Neuronal TCF7L2 in Lateral Habenula Is Involved in Stress-Induced Depression

Abstract

Depression is a complex psychiatric disorder that has substantial implications for public health. The lateral habenula (LHb), a vital brain structure involved in mood regulation, and the N-methyl-D-aspartate receptor (NMDAR) within this structure are known to be associated with depressive behaviors. Recent research has identified transcription factor 7-like 2 (TCF7L2) as a crucial transcription factor in the Wnt signaling pathway, influencing diverse neuropsychiatric processes. In this study, we explore the role of TCF7L2 in the LHb and its effect on depressive-like behaviors in mice. By using behavioral tests, AAV-mediated gene knockdown or overexpression, and pharmacological interventions, we investigated the effects of alterations in TCF7L2 expression in the LHb. Our results indicate that TCF7L2 expression is reduced in neurons within the LHb of male ICR mice exposed to chronic mild stress (CMS), and neuron-specific knockdown of TCF7L2 in LHb neurons leads to notable antidepressant activity, as evidenced by reduced immobility time in the tail suspension test (TST) and forced swimming test (FST). Conversely, the overexpression of TCF7L2 in LHb neurons induces depressive behaviors. Furthermore, the administration of the NMDAR agonist NMDA reversed the antidepressant activity of TCF7L2 knockdown, and the NMDAR antagonist memantine alleviated the depressive behaviors induced by TCF7L2 overexpression, indicating the involvement of NMDAR. These findings offer novel insights into the molecular mechanisms of depression, highlighting the potential of TCF7L2 as both a biomarker and a therapeutic target for depression. Exploring the relationship between TCF7L2 signaling and LHb function may lead to innovative therapeutic approaches for alleviating depressive symptoms.

Keywords: N-methyl-D-aspartate receptor; chronic mild stress; depression; lateral habenula; transcription factor 7-like 2.

Figure 1

TCF7L2 in LHb neurons was downregulated in CMS mice. (A) Timeline of the CMS and behavioral tests, including the OFT, the TST, the EPM, the FST, and the SPT. (B) Time spent in the center in the OFT, mean of Naive: 30.98, mean of CMS: 18.46. (C) Total distance traveled during the OFT, mean of Naive: 2773, mean of CMS: 3258. (D) Latency to the first immobility in the TST, mean of Naive: 108.07, mean of CMS: 106.71. (E) Total immobility time in the TST, mean of Naive: 51.2, mean of CMS: 138.1. (F) Time spent in the open arms in the EPM, mean of Naive: 54.63, mean of CMS: 42.79. (G) Latency to the first floating in the FST, mean of Naive: 96.36, mean of CMS: 85.21. (H) Total floating time in the FST, mean of Naive: 93, mean of CMS: 139.9. (I) Sucrose preference (%) of SPT, mean of Naive: 75.69, mean of CMS: 61.18, (J,K) Immunofluorescence staining of TCF7L2 in LHb neurons, mean of Naive: 37.34, mean of CMS: 17.47, (red: TCF7L2, green: NeuN, blue: DAPI, scale bar = 50 μm; n = 4). Comparison between the Naive and CMS groups was conducted using T-tests or Mann–Whitney U-tests. Data are expressed as means ± SEM. n = 14 per group. * p < 0.05, ** p < 0.01, *** p < 0.001, versus the Naive group.

