Abrogated Freud-1/Cc2d1a Repression of 5-HT1A Autoreceptors Induces Fluoxetine-Resistant Anxiety/Depression-Like Behavior

Abstract

Freud-1/Cc2d1a represses the gene transcription of serotonin-1A (5-HT1A) autoreceptors, which negatively regulate 5-HT tone. To test the role of Freud-1 in vivo, we generated mice with adulthood conditional knock-out of Freud-1 in 5-HT neurons (cF1ko). In cF1ko mice, 5-HT1A autoreceptor protein, binding and hypothermia response were increased, with reduced 5-HT content and neuronal activity in the dorsal raphe. The cF1ko mice displayed increased anxiety- and depression-like behavior that was resistant to chronic antidepressant (fluoxetine) treatment. Using conditional Freud-1/5-HT1A double knock-out (cF1/1A dko) to disrupt both Freud-1 and 5-HT1A genes in 5-HT neurons, no increase in anxiety- or depression-like behavior was seen upon knock-out of Freud-1 on the 5-HT1A autoreceptor-negative background; rather, a reduction in depression-like behavior emerged. These studies implicate transcriptional dysregulation of 5-HT1A autoreceptors by the repressor Freud-1 in anxiety and depression and provide a clinically relevant genetic model of antidepressant resistance. Targeting specific transcription factors, such as Freud-1, to restore transcriptional balance may augment response to antidepressant treatment.SIGNIFICANCE STATEMENT Altered regulation of the 5-HT1A autoreceptor has been implicated in human anxiety, major depression, suicide, and resistance to antidepressants. This study uniquely identifies a single transcription factor, Freud-1, as crucial for 5-HT1A autoreceptor expression in vivo Disruption of Freud-1 in serotonin neurons in mice links upregulation of 5-HT1A autoreceptors to anxiety/depression-like behavior and provides a new model of antidepressant resistance. Treatment strategies to reestablish transcriptional regulation of 5-HT1A autoreceptors could provide a more robust and sustained antidepressant response.

Keywords: 5-HT1A receptor; anxiety; major depression; raphe; repressor; serotonin.

Figure 1.

Loss of Freud-1 in 5-HT neurons increases 5-HT1A autoreceptors. A, Conditional KO strategy. To delete Freud-1 in 5-HT neurons, the cF1ko mouse was generated by crossing Cc2d1a(Freud-1)^flx/flx mice with TPH2-CreERT2 mice. At 8 weeks of age, mice were administered tamoxifen to activate CreERT2-induced recombination. To delete both Freud-1 and 5-HT1A autoreceptors in 5-HT neurons, the cF1ko mice were mated to the 5-HT1Aflx/flx mice, in which the 5-HT1A gene is flanked by LoxP sites and a YFP cassette to generate the Freud-1/5-HT1A double KO mice following tamoxifen administration. B, Tamoxifen-induced recombination specificity. Hippocampal and prefrontal cortex sections from tamoxifen-treated conditional Freud-1 KO/ROSA-GFP mice (cF1KO) show background GFP staining. C, Tamoxifen-induced recombination and loss of Freud-1 in DR. DR sections from tamoxifen-treated cF1KO mice were stained for GFP and either TPH or Freud-1 (10× magnification). Scale bar, 20 μm; inset, 20×. GFP was present in 92% of TPH⁺ cells, whereas 88% of Freud-1 was in GFP⁻ cells in cF1KO sections (n = 4). D, Loss of Freud-1 in 5-HT neurons. Freud-1/TPH-labeled cells in DR were almost absent in cF1ko compared with WT. By contrast, Freud-1⁺/TPH⁻ cells remained (white arrowheads) (n = 4). E, Quantification of GFP-, TPH-, and Freud-1-stained cells in DR of cF1KO and WT mice (n = 4), shown as mean ± SE (p < 0.001). F, Quantification of 5-HT1A-, TPH-, and Freud-1-stained cells in DR of cF1ko (non-ROSA-GFP) and WT mice (n = 4), shown as mean ± SE (p < 0.001). G, Loss of Freud-1 and increased 5-HT1A⁺ cells in DR of cF1ko mice. DR sections from tamoxifen-treated cF1ko versus F1wt (WT) mice (Scale bar, 50 μm; n = 4) were stained for TPH and 5-HT1A receptors (arrow, 5-HT1A in TPH⁻ cells); 5-HT1A receptors were increased in TPH⁺ cells. H, Increased 5-HT1A binding in raphe of cF1ko mice. Left, Representative images of ¹²⁵I-MPPI autoradiography of sections from cF1ko and WT mice in dorsal and median raphe (boxes) at two levels (bregma −4.60 and −4.72 mm) and hippocampus (bregma −1.70). Right, Quantification of ¹²⁵I-MPPI binding. Data are mean ± SEM (n = 4/group). *p < 0.05. **p < 0.01. 5-HT1A binding was increased in raphe of cF1ko mice: DR (unpaired two-tailed Student's t test, df = 6, t = 9.129, ***p < 0.001), MR (unpaired two-tailed Student's t test, df = 6, t = 6.635, *p < 0.05).

Figure 2.

Loss of Freud-1 augments 5-HT1A autoreceptor function and reduces 5-HT neuron activity and 5-HT levels. A, 5-HT1A-induced hypothermia. 8-OH-DPAT (0.75 mg/kg, i.p.) induced a greater body temperature reduction in cF1ko compared with F1wt (WT). Data are mean ± SEM (n = 3/group). *p < 0.05 versus WT-saline. **p < 0.001 versus WT-DPAT. ^#p < 0.05 versus cF1ko-saline. ^##p < 0.001 versus cF1ko-DPAT. B, Whole-cell voltage-clamp recordings (V_m = −55 mV) of 5-HT neurons in slices of DR in vitro, from cF1ko or F1wt mice (n = 4) in response to 5-CT (10 nm). No significant difference in 5-HT1A receptor-induced outward current was observed. C, Reduced 5-HT- and FosB-stained cells in cF1ko raphe. DR sections of cF1ko or F1wt (WT) mice stained for 5-HT, TPH, and FosB shown at 10× (Scale bar: left, 50 μm) or 20× magnification of boxed region (Scale bar: right, 20 μm). D, Reduced raphe 5-HT content in cF1ko mice. Tissue 5-HT and 5-HIAA content was quantified by HPLC for DR, hippocampus (Hippo), and PFC of cF1ko versus WT mice. Data are mean ± SEM (n = 3/group); reduced raphe 5-HT content in cF1ko versus WT mice (unpaired two-tailed Student's t test, df = 4, t = 6.675). **p < 0.01.

Figure 3.

Increased anxiety- and depression-like behavior in cF1ko mice. The cF1ko and WT littermates underwent the indicated behavioral tests or assays according to the timeline shown, 2 weeks after the last tamoxifen injection (11 weeks of age). A–C, Increased anxiety in cF1ko mice. A, EPM test. Compared with WT, cF1ko mice spent less time in open arms (unpaired two-tailed Student's t test, df = 47, t = 4.104, **p < 0.01), with no difference detected in closed arm time. B, OF. cF1ko mice displayed significantly reduced distance traveled in large center (unpaired two-tailed Student's t test, df = 47, t = 2.486, *p = 0.0165), with no change in total distance (data not shown). C, NSF test. cF1ko mice showed greater latency to approach food in the novel arena (unpaired two-tailed Student's t test, df = 48, t = 2.221 *p = 0.0311), but no difference in the home cage. D, E, Depression-like behavior in cF1ko mice. D, FST. The cF1ko mice showed significant greater immobility duration in the FST compared with F1wt mice (unpaired two-tailed Student's t test, df = 47, t = 2.962, *p < 0.05). E, TST. No difference in immobility duration between cF1ko and WT was seen. F, Locomotion test. Results from the BBK test showed no difference in total 30 min activity comparing cF1ko and F1wt (WT) mice. Data are mean ± SEM in cF1ko mice (n = 17) versus F1wt (WT) (n = 32).

Figure 4.

