mGluR5 in hippocampal CA1 pyramidal neurons mediates stress-induced anxiety-like behavior

Abstract

Pharmacological manipulation of mGluR5 has showed that mGluR5 is implicated in the pathophysiology of anxiety and mGluR5 has been proposed as a potential drug target for anxiety disorders. Nevertheless, the mechanism underlying the mGluR5 involvement in stress-induced anxiety-like behavior remains largely unknown. Here, we found that chronic restraint stress induced anxiety-like behavior and decreased the expression of mGluR5 in hippocampal CA1. Specific knockdown of mGluR5 in hippocampal CA1 pyramidal neurons produced anxiety-like behavior. Furthermore, both chronic restraint stress and mGluR5 knockdown impaired inhibitory synaptic inputs in hippocampal CA1 pyramidal neurons. Notably, positive allosteric modulator of mGluR5 rescued stress-induced anxiety-like behavior and restored the inhibitory synaptic inputs. These findings point to an essential role for mGluR5 in hippocampal CA1 pyramidal neurons in mediating stress-induced anxiety-like behavior.

Fig. 1. Chronic restraint stress leaded to anxiety-like behaviors and impaired the synaptic inputs in hippocampal CA1 pyramidal neurons.

a After 5 days of chronic restraint stress, the weight of the mice was decreased. n = 19 mice for control group, n = 18 mice for chronic restraint stress group. ****P < 0.0001 by two-tailed unpaired Student’s t-test. t = 11.15, d.f. = 35. b–f After 5 days of chronic restraint stress, the mice showed increased locomotion and anxiety-like behavior in the open field test. n = 9 mice for control group, n = 10 mice for chronic restraint stress group. Total distance: **P < 0.01 by two-tailed unpaired Student’s t-test. P = 0.0015, t = 3.782, d.f. = 17. Center time: ***P < 0.001 by two-tailed unpaired Student’s t-test. P = 0.0008, t = 4.078, d.f. = 17. Periphery/ total distance ratio: *P < 0.05 by two-tailed Mann Whitney test. P = 0.0101. Center/ total distance ratio: *P < 0.05 by two-tailed Mann Whitney test. P = 0.0101. Periphery/ total time ratio: ***P < 0.001 by two-tailed unpaired Student’s t-test. P = 0.0008, t = 4.078, d.f. = 17. g, h After 5 days of chronic restraint stress, the mice decreased open arm time in the elevated plus maze test. No difference in open arm entries. n = 9 mice for control group, n = 10 mice for chronic restraint stress group. Open arm time: *P < 0.05 by two-tailed unpaired Student’s t-test. P = 0.0495, t = 2.115, d.f. = 17. i–k Representative traces and quantitative analysis of sEPSC. n = 10 cells from 4 mice in the control group, n = 10 cells from 3 mice in the CRS group. **P < 0.01 by two-tailed unpaired Student’s t-test. P = 0.003, t = 3.425, d.f. = 18. ****P < 0.0001 by Kolmogorov-Smirnov test. Scale bar: 1 s, 30 pA. l–n Representative traces and quantitative analysis of mEPSC. n = 10 cells from 4 mice in the control group, n = 8 cells from 4 mice in the CRS group. *P < 0.05 by two-tailed unpaired Student’s t-test. P = 0.0386, t = 2.254, d.f. = 16. Cumulative probability: ****P < 0.0001 by Kolmogorov-Smirnov test. Scale bar: 1 s, 30 pA. o–q Representative traces and quantitative analysis of sIPSC. n = 11 cells from 4 mice for each group. *P < 0.05 by two-tailed unpaired Student’s t-test. P = 0.0107, t = 2.813, d.f. = 20. ****P < 0.0001 by Kolmogorov-Smirnov test. Scale bar: 1 s, 30 pA. r–t Representative traces and quantitative analysis of mIPSC. n = 15 cells from 4 mice in the control group, n = 10 cells from 4 mice in the CRS group. **P < 0.01 by two-tailed unpaired Student’s t-test. P = 0.007, t = 2.963, d.f. = 23. Cumulative probability: ****P < 0.0001 by Kolmogorov-Smirnov test. Scale bar: 1 s, 30 pA. Error bars, mean ± SEM.

