Key role of the 5-HT1A receptor addressing protein Yif1B in serotonin neurotransmission and SSRI treatment

Abstract

Background: Altered function of serotonin receptor 1A (5-HT1AR) has been consistently implicated in anxiety, major depressive disorder and resistance to antidepressants. Mechanisms by which the function of 5-HT1AR (expressed as an autoreceptor in serotonergic raphe neurons and as a heteroreceptor in serotonin [5-HT] projection areas) is altered include regulation of its expression, but 5-HT1AR trafficking may also be involved.

Methods: We investigated the consequences of the lack of Yif1B (the 5-HT1AR trafficking protein) on 5-HT neurotransmission in mice, and whether Yif1B expression might be affected under conditions known to alter 5-HT neurotransmission, such as anxious or depressive states or following treatment with fluoxetine (a selective serotonin reuptake inhibitor) in humans, monkeys and mice.

Results: Compared with wild-type mice, Yif1B-knockout mice showed a significant decrease in the forebrain density of 5-HT projection fibres and a hypofunctionality of 5-HT1A autoreceptors expressed on raphe 5-HT neurons. In addition, social interaction was less in Yif1B-knockout mice, which did not respond to the antidepressant-like effect of acute fluoxetine injection. In wild-type mice, social defeat was associated with downregulated Yif1B mRNA in the prefrontal cortex, and chronic fluoxetine treatment increased Yif1B expression. The expression of Yif1B was also downregulated in the postmortem prefrontal cortex of people with major depressive disorder and upregulated after chronic treatment with a selective serotonin reuptake inhibitor in monkeys.

Limitations: We found sex differences in Yif1B expression in humans and monkeys, but not in mice under the tested conditions.

Conclusion: These data support the concept that Yif1B plays a critical role in 5-HT1AR functioning and brain 5-HT homeostasis. The opposite changes in its expression observed in anxious or depressive states and after therapeutic fluoxetine treatment suggest that Yif1B might be involved in vulnerability to anxiety and depression, and fluoxetine efficacy.

Fig. 1

5-HT_1AR functionality in male Yif1B knockout (Yif1B-KO) versus wild-type (WT) mice. (A) Electrophysiological activity recording of serotonergic neurons in the dorsal raphe nucleus demonstrated no difference in basal firing frequency between genotypes (WT, n = 10; Yif1B-KO, n = 9), but the efficacy of the 5-HT_1AR agonist ipsapirone (60 nM) in inhibiting 5-HT cell activity was significantly lower in Yif1B-KO than in WT mice (WT, n = 7; Yif1B-KO, n = 13; t test, p < 0.01) as illustrated by representative integrated firing rate histograms (in spikes per 10 s) of neurons from WT and Yif1B-KO mice. (B) Quantification of the number of spines in terminal dendritic segment from pyramidal neurons of CA1 in WT and Yif1B-KO mice using DiI staining demonstrated a significant decrease in the Yif1B-KO mice (WT, n = 10; Yif1B-KO, n = 20; t test, p < 0.0001; see representative fluorescent dendrite images, scale bar 1 μm). (C) 8-OH-DPAT-induced hypothermia in Yif1B-KO versus WT mice. Mice were injected with saline (WT, n= 3; Yif1B-KO, n = 4) or 8-OH-DPAT (0.5 mg/kg subcutaneous; WT, n = 6; Yif1B-KO, n = 10), and rectal temperature was recorded for 2 hours. Data (means ± standard error of the mean [SEM]) represent changes in body temperature after injection. Repeated-measure 2-way analysis of variance for 8-OH-DPAT effects revealed an interaction effect for time × genotype effect (F_7,98 = 46.29, p < 0.001). Bonferroni post hoc tests revealed that the effect of 8-OH-DPAT was significantly different 15 to 55 minutes after injection (p < 0.05 for each time point) in Yif1B-KO compared to WT mice. Graphs represent means ± SEM; *p < 0.05; ****p < 0.0001. 5-HT_1AR = serotonin receptor 1A; 8-OH-DPAT = 8-hydroxy-2-di-n-propylamino) tetralin; CA1 = hippocampus section; DiI = 1,1-dioctadecyl-3,3,3′,3′-tetramethylindocarbo- cyanine perchlorate; NS = nonsignificant.