Figure 2

TCF7L2 knockdown in the LHb neurons caused antidepressant activity in mice. (A) Schematic of the experimental design of AAV-mediated TCF7L2 knockdown in the LHb neurons of mice. (B) Verification of TCF7L2 knockdown efficiency using fluorescence staining, mean of AAV-sh-scrambled: 93, mean of AAV-sh-TCF7L2: 139.9 (red: TCF7L2, green: GFP, blue: DAPI, scale bar = 50 μm). (C) Sucrose preference (%) of SPT, mean of AAV-sh-scrambled: 67.24, mean of AAV-sh-TCF7L2: 62.92. (D) Latency to eat in the NSF test, mean of AAV-sh-scrambled: 45.1, mean of AAV-sh-TCF7L2: 19.5. (E) Total intake of food in the NSF test, mean of AAV-sh-scrambled: 0.3667, mean of AAV-sh-TCF7L2: 0.3625. (F) Latency to the first immobility in the TST, mean of AAV-sh-scrambled: 107.2, mean of AAV-sh-TCF7L2: 116.5. (G) Total immobility time in the TST, mean of AAV-sh-scrambled: 66.86, mean of AAV-sh-TCF7L2: 37.53. (H) Latency to the first floating in the FST, mean of AAV-sh-scrambled: 59, mean of AAV-sh-TCF7L2: 104.8. (I) Total floating time in the FST, mean of AAV-sh-scrambled: 125.9, mean of AAV-sh-TCF7L2: 32.81. (J) Time spent in the center in the OFT, mean of AAV-sh-scrambled: 17.17, mean of AAV-sh-TCF7L2: 15.25. (K) Total distance traveled during the OFT, mean of AAV-sh-scrambled: 4039, mean of AAV-sh-TCF7L2: 3870. (L) Recognition index of NOR test, mean of AAV-sh-scrambled: 66.21, mean of AAV-sh-TCF7L2: 62.33. (M) Sniffing index in trial 1 of the three-chamber SIT, mean of AAV-sh-scrambled: 75.78, mean of AAV-sh-TCF7L2: 76.87. (N) Total sniffing time in trial 1 of the three-chamber SIT, mean of AAV-sh-scrambled: 85.82, mean of AAV-sh-TCF7L2: 77.41. (O) Preference index in trial 2 of the three-chamber SIT, mean of AAV-sh-scrambled: 35.31, mean of AAV-sh-TCF7L2: 35.61. (P) Total sniffing time in trial 2 of the three-chamber SIT, mean of AAV-sh-scrambled: 67.45, mean of AAV-sh-TCF7L2: 64.59. (Q) Analysis of the correlation between the total floating time in FST and density of TCF7L2⁺ cells in LHb/mm². Comparison between the AAV-sh-Scrambled and AAV-sh-TCF7L2 groups was conducted using the T-test or Mann–Whitney U-test. Data are expressed as means ± SEM. n = 10–23 per group. * p < 0.05, *** p < 0.001, versus the AAV-sh-Scrambled group.

Figure 3

TCF7L2 overexpression in the LHb neurons led to depressive-like behavior in mice. (A) Schematic of the experimental design of AAV-mediated TCF7L2 overexpression in the LHb neurons of mice. (B) Verification of AAV injection point using fluorescence staining, mean of AAV-EGFP: 67.45, mean of AAV-TCF7L2: 64.59 (red: TCF7L2, green: GFP, blue: DAPI, scale bar = 50 μm). (C) Sucrose preference (%) of SPT, mean of AAV-EGFP: 73.5, mean of AAV-TCF7L2: 74. (D) Latency to eat in the NSF test, mean of AAV-EGFP: 45.33, mean of AAV-TCF7L2: 124.27. (E) Total intake of food in the NSF test, mean of AAV-EGFP: 0.22, mean of AAV-TCF7L2: 0.16. (F) Latency to the first immobility in the TST, mean of AAV-EGFP: 97, mean of AAV-TCF7L2:88.06. (G) Total immobility time in the TST, mean of AAV-EGFP: 60.39, mean of AAV-TCF7L2: 98.89. (H) Latency to the first floating in the FST, mean of AAV-EGFP: 87.67, mean of AAV-TCF7L2: 64.11. (I) Total floating time in the FST, mean of AAV-EGFP: 50.06, mean of AAV-TCF7L2: 117.4. (J) Time spent in the center in the OFT, mean of AAV-EGFP: 17.61, mean of AAV-TCF7L2: 17.9. (K) Total distance traveled during the OFT, mean of AAV-EGFP: 2235, mean of AAV-TCF7L2: 2147. (L) Recognition index of NOR test, mean of AAV-EGFP: 59.34, mean of AAV-TCF7L2: 62.5. (M) Sniffing index in trial 1 of the three-chamber SIT, mean of AAV-EGFP: 79.49, mean of AAV-TCF7L2: 82.19. (N) Total sniffing time in trial 1 of the three-chamber SIT, mean of AAV-EGFP: 75.94, mean of AAV-TCF7L2: 78.22. (O) Preference index in trial 2 of the three-chamber SIT, mean of AAV-EGFP: 29.86, mean of AAV-TCF7L2: 25.55. (P) Total sniffing time in trial 2 of the three-chamber SIT, mean of AAV-EGFP: 71.94, mean of AAV-TCF7L2: 76.83. Comparison between the AAV-EGFP and AAV-TCF7L2 groups was conducted using T-test or Mann–Whitney U-test. (Q) Analysis of the correlation between the total floating time in FST and density of TCF7L2⁺ cells in LHb/mm². Data are expressed as means ± SEM. n = 18 per group. * p < 0.05, ** p < 0.01, *** p < 0.001, versus the AAV-sh-Scrambled group.

