Opioid suppression of an excitatory pontomedullary respiratory circuit by convergent mechanisms
- Department of Pharmacology and Therapeutics, Breathing Research and Therapeutics Center, University of Florida, United States
Figures
Figure 1
Dorsolateral pontine neurons express mu opioid receptors (MORs).
(A–D) Representative images of tdTomato expression, as an indicator of MOR expression, in coronal dorsolateral pontine slices from Oprm1-tdT mice (n = 3) across the rostral to caudal …
Figure 2 with 2 supplements
Oprm1+ Kölliker-Fuse (KF) neurons and neurites retrogradely labeled from the preBötzinger complex (preBötC) and rostral ventral respiratory group (rVRG).
(A) Schematic illustrating the approach to retrogradely label Oprm1+ KF neurons and neurites projecting to the preBötC or rVRG. (B) Images of coronal slices from the medulla with a control injection …
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Figure 2—source data 1
Quantification of spread at the injection sites.
- https://cdn.elifesciences.org/articles/81119/elife-81119-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Oprm1+ Kölliker-Fuse (KF) neurons project to the Bötzinger complex (BötC).
(A) Schematic illustrating the approach to retrogradely label Oprm1+ KF neurons projecting to the BötC. (B–, C) Images of coronal slices from the medulla with a control injection of …
Figure 2—figure supplement 2
Higher magnification images of retrogradely labeled Kölliker-Fuse (KF) neurons.
Images of GFP expression, as an indicator of retrograde-labeled Oprm1-expressing neurons, following injections into preBötzinger complex (preBötC) (A), rostral ventral respiratory group (rVRG) (B), …
Figure 3 with 1 supplement
Presynaptic opioid receptors inhibit glutamate release from Kölliker-Fuse (KF) terminals onto excitatory preBötzinger complex (preBötC) and rostral ventral respiratory group (rVRG) neurons.
(A) Schematic of approach to optogenetically stimulate KF terminals and drive optogenetically evoke excitatory postsynaptic currents (oEPSCs) in excitatory preBötC and rVRG neurons in an acute brain …
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Figure 3—source data 1
Presynaptic opioid receptors inhibit glutamate release from Kölliker-Fuse (KF) terminals onto excitatory preBötzinger complex (preBötC) and rostral ventral respiratory group (rVRG) neurons.
- https://cdn.elifesciences.org/articles/81119/elife-81119-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Kölliker-Fuse (KF) neurons send monosynaptic, glutamatergic projections to excitatory ventrolateral medullary neurons.
(A) Recording of optogenetically evoke excitatory postsynaptic currents (oEPSCs) from an excitatory (vglut2+) preBötzinger complex (preBötC) neuron in an acute brain slice at baseline (black), …
Figure 4 with 1 supplement
Opioids hyperpolarize Kölliker-Fuse (KF) neurons that project to the preBötzinger complex (preBötC) and rostral ventral respiratory group (rVRG).
(A) Schematic (left) of approach to retrogradely label KF neurons that project to the preBötC or rVRG with FluoSpheres in wild-type mice. Images (right) of FluoSpheres in the injection area (preBötC …
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Figure 4—source data 1
Opioid-mediated outward currents in Kölliker-Fuse (KF) neurons that project to the preBötzinger complex (preBötC) and rostral ventral respiratory group (rVRG).
- https://cdn.elifesciences.org/articles/81119/elife-81119-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Opioids hyperpolarize Kölliker-Fuse (KF) neurons that project to the Bötzinger complex (BötC).
(A) Schematic of approach to retrogradely label KF neurons that project to the BötC with FluoSpheres in wild-type mice. (B) Image of FluoSpheres injection into the BötC. (C) Quantification of …
Figure 5 with 1 supplement
Oprm1+ and Oprm1- dorsolateral pontine neurons project to the ventrolateral medulla.
(A) Schematic of approach injecting retrograde virus encoding Cre-dependent GFP expression and a retrograde virus encoding mCherry expression into the ventrolateral medulla of Oprm1Cre/+ mice to …
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Figure 5—source data 1
Oprm1+ and Oprm1- dorsolateral pontine neurons project to the ventrolateral medulla.
- https://cdn.elifesciences.org/articles/81119/elife-81119-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Medullary-projecting Oprm1+ neurons are mostly absent from the caudal Kölliker-Fuse (KF) and lateral parabrachial areas.
Retrograde-labeled neurons (both Oprm1+ and Oprm1-) were mostly lacking in caudal KF or lateral parabrachial area. The bregma coordinate and approximate location in KF/lateral parabrachial area …
Figure 6 with 1 supplement
Oprm1+, medullary-projecting Kölliker-Fuse (KF) neurons express Forkhead box protein P2 (FoxP2).
Oprm1+ neurons that project to the ventrolateral medulla were retrogradely labeled by injection of retrograde AAV-DIO-GFP into Oprm1Cre/+ mice. Immunohistochemistry was used to label FoxP2. (A, B) …
Figure 6—figure supplement 1
Forkhead box protein P2 (FoxP2) expression in caudal Kölliker-Fuse (KF), but not external lateral parabrachial subnucleus.
Oprm1+ neurons that project to the ventrolateral medulla were retrogradely labeled by injection of retrograde AAV-DIO-GFP into Oprm1Cre/+ mice. Immunohistochemistry was used to label FoxP2. (A) …
Figure 7
Oprm1+, medullary-projecting Kölliker-Fuse (KF) neurons do not express calcitonin gene-related peptide (CGRP).
Oprm1+ neurons that project to the ventrolateral medulla were retrogradely labeled by injection of retrograde AAV-DIO-GFP into Oprm1Cre/+ mice. Immunohistochemistry was used to label CGRP. (A, B) …
Figure 8
Summary schematic of mu opioid receptor (MOR) regulation of excitatory pontomedullary circuitry.
Kölliker-Fuse (KF): Somatodendritic MORs hyperpolarize KF neurons that project to the ventrolateral medulla. Ventrolateral medulla: presynaptic MORs inhibit glutamate release from KF axon terminals …
Author response image 1
Example intensity profile plots of the GFP, FoxP2 and DAPI signal across the neuron, indicated by the yellow line on the image.
Author response image 2
FoxP2 immunolabeling (magenta) in the KF area with (top row) and without (bottom row) the primary anti-FoxP2 antibody.
Neurotrace (green) is a fluorescent Nissl stain and labels neurons.
Author response image 3
FoxP2 immunolabeling in the rostral dorsolateral pons (~bregma -4.84).
See also, ISH for Foxp2 from the Allen Brain Atlas.
Tables
Table 1
Mice used in this study.
| Strain | Reference | Source information | Key gene |
|---|---|---|---|
| Oprm1-cre | Liu et al., 2021. | Jax 035574 https://www.jax.org/strain/035574 Dr. Richard Palmiter (University of Washington) | Cre recombinase expressed in neurons with mu-opioid receptors |
| Vglut2-cre | Vong et al., 2011 | Jax 028863 https://www.jax.org/strain/028863 | Cre recombinase expressed in excitatory glutamatergic neurons |
| Ai9, tdTomato Cre-reporter | Madisen et al., 2010 | Jax 007909 https://www.jax.org/strain/007909 | LoxP-flanked STOP cassette preceding transcription of CAG promoter-driven red fluorescent protein variant (tdTomato) inserted into the Gt(ROSA)26Sor locus |
| C57BL/6J (wild-type) | Simon et al., 2013 | Jax 000664 https://www.jax.org/strain/000664 |
Table 2
Key resources.
| Injectate | Strain used | Injection target | Figure | Source Information |
|---|---|---|---|---|
| FluoSpheres 580/605, diameter: 0.04 µm | C57BL/6J | BötC, preBötC, or rVRG | Figure 4 and Figure 4—figure supplement 1 | Invitrogen |
| Retrograde AAV-hSyn-DIO-EGFP | Oprm1Cre/+ | BötC, preBötC, or rVRG | Figure 2 and Figure 2—figure supplement 1 | Addgene |
| AAV2-hSyn-mCherry | Oprm1Cre/+ | BötC, preBötC, or rVRG | Figure 2 and Figure 2—figure supplement 1 | UNC Vector Core |
| Retrograde AAV-hSyn-mCherry | Oprm1Cre/+ | BötC, preBötC, and rVRG | Figure 5 | Addgene |
| AAV2-hSyn-DIO-EGFP | Oprm1Cre/+ | KF/PB | Figure 1E–H | Addgene |
| AAV2-hSyn-hChR2(H134R)-EYFP-WPRE-PA | vglut2-tdT | KF/PB | Figure 3 | UNC Vector Core |
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PreBötC, preBötzinger complex; rVRG, rostral ventral respiratory group; KF, Kölliker-Fuse; PB, parabrachial area.
Table 3
Antibodies used in this study.
| Antigen | Immunogen description | Source, host species, RRID | Concentration |
|---|---|---|---|
| Forkhead box P2 (FoxP2) | Targets human and mouse FoxP2 | R&D Systems, sheep polyclonal, Cat# AF5647, RRID:AB_2107133 | 1:1000 |
| Calcitonin gene-related peptide (CGRP) | Targets alpha-CGRP in canine, mouse, and rat | Peninsula, rabbit polyclonal, Cat# T-4032, RRID:AB_518147 | 1:1000 |
| Neurokinin 1 receptor (NK1R) | Targets C-terminal of NK1R in mouse, guinea pig, and human | Sigma-Aldrich, rabbit polyclonal, Cat# S8305 RRID:AB_261562 | 1:1000 |
