Serotonin inputs to the dorsal BNST modulate anxiety in a 5-HT1A receptor-dependent manner

Abstract

Serotonin (5-HT) neurons project from the raphe nuclei throughout the brain where they act to maintain homeostasis. Here, we study 5-HT inputs into the bed nucleus of the stria terminalis (BNST), a major subdivision of the extended amygdala that has been proposed to regulate responses to anxiogenic environments in humans and rodents. While the dorsal part of the BNST (dBNST) receives dense 5-HT innervation, whether and how 5-HT in the dBNST normally modulates anxiety remains unclear. Using optogenetics, we demonstrate that activation of 5-HT terminals in the dBNST reduces anxiety in a highly anxiogenic environment. Further analysis revealed that optogenetic inhibition of 5-HT inputs into the dBNST increases anxiety in a less anxiogenic environment. We found that 5-HT predominantly hyperpolarizes dBNST neurons, reducing their activity in a manner that can be blocked by a 5-HT_1A antagonist. Finally, we demonstrate that activation of 5-HT_1A receptors in the dBNST is necessary for the anxiolytic effect observed following optogenetic stimulation of 5-HT inputs into the dBNST. These data reveal that 5-HT release in the dBNST modulates anxiety-like behavior via 5-HT_1A receptors under naturalistic conditions.

Figure 1. Photostimulation of 5-HT terminals in the dBNST and CeA decreases new cage induced c-fos

(A) Diagram for new cage c-fos induction paradigm. (B) Representative images of c-fos staining in the dBNST (Top) and vBNST (Bottom) of control mice in home cage (Left) and in new cage (Center), and TPH-Chr2^RPH-dBNST mice in new cage (Right). Scale bar=100 μm. (C) Photostimulation in TPH-Chr2^RPH-dBNST mice reduces the number of c-fos positive cells in the dBNST but not in the vBNST in a new cage (one-way ANOVA dBNST: F_1,6=35.877, p<0.01; vBNST: F_1,6=1.257, p=0.3051) (n= 4–5/group). (D) Representative images of c-fos staining in the CeL (Top) and CeM (Bottom) of control mice in home cage (Left) and in new cage (Center), and TPH-Chr2^RPH-CeA mice in new cage (Right). Scale bar=50 μm (E) Photostimulation in TPH-ChR2^PHN-CeA mice results in fewer c-fos positive cells in the CeL and CeM in a new cage (one-way ANOVA CeL: F_1,6=82.778, p<0.01; CeM: F_1,6=34.508, p<0.01) (n= 4–5/group). Means are represented as ±SEM. (*p<0.05; **p<0.01). See also Supplementary Figure 1 and 6A, B.

Figure 2. Activation of 5-HT inputs into the dBNST results in anxiolysis

(A) Cartoon of TPH-ChR2^RPH-dBNST mice with fiber optics implanted over dBNST. (B) TPH-ChR2^RPH-dBNST mice spent more time in the center in the ON epoch compared to controls (two-way repeated measures ANOVA group x epoch interaction, F_2,32=7.332, p=0.0024; post hoc Bonferroni corrected t-test, p<0.05). (C) No effect on total distance traveled in the open-field was observed (two-way repeated measures ANOVA group x epoch interaction, F_2,32=0.614, p=0.5475). (D) TPH-ChR2^RPH-dBNST mice spent more time in the open arms (two-way repeated measures ANOVA group x epoch interaction, F_2,32=5.057, p=0.0124; post hoc Bonferroni corrected t-test, p<0.05) and (E) showed more open arm entries during photostimulation in the elevated-plus maze (two-way repeated measures ANOVA group x epoch interaction, F_2,32=4.551, p=0.0182; post hoc Bonferroni corrected t-test, p<0.05). (F) Photostimulation of dBNST 5-HT inputs in TPH-ChR2^RPH-dBNST mice decreased the latency to feed (two-way repeated measures ANOVA group x epoch interaction, F_1,16=41.783, p<0.01; post hoc Bonferroni corrected t-test, p<0.05). (G) No changes in home cage (HC) consumption was observed (two-way repeated measures ANOVA group x epoch interaction, F_1,16=0.540, p=0.4730). (n=8–10/group). Means are represented as ±SEM. (*p<0.05). See also Supplementary Figure 2 and 6A.

