Anatomically Defined and Functionally Distinct Dorsal Raphe Serotonin Sub-systems

Abstract

The dorsal raphe (DR) constitutes a major serotonergic input to the forebrain and modulates diverse functions and brain states, including mood, anxiety, and sensory and motor functions. Most functional studies to date have treated DR serotonin neurons as a single population. Using viral-genetic methods, we found that subcortical- and cortical-projecting serotonin neurons have distinct cell-body distributions within the DR and differentially co-express a vesicular glutamate transporter. Further, amygdala- and frontal-cortex-projecting DR serotonin neurons have largely complementary whole-brain collateralization patterns, receive biased inputs from presynaptic partners, and exhibit opposite responses to aversive stimuli. Gain- and loss-of-function experiments suggest that amygdala-projecting DR serotonin neurons promote anxiety-like behavior, whereas frontal-cortex-projecting neurons promote active coping in the face of challenge. These results provide compelling evidence that the DR serotonin system contains parallel sub-systems that differ in input and output connectivity, physiological response properties, and behavioral functions.

Keywords: 5-HT; Tph2; Vglut3; anxiety; central amygdala; depression; dorsal raphe; fiber photometry; iDISCO; orbital frontal cortex; serotonin.

Figure 1.. Spatial Organization of DR Serotonin Neurons according to Axonal Projections and Vglut3 Co-expression

(A) Schematic of retrograde labeling and 3D reconstruction of spatial locations of DR serotonin neurons. Cyan and red dots represent injection sites of HSV-Cre in Ai14 mice. Anti-Tph2 staining was performed on consecutive coronal sections containing DR (star). The positions of Tph2⁺/tdTomato⁺ cells were recorded in confocal images. Each section was registered to the Allen reference brain and reconstructed in 3D. DBSCAN was applied for spatial clustering to generate a 3D surface based on the location of Tph2⁺ neurons (STAR Methods). (B) Representative coronal confocal sections of the DR. Magenta, retrogradely labeled cells from eight projecting sites; yellow, anti-Tph2 staining. Dashed line, aqueduct border. Scale, 100 μm. Left insets, high-magnification images of neurons marked by arrows in individual channels. Scale, 25 μm. Right insets (top, coronal view; bottom, sagittal view): yellow, cyan, and red structures represent 3D surface of the clusters of, respectively, all DR Tph2⁺ neurons; those that project to PVH/CeA/LHb/dLGN; or those that project to OB/PIR/OFC/ENT. Scale, 500 μm. (C) Merged surface view of the DR^Tph2→SC (subcortical) cluster (cyan), →AC (anterior cortex) cluster (red), and →ENT cluster (brown) in coronal (C₁) and sagittal (C₂) view. Scale, 200 μm. C₃, coronal projection showing the location of individual cells from the DR^Tph2→SC and →AC groups. C₄, densities of DR^Tph2→SC and →AC neurons along D–V axis. The two clusters exhibit the same line density at a horizontal plane 3742 μm ventral to the brain surface (dashed line). 74% of DR^Tph2→SC neurons were dorsal to this plane, whereas 80% of DR^Tph2→AC neurons were ventral to this plane. (D) Representative coronal confocal sections of DR showing anti-Tph2 staining in Vglut3-Cre/Ai14 mice (green), which express tdTomato in Vglut3⁺ cells (red). (E) Coronal (E₁) and sagittal (E₂) view integrating projection-defined clusters and the cluster of Tph2⁺&Vlgut3⁺ neurons (yellow, 1730±219.8 neurons; n=3). Scale, 200 μm. (F) Representative coronal confocal sections of the DR showing retrograde labeling neurons from eight brain regions (magenta, pseudo-colored from rabies-derived GFP), anti-Tph2 staining (green), and tdTomato from Vglut3-Cre⁺ neurons (red). Scale, 100 μm. Insets: magnified images showing the neurons indicated with arrows in individual channels. Scale, 25 μm. (G) The proportion of GFP, Tph2, and Vglut3 triple positive neurons in GFP⁺/Tph2⁺ neurons for 8 projection brain regions. In this and all subsequent figures, abbreviations for anatomical regions can be found in Methods. Axis labels: A, anterior; P, posterior, D, dorsal; V, ventral; M, medial; L, lateral. Error bars, SEM. See Figure S1 for related data, and Table S4 for cell numbers labeled by retrograde tracers.

Figure 2.. Distinct Collateralization Patterns of DR^Sert→OFC and DR^Sert→CeA Neurons

(A) Schematic of viral-genetic tracing and whole-brain 3D imaging. w.d., working distance of the light-sheet microscope objective. (B) Overview of axonal projections from one representative brain each from the DR^Sert→OFC (blue) and DR^Sert→CeA (green) groups. Whole-mount imaging included the entire left hemisphere and the medial-most ~650 μm of the right hemisphere. Axes as indicated on right. See Movie S2 for a 3D rendering. (C, D) Sagittal view of single 5-μm optical sections from eight individual brains registered to the Allen Institute common coordinate framework. Axons from DR^Sert→OFC (C₁–C_4; merged in C₅) and DR^Sert→CeA (D₁–D₄; merged in D₅) neurons are shown in green and blue, respectively. (E) Sagittal brain atlas image (10 μm) from the Allen Institute that encompasses CeA and anterior insular cortex (AI). The red box indicates the displayed region of (C) and (D).

(F) Coronal density maps of DR^Sert→CeA (left) and DR^Sert→OFC (middle) projections generated by voxel-wise dilation of axons. Right, a p-value map highlighting individual voxels with p<0.05 between groups. See Movie S3 for the fly-through of coronal maps of the brain rostral to DR. (G) Heat map of relative labeling density (normalized to region volume and gross label content per brain) across 255 regions defined by the Allen Atlas. Additional columns show statistics for effect size, uncorrected p-values from two-tailed t-tests, and findings that pass false discovery rate at 10% (black bars). See Table S1 for the list of brain regions in the same sequence. See Figure S2 and Table S1 for related data.

Figure 3.. Biased Input Distributions for DR^Sert→OFC and DR^Sert→CeA Neurons

(A) Schematic of cTRIO experiments. (B) Confocal images of coronal sections containing the DR, showing OFC- and CeA-projecting serotoninergic starter cells (starter cell numbers for DR^Sert→OFC: 73±8.3, n=9 mice; for DR^Sert→CeA: 96±13.4, n=8 mice). Red, TVA-mCherry (TC) expression; green, GFP expression; cyan, anti-Tph2 staining. Scale, 100 μm. Insets: high magnification images of neurons indicated by arrows. Scale, 50 μm. (C) Quantification of whole-brain inputs to DR^Sert→OFC and DR^Sert→CeA neurons (n=9, 8). Y-axis presents percentage of total inputs counted for each brain. Error bars, SEM. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 (multiple t-tests with Holm-Sidak correction). (D) Representative GFP labeled ipsilateral input cells to DR^Sert→OFC and DR^Sert→CeA neurons. Scale, 250 μm. See Figures S3 and Table S2 for related data.

Figure 4.. DR^Sert→OFC and DR^Sert→CeA Neurons Are Both Activated by Reward but Show Opposite Responses to Punishment.

Fiber photometry recordings were performed on DR^Sert (A), DR^Sert→OFC (B) and DR^Sert→CeA (C) neurons. (A₁–C₁) Schematic of viral injection and optical fiber (cyan) implantation. (A₂–C₂) Confocal images of coronal sections showing fiber optic placement (dotted rectangle) and the expression of GCaMP6m (green) with Tph2 staining (red, A₂’–C₂’) in the DR. Vertical dashed lines represent the midline. Scale, 100 μm. Estimate of GCaMP6m⁺ serotonin neurons under optical fiber: DR^Sert group, 204±39.0, n=7 mice; DR^Sert→OFC group, 112±28.0, n=7 mice; DR^Sert→CeA group, 115±22.1, n=8 mice. (A₃–C₃) Mean responses of individual mice to sucrose consumption after lever press. Time 0 is aligned to lick initiation (vertical dashed line). Red traces correspond to the mice shown in (A_2–C₂) and Figure S4. (A₃’–C₃’) Group data from all trials of individual mice showing quantification of the peak ΔF/F during sucrose water licking comparing to the ΔF/F at time 0 (paired t test; p<0.001 for all comparisons; n≥20 trials). (A₄–C₄) Mean responses of individual mice from the three groups to electrical shock. Time 0 is aligned to onset of 1-sec electric shock delivery. (A₄’–C₄’) Group data from all the trials of individual mice showing quantification of the peak ΔF/F (positive extreme) and trough ΔF/F (negative extreme) recorded after electric shock delivery comparing to the ΔF/F at time (B₄’, two-tailed paired t test, p<0.01 for all comparisons in inhibition. C₄’, one-way ANOVA followed by multiple t-test, p<0.05 for all comparisons in activation. n=12 trials). (D) Quantification of integrated ΔF/F signals between 0.13s to 0.65s before lever press (after subtracting integrated signals between 0.65s and 0.117s before lever press) comparing DR^Sert→OFC and DR^Sert→CeA neurons. (E) Quantification of the peak ΔF/F recorded during sucrose water licking from DR^Sert→OFC and DR^Sert→CeA neurons. (F) Quantification of the peak ΔF/F (negative or positive extreme) recorded after electric shock delivery from DR^Sert→OFC and DR^Sert→CeA neurons. Error bars, SEM; n.s., not significant; *p<0.05; **p<0.01; ***p<0.001; ***p<0.001; ****p<0.0001. (For D–F, unpaired t-test; n=7, 8 mice for DR^Sert→OFC and DR^Sert→CeA groups, respectively). See Figures S4 and Table S3 for related data.

Figure 5.. Chemogenetic Activation and Conditional Tph2 Knockout Reveal that Both DR^Sert→OFC and DR^Sert→CeA Neurons Suppress Locomotion.

(A, B) Chemogenetic activation of DR^Sert→OFC (A) and DR^Sert→CeA (B) neurons. (A₁, B₁) Schematic for experimental (Exp) groups. Conditions for the two controls (Ctrl) are listed below. (A₂, B₂) Confocal images of coronal sections showing the expression of hM3Dq-2A-mCherry (red), and co-labeling with Tph2 staining (green) in the DR. Dotted lines are aqueduct borders. Scale, 100 μm. Right, high magnification images of neurons indicated by arrows. Scale, 50 μm. See Table S4 for cell counts. (C, D) DR^Sert→OFC (C) and DR^Sert→CeA (D) neurons were Tph2 depleted by bilaterally injecting AAV_retro-Cre into OFC (C₁) or CeA (D₁) of the Tph2^flox/flox mice. (C₂, D₂) Confocal images of coronal sections showing the expression of Cre-2A-GFP (green), almost all of which were negative from Tph2 staining (red) in the DR. (Cells counts: Exp DR^Sert→OFC group, 272±67.9, 98.6%±0.43% were Tph2⁻; Exp DR^Sert→CeA group, 331±76.6, 98.7%±0.33% were Tph2^–. Ctrl DR^Sert→OFC group, 160±8.5, 16.7%±0.18% were Tph2^–; Ctrl DR^Sert→CeA group, 177±42.2, 39.2%±0.16% were Tph2^–; n=3 mice for all groups). Scale, 100 μm. Right, high magnification images showing neurons indicated by arrows. Scale, 25 μm. (E) Chemogenetic activation of DR^SertĺOFC neurons decreases distance traveled (one-way ANOVA followed by multiple t-tests; F(2, 31) = 4.42, t=2.672, 2.29. n= 9, 11, 14). *p<0.05. (F) Conditionally knocking out Tph2 from DR^SertĺOFC neurons does not have a significant effect on distance traveled (two-tail unpaired t-test, t=1.24, df=17; n=9, 10). (G) Activation of DR^Sert→CeA neurons decreases distance traveled (one-way ANOVA followed by multiple t-test; F(2, 30) = 4.516; t=2.21, 2.83. n= 10, 11, 12). *p<0.05. (H) Conditionally knocking out Tph2 from DR^Sert→CeA neurons increases distance traveled (two-tail unpaired t-test, t=5.07, df=14. n=8, 8). ***p<0.001. Error bars, SEM. See Figures S5 and S6 for related data.

Figure 6.. DR^Sert→CeA but Not DR^Sert→OFC Neurons Promote an Anxiety-like State.

(A) Chemogenetic activation of DR^Sert→OFC neurons does not affect the number of center entries (A₁) or time spent in the center (A₂) of the open field (one-way ANOVA, A₁, F(2, 31) = 1.23; A₂, F(2, 31) = 0.757. n= 9, 11, 14). (B) Conditionally knocking out Tph2 from DR^Sert→OFC neurons decreases the number of center entries (B₁) and the time spent in the center (B₂) (two-tail unpaired t-test; B₁, t=2.22, df=17; B₂, t=2.54, df=17. n=9, 10). (C) Activation of DR^Sert→CeA neurons decreases the number of center entries (C₁) and the time spent in the center (C₂) (one-way ANOVA followed by multiple t-tests; C₁, F(2, 30) = 6.54, t=3.02, 3.17; C₂, F(2, 30) = 6.54, t=2.85, 3.30. n= 10, 11, 12). (D) Conditionally knocking out Tph2 from DR^Sert→CeA neurons increases the number of center entries (D₁), but not the time spent in the center (D₂) (two-tail unpaired t-test; D₁, t=4.31, df=14; D₂, t=1.13, df=14. n=8, 8). (E) Activation of DR^Sert→OFC neurons does not affect the number of entries to the open arm (E₁) or the time spent in the open arm in the EPM (E₂) (one-way ANOVA, E₁, F(2, 31) = 0.290; E₂, F(2, 31) = 0.341; n= 9, 11, 14). (F) Conditionally knocking out Tph2 from DR^Sert→OFC neurons does not affect the number of open arm entries (F₁), but increases the time spent in the open arm (F₂) (two-tail unpaired t-test; F₁, t=0.337, df=17; F₂, t=2.45, df=17. n=9, 10). (G) Activation of DR^Sert→CeA neurons decreases the number of open arm entries (G₁), and decreases the time spent in the open arm (G₂) (one-way ANOVA followed by multiple t-tests; G₁, F(2, 30) = 7.59, t=3.00, 3.60.; G₂, F(2, 30) = 3.43, t=1.51, 2.60. n= 10, 11, 12. n.s., p=0.142). (H) Conditionally knocking out Tph2 from DR^Sert→CeA neurons does not affect the number of open arm entries (H₁), but increased the time spent in the open arm (H₂) (two-tail unpaired t-test; H1, t=0.045, df=14; H2, t=2.55, df=14. n=8, 8). *p<0.05; **p<0.01; ***p<0.001. Error bars, SEM.

Figure 7.. DR^Sert→OFC but Not DR^Sert→CeA Neurons Promote Escape Behavior in the Forced-Swim Test.

(A) Activation of DR^Sert→OFC neurons decreases the immobility time on Day 2 testing session (one-way ANOVA followed by multiple t-tests; F(2, 31) = 6.84; t=2.97, 3.24. n= 9, 11, 14). **p<0.01. See Figures S7I for related data. (B) Conditionally knocking out Tph2 from DR^Sert→OFC neurons increases the immobility time (two-tail unpaired t-test, t=2.21, df=17. n=9, 10). *p<0.05. (C) Activation of DR^Sert→CeA neurons does not affect the immobility time (one-way ANOVA; F(2, 29) = 2.50. n= 10, 11, 11). (D) Conditionally knocking out Tph2 from DR^Sert→CeA neurons decreases the immobility time (two-tail unpaired t-test; t=3.58, df=14. n=8, 8). **p<0.01. Error bars, SEM. (E) Summary of gain- and loss-of-function results.