Fig. 2. Chronic restraint stress decreases mGluR5 expression in hippocampal CA1 and mGluR5 knockdown produced anxiety-like behaviors.

a–c mGluR5 in CA1 was downregulated after chronic restraint stress. No difference in CA3 or DG. n = 4 mice for each group. CA1: **P < 0.01 by two-tailed unpaired Student’s t-test. P = 0.0086, t = 3.84, d.f. = 6. d Western analysis of mGluR5 expression after chronic restraint stress. n = 4 mice for each group. *P < 0.05 by two-tailed unpaired Student’s t-test. P = 0.0295, t = 2.842, d.f. = 6. e The diagram of virus injected sites. f Representative images of the distribution of AAV injected mice. Scale bar: 500 μm. g qPCR analysis of mGluR5 expression in AAV-CamKII-Cre injected mice. n = 5 mice for control group, n = 4 mice for AAV-CamKII-Cre group. *P < 0.05 by two-tailed unpaired Student’s t-test. P = 0.0134, t = 3.285, d.f. = 7. h Western blot analysis of mGluR5 expression after AAV-CamKII-Cre injection. n = 5 mice for each group. *P < 0.05 by two-tailed unpaired Student’s t-test. P = 0.0148, t = 3.095, d.f. = 8. i–m The AAV-CamKII-Cre injected mice showed decreased locomotion and increased anxiety-like behavior in the open field test. n = 8 mice for control group, n = 10 mice for AAV-CamKII-Cre group. Total distance: *P < 0.05 by two-tailed unpaired Student’s t-test. P = 0.0334, t = 2.328, d.f. = 16. Center time: *P < 0.05 by two-tailed unpaired Student’s t-test. P = 0.0207, t = 2.567, d.f. = 16. Periphery/ total distance ratio: *P < 0.05 by two-tailed unpaired Student’s t-test. P = 0.0338, t = 2.321, d.f. = 16. Center/ total distance ratio: *P < 0.05 by two-tailed unpaired Student’s t-test. P = 0.0338, t = 2.321, d.f. = 16. Periphery/ total time ratio: *P < 0.05 by two-tailed unpaired Student’s t-test. P = 0.0207, t = 2.567, d.f. = 16. n, o The AAV-CamKII-Cre injected mice showed decreased open arm time, open arm entries in the elevated plus maze test. n = 8 mice for control group, n = 10 mice for AAV-CamKII-Cre group. Open arm time: **P < 0.01 by two-tailed unpaired Student’s t-test. P = 0.0016, t = 3.794, d.f. = 16. Open arm entries: *P < 0.05 by two-tailed unpaired Student’s t-test. P = 0.0145, t = 2.74, d.f. = 16. Error bars, mean ± SEM.

Fig. 3. Knockdown of mGluR5 in hippocampal CA1 pyramidal neurons impairs inhibitory synaptic inputs.

a–c Representative traces and quantitative analysis of sEPSC. n = 9 cells from 4 mice in the control group, n = 10 cells from 4 mice in the AAV-CamkII-Cre group. Cumulative probability: ****P < 0.0001 by Kolmogorov-Smirnov test. Scale bar: 1 s, 30 pA. d–f Representative traces and quantitative analysis of mEPSC. n = 9 cells from 4 mice in the control group, n = 10 cells from 4 mice in the AAV-CamkII-Cre group. Cumulative probability: ****P < 0.0001 by Kolmogorov-Smirnov test. Scale bar: 1 s, 30 pA. g–i Representative traces and quantitative analysis of sIPSC. n = 10 cells from 4 mice in the control group, n = 8 cells from 4 mice in the AAV-CamkII-Cre group. **P < 0.01 by two-tailed unpaired Student’s t-test. P = 0.0049, t = 3.259, d.f. = 16. ****P < 0.0001 by Kolmogorov-Smirnov test. Scale bar: 1 s, 30 pA. j–l Representative traces and quantitative analysis of mIPSC. n = 9 cells from 4 mice in each group. *P < 0.05 by two-tailed unpaired Student’s t-test. P = 0.0216, t = 2.545, d.f. = 16. ****P < 0.0001 by Kolmogorov-Smirnov test. Scale bar: 1 s, 30 pA. Error bars, mean ± SEM.

