nNOS-expressing interneurons control basal and behaviorally evoked arterial dilation in somatosensory cortex of mice
- Bioengineering Graduate Program, Pennsylvania State University, United States
- Molecular, Cellular, and Integrative Biology Graduate Program, Pennsylvania State University, United States
- Cell and Developmental Biology Graduate Program, Pennsylvania State University, United States
- Departments of Engineering Science and Mechanics, Biomedical Engineering, and Neurosurgery, Pennsylvania State University, United States
Figures
Figure 1 with 5 supplements
Local neural activity controls basal and evoked arteriole diameter.
(A) Top, schematic of imaging window. Bottom, photo of mouse on spherical treadmill. (B) Top, photo of the pial vasculature of the somatosensory cortex through the PoRTs window. Cytochrome oxidase …
Figure 1—figure supplement 1
Example neural responses in forelimb/hindlimb representation of somatosensory cortex during voluntary locomotion.
(A) Schematic showing experimental setup of electrophysiological measurements taken with multi-electrode array. (B) Average locomotion-evoked multi-unit spiking from a single site in the FL/HL …
Figure 1—figure supplement 2
Diameter changes in penetrating arterioles.
All data is taken from penetrating arterioles imaged ~100 µm below the pial surface. Statistical comparisons of locomotion triggered averages are of the control and manipulation are done with both …
Figure 1—figure supplement 3
Muscimol infusion did not significantly affect capillary diameter.
(A) Representative images of the same capillary after vehicle infusion (left) and after muscimol infusion (right). Scale bar 5 μm. (B) Capillary diameter measured after vehicle infusion (x-axis) vs. …
Figure 1—figure supplement 4
Muscimol infusion did not significantly affect basal arteriole diameter variance.
(A) Scatter plot of basal arteriole diameter variance during stationary periods following vehicle infusion (x-axis) versus muscimol infusion (y-axis). There was no significant change in arterial …
Figure 1—figure supplement 5
Effects of chemogenetic and pharmacological manipulation on locomotion behavior.
Plot showing the amount of time locomoting during 2PLSM imaging for each manipulation normalized by the amount of time locomoting with the vehicle. No manipulation resulted in a significant change …
Figure 2 with 2 supplements
Neural activity bidirectionally controls basal arteriole diameter.
(A) Left, image through the polished and reinforced thinned-skull window showing AAV expression (red) in the somatosensory cortex (scale bar 1 mm). Right, vasculature within box of A. (B) …
Figure 2—figure supplement 1
No significant effect of CNO on basal arteriole diameters or neural activity.
(A) Representative cortical sections showing mCherry expressing neurons (red) and cell nuclei (DAPI, blue). Scale bar is 100 μm. Data in B-D are from mice injected with AAV-CMV-TurboRFP-WPRE-rBG …
Figure 2—figure supplement 2
hM4D-G(i) DREADDs cause bursts on neural activity.
(A) Representative trial after vehicle (top) or CNO (bottom) injection in hSyn-G(i) DREADDs mice. The gamma band power of the local field potential (LFP) showed that the CNO induced bursting …
Figure 3
Activity, but not of pyramidal neurons, bidirectionally controls basal arteriole diameter.
(A) Representative image of AAV-CaMKIIa-hM3D(Gq)-mCherry infected cortex, where DREADDs are expressed under a CaMKIIa promoter. Top image is wide field image of CaMKIIa-mCherry DREADDs virus (red) …
Figure 4 with 1 supplement
nNOS expressing neurons controls arteriole diameter independent of overall neural activity.
(A) Representative image of cortex taken of AAV-hSyn-DIO-hM3D(Gq)-mCherry in nNOS-cre mice, where DREADDs expressed in nNOS+ cells. The mCherry label is magenta, nNOS antibody is green. Co-labeling …
Figure 4—figure supplement 1
NO produced by nNOS expressing neurons controls arteriole diameter independent of overall neural activity.
Data in A-C are from mice after local L-NAME infusions through a chronically implanted cannula. (A) LFP power spectra during stationary (basal) and locomotion periods after L-NAME infusion, …
Figure 5 with 2 supplements
Type I nNOS expressing neurons control basal arteriole diameter.
Data in A-C are from mice after local CP-99994 infusions, which will block the excitatory Substance P receptors on type1 nNOS neurons, data in D-E is from mice after Substance P infusions, which …
Figure 5—figure supplement 1
No significant effect of chemogenetic manipulation of astrocytes on basal vessel diameter or neural activity.
(A) Representative cortical sections showing mCherry expressing cells (red), GFAP staining (green), and cell nuclei (DAPI, blue). Scale bar is 100 μm. AAV-hSyn-DIO-hM3D(Gq)-mCherry in Aldh1L1-cre …
Figure 5—figure supplement 2
Kir-channel blockers increase in neural activity and basal arterial diameter.
(A) Representative trial after vehicle (left) or BaCl2 (right) infusion (Kir-channel blocker). BaCl2 infusion-induced epileptic-like activity. Locomotion events are denoted with shading. (B) LFP …
Figure 6 with 2 supplements
Comparisons of the effects of various manipulations on the basal diameters of pial and penetrating arterioles.
(A) Comparison of change in resting vessel diameter in penetrating and pial arteries after Muscimol infusion. There was a significant difference in the change of baseline diameter between pial and …
Figure 6—figure supplement 1
Schematic showing the calculation of the locomotion-evoked component of the diameter change.
In this schematic, the locomotion-evoked diameter change from the pre-locomotion is shown. Left, the manipulation causes an 10% baseline constriction relative to the control. During locomotion, in …
Figure 6—figure supplement 2
Locomotion triggered average diameter changes relative to locomotion diameter.
Baseline diameters for both vehicle and treatment conditions were calculated as the average diameter over the 2 s prior to locomotion onset for pial and penetrating arterioles. The steady-state …
Figure 7 with 3 supplements
Locomotion-evoked Ca2+ signals in neurons.
(A–C) Left, a representative image of cortex taken of AAV-hSyn-GCaMP6s in C57BL/6J mice (A), AAV-CaMKII-GCaMP6s in C57BL/6J mice (B) and AAV-Syn-FLEX-GCaMP6s in nNOS-cre mice. Note that there are …
Figure 7—figure supplement 1
Locomotion-evoked Ca2+ signals in nNOS neurons, correction of photobleaching and hemodynamic attenuation of GCaMP signals.
(A) Representative example of raw GFP (green) and TRITC (magenta) fluorescence obtained using fiber photometry in B6.CAG-eGFP mice, which express GFP ubiquitously in their tissues. TRITC …
Figure 7—figure supplement 2
Cross-correlation, coherence, and signal-to-noise comparisons for fiber photometry signals.
(A) Population averaged peak correlation coefficient between blood volume and GCaMP6s fluorescence signals across all behaviors. Bars are population mean of each GCaMP6s sub-type; circles are …
Figure 7—figure supplement 3
Sustained locomotion-evoked Ca2+ signals in nNOS neurons during long duration locomotion events.
(A) Population average locomotion-evoked blood volume change of events of increasing duration. Bars are population mean of each GCaMP6s sub-type; circles are individual animal averages. Population …
Figure 8 with 3 supplements
Relationship between neural activity and arterial diameter.
(A) Summary showing the gamma-band power vs. arteriole diameter, normalized to the relevant vehicle control for each condition. Lines connect mean basal and locomotion neural/arterial responses for …
Figure 8—figure supplement 1
Examples of chemogenetic and pharmacological effects on arteriole diameters.
Examples of two-photon images of arterioles (scale bar 30 μm) after vehicle treatment (gray box) and after chemogenetic/pharmacological treatment (red box). The white arrow shows region where the …
Figure 8—figure supplement 2
Relationship between arterial diameter and LFP power at lower frequencies.
(A) Summary showing the change in 10–40 Hz band power vs. arteriole diameter, normalized to the relevant vehicle control for each condition. Lines connect basal and locomotion for each condition. A …
Figure 8—figure supplement 3
The effects of chemogenetic and pharmacological infusions on evoked arterial diameter changes normalized to the baseline within condition.
For the locomotion-triggered averages here, instead of being normalized to the arteriole baseline for the vehicle condition, the baseline dimeter during the manipulation (e.g. CNO or drug) was used. …
Videos
Video 1
Locomotion produces a rapid dilation in pial arterioles.
This movie shows in vivo imaging of a surface arteriole diameter in the somatosensory cortex through the PoRTs window using two-photon microscopy. Left, behavioral camera. Right, FITC-filled …
Video 2
Locomotion produces a rapid dilation in pial arterioles.
This movie shows in vivo imaging of a surface arteriole diameter in the somatosensory cortex through the PoRTs window using two-photon microscopy. Left, behavioral camera. Right, FITC-filled …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL/6J | Jackson Labs | #000664 | |
| Strain, strain background (Mus musculus) | B6.129-Nos1tm1(cre)Mgmj/J | Jackson Labs | #017526 | |
| Strain, strain background (Mus musculus) | FVB-Tg(Aldh1l1-cre/ERT2) 1Khakh/J | Jackson Labs | #023748 | |
| Strain, strain background (Mus musculus) | C57BL/6-Tg(CAG-EGFP) 131Osb/LeySopJ | Jackson Labs | #006567 | |
| Genetic reagent (Adeno-associated virus) | AAV5-CMV-Turbo RFP-WPRE-rBG | UNC vector core | ||
| Genetic reagent (Adeno-associated virus) | AAV8-hSYN-HA-hM3D (Gq)-mCherry | Addgene | #50474-AAV8 | |
| Genetic reagent (Adeno-associated virus) | AAV5-hSYN-HA-hM4D (Gi)- mCherry | UNC Vector Core | ||
| Genetic reagent (Adeno-associated virus) | AAV5-CaMKIIa-hM3D (Gq)- mCherry | Addgene | #50476-AAV5 | |
| Genetic reagent (Adeno-associated virus) | AAV5-CaMKIIa-hM4D (Gi)- mCherry | UNC Vector Core | ||
| Genetic reagent (Adeno-associated virus) | AAV8-DIO-hM3D (Gq)-mCherry | Addgene | #44361-AAV8 | |
| Genetic reagent (Adeno-associated virus) | AAV8-DIO-hM4D (Gi)-mCherry | Addgene | #44362-AAV8 | |
| Genetic reagent (Adeno-associated virus) | AAV9-CaMKII-GCaMP6s | Addgene | #107790-AAV9 | |
| Genetic reagent (Adeno-associated virus) | AAV8-syn-GCaMP-WPRE | Vigene | AAV8-syn-GCaMP-WPRE | |
| Genetic reagent (Adeno-associated virus) | AAV9-CaMKII-GCaMP6s | Addgene | #107790-AAV9 Viagene | |
| Antibody | Mouse monoclonal GAD-65 Antibody (A-3) | Santa Cruz Biotechnology | sc-377145 | (1:200) |
| Antibody | Mouse monoclonal CaMKII Antibody (G-1) | Santa Cruz Biotechnology | sc-5306 | (1:200) |
| Antibody | Rabbit polyclonal to GFAP | Abcam | ab7260 | (1:200) |
| Antibody | Alexa Fluor 488 Goat Anti-Mouse IgG H and L | Abcam | ab150113 | (1:500) |
| Antibody | Goat Anti-Rabbit IgG H and L | Abcam | ab6702 | (1:500) |
| Antibody | Goat polyclonal nNOS | Abcam | ab1376 | (1:200) |
| Chemical compound, drug | muscimol | Sigma-Aldrich | M1523 | 10 mM |
| Chemical compound, drug | L-NAME | Sigma-Aldrich | N5751 | 1 mM |
| Chemical compound, drug | Substance P | Tocris | 1156 | 1 mM |
| Chemical compound, drug | CP-99994 | Tocris | 3417 | 8 mM |
| Chemical compound, drug | ML-133 | Sigma-Aldrich | SML0190 | 10 µM |
| Chemical compound, drug | DMSO | Sigma-Aldrich | D8418 | 1% |
| Chemical compound, drug | BaCl2 | EMD Millipore | B1493816 | 100 µM |
| Chemical compound, drug | CNO | Sigma-Aldrich | C0832 | 2.5 mg/kg |
| Chemical compound, drug | FITC-dextran 70 kDa | Sigma-Aldrich | 46945 | 50 µL at 5% |
| Chemical compound, drug | TRITC dextran 70kD | Sigma-Aldrich | T1162 | 50 µL at 5% |
| Software, algorithm | Pial vessel dimeter measurements | https://github.com/DrewLab/ Surface-Vessel-FWHM-Diameter | ||
| Software, algorithm | Penetrating vessel dimeter measurements | https://github.com/DrewLab/ Thresholding_in_Radon_Space | ||
| Software, algorithm | Red blood cell velocity measurements | https://github.com/ DrewLab/MCS_Linescan | ||
| