PBN-PVT projections modulate negative affective states in mice
- Department of Anesthesiology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, China
- The First Rehabilitation Hospital of Shanghai, Tongji University School of Medicine, China
- Department of Respiratory, The First Affiliated Hospital of Guangzhou Medical University, China
- SUSTech Center for Pain Medicine, School of Medicine, Southern University of Science and Technology, China
Figures
Figure 1 with 3 supplements
Functional connectivity pattern of parabrachial nucleus-paraventricular nucleus (PBN-PVT) projections.
(A) The schematic for virus injection of AAV2/8-hSyn-ChR2-mCherry into the PBN nucleus and the slice recording with 473 nm laser stimulation. (B) The projection fibers in the PVT nucleus. Scale bar: …
Figure 1—figure supplement 1
Characterization of parabrachial nucleus-paraventricular nucleus (PBN-PVT) neurons.
(A−O) Double staining of tdTomato signals (red) with Tacr1 mRNA (A−C), Tac1 mRNA (E−G), Pdyn mRNA (I−K), and CGRP protein (M−O). Scale bar: 100 μm. (D, H, L) The proportions of co-expressing neurons …
Figure 1—figure supplement 2
The distribution pattern of the parabrachial nucleus-paraventricular nucleus (PBN-PVT) glutamatergic projection.
(A) The illustration for virus injection of AAV2/8-EF1α-DIO-EGFP into the PBN nucleus on Vglut2-ires-Cre mice. (B−D) The virus expression in the anterior (B), the middle (C), and the posterior PBN (D…
Figure 1—figure supplement 3
The distribution pattern of collateral projection fibers from parabrachial nucleus-paraventricular nucleus (PBN-PVT) neurons.
(A) The illustration shows the injection of retroAAV2/2-hSyn-Cre into the PVT and AAV2/8-EF1α-DIO-EGFP into the PBN to label the PBN-PVT neurons. (B) Examples of AAV2/8-EF1α-DIO-EGFP expression in …
Figure 2 with 4 supplements
Optogenetic activation of parabrachial nucleus-paraventricular nucleus (PBN-PVT) projections induced negative affective states.
(A) The illustration shows the injection of AAV2/9-EF1α-DIO-ChR2-mCherry virus into the PBN nucleus and the optic fiber above the PVT on the Vglut2-ires-Cre mice. (B, C) The virus injection sites of …
Figure 2—figure supplement 1
Virus expression in the parabrachial nucleus (PBN) and the optic fiber position in the paraventricular nucleus (PVT) of Vglut2-ires-Cre mice injected with AAV2/9-EF1α-DIO-ChR2-mCherry virus or AAV2/9-EF1α-DIO-mCherry virus.
(A) Histological map showing area of ChR2 expression in the PBN at bregma −5.20 mm in eight mice. Scale bar: 400 μm. (B) The position of optic fiber (rectangle) in the PVT in the …
Figure 2—figure supplement 2
Effects of optogenetic activation of parabrachial nucleus-paraventricular nucleus (PBN-PVT) projections in the open field test (OFT) and the conditioned place aversion (CPA).
(A, B) Quantification of the immobility time (A) and the distance (B) in the OFT (mCherry group: n = 7 mice; ChR2 group: n = 8 mice). (C−F) Quantification of the velocity (C), the immobility time (D)…
Figure 2—video 1
Optogenetic activation of parabrachial nucleus-paraventricular nucleus (PBN-PVT) projections in open field test (OFT).
The 473 nm laser (20 Hz, 5 mW, 5 ms) was delivered from 00:10 to 05:10 in the video.
Figure 2—video 2
Optogenetic activation of parabrachial nucleus-paraventricular nucleus (PBN-PVT) projections in real-time place aversion (RTPA).
The 473 nm laser (20 Hz, 5 mW, 5 ms) was delivered when the mouse entered the laser-paired chamber and withdrew when the mouse exited the laser-paired chamber during the 10 min. The video was played …
Figure 3 with 2 supplements
Pharmacogenetic activation of parabrachial nucleus-paraventricular nucleus (PBN-PVT) neurons induced anxiety-like behaviors and fear-like behaviors.
(A) The illustration shows virus injection of retroAAV2/2-hSyn-Cre into the PVT nucleus and bilateral injection of AAV2/9-hSyn-DIO-hM3Dq-mCherry into the PBN nucleus. (B, C) Clozapine N-oxide …
Figure 3—figure supplement 1
Virus expression in the parabrachial nucleus (PBN) of mice injected with AAV2/9-hSyn-DIO-hM3Dq-mCherry or AAV2/9-EF1α-DIO-mCherry in the pharmacogenetic manipulation experiment.