Fig. 4. The CDPPB restored stress-impaired inhibitory synaptic inputs.

a Representative traces of sIPSCs. Scale bar: 2 s, 30 pA. b, c Quantitative analysis of sIPSCs after CDPPB application. sIPSC frequency: n = 9 cells from 4 mice in the control group, n = 11 cells from 4 mice in the CRS group. *P < 0.05, **P < 0.01 by Repeated measure two-way ANOVA. Con(before) versus CRS(before): P = 0.0048. CRS(before) versus CRS(after): P = 0.0366. Con(before) versus CRS(after): P = 0.1037. No difference in sIPSC amplitude. d, e Quantitative analysis of △sIPSC after CDPPB application. △sIPSC frequency was calculated by (f2 - f1)/f1, where f1 and f2 were the frequencies of sIPSCs recorded before and after CDPPB treatment, respectively. △sIPSC amplitude was calculated by (a2 - a1)/a1, where a1 and a2 were the amplitudes of sIPSCs recorded before and after CDPPB treatment, respectively. △sIPSC frequency: n = 9 cells from 4 mice in the control group, n = 11 cells from 4 mice in the CRS group. **P < 0.01 by two-tailed unpaired Student’s t-test. P = 0.0042. No difference in △sIPSC amplitude. Cumulative frequency distribution curves of sIPSC frequency: Con versus CRS: ****P < 0.0001 by Kolmogorov-Smirnov test. CRS versus CRS + CDPPB: P = 0.0188 by Kolmogorov-Smirnov test. f Representative traces of mIPSCs. Scale bar: 2 s, 30 pA. g, h Quantitative analysis of mIPSCs after CDPPB application. mIPSC frequency: n = 10 cells from 4 mice in the control group, n = 11 cells from 4 mice in the CRS group. *P < 0.05, **P < 0.01 by Repeated measure two-way ANOVA. Con(before) versus CRS(before): P = 0.0217. CRS(before) versus CRS(after): P = 0.0046. Con(before) versus CRS(after): P = 0.4639. No difference in mIPSC amplitude. i, j Quantitative analysis of △mIPSC after CDPPB application. △mIPSC frequency: n = 10 cells from 4 mice in the control group, n = 11 cells from 4 mice in the CRS group. *P < 0.05 by Mann-Whitney test. P = 0.0127. No difference in △mIPSC amplitude. Cumulative frequency distribution curves of mIPSC frequency: Con versus CRS: ****P < 0.0001 by Kolmogorov-Smirnov test. CRS versus CRS + CDPPB: ****P < 0.0001 by Kolmogorov- Smirnov test. Error bars, mean ± SEM.

Fig. 5. The CDPPB restored stress-induced anxiety-like behavior.

a–e The CDPPB did not rescue stress-induced anxiety-like behavior in the open field test. n = 10 mice for control group, n = 10 mice for CRS group, n = 11 mice for CRS + CDPPB group. f Representative traces in elevated plus maze test after CDPPB treatment. g, h The CDPPB treated mice showed restored open arm time and open arm entries in the elevated plus maze test. n = 10 mice for control group, n = 10 mice for CRS group, n = 11 mice for CRS + CDPPB group. Open arm time: *P < 0.05 by One-way ANOVA, F_2,28 = 5.169. Con versus CRS: P = 0.0397, q = 3.648, d.f. = 28. CRS versus CRS + CDPPB: P = 0.0162, q = 4.199, d.f. = 28. Open arm entries: *P < 0.05 by One-way ANOVA. F_2,28 = 4.447. Con versus CRS: P = 0.0326, q = 3.772, d.f. = 28. CRS versus CRS + CDPPB: P = 0.0461, q = 3.552, d.f. = 28. i Representative infusion site (as indicated by the arrow) in the mouse dorsal hippocampal CA1 area. j–n Local infusion of CDPPB in dorsal CA1 did not rescue stress-induced anxiety-like behavior in the open field test. n = 9 mice for control group, n = 9 mice for Con+CDPPB group, n = 10 mice for CRS group, n = 9 mice for CRS + CDPPB group. Center time: *P < 0.05 by Two-way ANOVA. Con versus CRS: P = 0.0145, t = 3.284, d.f. = 33. Con+CDPPB versus CRS: P = 0.0206, t = 3.15, d.f. = 33. o, p Local infusion of CDPPB in dorsal CA1 rescued reduced open arm time, but not open arm entries, in the elevated plus maze test. n = 9 mice for control group, n = 9 mice for Con+CDPPB group, n = 10 mice for CRS group, n = 9 mice for CRS + CDPPB group. Open arm time: *P < 0.05, **P < 0.01 by Two-way ANOVA. Con versus CRS: P = 0.0196, t = 3.1, d.f. = 33. CRS versus CRS + CDPPB: P = 0.0033, t = 3.83, d.f. = 33. q Schematic diagram of the involvement of mGluR5 in the hippocampus in anxiety-like behavior. Chronic restraint stress decreases mGluR5 activity in the hippocampus, leading to deficits of firing and the development of anxiety-like behavior. CDPPB reversed the chronic restraint stress-induced decrease in firing in the hippocampus and chronic restraint stress-induced increase in anxiety-like behavior. Error bars, mean ± SEM.