Figure 4

NMDAR was involved in TCF7L2-mediated depressive-like behavior. (A) Experimental timeline for NMDAR agonist-NMDA administration and behavioral tests. (B) Effects of NMDA administration on latency to the first immobility in the TST with LHb neurons special TCF7L2 knockdown, mean of AAV-sh-scrambled + Saline: 106, mean of AAV-sh-scrambled + NMDA: 111.9, mean of AAV-sh-TCF7L2 + Saline: 111.1, mean of AAV-sh-TCF7L2 + NMDA: 108.8. (C) Total immobility time in the TST, mean of AAV-sh-scrambled + Saline: 57.75, mean of AAV-sh-scrambled + NMDA: 52.45, mean of AAV-sh-TCF7L2 + Saline: 8.143, mean of AAV-sh-TCF7L2 + NMDA: 62.63. (D) Effects of NMDA administration on latency to the first floating in the FST, mean of AAV-sh-scrambled + Saline: 72.36, mean of AAV-sh-scrambled + NMDA: 95.45, mean of AAV-sh-TCF7L2 + Saline: 110.9, mean of AAV-sh-TCF7L2 + NMDA: 102.6. (E) Total floating time in the FST, mean of AAV-sh-scrambled + Saline: 106.1, mean of AAV-sh-scrambled + NMDA: 120.7, mean of AAV-sh-TCF7L2 + Saline: 45, mean of AAV-sh-TCF7L2 + NMDA: 134.7. (F) Experimental timeline for NMDAR antagonist-memantine administration and behavioral tests. (G) Effects of memantine administration on latency to the first immobility in the TST with LHb neurons special TCF7L2 overexpression, mean of AAV-EGFP + Saline: 85.25, mean of AAV-EGFP + Memantine: 88.25, mean of AAV-TCF7L2 + Saline: 50.75, mean of AAV-TCF7L2 + Memantine: 80.05. (H) Total immobility time in the TST, mean of AAV-EGFP + Saline: 79.75, mean of AAV-EGFP + Memantine: 75.38, mean of AAV-TCF7L2 + Saline: 131.5, mean of AAV-TCF7L2 + Memantine: 72.38. (I) Effects of memantine administration on latency to the first floating in the FST, mean of AAV-EGFP + Saline: 91.5, mean of AAV-EGFP + Memantine: 100.8, mean of AAV-TCF7L2 + Saline: 35, mean of AAV-TCF7L2 + Memantine: 82.5. (J) Total floating time in the FST, mean of AAV-EGFP + Saline: 32.25, mean of AAV-EGFP + Memantine: 44.63, mean of AAV-TCF7L2 + Saline: 157.5, mean of AAV-TCF7L2 + Memantine: 77.88. Comparisons between groups were conducted using one-way ANOVA. Data are expressed as means ± SEM. n = 7–11 per group. * p < 0.05, ** p < 0.01, *** p < 0.001.