(A) Representative histological images of hM3Dq expression in an AAV2/9-hSyn-DIO-hM3Dq-mCherry-injected mouse at brain level from bregma −5.02 mm to bregma −5.34 mm. Scale bar: 1 mm. (B) Depiction …
Figure 3—figure supplement 2
Pharmacogenetic activation of parabrachial nucleus-paraventricular nucleus (PBN-PVT) neurons did not affect depressive-like behaviors, basal nociceptive thresholds, formalin-induced licking behavior, or motor function.
(A) Immobility time in the tail suspension test (TST), n = 7 mice per group. (B) Immobility time in the forced swimming test (FST), mCherry group: n = 7 mice; hM3Dq group: n = 8 mice. (C, D) Effects …
Figure 4 with 3 supplements
Optogenetic inhibition of parabrachial nucleus-paraventricular nucleus (PBN-PVT) projections reduced aversion-like behavior and fear-like behaviors.
(A) The illustration shows the bilateral injection of AAV2/9-EF1α-DIO-NpHR3.0-EYFP virus into the PBN and placement of optic fiber above the PVT on Vglut2-ires-Cre mice. (B, C) Examples of …
Figure 4—figure supplement 1
Virus expression in the parabrachial nucleus (PBN) and the optic fiber position in the paraventricular nucleus (PVT) of Vglut2-ires-Cre mice injected with AAV2/9-EF1α-DIO-NpHR3.0-EYFP or AAV2/8-EF1α-DIO-EGFP.
(A) Histological map showing area of NpHR3.0 expression in the PBN at bregma −5.20 mm in seven mice. Scale bar: 400 μm. (B) The position of optic fiber (rectangle) in the PVT in the …
Figure 4—figure supplement 2
Optogenetic inhibition of parabrachial nucleus-paraventricular nucleus (PBN-PVT) projections did not affect associative fear memory acquisition and retrieval.
(A) The protocol of auditory fear conditioning experiments with optogenetic inhibition of the PBN-PVT projection. (B−D) Quantification of freezing levels during condition trials (B), contextual test …
Figure 4—figure supplement 3
Optogenetic inhibition of the parabrachial nucleus-paraventricular nucleus (PBN-PVT) neurons reduced aversion-like behavior and fear-like freezing behavior.
(A) The illustration shows virus injection of retroAAV2/2-hSyn-Cre into the PVT, bilateral injection of AAV2/9-EF1α-DIO-NpHR3.0-EYFP into the PBN, and bilateral placement of optic fiber above the …
Figure 5 with 1 supplement
Activation of PVTPBN neurons by diverse aversive stimuli.
(A) The illustration shows the injection of AAV2/1-hSyn-Cre into the parabrachial nucleus (PBN) of Rosa26-tdTomato mice. (B−D) The distribution of the neurons in the paraventricular nucleus (PVT) at …
Figure 5—figure supplement 1
Calcium signals of paraventricular nucleus (PVT) neurons in response to aversive stimuli.
(A) Schematic shows injection of AAV2/8-hSyn-GCaMP6s into the PVT and placement of the optic fiber above the PVT. (B) Representative of GCaMP6s expression and the position of optic fiber in the PVT. …
Figure 6 with 2 supplements
Neuronal activity of paraventricular nucleus (PVT) neurons in response to the footshock was modulated by parabrachial nucleus (PBN)-PVT projections.
(A) Top: schematic shows injection of AAV2/9-EF1α-DIO-ChR2-mCherry into the PBN and placement of the optoelectrode above the PVT of Vglut2-ires-Cre mice. Bottom: the protocol of 10 sweeps of laser …
Figure 6—figure supplement 1
Dual Fos staining detecting Fos protein and fos mRNA induced by laser stimulation and footshock.
(A) Top: schematic shows injection of AAV2/9-EF1α-DIO-ChR2-mCherry into the parabrachial nucleus (PBN) and placement of the optic fiber above the paraventricular nucleus (PVT) of Vglut2-ires-Cre …
Figure 6—figure supplement 2
The response latency of paraventricular nucleus (PVT) neurons to laser activation and footshock activation.
(A) Response latency of laser-activated PVT neurons (n = 23 neurons). (B) The histogram of response latency of laser-activated PVT neurons (less than 14 ms, n = 12 neurons). (C) Response latency of …
Figure 7 with 1 supplement
Activation of PVTPBN neurons induced anxiety-like behaviors.
(A) The illustration shows injection of AAV2/1-hSyn-Cre into the parabrachial nucleus (PBN) and AAV2/9-hSyn-DIO-hM3Dq-mCherry into the paraventricular nucleus (PVT). (B, C) Clozapine N-oxide …
Figure 7—figure supplement 1
Distribution pattern of projection fibers of PVTPBN neurons.