Fig. 2

Biomarkers of the brain serotonergic system in male Yif1B knockout (Yif1B-KO) and wild-type (WT) mice. (A1) 5-HT levels (ng/g of tissue) were significantly lower in the hippocampus (t test, p = 0.003), the frontal cortex (t test, p = 0.003) and the remaining cortex (t test, p = 0.042) in Yif1B-KO (n = 10) compared with WT (n = 10) mice. (A2) 5-hydroxy-indoleacetic acid (5-HIAA) levels (ng/g of tissue) in brain areas from Yif1B-KO (n = 10) versus WT (n = 10) mice followed the same trends; however, differences reached statistical significance only for the hippocampus and the cortex (t test, p = 0.013). (A3) The 5-HT turnover index (determined as the [5-HIAA]/[5-HT] ratio) in the same brain areas revealed no difference between genotypes. (B1) 5-HT immunoreactive neurons in the dorsal raphe nucleus showed similar antero-posterior distribution (−4.24 to −4.96 from the bregma; Franklin and Paxinos atlas27) in both genotypes (n = 3 per genotype). (B2) Two-way analysis of variance, antero-posterior level effect (F_2,11 = 31.52, p < 0.0001), genotype effect (F_1,11 = 1.116, p = 0.31) and no interaction of geno-type × antero-posterior level (F_2,11 = 1.093, p = 0.36). (C1) Serotonergic innervation in the prefrontal cortex of WT (n = 4) and Yif1B-KO (n = 3) mice revealed by SERT immunostaining. (C2) Quantification of total serotonin (5-HT) immunoreactive fibre length demonstrated a significant effect of genotype (t test, p = 0.049). Graphs represent means ± standard error of the mean (SEM); *p < 0.05, **p < 0.01. Scale bars, 250 μm (B1), 100 μm (C1).

Fig. 3

Phenotypic characterization of male Yif1B knockout (Yif1B-KO) versus wild-type (WT) littermate mice with anxiety- and depressive-like tests. (A) Behavioural performance in the open field (OF) test during a 20-minute session (total locomotion, number of entries in the centre, time spent in the centre) was similar in WT (n = 14) and Yif1B-KO (n = 16) mice. (B) Behavioural performance in the elevated plus maze (EPM) test: total locomotion and percentage of entries in open arms were similar in Yif1B-KO (n = 6) and WT (n = 7) mice. (C) Total immobility recorded during the 6 minutes of the forced swim test (FST) was identical in WT (n = 8) and Yif1B-KO (n = 14) mice. (D) The social anxiety profile of the Yif1B-KO mice was revealed by a decrease in time spent in active social interaction (SI) (WT, n = 6; Yif1B-KO, n = 7; t test, p = 0.022). Graphs are means ± standard error of the mean (SEM); *p < 0.05.

Fig. 4

Yif1B expression in depressive conditions and after chronic selective serotonin reuptake inhibitor (SSRI) treatment. (A, B) Quantification of Yif1B protein levels (normalized to actin relative optical density [ROD]) in the prefrontal cortex, targeting a specific region of serotonergic innervation, in humans and monkeys. (A) Quantification in healthy controls (men and women; n = 9–10) and in people with major depressive disorder (MDD) (n = 9–10) demonstrated an effect of MDD in women (paired t test, 2-tailed, p = 0.031 compared with sex-matched controls), but not in men (MDD v. controls, p = 0.05). (B) After 9 months of fluoxetine treatment in monkeys (n = 5–6), quantification of Yif1B protein levels revealed an effect of the drug in females (paired t test, 2-tailed, p = 0.011) but no change in males (p = 0.51). Representative Western blot bands are presented below each graph (Yif1B, 34 kDa; actin, 46 kDa). (C, D) Quantification of Yif1B mRNA in the prefrontal cortex of wild-type (WT) mice after SSRI treatment and in social-stressed mice. (C) A 22 day-treatment with fluoxetine (10 mg/kg daily) in male and female WT mice (n = 10–12) revealed an effect of the drug (2-way analysis of variance; drug effect, F_1,42 = 18.92, p < 0.0001; no sex effect and no interaction effect of genotype × gender, p = 0.12 for both). (D) After 10 days of social stress (1 session per day) in WT male mice (n = 13–18), we observed an effect of stress (t test, p = 0.020). Graphs represent means ± standard error of the mean (SEM). *p < 0.05; **p < 0.01; ****p < 0.0001.

Fig. 5

Behavioural immobility of wild-type (WT) and Yif1B knockout (Yif1B-KO) mice in the repeated forced swim test. (A) Male mice (WT, n = 17; Yif1B KO, n = 22) were sensitized by immersion in water over 3 consecutive days during 6-minute sessions to induce an increase in resignation. Three-way analysis of variance revealed no effect of genotype (F = 0.243, p = 0.62) and no interaction of day × period × geno-type (F = 1.093, p = 0.36). (B) Response to acute injection of fluoxetine (20 mg/kg intraperitoneally, 30 minutes before the test) on the fourth day differed markedly in Yif1B-KO mice compared with WT mice (WT saline, n = 9; Yif1B-KO saline, n = 9; WT fluoxetine, n = 8, Yif1B-KO fluoxetine, n = 13). Three-way analysis of variance revealed a significant effect on the interaction of period × genotype × treatment (F = 2.8072, p = 0.042). Two-way analysis of variance and post hoc tests on each period showed that response to fluoxetine was significantly different between genotypes during the first minute of the test (multiple comparisons with WT mice injected with saline, p = 0.002 for fluoxetine in WT mice, p = 0.97 for saline in Yif1B-KO mice and p = 0.54 for fluoxetine in Yif1B-KO mice). Graphs represent mean ± standard error of the mean (SEM); **p < 0.01. MDD = major depressive disorder; ROD = relative optical density; SSRI = selective serotonin reuptake inhibitor.