.4 ± 0.97% vs. 196.9.2 ± 34.6%, P = 0.005 < 0.01, fig.1 e). Both the loss-of-function and gain-of-function studies supported the critical role of CCK signaling in LTP induction or neuroplasticity in the BLA.
Figure 1. CCK signaling is critical in LTP induction in the BLA. a. Workflow of in-vitro recording and CCK4 administration, after 15min baseline recording, perfused artificial cerebrospinal fluid (ACSF) diluted with CCK4 for 5min with LFS, then fresh ACSF washed out CCK4 after. b. Light microscopy photograph showing the location of a 4*4 microelectrode array placed on the BLA region. c. Schematic diagram of TBS and LFS. d. Time courses of field excitatory postsynaptic potentials (fEPSPs) changes in response to TBS of wild-type mice (WT) and CCK-KO mice in the BLA (left). Representative fEPSPs traces before (gray) and after LTP induction (right). e. Time courses of fEPSPs changes in response to CCK4/vehicle with LFS in the BLA of WT mice (left). Representative fEPSPs traces before (gray) and after LTP induction (right). All data are the mean ± SEM.
Optogenetic activation of the ECcck–BLA pathway promoted stress susceptibility.
CCK signaling is critical in neuroplasticity in the amygdala. The fear memory can be inactivated and reactivated by LTD and LTP (Nabavi, Fox et al. 2014). Recently, we found that activating CCK-positive projection from the EC to LA by a high-frequency laser stimulation modulated the neuronal plasticity, and potentiated auditory response in LA neurons (Feng, Su et al. 2021). We implanted an optical fiber in the BLA after virus injection into the EC of CCK-Cre mice (AAV-EF1a-DIO-ChETA-EYFP in the experimental group, ChETA; AAV-EF1a-DIO- EYFP in the control group, EYFP; fig. 2 a b). To test whether CCK from the EC to BLA enhances the development of depression, we adopted the two-trial subthreshold social defeat stress (SSDS) model behaviorally. As described previously (Chaudhury, Walsh et al. 2013, Shen, Zheng et al. 2019), an intruder CCK-Cre mouse was introduced into the home cage of an aggressive novel resident CD1 mouse for 10 min physically defeat, then, they were maintained in sensory contact for 10 min. After a 5 min interval, the second trial was carried out.During the sensory stress phases of SSDS, we applied bilateral intra-BLA high-frequency laser stimulation (HFLS, 20HZ, duration 5ms, 600 pulses) to activate the ECcck-BLA projections (fig. 2 c).
We confirmed the injection site of the AAV virus in the EC and their projections in the BLA (fig. 2 d). The duration spent in the social interaction zone when a novel social target was present in the SI test showed no difference in the two groups (two-way RM ANOVA, F [1,26] = 0.40, p > 0.05; pairwise comparison, post hoc Bonferroni test; target phase, ChETA vs. EYFP, 52.6 ± 13.0s vs. 52.9 ± 8.0s, P > 0.05, fig. 2 e). However, the preference for sucrose solution of the ChETA group reduced significantly, compared with that of the control mice (two-sample t-test, ChETA vs. EYFP, 66.9 ± 3.9% vs. 78.1 ± 2.7%, t = -2.4, P = 0.02 < 0.05, fig. 2 f). These results indicated that the activation of CCK projections from the EC to BLA facilitated stress susceptibility and induced anhedonia phenotype, a loss of capability to perceive pleasure, one of the most stringent parameters in depression.
Figure 2. Optogenetic activation of the ECcck–BLA in SSDS induced depressive behaviors. a. The workflow of the SSDS model with optogenetic manipulation. Day 1, virus injection into the bilateral EC of CCK-Cre mice. Day 28, fibers implantation to the bilateral BLA. Day 35, SSDS paradigm with optogenetic activations. Day 36, SI, SPT. Day 43, BLA tissue collection for the immunohistochemistry (IHC) test. b. Schematic diagram of virus injection of EC and optical fiber implantation of BLA. c. Paradigms of 20 Hz optogenetic activation of CCK fibers in the EC-BLA circuit during two sensory phases of SSDS. d. CHETA-EYFP or EYFP was specifically expressed in CCK neurons at EC (up) and projections to BLA (bottom), scale bar, 500 μm. e. Schematic diagram of SI (left). Time that mice spent in social interaction zone, two-way RM ANOVA, pairwise comparison, post hoc Bonferroni test (right). f. Sucrose preference percentage in the SPT, two-sample t-test. * P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant. All data are the mean ± SEM.
CSDS activated CCKBR neurons at BLA.
Activity in the amygdala is higher when a person is sad or clinically depressed (Schneider, Grodd et al. 1997, Peluso, Glahn et al. 2009, Yang T T 2010). The CSDS is a widely accepted model for depression study (Golden, Covington et al. 2011). In the next experiment, we examined if CSDS could induce the hyperactivated amygdala. As CCKBRs are expressed intensively in the amygdala (Honda, Wada et al. 1993, Feng, Su et al. 2021), we examined whether the activated neurons correlate with CCKBRs.
We adopted a 10-day protocol of CSDS in depression induction in the mice (supplementary fig. 1 a). The time spent in the social interaction zone in the presence of a social target of the mice who underwent the CSDS is significantly less than that of sham group mice (two-way RM ANOVA, F [1,13] = 4.49, p = 0.05; pairwise Comparison, post hoc Bonferroni test; with the target, CSDS vs. sham, 64.1.8 ± 2.3s vs. 106.8 ± 4.6s, P = 0.03 < 0.05; supplementary fig. 1 c). We measured c-Fos and CCKBR expression in the BLA when the mice were re-exposed to novel CD1 mice. Notably, c-Fos expression in the BLA was significantly higher than in the sham group mice (two-sample test, t = -7.0, P = 1.9E-7 < 0.001; supplementary fig. 1 d-e). Furthermore, 91.5 ± 2.1% of c-Fos-positive neurons activated by stress in CSDS mice were colocalized with CCKBR neurons (supplementary fig. 1 f). These results indicated that CCKBR correlated with the hyperactivation of the BLA of CSDS mice.
CCKBR antagonist suppressed LTP induction in the BLA
Previously, we have shown that CCKBR antagonists block the LTP induction in the neocortex and the encoding of sound-sound and visuoauditory associative memory (Li, Yu et al. 2014, Chen, Li et al. 2019). Here, we investigated the role of CCKBR in LTP induction in the BLA with brain-slice preparation.
Transgenic mice lacking the CCKBR gene (CCKBR-KO) exhibited a deficit of TBS-induced LTP in the BLA, as compared to their wild-type (WT, 129S1 mice) control (two-way RM ANOVA, significant interaction, F [1,17] = 24.4, p = 1.2E-4 < 0.001; pairwise comparison, post hoc Bonferroni test; in CCKBR-KO mice, first 10 min before vs. last 10 min after TBS, 100.0 ± 0.3% vs. 102.7 ± 2.4% , P = 0.57 > 0.05; in WT mice, first 10 min before vs. last 10 min after TBS, 99.8 ± 0.5% vs. 135.4 ± 6.0%, P = 2.1E-6 < 0.001, fig. 3 b).
A natural question was whether a CCKBR antagonist could block or suppress the LTP induction in the BLA. YM022 is a selective CCKBR antagonist, with an IC50 for CCKBR of 68 pM vs 63 nM for CCKAR (Nishida, Miyata et al. 1994). Perfusion of YM022 (1 nM/10 nM) suppressed the LTP induction in the BLA of C57 mice, whereas perfusion of the vehicle did not (two-way RM ANOVA, significant interaction, F[2, 23] = 9.78, P = 8.4E-4 < 0.001; pairwise comparison, post hoc Bonferroni test; with vehicle, first 10 min before vs. last 10 min after TBS, 99.20 ± 0.3% vs. 172.3 ± 18.0%, P = 3.9E-6 < 0.001; 1 nM of YM022, first 10 min before vs. last 10 min after TBS, 99.6 ± 0.9% vs. 122.1 ± 2.8%, P > 0.05; 10 nM of YM022, first 10 min before vs. last 10 min after TBS, 99.6 ± 0.4% vs. 110.2 ± 5.6%, P > 0.05; last 10 min after TBS, between groups, vehicle vs. 1 nM of YM022, 172.3 ± 18.0% vs. 122.1 ± 2.8%, p = 8.5E-4 < 0.001, vehicle vs. 10 nM of YM022, 172.3 ± 18.0% vs. 110.2 ± 5.6%, P = 2.3E-5 < 0.001, fig. 3 c).
Another selective CCKBR antagonist YF476 with an IC50 for CCKBR of 0.1 nM vs 502 nM for CCKAR (Semple, Ryder et al. 1997) , 10nM of YF476 fully blocked TBS-induced LTP in the BLA of C57 mice (two-way RM ANOVA, significant interaction, F[1, 18] = 14.6, P = 0.001; pairwise comparison, post hoc Bonferroni test ; for YF476, first 10 min before vs. last 10 min after TBS, 101.0 ± 0.7% vs. 106.5 ± 9.0%, P > 0.05; for vehicle, first 10 min before vs. last 10 min after TBS, 101.1 ± 0.8% vs. 187.5 ± 17.4%, P = 2.67E-5 < 0.001; last 10 min after TBS, between groups, vehicle vs. YF476, 187.5 ± 17.4% vs. 106.5 ± 9.0%, P = 1.1E-4 < 0.001, supplementary fig. 2 b).
Figure 3. LTP deficit in CCKBR-KO mice and YM022 blocked LTP in the BLA. a. Workflow of in-vitro recording and drug administration, after 15min baseline recording, perfused ACSF diluted with drug for 10min, then fresh ACSF washed out drug after TBS. b. Time courses of fEPSPs changes in response to TBS of wild-type mice (WT) and CCKBR-KO mice in the BLA (left). Representative fEPSPs traces before (gray) and after LTP induction (right). c. Time courses of fEPSPs changes of C57 wild-type mice (WT) in response to YM022/vehicle with TBS in the BLA (left). Representative fEPSPs traces before (gray) and after LTP induction (right). All data are the mean ± SEM.
CCKBR-KO and CCK-KO transgenic mice demonstrated lower levels of depression-like behaviors
Before testing CCKBR antagonists in the behavioral animal model, we examined how CCKBR-KO and CCK-KO mice performed in the TST and FST in terms of the total immobility time. The shorter total immobility time indicates less despair behavior, indicating less depression-like activity (Vincent Castagn´e 2010). The CCKBR-KO mice had significantly less immobility time in the FST and TST than the wild-type (ICR mice) (fig. 4 b-c; CCKBR-KO vs. wild-type, in FST, 118.8 ± 17.3s vs.197.4 ± 12.3s, two-sample t-test, t = 3.7, P = 0.002 < 0.01; in TST, 67.1 ± 15.5s vs. 131.8 ± 21.2s, two-sample t-test, t = 2.5, P = 0.02 < 0.05). However, no significant difference in immobility times was observed in the FST and TST for the CCK-KO mice compared with the wild-type background (supplementary fig 3. a b; CCK-KO vs. wild-type, in FST, 180.7 ± 4.3s vs. 185.8 ± 3.6s, two-sample t-test, t = 0.9, P > 0.05; in TST, 186.1 ± 15.6s vs. 208.6 ± 16.1s, two-sample t-test, t =1.0, P > 0.05).
The OFT is the most widely used technique to investigate the anxiolytic or anxiogenic effects of pharmacological compounds (Seibenhener and Wooten 2015). Compared with WT mice, the CCKBR-KO mice spent significantly more time in the center zone (fig. 4 d, CCKBR-KO vs. wild-type, 17.7 ± 1.2% vs.12.4 ± 1.1%, two-sample t-test, t = -3.3, P = 0.005 < 0.01), and traveled significantly more distance (fig. 4 e, CCKBR-KO vs. wild-type, 49.6 ± 1.8m vs. 29.1 ± 1.8m, two-sample t-test, t = -8.0, P = 1.4E-6 < 0.001) and defecated significantly fewer feces than the wild-type in the open field (fig. 4 f, CCKBR-KO vs. wild-type, 1.7 ± 0.5 vs. 5.4 ± 0.6, two-sample t-test, t = 4.8, P = 2.9E-4 < 0.001). Notably, the CCK-KO mice spent significantly more time in the central area than their wild-type control (supplementary fig. 3 d, CCK-KO vs. wild-type, 5.8 ± 1% vs. 2.4 ± 0.5%, two-sample t-test, t= -3.0, P = 0.01 < 0.05). We observed no significant difference in fecal boil deposits and total distance in the OFT (supplementary fig 3. e f: CCK-KO vs. wild-type, total distance, 17.5 ± 0.8m vs. 17.5 ± 1.5m, two-sample t-test, t = 0.007, P > 0.05; fecal boil, 3.6 ± 0.9 vs. 4.9 ± 0.7, two-sample t-test, t = 1.0, P > 0.05). In summary, the CCKBR-KO and CCK-KO mice showed a lower level of anxiety-like behavior as compared with their WT controls.
Figure 4. Antidepressant-related behavioral responses of CCKBR-KO mice and antidepressant effect of YM022 in acute stress model. a. Schematic of the TST and FST. b.Total immobility time in the last 4 min of the FST. wild-type mice (WT, N= 8) vs. CCKBR-KO mice (N=9 ), two-sample t-test. c. Total immobility time in 6 min of the TST, WT( N= 8) vs. CCKBR-KO (N=9 ), two-sample t-test. d-g. The anxiolytic behavior of CCKBR-KO mice (N=7) in the OFT, WT group (N=9). d. Time percentage spent in the center percentage, two-sample t-test. e. Total distance traveled, two-sample t-test. f. Fecal deposits, two-sample t-test. g. Representative traces in OFT (5min). h. YM022 administration 30 min before TST test by IP injection. i, Total immobility time in 6 min of the TST, saline group (N=16); YM022 groups, 0.3 μg/kg (N= 9), 0.6 μg/kg (N= 10), 3.0 μg/kg (N= 10), 30 μg/kg (N= 10), fluoxetine 10mg.kg (N= 10), one-way ANOVA, pairwise comparison, post hoc Bonferroni test. j. YM022 administration 30 min before the OFT test by IP injection. k-m. The anxiolytic effect of YM022 (3.0ug/kg, N=10) in the OFT, vehicle group (N=9). k. Time percentage spent in the center, two-sample t-test. i. Total distance traveled in the OFT, two-sample t-test. m. Representative traces in OFT (10min test time).* P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant. All data are the mean ± SEM.
Blood-brain barrier penetration of CCKBR antagonists, YM022 and YF476
Depression is associated with the hyperactivity of the amygdala (Mayo_Clinic , Siegle, Thompson et al. 2007, Peluso, Glahn et al. 2009, Yang T T 2010). Most c-Fos expressed neurons in the BLA after CSDS were CCKBR positive neurons. CCKBR mediates the CCK signaling of EC-LA projections and LTP in the BLA. Our previous study demonstrated that the EC-LA CCK projection enabled the association between the auditory cortex and the amygdala, linking the fear memory with the sensory signal (Feng, Su et al. 2021). Up to this point, it is reasonable to examine whether CCKBR antagonists can alleviate the depression symptoms, by suppressing the LTP induction that encodes/consolidate aversive memory in the BLA. To show an antidepressant-like response, a drug must cross the blood-brain barrier (BBB) so that it can reach the target. According to Pardridge, around 98% of small molecules cannot penetrate the blood-brain barrier (BBB) and work on the CNS [20]. Before we apply the CCKBR antagonists, YM022 and YF476, for behavioral assay, we examined their BBB permeability and pharmacokinetics.
After single oral administration of YM022 (5 mg/kg) and YF476 (5 mg/kg), the plasma and brain samples were obtained at the several time points after dosing: 15, 30 min, and 1, 2, 4, 6, 8, 24 hours, and analyzed by LC-MS/MS analysis (supplementary fig. 4 a). The peak concentration of YM022 in brain tissue was 6.0 ± 1.8 ug/L at 30min post-dose; pharmacokinetic profiles of YM022 in the plasma showed that maximum concentration(Cmax) was 215.2 ug/L, time for Cmax to occur (Tmax) was 0.38 hour, half-life (T1/2) was 1.3 hours, and area under the plasma concentration-time curve AUC (0-∞) was 321.8 ug/L*h (supplementary fig. 4 b-c). The peak concentration of YF476 in brain tissue was 32.7 ± 6.8 ug/L at 15min post-dose; pharmacokinetic parameters of YF476 in plasma Cmax was 341.5 ug/L, Tmax was 0.38 hour, T1/2 was 0.5 hours, and AUC (0-∞) was 228.1 ug/L*h (supplementary fig. 4 d-e). The results indicated that YM022 and YF476 can penetrate the BBB.
CCKBR antagonists showed antidepressant effects in the acute TST and OFT
Determination of the total immobility time (despair behavior) in the TST allows a quick assessment of potential antidepressant effects, various classes of antidepressant drugs decrease immobility time in the TST. (Cryan, Mombereau et al. 2005, Vincent Castagn´e 2010). We examined the effects of acute administration of CCKBR antagonists on the duration of immobility in the TST.
We compared the effects of the CCKBR antagonists, YM022 and YF476, with the saline control and current antidepressant, fluoxetine (a selective serotonin receptor inhibitors, SSRIs) as the positive control. The fluoxetine at a dose of 10 mg/kg significantly decreased the total immobility time in the TST; of four different concentrations of YM022 (0.3 μg/kg, 0.6 μg/kg, 3.0 μg/kg, and 30 μg/kg) that were tested, mice showed a considerable less immobility time with doses of 0.6 μg/kg and 3.0 μg/kg (fig. 4 i, one-way ANOVA, F [5,59] = 5.6, P = 2.81E-4 < 0.001; means comparisons, post hoc Bonferroni test; fluoxetine vs. saline, 92.2 ± 15.8s vs. 192.3 ± 16.9s, P = 0.001 < 0.01; for YM022, 0.3 μg/kg vs. saline, 186.1 ± 14.3s vs.192.3 ± 16.9s, P > 0.05, 0.6 μg/kg vs. saline, 117.6 ± 13.6s vs.192.3 ± 16.9s, P = 0.035 < 0.05, 3.0μg/kg vs. saline, 118.1 ± 13.4s vs.192.3 ± 16.9s, P = 0.038 < 0.05, 30μg/kg vs. saline, 132.3 ± 26.3s vs.192.3 ± 16.9s, P > 0.05). Moreover, mice treated with YF476 also showed a significant decreased immobility time with a doses of 1.3 μg/kg and 3.0 μg/kg (one-way ANOVA, F [4,45] = 9.0, P = 2.0E-5 < 0.001, means comparisons, post hoc Bonferroni test; for YF476, 0.3 μg/kg vs. saline, 231.3 ± 13.6s vs. 195 ± 11.3s, P > 0.05, 1.3 μg/kg vs. saline, 111.1 ± 15.4s vs. 195 ± 11.3s, p = 0.012 < 0.05, 3.0μg/kg vs. saline, 114.8 ± 21.7s vs. 195 ± 11.3s, p = 0.024 < 0.05, 30μg/kg vs. saline, 137.9 ± 23.2s vs. 195 ± 11.3s, p > 0.05, supplementary fig. 5 b). As the period of immobility is an indicator of hopelessness during the TST test, CCKBR antagonists, both YM022 and YF476, demonstrated a similar beneficial effect as compared to fluoxetine a positive control.
Next, we adopted the OFT to evaluate the anxiolytic effect of YM022. We administrated intraperitoneally YM022 (3.0 ug/kg) 30 min before the OFT. We observed no difference in the total distance traveled between vehicle and YM022 treated mice during the 10-min test time (fig. 4 l, vehicle vs. YM022, 63.6 ± 4.3m vs. 63.3 ± 2.8m, two-sample t-test, t = 0.06, P > 0.05), but YM022 treated mice spent significantly more time in the center (fig. 4 k, vehicle vs. YM022, 14.9 ± 0.86% vs. 18.8 ± 1.4%, two-sample t-test, t = -2.4, P = 0.03 < 0.05). This result revealed that CCKBR antagonist YM022 has anxiolytic effects at the dose of 3.0 ug/kg.
Figure 4. Antidepressant-related behavioral responses of CCKBR-KO mice and antidepressant effect of YM022 in acute stress model. a. Schematic of the TST and FST. b. Total immobility time in the last 4 min of the FST. wild-type mice (WT, N= 8) vs. CCKBR-KO mice (N=9 ), two-sample t-test. c. Total immobility time in 6 min of the TST, WT( N= 8) vs. CCKBR-KO (N=9 ), two-sample t-test. d-g. The anxiolytic behavior of CCKBR-KO mice (N=7) in the OFT, WT group (N=9). d. Time percentage spent in the center percentage, two-sample t-test. e. Total distance traveled, two-sample t-test. f. Fecal deposits, two-sample t-test. g. Representative traces in OFT (5min). h. YM022 administration 30 min before TST test by IP injection. i. Total immobility time in 6 min of the TST, saline group (N=16); YM022 groups, 0.3 μg/kg (N= 9), 0.6 μg/kg (N= 10), 3.0 μg/kg (N= 10), 30 μg/kg (N= 10), fluoxetine 10mg.kg (N= 10), one-way ANOVA, pairwise comparison, post hoc Bonferroni test. j. YM022 administration 30 min before the OFT test by IP injection. k-m. The anxiolytic effect of YM022 (3.0ug/kg, N=10) in the OFT, vehicle group (N=9). k. Time percentage spent in the center, two-sample t-test. l. Total distance traveled in the OFT, two-sample t-test. m. Representative traces in OFT (10min test time).* P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant. All data are the mean ± SEM.
Antidepressant effects of CCKBR antagonists in the CSDS model
Finally, we used the CSDS to examine whether CCKBR antagonists have a possible antidepressant effect. We administrated YM022 30 min before each physical defeat to prevent the development of depressive-like behavior in mice. We conducted SI and SPT respectively, 24 hours after the final social defeat(fig 5. a).
YM022 (3.0 μg/kg) and positive control fluoxetine (10mg/kg) treated mice spent significantly more time at the social interaction zone with the social target than vehicle treated mice (fig 5. b, two-way RM ANOVA , F [3,78] = 7.5, p = 1.9E-4 < 0.001; pairwise comparison, post hoc Bonferroni test; target phase, vehicle vs. sham, 58.5 ± 5.6s vs. 79.8.8 ± 3.4s, p = 0.058, vehicle vs YM022, 58.5 ± 5.6s vs. 79.3 ± 5.3s, p = 0.03 < 0.05, vehicle vs fluoxetine, 58.5 ± 5.6s vs. 88.8 ± 6.2s, p = 7.28148E-4 < 0.001), and the social interaction ratio in drug treated group is also increased significantly (fig 5. c, one-way ANOVA, F [3,78] = 5.49, P = 0.002 < 0.01; means comparison, post hoc Bonferroni test; vehicle vs. sham, 0.99 ± 0.11 vs. 1.6 ± 0.1, P = 0.003 < 0.01, vehicle vs. YM022, 0.99 ± 0.11 vs. 1.47 ± 0.1; P = 0.02 < 0.05, vehicle vs fluoxetine, 0.99 ± 0.11 vs. 1.5 ± 0.15, P = 0.026 < 0.05).
Besides, it has been found that only the vehicle group mice spent significantly more time in corners than the sham, YM022, and fluoxetine treated mice, to actively avoid the novel social target (fig 5. d, two-way RM ANOVA, F [3,78] = 8.83, p = 4.1E-5 < 0.001; pairwise comparison, post hoc Bonferroni test; vehicle vs. sham , 35.8 ± 5.2s vs. 16.1 ± 1.6s, P = 5.9E-4 < 0.001, vehicle vs. YM022, 35.8 ± 5.2s vs. 15.9 ± 2.9s, p = 1.34E-4 < 0.001, vehicle vs. fluoxetine, 35.8 ± 5.2s vs. 17.0 ± 3.2s p = 0.002 < 0.01). The corner ratio showed a significant difference accordingly (fig 5. e, one-way ANOVA, F [3,78] = 13.7, P =2.9E-7 < 0.001; means comparison, post hoc Bonferroni test; vehicle vs. sham, 2.15 ± 0.29 vs. 0.73 ± 0.09, P = 1.9E-5 < 0.001, vehicle vs YM022, 2.2 ± 0.29 vs. 0.65 ± 0.14, P = 9.95E-7< 0.001, vehicle vs fluoxetine, 2.2 ± 0.29 vs. 0.93 ± 0.16, P = 3.6E-4 < 0.001).
Consistent with social interaction behavior results, sham, YM022, and fluoxetine groups consumed significantly more sucrose liquid than vehicle group mice (fig 5. g, one-way ANOVA, F [3,78] = 8.25734 , P = 7.7E-5 < 0.001; means comparison, post hoc Bonferroni test; vehicle vs. sham, 66.9 ± 1.7% vs. 78.9 ± 1.8%, P = 4.6E-5 < 0.001, vehicle vs YM022, 66.9 ± 1.7% vs. 74.4 ± 1.8%, P = 0.01 < 0.05, vehicle vs fluoxetine, 66.9 ± 1.7% vs. 74.6 ± 1.6%, P = 0.019 < 0.05).
These results demonstrated that CCKBR antagonist YM022 effectively prevented CSDS-induced social avoidance and anhedonia in mice.
Figure 5. Antidepressant effects of YM022 in CSDS model. a. The workflow of CSDS with vehicle/YM022(3ug/kg)/fluoxetine(10mg/kg) injection 30min before every social defeat (left) and sham group(right). b. Time spent in social interaction zone in the absence and presence of a social target. Two-way RM ANOVA, pairwise comparison, post hoc Bonferroni test. c. Social interaction ratio = time spent in the social interaction zone with target/time spent in the social interaction zone without a target. One-way ANOVA, pairwise comparison, post hoc Bonferroni test. d. The time that mice spent in corners in the absence and presence of a social target. Two-way RM ANOVA, pairwise comparison, post hoc Bonferroni test. e. Corner ratio = time spent in the corner with target/time spent in the corner without a target, One-way ANOVA, pairwise comparison, post hoc Bonferroni test. f. Representative sample track traces of social interaction test no-target phase (top), and the target phase (bottom). g. Sucrose preference percentage in SPT. One-way ANOVA, pairwise comparison, post hoc Bonferroni test. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant. All data are the mean ± SEM.