Figure 3. Activation of 5-HT terminals in the CeA has no effect on anxiety

(A) Diagram illustrating TPH-ChR2^RPH-CeA mice. (B, C) No effect on center time (two-way repeated measures ANOVA group x epoch interaction, F_2,22=0.043, p=0.9581) or total distance traveled in the open-field was observed (two-way ANOVA group x epoch interaction, F_2,22=1.127, p=0.3420). (D, E) No differences were detected between TPH-ChR2^RPH-CeA and control mice in time on the open arms (two-way repeated measures ANOVA group x epoch interaction, F_2,22=0.026, p=0.9743) or open arm entries in the elevated-plus maze (two-way repeated measures ANOVA group x epoch interaction, F_2,22=0.303, p=0.7417). (F) No effect was detected on latency to eat (two-way repeated measures ANOVA group x epoch interaction, F_2,22=0.367, p=0.557) or (G) in home cage (HC) consumption (two-way repeated measures ANOVA group x epoch interaction, F_2,22=0.287, p=0.6026). (n=6–7/group). Means are represented as ±SEM. See also Supplementary Figure 3 and 6B.

Figure 4. Inhibition of 5-HT inputs in the dBNST increases anxiety-like behaviors

(A) Diagram illustrating Pet-Arch^RPH-dBNST mice. (B) Representative images of c-fos staining in the dBNST (Top) and vBNST (Bottom) of control mice in home cage (Left) and in new cage (Center), and Pet-Arch^RPH-dBNST mice in new cage (Right). Scale bar=100 μm. (C) Green light illumination of Pet-Arch^RPH-dBNST mice increases c-fos+ cells in the dBNST but not in the vBNST in a new cage (one-way ANOVA dBNST: F_1,8=6.574, p<0.05; vBNST: F_1,8=0.450, p=0.5214). (n=5/group) (D) During ON epochs, Pet-Arch^RPH-dBNST mice spent significantly less time in the center (unpaired Student’s t test, OFF: DF=17, t=0.184, p=0.8656; ON: DF=17, t=2.416, p<0.05). (E) Group or light had no effect on total distance traveled in the open-field (unpaired Student’s t test, OFF: DF=17, t=0.013, p=0.9896; ON: DF=17, t=0.774, p=0.4670). (F) Pet-Arch^RPH-dBNST mice spent less time in the open arms (unpaired Student’s t test, OFF: DF=17, t=0.290, p=0.7754; ON: DF=17, t=2.447, p<0.05) and (G) showed a lower number of entries into the open arms during the illumination epoch (unpaired Student’s t test, OFF: DF=17, t=0.421, p=0.6794; ON: DF=17, t=2.313, p<0.05). (n=8–11/group). Means are represented as ±SEM. (*p<0.05). See also Supplementary Figure 4 and 6C.

Figure 5. The behavioral effects of 5–HT release in the dBNST are mediated by activation of 5–HT_1A receptors

(A) Model depiction of electrophysiological recordings. (B) Representative traces from electrophysiological recordings showing a delayed response to photostimulation of 5-HT inputs (20 Hz, 5 ms pulses for 15s) to dBNST neurons in TPH-CHR2 mice but not in controls or TPH-ChR2 mice pretreated with WAY 100,635. (C) Average magnitude of hyperpolarization in the dBNST neurons after photostimulation of 5-HT inputs (one-way ANOVA F_2,19=21.152, p<0.01; Fisher post hoc TPH-ChR2 and control: p<0.01; TPH-ChR2 and TPH-ChR2+WAY: p<0.01) (n=7–8 cells/group). (D) Saline or the 5-HT_1A receptor antagonist WAY 100,635 were locally infused into the dBNST of TPH-Chr2^RPH-dBNST mice using a guide cannula 30 min before behavior. (E) TPH-Chr2^RPH-dBNST mice pre-treated with WAY spend less time in the center of the open field during the ON epoch than the saline treated mice (two-way repeated measures ANOVA treatment x epoch interaction, F_2.28=4.933, p=0.0146; post hoc Bonferroni corrected t-test, p<0.05). (F) No changes were detected in total distance traveled (two-way repeated measures ANOVA treatment x epoch interaction, F_2.28=0.236, p=0.7193). (G) TPH-Chr2^RPH-dBNST mice treated with WAY spend less time in the open arms (two-way repeated measures ANOVA treatment x epoch interaction, F_2.28=11.326, p=0.0002; post hoc Bonferroni corrected t-test, p<0.05) and (H) show less open arm entries in the elevated-plus maze during the ON epoch when compared to saline treated mice (two-way repeated measures ANOVA treatment x epoch interaction, F_2.28=4.100, p=0.0274; post hoc Bonferroni corrected t-test, p<0.05). (I) 5-HT_1A antagonist injection into the dBNST attenuated the light-induced decrease in latency as seen after saline treatment (paired Student’s t test, DF=7, t=6.903, p=0.0002) with (J) no changes in home cage (HC) consumption (paired Student’s t test, DF=7, t=1.102, p=0.3451). (n=8/group). Means are represented as ±SEM. (*p<0.05; **p<0.01 vs. control or saline; ^^p<0.01 vs. WAY in recordings). See also Supplementary Figure 5 and 6D, E.