(A) The illustration shows injection of AAV2/1-hSyn-Cre into the parabrachial nucleus (PBN) and AAV2/8-EF1α-DIO-EGFP into the paraventricular nucleus (PVT) of Rosa26-tdTomato mice. (B) The …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (Ai9) | Jackson Laboratory | Stock# 007909;RRID:MGI:3813511 | Dr. Hua-Tai Xu (Institutes of Neuroscience, Chinese Academic of Sciences) |
| Genetic reagent (M. musculus) | STOCK Slc17a6tm2(cre)Lowl/J (Vglut2-ires-Cre) | Jackson Laboratory | Stock# 016963;RRID:MGI:5300532 | Dr. Yan-Gang Sun (Institutes of Neuroscience, Chinese Academic of Sciences) |
| Genetic reagent (Dependoparvovirus) | AAV2/8-hSyn-ChR2-mCherry | Obio Technology | Cat# AG26976 | 4 × 1012 v.g./mL |
| Genetic reagent (Dependoparvovirus) | AAV2/8-EF1α-DIO-EGFP | Taitool Bioscience | Cat# S0270 | 4 × 1012 v.g./mL |
| Genetic reagent (Dependoparvovirus) | retroAAV2/2-hSyn-Cre | Taitool Bioscience | Cat# S0278-2RP-H20 | 4 × 1012 v.g./mL |
| Genetic reagent (Dependoparvovirus) | AAV2/9-EF1α-DIO-ChR2-mCherry | Taitool Bioscience | Cat# S0170-9-L20 | 4 × 1012 v.g./mL |
| Genetic reagent (Dependoparvovirus) | AAV2/9-EF1α-DIO-mCherry | Obio Technology | Cat# AG20299 | 4 × 1012 v.g./mL |
| Genetic reagent (Dependoparvovirus) | AAV2/9-hSyn-DIO-hM3Dq-mCherry | BrainVTA | Cat# PT-0019 | 4 × 1012 v.g./mL |
| Genetic reagent (Dependoparvovirus) | AAV2/9-EF1α-DIO-NpHR3.0-EYFP | Obio Technology | Cat# AG26966 | 4 × 1012 v.g./mL |
| Genetic reagent (Dependoparvovirus) | AAV2/8-hSyn-GCaMP6s | Taitool Bioscience | Cat# S0225-8 | 4 × 1012 v.g./mL |
| Genetic reagent (Dependoparvovirus) | AAV2/1-hSyn-Cre | Taitool Bioscience | Cat# S0278-1-H50 | 1.5 × 1013 v.g./mL |
| Sequence-based reagent | RNAscope Probe-fos-C2 | Advanced Cell Diagnostics | Cat# 316921-C2 | |
| Sequence-based reagent | RNAscope Probe-Tac1-C2 | Advanced Cell Diagnostics | Cat# 410351-C2 | |
| Sequence-based reagent | RNAscope Probe-Tacr1-C2 | Advanced Cell Diagnostics | Cat# 428781-C2 | |
| Sequence-based reagent | RNAscope Probe-Pdyn | Advanced Cell Diagnostics | Cat# 318771 | |
| Sequence-based reagent | RNAscope Probe- Slc17a6-C2 | Advanced Cell Diagnostics | Cat# 319171-C2 | |
| Antibody | Anti-Fos (rabbit polyclonal) | Abcam | Cat# ab190289; RRID:AB_2737414 | IF (1:4000) |
| Antibody | Anti-CGRP (goat polyclonal) | Abcam | Cat# ab36001; RRID:AB_725807 | IF (1:1000) |
| Antibody | Anti-DsRed (goat polyclonal) | Takara Bio | Cat# 632496; RRID:AB_10013483 | IF (1:500) |
| Antibody | Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG (H+L) | Jackson ImmunoResearch Labs | Cat# 711-545-152; RRID:AB_2313584 | IF (1:400) |
| Antibody | Cy3 AffiniPure Donkey Anti-Rabbit IgG (H+L) | Jackson ImmunoResearch Labs | Cat# 711-165-152; RRID:AB_2307443 | IF (1:400) |
| Antibody | Alexa Fluor 488 AffiniPure F(ab’)2 Fragment Donkey Anti-Goat IgG (H+L) | Jackson ImmunoResearch Labs | Cat# 705-546-147; RRID:AB_2340430 | IF (1:400) |
| Commercial assay or kit | RNAscope Multiplex Fluorescent Reagent Kit v2 | Advanced Cell Diagnostics | Cat# 320293 | |
| Software, algorithm | ImageJ | NIH | ||
| Software, algorithm | LabState | AniLab |
