The DMSO group (M = 17.75, SD = 13.63) and saline group (M = 23.86, SD = 17.24) showed similar immobility times in FST-2 (t(8) = 0.62, p = 0.55, Student’s t-test). There were also no differences in locomotor activity (t(8) = 0.55, p = 0.59) or time spent in the center of the OFT (t(8) = 0.63, p = 0.54, Student’s t-test). In addition, both groups displayed similar levels of anxiety in the EPM (t(8) = 0.77, p = 0.46, Student’s t-test). These findings are in line with earlier behavioral studies that utilize saline and DMSO or Tween-80 as vehicle groups, and demonstrate comparable durations in locomotor activity (Amiri et al. 2015; Castro et al. 1995; Jesse et al. 2008; Konieczny et al. 1998) and behavioral despair (Amiri et al. 2015; Tanyeri et al. 2013; Zomkowski et al. 2005).

We confirmed the antidepressant effect of the 10 mg/kg dose of ketamine (i.e. Ket10 group) in the FST, which produced a significant reduction in immobility (M = 18.23, SD = 11.18) compared to the vehicle group (M = 44.49, SD = 21.90; t(14) = 3.01, p = 0.009, d = 1.51, Student’s t-test). In contrast, the sub-effective dose used in the Ket1 group led to similar immobility levels (M = 31.54, SD = 26.42) with the control animals (t(14) = 1.06, p = 0.304, Student’s t-test).

We analyzed FST-1 by dividing it into three 5-min periods to confirm the induction of behavioral despair. We found a significant increase in immobility behavior over time (F(2,110) = 98.07, p < 0.001, R2 = 0.64, repeated measures one-way ANOVA), consistent with earlier protocols (Porsolt et al. 1977; Yankelevitch-Yahav et al. 2015). Immobility time during the last five minutes (M = 78.53, SD = 38.39) was higher than in the second five minutes (M = 62.59, SD = 29.43; t(110) = 4.00, p < 0.001, d = 0.46), and the first five minutes (M = 24.27, SD = 14.69; t(110) = 13.62, p < 0.001, d = 1.86). Similarly, immobility time significantly increased from the first five minutes to the second five minutes of FST-1 (t(110) = 9.62, p < 0.001, d = 1.64, Sidak’s corrected). We also compared the immobility time in the first five minutes of FST-1 to FST-2 for each group, and found that MTEP led to an elevated immobility duration in FST-2 (M = 37.35, SD = 21.00) compared to the initial phase of FST-1 (M = 20.91, SD = 8.91; t(7) = 3.04, p = 0.019, Student’s t-test).

In following experiments, pre-treatment with mGluR5 agents was done 10 min before ketamine administration as explained earlier (Fig. 2A). We observed a significant main effect of ketamine following mGluR5 activation via CDPPB pre-treatment (F(1,28) = 12.90, p = 0.001, R2 = 19.41, 2 × 2 two-way ANOVA; Fig. 2B), as the Ket10 group displayed less immobility compared to the vehicle group (M = 44.49, SD = 21.90; t(28) = 2.90, p = 0.021). The immobility scores of CDPPB + Ket10 (M = 25.76, SD = 19.93) did not differ from the vehicle group (M = 44.49, SD = 21.90), t(28) = 2.07, p = 0.137) or the Ket10 group (t(28) = 0.83, p = 0.797, Sidak’s corrected; Fig. 2B), suggesting a partial attenuation (Koike et al. 2011; Li et al. 2015) or occlusion (Gerhard et al. 2020) of the antidepressant effect of ketamine by CDPPB pre-treatment.

The forced swim test (FST). A) The experimental design and treatment schedule of the FST, and the legend for all experimental groups. B) The duration of immobility. C) The duration of struggling behavior. D) The duration of swimming. E) The number of headshaking behaviors. Asterisks denote statistically significant (p < .05) main effects and post-hoc comparisons (brackets). Error bars represent SEM

The combination of the sub-effective dose of ketamine (1 mg/kg) with MTEP (1.25 mg/kg) also revealed the effect of ketamine (F(1,28) = 4.35, p = 0.046, R2 = 12.72; Fig. 2B). The MTEP + Ket1 group (M = 18.93, SD = 13.85) exhibited significantly less immobility compared to the vehicle group (M = 44.49, SD = 21.90; t(28) = 2.40, p = 0.046, d = 1.39; Fig. 2B). In contrast, animals receiving MTEP alone (M = 37.35, SD = 20.99) showed a similar immobility duration to the vehicle group (M = 44.49, SD = 21.90). Likewise, the Ket1 group (M = 31.54, SD = 26.42) did not differ from the vehicle group (M = 44.49, SD = 21.90; t(28) = 1.21, p = 0.413, Sidak’s corrected). These findings collectively indicate a synergistic antidepressant effect of MTEP and ketamine.