Resistance to chronic SSRI treatment in cF1ko mice. WT or cF1ko (KO) mice were treated with FLX or vehicle (V) for 3 weeks and throughout the behavioral assays (timeline). A–C, Anxiety phenotype: chronic FLX reduced anxiety in WT mice but did not affect the increased anxiety seen in cF1ko mice. A, EPM, significant changes in time spent in open arms in EPM test (one-way ANOVA treatment × genotype interaction, F_(3,36) = 27.01, p < 0.01; post hoc Tukey). **p < 0.01 versus WT-V. ^##p < 0.01 versus WT-FLX. B, OF, changes in distance traveled in large center (one-way ANOVA treatment × genotype interaction, F_(3,36)) = 14.53, p < 0.01; post hoc Tukey). **p < 0.01 versus WT-V, ^##p < 0.01 versus WT-FLX. C, NSF, latency to approach food in novel arena (one-way ANOVA treatment × genotype interaction, F_(3,36) = 14.16, p < 0.01; post hoc Tukey). **p < 0.01 versus WT-V. ^##p < 0.01 versus WT-FLX. D, E, Depression phenotype. D, FST. Depression-like behavior in cF1ko (V) compared with F1wt (V) was indicated by increased immobility in the FST. Chronic FLX significantly reduced immobility in F1wt, but not in cF1ko mice (one-way ANOVA treatment × genotype interaction, F_(3,36) = 13.62, p < 0.01; post hoc Tukey). **p < 0.01 versus WT-V. ^##p < 0.01 versus WT-FLX. E, TST, no significant changes were observed. F, BBK test. No differences in 30 min activity in novel arena were observed. Data represent individual animals with mean ± SEM; n = 10/group. **p < 0.01 versus WT-V. ^##p < 0.01 versus WT-FLX.

Figure 5.

Reversal of anxiety- and depression-like phenotypes upon loss of Freud-1 in the absence of 5-HT1A autoreceptors. Tissues from mice with conditional KO of Freud-1 and 5-HT1A receptor in adult serotonin neurons (F1/1A dKO) were compared with 5-HT1A conditional KO (1AKO) or WT littermates (WT/WT). A, Tamoxifen-induced recombination. Costaining for YFP and TPH in the DR. Recombination occurred exclusively in 5-HT cells; 92% of TPH⁺ cells were YFP⁺. Scale bar, 100 μm. B, Loss of 5-HT1A autoreceptors. Immunostaining for 5-HT1A and TPH showed significant loss of 5-HT1A immunostaining in TPH⁺ cells of cF1/1A dko mice. White arrows indicate the presence of 5-HT1A receptors in non-TPH⁺ cells in DR. Scale bar, 100 μm. C, 5-HT1A autoradiography. 5-HT1A receptor autoradiography using ¹²⁵I-MPPI of representative midbrain sections from WT/WT and cF1/1A dko mice, including dorsal (DR) and median (MR) raphe (boxes) and hippocampal sections. DR and MR showed significant decreases in 5-HT1A autoreceptor binding: DR (unpaired two-tailed Student's t test, df = 4, t = 11.24, ***p < 0.001), MR (unpaired two-tailed Student's t test, df = 4, t = 5.235, ***p < 0.001). D–H, Behavioral studies were done in either WT or conditional KO for Freud-1 (F1) and/or 5-HT1A receptor (1A) in littermate mice. D, EPM. No change in anxiety-like behavior (open arm time) was observed. E, OF. No difference among groups was observed in time spent in the large center. F, NSF. The latency to feed was not altered. G, FST. Immobility duration was reduced in F1/1A dKO versus WT and 1AKO mice (one-way ANOVA genotype × genotype interaction, F_(2,25) = 4.463, p = 0.0067; post hoc Tukey). *p < 0.05 versus WT. ^#p < 0.05 versus 1AKO. H, TS. Immobility time was reduced in F1/1AKO compared with WT or 1AKO (one-way ANOVA genotype × genotype interaction, F_(2,29) = 6.998, p = 0.0033; post hoc Tukey). ^#p < 0.05 versus F1/1AKO. I, BBK test. No difference in 30 min activity in novel arena was observed. Data points represent individual mice, with mean ± SEM: WT, n = 10; F1WT/1AKO, n = 11; F1/1AKO, n = 11.