Circuit and synaptic organization of forebrain-to-midbrain pathways that promote and suppress vocalization
- Department of Neurobiology, Duke University Medical Center, United States
- Department of Biostatistics & Bioinformatics, Duke University Medical Center, United States
- Department of Psychology, Cornell University, United States
Figures
Figure 1 with 4 supplements
Inhibitory neurons in the hypothalamus and amygdala provide input to the periaqueductal gray (PAG) vocal gating circuit.
(A) (Left) Viral strategy shown for transsynaptic labeling of direct inputs to PAG-USV neurons (performed in N = 4 males). (Right) Confocal images are shown of starter PAG-USV neurons, upstream …
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Figure 1—source data 1
Source data for Figure 1A–C.
- https://cdn.elifesciences.org/articles/63493/elife-63493-fig1-data1-v3.docx
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Figure 1—source data 2
Source data for Figure 1D.
- https://cdn.elifesciences.org/articles/63493/elife-63493-fig1-data2-v3.xlsx
Figure 1—figure supplement 1
Monosynaptic rabies-based tracing reveals preoptic and amygdala inputs to the midbrain vocalization circuit.
(A) Example confocal images showing transsynaptic labeling of neurons in the preoptic area of the hypothalamus (preoptic area [POA], green) that provide monosynaptic input to GABAergic …
Figure 1—figure supplement 2
Monosynaptic rabies-based tracing reveals cortical inputs to the midbrain vocalization circuit.
(A) Example confocal images showing transsynaptic labeling of neurons in the infralimbic cortex (green) that provide monosynaptic input to GABAergic periaqueductal gray (PAG) neurons (left-most …
Figure 1—figure supplement 3
Monosynaptic rabies-based tracing reveals additional hypothalamic inputs to the midbrain vocalization circuit.
(A) Example confocal images showing transsynaptic labeling of neurons in the anterior hypothalamus (green) that provide monosynaptic input to GABAergic periaqueductal gray (PAG) neurons (left-most …
Figure 1—figure supplement 4
Monosynaptic rabies-based tracing reveals additional subcortical inputs to the midbrain vocalization circuit.
(A) Example confocal images showing transsynaptic labeling of neurons in the ventral pallidum (green) that provide monosynaptic input to GABAergic periaqueductal gray (PAG) neurons (left-most …
Figure 2 with 3 supplements
Activating periaqueductal gray (PAG)-projecting POA neurons elicits ultrasonic vocalizations (USVs) in the absence of social cues.
(A) (Left) Viral strategy to express ChR2 in POAPAG neurons. (Right) Example trial showing that optogenetic activation of POAPAG neurons elicits USV production in an isolated animal. (B) (Left) …
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Figure 2—source data 1
Source data for Figure 2C and F.
- https://cdn.elifesciences.org/articles/63493/elife-63493-fig2-data1-v3.xlsx
Figure 2—figure supplement 1
Additional characterization of ultrasonic vocalizations (USVs) elicited by optogenetic activation of preoptic area (POA) neurons.
(A) Mean USV rate across trials aligned to delivery of blue light pulses plotted for N = 8 individual mice following optogenetic activation of POAPAG neurons. Plots represent the individual animals …
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Figure 2—figure supplement 1—source data 1
Source data for panels B and C of Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/63493/elife-63493-fig2-figsupp1-data1-v3.xlsx
Figure 2—figure supplement 2
Additional information related to the optogenetic activation of POAPAG, POA-Esr1+ neurons, and AmgC/M-PAG neurons.
(A) Real-time place preference tests were performed in which either POA or AmgC/M neurons were optogenetically activated when mice were in one of two sides of a test chamber (see Materials and method…
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Figure 2—figure supplement 2—source data 1
Source data for panel A of Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/63493/elife-63493-fig2-figsupp2-data1-v3.xlsx
Figure 2—figure supplement 3
Dual tracing of the axonal projections of POAPAG and AmgC/M-PAG neurons.
POAPAG neurons are labeled with GFP, and AmgC/M-PAG neurons are labeled with tdTomato. (Top left) Plane of section through the preoptic area (POA) shows the beginning of POAPAG cell body labeling in …
Figure 3
Acoustic characterization of ultrasonic vocalizations (USVs) elicited by optogenetic activation of preoptic area (POA) neurons.
(A) The variational autoencoder (VAE) takes spectrograms as input (left), maps the spectrograms to low-dimensional latent representations using an encoder (middle), and approximately reconstructs …
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Figure 3—source data 1
Source data for Figure 3D.
- https://cdn.elifesciences.org/articles/63493/elife-63493-fig3-data1-v3.xlsx
Figure 4 with 2 supplements
Activating AmgC/M-PAG neurons transiently suppresses ultrasonic vocalization (USV) production.
(A) (Left) Viral strategy used to express ChR2 in AmgC/M-PAG neurons. (Right) Confocal image of representative AmgC/M-PAG cell body labeling achieved with this viral strategy. (B) (Left) Spectrogram …
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Figure 4—source data 1
Source data for Figure 4C and D.
- https://cdn.elifesciences.org/articles/63493/elife-63493-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
Extent of cell body labeling of AmgC/M-PAG neurons.
AmgC/M-PAG neurons were labeled by injecting AAV-retro-Cre into the caudolateral periaqueductal gray (PAG) and AAV-FLEX-GFP into the AmgC/M. (A–C) Three planes of coronal section are shown that …
Figure 4—figure supplement 2
Comparison of hypothalamus and amygdala cell body labeling achieved after transsynaptic tracing from the periaqueductal gray (PAG) vocal gating circuit versus.
AAV-retro-Cre injection into the caudolateral PAG. Representative coronal sections are shown for POA and amygdala cell body labeling observed after monosynaptic rabies-based tracing from GABAergic …
Figure 5
AmgC/M neurons provide direct inhibition onto PAG-USV neurons.
(A) Viral strategy (left) and schematic (right) for whole-cell patch clamp recordings from fluorescently identified CANE-tagged PAG-USV neurons while optogenetically activating AmgC/M-PAG axons. (B) …
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Figure 5—source data 1
Source data for Figure 5D and F.
- https://cdn.elifesciences.org/articles/63493/elife-63493-fig5-data1-v3.xlsx
Figure 6 with 1 supplement
POA neurons provide direct inhibition onto VGAT+ PAG neurons, which provide direct inhibition onto PAG-USV neurons.
(A) Viral strategy (left) and schematic (right) for whole-cell patch clamp recordings from fluorescently identified VGAT+ PAG cells while optogenetically activating POAPAG axons. (B) Example image …
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Figure 6—source data 1
Source data for Figure 6D and H.
- https://cdn.elifesciences.org/articles/63493/elife-63493-fig6-data1-v3.xlsx
Figure 6—figure supplement 1
POA neurons provide direct inhibition onto few PAG-USV neurons.
(A) Viral strategy (left) and schematic (right) for whole-cell patch clamp recordings from fluorescently identified PAG-USV cells while optogenetically activating POAPAG axons. (B) Light-evoked …
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Figure 6—figure supplement 1—source data 1
Source data for panel B of Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/63493/elife-63493-fig6-figsupp1-data1-v3.xlsx
Figure 7
Model of bidirectional descending control of the periaqueductal gray (PAG) vocal gating circuit.
Inhibitory neurons within the POA provide direct input to inhibitory neurons within the PAG, which in turn provide direct input to PAG-USV neurons. In this manner, activation of POAPAG neurons …
Videos
Video 1
Optogenetic activation of POAPAG neurons elicits ultrasonic vocalizations (USVs).
An isolated male mouse is shown which has ChR2 is expressed in POAPAG neurons. Optogenetic activation of these neurons with pulses of blue light elicits USV production. Video is shown at the top, a …
Video 2
Optogenetic activation of AmgC/M-PAG neurons causes no obvious behavioral effects in the absence of a social partner.
An example male mouse with ChR2 expression in AmgC/M-PAG neurons is shown alone in a chamber with no social partner. Optogenetic activation of AmgC/M-PAG neurons with pulses of blue light does not …
Video 3
Optogenetic activation of AmgC/M-PAG neurons causes no obvious behavioral effects in the absence of a social partner.