The struggling behavior observed in the FST was affected by the administration of ketamine (10 mg/kg) (F(1,28) = 4.67, p = 0.039, R2 = 14.08, 2 × 2 two-way ANOVA). However, CDPPB (F(1,28) = 0.13, p = 0.713) or its interaction with ketamine (F(1,28) = 0.38 p = 0.539) did not have an impact on the struggling behavior. Similarly, MTEP or ketamine (1 mg/kg) did not produce a main effect (F(1,28) = 2.10, p = 0.158 and F(1,28) = 0.02, p = 0.876, respectively) or revealed a significant interaction (F(1,28) = 0.26, p = 0.61, 2 × 2 two-way ANOVA; Fig. 2C).

Swimming performance in the FST remained unaffected by the presence of mGluR5 agents or ketamine. There was no main effect of CDPPB (F(1,28) = 0.33, p = 0.569, 2 × 2 two-way ANOVA) or ketamine (10 mg/kg; F(1,28) = 2.14, p = 0.155) or their interaction (F(1,28) = 0.01, p = 0.897) on swimming duration. Likewise, we found no main effect of MTEP (1.25 mg/kg; F(1,28) = 1.15, p = 0.293), ketamine (1 mg/kg; F(1,28) = 1.08, p = 0.308), or their interaction (F(1,28) = 0.18, p = 0.674, 2 × 2 two-way ANOVA; Fig. 2D).

The frequency of diving and headshaking behavior were also not influenced by the administration of CDPPB or ketamine (all p values > 0.5, 2 × 2 two-way ANOVA). In the MTEP experiments, diving behavior did not show a dependence on MTEP (F(1,28) = 0.22, p = 0.637), ketamine (1 mg/kg; F(1,28) = 1.53, p = 0.225), or their interaction (F(1,28) = 0.44, p = 0.510, 2 × 2 two-way ANOVA). In contrast, pre-treatment with MTEP had a significant effect on headshaking (F(1,28) = 6.20, p = 0.019, R2 = 17.96, 2 × 2 two-way ANOVA; Fig. 2E).

The activation of mGluR5 via CDPPB elevated locomotor activity (F(1,28) = 6.02, p = 0.021, R2 = 17.41; Fig. 3A). This observation was not affected by ketamine (10 mg/kg; F(1,28) = 0.57, p = 0.456), and there was no interaction between CDPPB and ketamine (F(1,28) = 0, p = 0.984). In the MTEP experiments, in contrast, ketamine (1 mg/kg) exerted a main effect on locomotor activity (F(1,28) = 6.87, p = 0.014, R2 = 12.76), along with an interaction between ketamine and MTEP (F(1,28) = 16.81, p < 0.001, R2 = 31.21, 2 × 2 two-way ANOVA). The Ket1 group (M = 109.40, SD = 17.54) displayed hyperlocomotion compared to the vehicle group (M = 45.10, SD = 22.92; t(28) = 4.75, p < 0.001, d = 3.15). Importantly, combinatorial treatment of MTEP and ketamine (M = 56.03, SD = 38.46) restored locomotion to baseline (vehicle) levels (t(28) = 0.88, p = 0.964) and prevented the hyperlocomotion observed in ketamine receiving animals (t(28) = 3.94, p = 0.003, d = 1.78; Sidak’s corrected, Fig. 3A).

The open filed test (OFT). A) The duration of overall locomotor activity. B) The time spent in the center zone of the maze. C) Movement trajectories of representative animals (black data points in Panels A and B) from each group. D) The number of unsupported rearing. E) The number of supported rearing. Asterisks denote statistically significant (p < .05) main effects and post-hoc comparisons (brackets). Error bars represent SEM

The time spent in the center of the OFT was not influenced by mGluR5 agents or ketamine (all p values > 0.5, 2 × 2 two-way ANOVA; Fig. 3B). Accordingly, all groups exhibited comparable levels of thigmotaxis (Fig. 3C). Rearing behavior, in contrast, differed among experimental conditions. CDPPB activation of mGluR5 had a main effect on unsupported rearing behavior (F(1,28) = 6.487, p = 0.017, R2 = 18.79, 2 × 2 two-way ANOVA; Fig. 3D). In the case of supported rearing, the administration of ketamine (10 mg/kg) exhibited a main effect (F(1,28) = 4.21, p = 0.049, R2 = 12.25, 2 × 2 two-way ANOVA; Fig. 3E). The low dose of ketamine (F(1,28) = 6.44, p = 0.01, R2 = 12.87) and MTEP also had a main effect (F(1,28 = 8.41, p = 0.007, R2 = 16.71) on supported rearing, with a significant interaction (F(1,28) = 7.19, p = 0.012, R2 = 14.38, 2 × 2 two-way ANOVA; Fig. 3E). The Ket1 group (M = 15.00, SD = 6.07) displayed higher frequency of supported rearing compared to the vehicle group (M = 6.00, SD = 2.92; t(28) = 3.69, p = 0.006, d = 2.09), MTEP group (M = 5.62, SD = 5.42; t(28) = 3.84, p = 0.004, d = 1.63), and the MTEP + Ket1 group (M = 5.37, SD = 4.50; t(28) = 3.94, p = 0.003, d = 1.80; Fig. 3E).

Anxiety-like behavior was subsequently evaluated using the elevated plus maze. Locomotor activity levels in the EPM were comparable across all groups (all p values > 0.5, 2 × 2 two-way ANOVA; Fig. 4A). In the CDPPB experiments, the administration of ketamine had a main effect on the time spent in the open arms (F(1,28) = 4.31, p = 0.047, R2 = 12.87; Fig. 4B). In the MTEP experiments, a significant interaction was observed between MTEP and ketamine regarding anxiety-like behavior (F(1,28) = 15.15, p < 0.001, R2 = 34.51, 2 × 2 two-way ANOVA; Fig. 4B). The Ket1 group spent a significantly longer duration in the open arms (M = 180.2, SD = 131.81) compared to the vehicle group (M = 0.53, SD = 1.41; t(28) = 3.29, p = 0.016, d = 1.92). Similarly, the MTEP group (M = 166.9, SD = 141.50) exhibited an increased duration in the open arms compared to the vehicle group (t(28) = 3.05, p = 0.029, d = 1.66). However, the time spent in the open arms of the MTEP + Ket1 group (M = 46.3, SD = 101.04) did not differ from the vehicle group (t(28) = 0.83, p = 0.957, Sidak’s corrected; Fig. 4B).

The elevated plus maze (EPM). A) The duration of overall locomotor activity. B) The time spent in the open arms of the maze. C) The number of unsupported rearing. D) The number of supported rearing. Asterisks denote statistically significant (p < .05) main effects and post-hoc comparisons (brackets). Error bars represent SEM

There were no group-level differences in the frequency of unsupported rearing observed in the EPM (all p values > 0.5, 2 × 2 two-way ANOVA; Fig. 4C). In contrast, mGluR5 activation via CDPPB resulted in a higher number of supported rearings compared to the vehicle and Ket10 groups (F(1,28) = 9.72, p < 0.004, R2 = 25.53, 2 × 2 two-way ANOVA; Fig. 4D). In the other set of experiments, MTEP (F(1, 28) = 0.53, p = 0.471), ketamine (1 mg/kg; F(1,28) = 2.05, p = 0.163), or their interaction (F(1,28) = 2.22, p = 0.147) did not yield any difference.

The 3-min acclimation periods of the conditioning (Fig. 5A) were used to record baseline freezing, which were similar for all groups in the CDPPB and MTEP experiments (all p values > 0.5, 2 × 2 two-way ANOVA). In the CDPPB experiments, the CS had a main effect on freezing (F(1,28) = 19.75, p < 0.001, R2 = 21.94, 2 × 2x2 three-way mixed ANOVA; Fig. 5B), indicating that the freezing response towards the cue was increased following the first tone-shock pairing. There was no main effect of the CDPPB (F(1,28) = 1.21, p = 0.280), ketamine (F(1,28) = 3.52, p = 0.071) or their interaction (F(1,28) = 0.27, p = 0.605). Similarly, there was no interaction of the CS with the CDPPB (F(1,28) = 1.14, p = 0.295), or with ketamine (F(1,28) = 0, p = 0.985; Fig. 5B).

