1. Organization of Functional Long-Range Circuits Controlling the Activity of Serotonergic Neurons in the Dorsal Raphe Nucleus 
Li Zhou, Ming-Zhe Liu, Qing Li, Juan Deng, Di Mu, Yan-Gang Sun 
Cell Reports 
Volume 18, Issue 12, Pages (March 2017)
DOI: /j.celrep
Copyright © 2017 The Author(s) Terms and Conditions

2. Cell Reports 2017 18, 3018-3032DOI: (10.1016/j.celrep.2017.02.077)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
DRN Serotonergic Neurons Receive Monosynaptic Glutamatergic Input from the LHb
(A) Schematic graph of the experiment.
(B) Micrograph showing mCherry+ axons (red) and serotonergic neurons (green; 5-HT) in the DRN. The scale bar represents 200 μm. The inset shows the enlargement of the boxed area. The scale bar represents 5 μm.
(C) In Pet1-tdTomato mice, whole-cell patch-clamp recordings were made from tdTomato+ neurons. Biocytin-filled cells (green) were reconstructed after recording. The scale bar represents 100 μm.
(D) Post hoc staining of a recorded tdTomato+ neuron for Tph2 (green) and biocytin (blue). The scale bar represents 10 μm.
(E) The response of a tdTomato+ neuron to current steps.
(F) A light-evoked EPSC recorded in a DRN tdTomato+ neuron in ACSF and following sequential application of TTX, 4-AP, and NBQX. Blue bar, LED stimulation (475 nm; 1 ms).
(G) Distribution of the latency of EPSCs in the DRN tdTomato+ neurons (n = 146 neurons). The inset shows the averaged EPSC latency.
(H) Summary showing the EPSC amplitude before and following application of TTX (1 μM) and 4-AP (100 μM; n = 5).
(I) An example experiment showing the NBQX-induced blockade of light-evoked EPSC.
(J) Summary data showing the EPSC amplitude before and after NBQX (10 μM; p < 0.001; Wilcoxon signed rank test; n = 12).
(K) Summary of the spatial location and connectivity (pie graph; 147/210 neurons received synaptic inputs).
(L) Distribution of the EPSC amplitude. The inset shows the averaged EPSC amplitude.
Data are represented as mean ± SEM. See also Figure S1.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

4. Figure 2
The DRN Serotonergic Neurons Receive Functional Inputs from Multiple Brain Areas
(A) (Top) Schematic diagram of the experiment to examine the functional connection from PFC to DRN serotonergic neurons. (Bottom) A representative experiment shows light-evoked EPSCs before (black; in ACSF) and after bath application of NBQX (10 μM; red). Blue bar, LED stimulation (475 nm; 1 ms); same for (E), (I), (M), and (Q).
(B) Summary of the spatial location and connectivity of the recorded serotonergic neurons (n = 82 neurons).
(C) Summary data showing the amplitude of EPSC evoked by photostimulaton of PFC ChR2+ fibers before and after bath application of NBQX (n = 10 neurons).
(D) Distribution of the EPSC amplitude of the input from the PFC (n = 65 neurons).
(E) Schematic graph of the experiment to examine the functional input from LH to DRN serotonergic neurons. EPSC (black) and IPSC (blue) recorded from an example neuron in ACSF were blocked following sequential application of NBQX (10 μM; red) and picrotoxin (PTX; 50 μM; gray).
(F) Summary of the spatial location of recorded neurons and connectivity of input from LH (n = 106 neurons).
(G) Summary data showing the EPSC amplitude from LH before and after applying NBQX (left; LH-DRN:5-HT neurons; n = 6), as well as the IPSC amplitude from LH before and after applying picrotoxin (right; LH-DRN:5-HT neurons; n = 5).
(H) Distribution of the EPSC amplitude (left; LH-DRN:5-HT neurons; n = 52) and IPSC amplitude (right; LH-DRN:5-HT neurons; n = 58).
(I–L) Experiments to examine POA-DRN:5-HT neurons connection. n = 89 neurons (J). The EPSC and IPSC were recorded from different neurons. The IPSC (blue) was recorded in the presence of NBQX. This is the same layout as (E)–(H).
(M–P) Experiments to examine SN-DRN:5-HT neurons connection. n = 51 neurons (N). The EPSC and IPSC were recorded from different serotonergic neurons, and the control EPSC and IPSC were recorded in ACSF. This is the same layout as (E)–(H).
(Q–T) Experiments to examine AMY-DRN:5-HT neurons connection. n = 94 neurons (R). EPSC and IPSC recorded from an example neuron in ACSF were blocked following sequential application of NBQX and picrotoxin. This is the same layout as (E)–(H).
(U) Summary of connectivity of different inputs to DRN serotonergic neurons. Open bars, the proportion of neurons receiving exclusively EPSC; gray bars, the proportion of neurons receiving exclusively IPSC; black bars, the proportion receiving both EPSC and IPSC.
(V and W) Summary showing latency of EPSC (V) and IPSC (W) of different inputs to DRN serotonergic neurons.
Wilcoxon signed rank test was used for statistical analysis. Note that some p values are the same due to the same sampling number when comparing the data by Wilcoxon signed rank test. Data are represented as mean ± SEM. See also Figure S2.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

5. Figure 3
Unilateral and Bilateral Patterns of Axonal Topographic Innervation to Serotonergic Neurons
(A) The injection sites and axonal projection pattern within the DRN from different inputs. (Upper row) Viral injection sites are shown; (bottom row) mCherry+ axons in the DRN are shown. dm, dorsal medial region; iwi, ipsilateral lateral wing; lwc, contralateral lateral wing; vm, ventral medial region. Rectangular dotted areas indicate the area for measuring axonal density in the lateral wing. The scale bars in the top row represent 500 μm for PFC and AMY; 100 μm for LHb; and 200 μm for POA, LH, and SN. The scale bar in the bottom row represents 100 μm.
(B–G) Quantification of the relative density of axons from PFC (B), LHb (C), POA (D), LH (E), SN (F), and AMY (G) within the DRN. Paired t test is shown. n = 3–6 mice.
(H–J) Experiments to examine the PFC input to serotonergic neurons in each subregion of DRN. Schematic of the experiment is shown in (H). Example traces recorded from different areas of DRN are shown in (I). The location of recorded cells is shown in (J). This dataset has been included in Figures 2A–2D. Data from three mice are shown. Blue bar, LED stimulation (475 nm; 1 ms; I).
(K–M) Experiments to examine the POA input to serotonergic neurons in each subregion of DRN. Schematic of the experiment is shown in (K). Examples traces recorded from different areas of DRN are shown in (L). The location of recorded cells is shown in (M). This dataset has been included in Figures 2I–2L. Data from four mice are shown. Blue bar, LED stimulation (475 nm; 1 ms; L).
(N and O) Summary of the connectivity (N; p = 0.24; Chi-square test) and EPSC amplitude (O) from PFC to DRN serotonergic neurons in each subregion.
(P) Summary of the POA-DRN serotonergic neurons connectivity in each subregion (p = 0.002; Chi-square test).
(Q and R) Summary of the amplitude of EPSC (Q) and IPSC (R) from POA to DRN serotonergic neurons in each subregion.
Data are represented as mean ± SEM. See also Figure S3.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

6. Figure 4
LH Provides Convergent Excitation and Inhibition to Individual DRN Serotonergic Neurons
(A–D) Experiments to examine the projection from LH glutamatergic neurons to DRN serotonergic neurons. (A) Schematic diagram of the experiment is shown. (B) Post hoc identification of a recorded DRN serotonergic neuron by immunostaining for Tph2 is shown. The scale bar represents 10 μm. (C) Example responses of a DRN serotonergic neuron to photostimulation of LH glutamatergic terminals are shown. Blue bar, LED stimulation (475 nm; 1 ms). (D) Summary of the amplitude of EPSCs and IPSCs is shown (p < 0.001; Wilcoxon signed rank test; n = 18).
(E–H) Experiments to examine the projection from LH GABAergic neurons to DRN serotonergic neurons. The scale bar in (F) represents 10 μm. Blue bar, LED stimulation (475 nm; 1 ms; G).
(I–K) Analysis of the LH-DRN:serotonergic neurons inputs. (I) Examples show that serotonergic neurons received three types of functional input from the LH. Blue bar, LED stimulation (475 nm; 1 ms). (J) The connectivity for the serotonergic neurons receiving different inputs from LH is shown. (K) The spatial distribution for the serotonergic neurons with different inputs or no input is shown (using dots with same color as in J). This is the same dataset (I–K) as shown in Figures 2E–2H.
Data are represented as mean ± SEM. See also Figure S4.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

7. Figure 5 DRN GABAergic Neurons Receive Functional Input from LHb
(A) Schematic showing configuration of the experiment.
(B) Micrograph showing mCherry+ axons (red) from LHb and DRN GABAergic neurons (green) in the DRN. The scale bar represents 200 μm.
(C) Enlarged graph of the boxed area in (B). The scale bar represents 5 μm.
(D) Response of a GABAergic neuron to current steps.
(E) A light-evoked EPSC recorded in a DRN GABAergic neuron in ACSF and following sequential application of TTX, 4-AP, and NBQX. Blue bar, LED stimulation (475 nm; 1 ms).
(F) Distribution of the latency of light-evoked EPSC. The inset shows the averaged EPSC latency.
(G) Summary data showing the EPSC amplitude before and after sequential bath application of TTX (1 μM) and 4-AP (100 μM; n = 5).
(H) An example experiment showing the NBQX-induced blockade of light-evoked EPSC.
(I) Summary data showing the EPSC amplitude before and after bath application of NBQX (10 μM; p = 0.001; Wilcoxon signed rank test; n = 11).
(J) Summary of the spatial location and connectivity (pie graph; 60/86 GABAergic neurons received synaptic input).
(K) Distribution of the EPSC amplitude of the recorded GABAergic neurons. The inset shows the averaged EPSC amplitude.
Data are represented as mean ± SEM. See also Figure S5.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