An example male mouse with ChR2 expression in AmgC/M-PAG neurons is shown alone in a chamber with no social partner. Optogenetic activation of AmgC/M-PAG neurons with pulses of blue light does not …
Video 4
Optogenetic activation of AmgC/M-PAG neurons causes no obvious behavioral effects in the absence of a social partner.
An example male mouse with ChR2 expression in AmgC/M-PAG neurons is shown alone in a chamber with no social partner. Optogenetic activation of AmgC/M-PAG neurons with pulses of blue light does not …
Video 5
Optogenetic activation of AmgC/M-PAG neurons transiently suppresses ultrasonic vocalization (USV) production.
A male mouse which has ChR2 expressed in AmgC/M-PAG neurons is shown interacting with and producing USVs directed at a female social partner. Optogenetic activation of these neurons with pulses of …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus, C57BL/6J) | C57 | Jackson Labs | RRID:IMSR_JAX:000664 | |
| Strain, strain background (Mus musculus, B6N.129S6(Cg)-Esr1tm1.1(cre)And/J) | Esr1-Cre | Jackson Labs | RRID:IMSR_JAX:017911 | |
| Strain, strain background (Mus musculus, B6J.129S6(FVB)-Slc32a1tm2(cre)Lowl/ MwarJ) | VGAT-Cre | Jackson Labs | RRID:IMSR_JAX:016962 | |
| Strain, strain background (Mus musculus, B6;129S6-Gt(ROSA)26Sortim14(Cag-tdTomato)Hze/J) | Ai14 | Jackson Labs | RRID:IMSR_JAX:007908 | |
| Strain, strain background (Mus musculus, B6;129-Fostm1.1Fawa/J) | Fos-dsTVA | Jackson Labs | RRID:IMSR_JAX:027831 | |
| Recombinant DNA reagent | AAV2/1-hSyn-Flex-Chr2-eYFP | Addgene (K. Deisseroth) | RRID:Addgene_26973 | |
| Recombinant DNA reagent | AAV-pgk-retro-Cre | Addgene (P. Aebischer) | RRID:Addgene_24593 | |
| Recombinant DNA reagent | AAV2/1-pCAG-flex-GFP | Addgene (H. Zeng) | RRID:Addgene_51502 | |
| Recombinant DNA reagent | AAV2/1-pCAG-flex-Tdtomato | Addgene (H. Zeng) | RRID:Addgene_51503 | |
| Recombinant DNA reagent | AAV-flex-oG | Duke Viral Vector Core | ||
| Recombinant DNA reagent | EnvA-G-RV-GFP | Rodriguez et al., 2017 (DOI: 10.1038/s41593-017-0012-1), Sakurai et al., 2016 (DOI: 10.1016/j.neuron.2016.10.015) | ||
| Recombinant DNA reagent | CANE-RV-mCherry | Rodriguez et al., 2017 (DOI: 10.1038/s41593-017-0012-1), Sakurai et al., 2016 (DOI: 10.1016/j.neuron.2016.10.015) | ||
| Recombinant DNA reagent | AAV-flex-TVA-mCherry | Rodriguez et al., 2017 (DOI: 10.1038/s41593-017-0012-1), Sakurai et al., 2016 (DOI: 10.1016/j.neuron.2016.10.015) | ||
| Commercial assay or kit | HCR v3.0 | Molecular Instruments | ||
| Chemical compound, drug | Gabazine | Tocris | Cat# 1262 | (10 µM) |
| Chemical compound, drug | TTX | Tocris | Cat# 1069 | (2 µM) |
| Chemical compound, drug | 4AP | Sigma-Aldrich | Cat# 275875 | (100 µM) |
| Software, algorithm | MATLAB | Mathworks | RRID:SCR_001622 | |
| Software, algorithm | ImageJ | NIH | RRID:SCR_003070 | |
| Software, algorithm | ZEN | Zeiss | RRID:SCR_013672 | |
| Software, algorithm | Spike7 | CED | RRID:SCR_000903 | |
| Software, algorithm | pClamp | Molecular Devices | RRID:SCR_011323 | |
| Software, algorithm | IGOR Pro | WaveMetrics | RRID:SCR_000325 | |
| Other | NeuroTrace 435/455 | Invitrogen/Thermo Fischer Scientific | Cat# N21479 | (1:500) |