Auditory fear conditioning. A) The experimental design and treatment schedule of auditory fear conditioning, and the legend for all experimental groups. B) Freezing levels during CS-US pairing in the CDPPB experiments. C) Freezing levels during CS-US pairing in the MTEP experiments. D) Extinction I freezing levels in the CDPPB experiments. E) Extinction I freezing levels in the MTEP experiments. F) Extinction II freezing levels in the CDPPB experiments. G) Extinction II freezing levels in the MTEP experiments. The same vehicle group was used and plotted for conditioning (B, C), Extinction I (D, E), and Extinction II (F, G). Asterisks denote statistical significance (p < .05). Error bars represent SEM

In the MTEP experiments, a main effect of the CS on freezing was evident (F(1,28) = 23.09, p < 0.001, R2 = 18.64, 2 × 2x2 three-way mixed ANOVA; Fig. 5C), indicating successful conditioning. Concurrently, a main effect of MTEP was observed (F(1,28) = 6.80, p = 0.014, R2 = 10.82). However, there was no effect of ketamine (F(1,28) = 1.81, p = 0.189), and no observable interactions between the CS and ketamine (F(1,28) = 0.09, p = 0.756), MTEP and ketamine (F(1,28) = 0.07, p = 0.782; Fig. 5C), or CS and MTEP (F(1,28) = 0.193, p = 0.664).

In the Extinction session I, baseline freezing levels were similar between the groups in the CDPPB (Fig. 5D) and MTEP experiments (Fig. 5E; all p values > 0.5, 2 × 2 two-way ANOVA). In the CDPPB experiments, the main effect for the CS was significant (F(9, 252) = 3.17, p = 0.001, R2 = 6.71, 2 × 2x10 three-way mixed ANOVA; Fig. 5D), indicating successful fear extinction. There was no interaction of the CS with CDPPB (F(9,252) = 1.79, p = 0.069), or with ketamine (F(9,252) = 1.73, p = 0.082). Likewise, we did not observe effects of the CDPPB (F(1,28) = 0.30, p = 0.584), or ketamine (F(1,28) = 0.58, p = 0.451) or an interaction (F(1,28) = 0.43, p = 0.515).

Extinction of the conditioned response was also observed in the MTEP experiments, with a main effect of the CS (F(9,252) = 2.74, p = 0.004, R2 = 5.83, 2 × 2x10 three-way mixed ANOVA; Fig. 5E). Like the CDPPB experiments, no interaction of the CS and MTEP (F(9,252) = 1.72, p = 0.084), or CS and ketamine was found (F(9,252) = 0.46, p = 0.898). No individual main effect of the MTEP (F(1,28) = 3.97, p = 0.56) or ketamine (F(1,28) = 1.45, p = 0.237) was observed. However, there was an interaction between the MTEP and ketamine (F(1,28) = 4.42, p = 0.045, R2 = 3.28, 2 × 2x10 three-way mixed ANOVA; Fig. 5E).

In the Extinction session II, CDPPB administration resulted in lower baseline freezing levels (F(1,28) = 6.20, p = 0.019, R2 = 17.33, 2 × 2 two-way ANOVA; Fig. 5F). Both the CDPPB (M = 20.35, SD = 19.33) and the CDPPB + Ket10 (M = 20.23, SD = 9.14) groups showed lower freezing compared to the vehicle group (M = 37.28, SD = 14.86; t(28) = 2.38, p = 0.048, d = 0.98 and t(28) = 2.40, p = 0.046, d = 1.38, respectively). Similarly, the MTEP groups also displayed lower baseline freezing levels (F(1,28) = 5.32, p = 0.029, R2 = 15.63, 2 × 2 two-way ANOVA; Fig. 5G).

Freezing levels in the CDPPB experiments did not change in this session (Fig. 5F; all p values > 0.5, 2 × 2x10 three-way mixed ANOVA), indicating lack of further extinction. In the MTEP experiments, ketamine administration exhibited an effect on freezing (F(1,28) = 9.06, p = 0.005, R2 = 6.14). There was no main effect of the CS (F(9,252) = 1.00, p = 0.435), or MTEP (F(1,28) = 0.06, p = 0.793), and no interaction of the MTEP and ketamine (F(1,28) = 0.60, p = 0.443), CS and MTEP (F(9,252) = 1.29, p = 0.239), or CS and ketamine (F(9,252) = 1.71, p = 0.086).

Finally, we compared the average freezing levels of the two extinction sessions. Overall freezing decreased from Extinction I to Extinction II in both the CDPPB (F(1,28) = 14.62, p < 0.001, R2 = 9.55, 2 × 2x2 three-way mixed ANOVA) and MTEP experiments (F(1,28) = 20.15, p < 0.001, R2 = 13.38, 2 × 2x2 three-way mixed ANOVA), indicating a significantly better extinction in the second session. There was no group-level difference in the CDPPB experiments (all p values > 0.5, 2 × 2x2 three-way mixed ANOVA). However, in the MTEP experiments, the Ket1 and MTEP + Ket1 groups demonstrated a greater reduction in freezing during Extinction II when compared to the MTEP only and vehicle groups (F(1,28) = 6.56, p = 0.016, R2 = 10.68; 2 × 2x2 three-way ANOVA).