8. Figure 6
DRN GABAergic Neurons Receive Functional Inputs from Multiple Brain Areas
(A) (Top) Schematic diagram of the experiment. (Bottom) A representative experiment shows light-evoked EPSC before (black; in ACSF) and after NBQX (10 μM; red). Blue bar, LED stimulation (475 nm; 1 ms); same for (E), (I), (M), and (Q).
(B) Summary of the spatial location and connectivity of the DRN GABAergic neurons (n = 58).
(C) Summary data showing the amplitude of EPSC evoked by photostimulaton of PFC fibers before and after NBQX (p = 0.063; n = 5).
(D) Distribution of the EPSC amplitude of the input from PFC (n = 35).
(E) Schematic graph of the experiment. EPSCs (black; in ACSF) were blocked following application of NBQX (red). In another neuron, IPSCs (blue; in ACSF) were blocked following application of picrotoxin (gray).
(F) Summary of the spatial location and connectivity of DRN GABAergic neurons receiving input from LH (n = 37 neurons).
(G) Summary data of the EPSC amplitude from LH before and after NBQX (left; LH-DRN:GABAergic neurons; p = 0.031; n = 6) and IPSC amplitude from LH before and after applying picrotoxin (right; LH-DRN:GABAergic neurons; p = 0.031; n = 6).
(H) Distribution of the amplitude of EPSCs (left; LH-DRN:GABAergic neurons; n = 14) and IPSCs (right; LH-DRN:GABAergic neurons; n = 22).
(I–L) Experiments to examine POA-DRN:GABAergic neurons connection. n = 45 neurons (J). EPSC and IPSC recorded from an example neuron in ACSF were blocked following sequential application of NBQX and picrotoxin. This is the same layout as (E)–(H).
(M–P) Experiments to examine SN-DRN:GABAergic neurons connection. n = 26 neurons (N). IPSC and EPSC recorded from an example neuron in ACSF were blocked following sequential application of picrotoxin and NBQX. This is the same layout as (E)–(H).
(Q–T) Experiments to examine AMY-DRN:GABAergic neurons connection. n = 58 neurons (R). The EPSC and IPSC were recorded from different GABAergic neurons, and the control EPSC and IPSC were recorded in ACSF. This is the same layout as (E)–(H).
(U) Summary of connectivity of different inputs to DRN GABAergic neurons. Open bars, the proportion of neurons receiving exclusively EPSC; gray bars, the proportion of neurons receiving exclusively IPSC; black bars, the proportion receiving both EPSC and IPSC.
(V and W) Summary showing latency of EPSCs (V) and IPSCs (W) of different inputs to DRN GABAergic neurons.
Data are represented as mean ± SEM. Wilcoxon signed rank test was used for statistical analysis. See also Figure S6.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

9. Figure 7
Feedforward Inhibition of DRN Serotonergic Neurons by LHb-DRN Inputs
(A) Schematic configuration of the experiment.
(B) An example experiment showing that photostimulation of the LHb ChR2+ fibers evoked a short-latency EPSC (red) and a long-latency IPSC (black) in a DRN 5-HT neuron. Blue bar, LED stimulation (475 nm; 1 ms).
(C) Summary of the latencies of light-evoked EPSCs and IPSCs (p < 0.001; n = 13).
(D) An experiment showing the blockade of the light-evoked IPSC by NBQX (10 μM). The inset shows the IPSC before (black) and after NBQX (red). Blue bar, LED stimulation (475 nm; 1 ms).
(E) Summary of the IPSC amplitude before and after NBQX (p = 0.063; n = 5 neurons).
(F) Activation of LHb fibers evoked reliable spikes in an example DRN GABAergic neuron. Example trace (top; scale bars: 400 pA and 100 ms), raster plot of the first ten trials (middle), and summary of all 33 trials (bottom) are shown. Blue bar, LED stimulation (475 nm; 1 ms).
(G) Schematic of the experiment to examine the local GABAergic inputs to DRN serotonergic neurons.
(H) An example experiment showing the blockade of light-evoked IPSC in a serotonergic neuron by picrotoxin (50 μM). The inset shows the IPSC before (black) and after picrotoxin (red). Blue bar, LED stimulation (475 nm; 1 ms).
(I) Summary of the IPSC amplitude evoked by local GABAergic inputs before and after picrotoxin (p = 0.016; n = 7).
Data are represented as mean ± SEM. Wilcoxon signed rank test was used for statistical analysis. See also Figure S7.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions