Chemo- and optogenetic activation of hypothalamic Foxb1-expressing neurons and their terminal endings in the rostral-dorsolateral PAG leads to tachypnea, bradycardia, and immobility
- Anatomy and program in Neuroscience, Faculty of Science and Medicine, University of Fribourg, Switzerland
- Zurich integrative Rodent Physiology (ZIRP), University of Zürich, Switzerland
- Institute of Anatomy and Cell Biology, University of Heidelberg, Germany
Figures
Figure 1
Left part: Schematic drawing of a horizontal section of the hypothalamus in which the Foxb1-neurons of the parvafox and of the PMd are located (modified after Alvarez-Bolado et al., 2000).
Their chemogenetic (DREADD) stimulation leads to an increase in breaths / minutes. Right part: dorsolateral PAG (dlPAG), seen in a cross-section of the midbrain. Axon terminals of parvafoxFoxb1 and PMdFoxb1 converge in the rostral part of the dlPAG. Optogenetic activation of these terminals lead to immobility and bradycardia.
Figure 2 with 1 supplement
Chemogenetic activation of lateral hypothalamic Foxb1-expressing neurons alters multiple respiratory parameters.
(a) Data across all DREADD WBP recordings was plotted as averaged line plots for each animal group (DREADD_neg, hM3Dq and hM4Di) and condition (Saline (red), Clozapine (green) or CNO (blue) i.p.injection). Each animal was recorded three times within each condition (nine recordings in total). Respiratory parameters do not differ between condition in DREADD_neg animals. Injection of CNO or Clozapine in hM3Dq mice significantly alters several respiratory parameters. The effects of Clozapine and CNO injections on respiratory parameters largely overlap and clearly separate from the effects saline injections. In hM4Di animals, more variability within respiratory parameters compared to DREADD_neg controls, however, no statistically significant effect is detected. Data shows the condition mean and standard error of the mean (s.e.m.) (b) Violin plots with integrated boxplots for each group and condition. The width of the violin plot represents the data distribution density. The boxplot ‘s lower and upper limits represent the 25% quantile and 75% quantile, respectively. The horizontal bar inside the boxplot represents the median. The whiskers of the boxplot display 1.5 x the interquartile range. (c) Comparison of gross locomotion as assessed by an open field test. hM3Dq animals show statistically significant reduction in distance moved, while there are no differences observed in the DREADD_neg and hM4Di animals. It is important to note, that the effect size for this reduction in distance moved is small. (d) A statistically significant reduction in time spent in immobility is observed in DREADD_neg animals, however, the effect size is negligible. Time spent in an immobile state does not differ in hM3Dq and hM4Di animals. Track visualization with underlying density maps and zone visit diagrams during open field tests for all DREADD mice can be found in Supplementary files 1 and 2. I = BPM (Breaths per minute); II = IT (Inspiratory time); III = ET (Expiratory time); IV = TT (Total respiratory cycle time); V = TVadjPerGram (Tidal volume normalized to bodyweight in microliters per gram); VI = MVadjPerGram (Minute volume normalized to bodyweight in milliliters per gram); VII = PIFadj (Peak inspiratory flow); VIII = PEFadj (Peak expiratory flow); CNO: clozapine-N-Oxide. Number of mice per condition: DREADD_neg n=4, hM3Dq n=5, hM4Di n=5.
Figure 2—figure supplement 1
Representative examples of DREADD expression and cFos staining in four mice of each condition (DREADD_neg, hM3Dq, and hM4Di) used for chemogenetic experiments.
Figure 3
Optogenetic activation of hypothalamic Foxb1+ terminals in the rostral dlPAG induces freezing-like behavior.
(a) A representative example of track visualizations with underlying density maps for a ChR2-expressing Foxb1-Cre mouse belonging to the ‘OnTarget_antPAG’ group during 3 min of baseline (left) and 3 min of optogenetic stimulation (right). Note that the mouse remained immobile for the entire duration of the stimulation period. The color coding scales are not fixed between the two conditions. Open field arena dimensions are 40x40cm. Track visualization with underlying density maps for all other mice can be found in Supplementary file 3. (b) A representative example of a zone visit diagram taken from the same recording as the track visualizations in (a). Note, that the zone transitions stop completely after the onset of optogenetic stimulation. Zone visit diagrams for all other mice can be found in Supplementary file 4. (c) A visual representation of the arena partitioning into the different zones (i.e. arena, periphery, center, and four corners). (d-h) Comparison of multiple parameters extracted from the open field experiments. Optogenetic activation of parvafoxFoxb1 terminals in the PAG reduces distance moved (d), speed.moving (e), and time in center (h), while time spent immobile (f) and time in periphery (g) increases. Optogenetic silencing of parvafoxFoxb1 terminals in the PAG induces opposing effects.
Figure 4 with 1 supplement
Single-cell RNA sequencing reveals differential expression profiles of Foxb1 and Cck in the PMd.
(a) Single-cell transcriptomic analysis of murine ventral-posterior hypothalamic PMd cells into a distinct cluster (Cluster 9). (b) Cluster identity of the PMd cluster is confirmed by expression pattern analysis of genes known to be upregulated in the PMd (Cck, Foxb1, Synpr, Ebf3, Dlk1, and Stxpb6). (c) In situ hybridization photomicrographs from the Allen Brain Atlas show the localization of Cck and Foxb1 transcripts in the PMd. (d) A magnified UMAP plot representation of the PMd cluster highlights the differential expression profiles of Cck and Foxb1 within the PMd cluster. While cells expressing high transcript levels of Cck (middle column, red) preferentially localize to the left side of the PMd cluster, cells with high levels of Foxb1 transcripts (middle column, green) preferentially localize to the opposite (i.e. right) side of the PMd cluster. Analysis of co-expression of Cck and Foxb1 transcripts identifies only few cells as strongly double positive (yellow; see color coding representation), while most cells with high expression levels for one of the two genes have very low to non-existing expression levels of the other gene.
Figure 4—figure supplement 1
Collection of in-situ hybridization data from the Allen Institute Mouse Brain Atlas for genes used to identify the PMd cell cluster in the scRNA-seq dataset.
Figure 5
Results from hot place experiments performed on Foxb1-Cre animals does not reveal statistically significant differences in (a) latency until endpoint behavior (i.e. hindpaw lick(-attempt) or jumping) nor in (b) number of shakes before endpoint behavior in any of the three tested groups.
These results contradict the hypothesis of a reciprocal effect of the parvafoxPvalb and parvafoxFoxb1 on pain sensation.
Figure 6
Optogenetic activation of Foxb1+ terminals in the rostral dlPAG alters heart rate.
(a) Telemetric recording of heart rate of mouse 34-21/10 in an open field. Average every 10 s, 40 min long recording. Heart rate increases from 700 to 740 during the first few minutes and remain at this level until the start of the optogenetic activation of the Foxb1 endings in the dlPAG. During the 2 min of flashing blue light, heart rate drops to under 640 but has an initial, short recovery to 690. During the following 10 min of baseline recording, the cardiovascular system is slightly dysregulated, with an increased variability in heart rate. During the second photo-stimulation, the drop in heartbeats has an amplitude comparable to the one during the first stimulation. (b) Telemetric recording of heart rate (blue curve) and movements (red curve) of mouse 106-21/10 in an open field. Average every 10 s, 20 min long recording. Heart rate values are around 740 during the first minutes (baseline condition), while the mouse is continuously moving around in the open field. Shortly after the beginning of the optogenetic activation of the Foxb1 endings in the dlPAG, the heartbeat drops massively (by ~40 %) from 740 to 450 bpm and remains around this value during the 2 min of blue light flashing (photo-stimulation). Notice the complete immobility of the mouse during this period. At the end of the optogenetic activation, heart rate rapidly returns to the baseline level of approximately 740 bpm.
Figure 7
Localization of the glass fibers during optogenetic activation of axon terminals in the PAG leading to immobility (a), and bradycardia (b), respectively.
The effects were achieved by inserting the fibers in the rostral part of the dlPAG (Bregma −3.28, –4.04) Glass fibers colored in yellow, with their tip located over the dlPAG (a), provoked immobility. Glass fibers colored in black had their tip below the dlPAG and did not evoke changes in mobility. Glass fibers indicated by a green arrow, with their tips over the dlPAG, provoked bradycardia. Glass fibers indicated by black arrows, with their tip below dlPAG or distal to bregma –4.04., did not affect the cardiovascular system (b). (c) Cross-sections of the rostral PAG with eYFP-labelled Foxb1-terminals located in the dlPAG (mouse 106-21/10). The position of the obliquely inserted glass cannula is indicated with a large white arrow and the flat tip of the cannula is positioned over the dlPAG on the right at bregma –3.40 (left image), and on the left at bregma –3.64 (right image). Aq: Aqueductus cerebri; Dk: Darkschewitsch Nucleus; dlPAG: dorsolateral periaqueductal gray; EW: Edigener-Westphal nucleus; lPAG: lateral PAG; Ma3: medial accessory oculomotor nucleus; PrEW: pre-Edinger-Westphal nucleus.
Author response image 1
Videos
Video 1
A representative movie for a ChR2-stimulated mouse that was still capable of moving its head as a sign of attentive behavior towards its surroundings, while all four limbs remained largely immobile.
The video shows the time window from 1 min before the onset of light stimulation, 5 min of light stimulation, and 1 min following light stimulation offset. The time window of light stimulation is marked by the ‘Light on’ indicator on the top right. The video is played at 5 x of the recording speed.
Video 2
A representative movie for a ChR2-stimulated mouse that was completely immobilized during optogenetic activation of the parvafoxFoxb1 terminals in the dlPAG.
The video shows the time window from 1 min before the onset of light stimulation, 5 min of light stimulation, and 1 min following light stimulation offset. The time window of light stimulation is marked by the ‘Light on’ indicator on the top right. The video is played at 5 x of the recording speed.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mus musculus) | Foxb1 | NCBI Gene | Gene ID: 64290 | |
| Genetic reagent (M. musculus) | Foxb1tm1(cre)Gabo | MGI | MGI ID: 3772366 | |
| Genetic reagent (M. musculus) | 129P2-Pvalbtm1(cre)Arbr/J | MGI | MGI ID: 3773708 | |
| Genetic reagent (adeno-associated virus) | AAV5-EF1α-DIO-hChR2(H134R)-eYFP | Addgene | Catalog #: 20298-AAV5 | |
| Genetic reagent (adeno-associated virus) | AAV2-EF1α-DIO-eArchT3.0-eYFP | University of North Carolina, Vector Core | Kind gift from Adamantidis Lab | |
| Genetic reagent (adeno-associated virus) | AAV2-hSyn-DIO-hM3D(Gq)-mCherry | Addgene | Catalog #: 44361-AAV2 | |
| Genetic reagent (adeno-associated virus) | AAV2-hSyn-DIO-hM4D(Gi)-mCherry | Addgene | Catalog #: 44362-AAV2 | |
| Antibody | anti-mCherry (Rabbit polyclonal) | Abcam plc. | product code: ab167453 | IF(1:1000) |
| Antibody | anti-c-Fos (Mouse monoclonal) | Abcam plc. | product code: ab208942 | IF(1:2000) |
| Antibody | anti-GFP (Chicken polyclonal) | Aves Labs, Inc. | Product code: GFP-1020 | IF(1:200) |
| Antibody | anti-mouse IgG (Horse polyclonal, biotinylated) | Vector Laboratories | Product code: BA-2000 | IF(1:200) |
| Antibody | anti-chicken IgG (Donkey polyclonal, Cy2-conjugated) | Jackson ImmunoResearch | Product code: 703-225-155 | IF(1:200) |
| Antibody | anti-Rabbit IgG (Donkey polyclonal, Cy3-conjugated) | Jackson ImmunoResearch | Product code: 711-165-152 | IF(1:200) |
| Peptide, recombinant protein | Streptavidin (Alexa Fluor 647-conjugated) | Jackson ImmunoResearch | Product code: 016-600-084 | IF(1:200) |
| Sequence-based reagent | EGFP-f | This paper | PCR primer | CTC GGC ATG GAC GAG CTG TAC AAG |
| Sequence-based reagent | GAB20 | This paper | PCR primer | CAC TGG GAT GGC GGG CAA CGT CTG |
| Sequence-based reagent | GAB22 | This paper | PCR primer | CAT CGC TAG GGA GTA CAA GAT GCC |
| Chemical compound, drug | DAPI | Life Technologies Corporation | Product code: D1306 | IF(1:5000) |
| Chemical compound, drug | Clozapine | Sigma-Aldrich | Product code: C6305 | |
| Chemical compound, drug | CNO | Sigma-Aldrich | Product code: SML2304 | |
| Software, algorithm | Ponemah Software | DSI | PNM-P3P-CFG | |
| Other | Whole-body barometric plethysmography chambers | DSI | Product code: 601-0001-011 | Used in respiration-related experiments |
| Other | Buxco Bias flow pump | DSI | Product code: 601-2201-001 | Used in respiration-related experiments |
| Other | Buxco differential pressure transducer | DSI | Product code: 600-1114-002 | Used in respiration-related experiments |
| Other | Temperature and humidity probe | DSI | Product code: 600-2249-001 | Used in respiration-related experiments |
| Other | ACQ-7700 USB amplifier | DSI | Product code: PNM-P3P-7002SX | Used in respiration-related experiments |
| Other | Dual LED light source | Prizmatix Ltd. | Product code: 34117 | Used for optogenetic experiments. blue (peak λ=453 nm) and lime green (peak λ=536 nm) |
| Other | 1.5 mm optical fiber | Prizmatix Ltd. | Product code: 34131 | Used for optogenetic experiments. Connecting LED light source to rotary joint |
| Other | Rotary joint | Prizmatix Ltd. | Product code: 43043 | Used for optogenetic experiments. |
| Other | Dual fiber patch cord | Prizmatix Ltd. | Product code: 34115 | Used for optogenetic experiments. 2x500 μm diameter |
| Other | Ceramic cannulas | Prizmatix Ltd. | Product code: 43071 | Used for optogenetic experiments. 1.25 mm outer diameter; 230 μm inner diameter |
| Other | 0.66 NA optical fibers | Prizmatix Ltd. | Used for optogenetic experiments. 200 μm outer diameter. | |
| Other | 473 nm DPSS laser | Laserglow technologies | Product code: LRS-0473-PFO-00500–01 | Used for optogenetic experiments. |
Additional files
-
Supplementary file 1
Track visualizations with underlying density maps of all tested DREADD animals.
- https://cdn.elifesciences.org/articles/86737/elife-86737-supp1-v1.pdf
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Supplementary file 2
Zone visit diagrams of all tested DREADD animals.
- https://cdn.elifesciences.org/articles/86737/elife-86737-supp2-v1.pdf
-
Supplementary file 3
Track visualizations with underlying density maps of all tested optogenetic animals.
- https://cdn.elifesciences.org/articles/86737/elife-86737-supp3-v1.pdf
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Supplementary file 4
Zone visit diagrams of all tested optogenetic animals.
- https://cdn.elifesciences.org/articles/86737/elife-86737-supp4-v1.pdf
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Supplementary file 5
Heart rate variability (HRV) during the optogenetic activation of the Foxb1-terminal endings in the dlPAG of mouse 106-21/10.
Comparison between the one-minute period before (green rectangle) and after (red rectangle) the start of the optogenetic activation. While all 4 parameters changed significantly during optogenetic stimulation (heart rate, systolic BP, diastolic BP, mean BP), the SD of heart cycle duration changed by a factor of more than five, from 3.8ms during the baseline period to 20.6ms during optogenetic activation.
- https://cdn.elifesciences.org/articles/86737/elife-86737-supp5-v1.tif
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Supplementary file 6
Antibodies and fluorescent labels used for histological processing of brain sections.
- https://cdn.elifesciences.org/articles/86737/elife-86737-supp6-v1.pdf
-
MDAR checklist
- https://cdn.elifesciences.org/articles/86737/elife-86737-mdarchecklist1-v1.pdf
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Chemo- and optogenetic activation of hypothalamic Foxb1-expressing neurons and their terminal endings in the rostral-dorsolateral PAG leads to tachypnea, bradycardia, and immobility
eLife 12:RP86737.
https://doi.org/10.7554/eLife.86737.3
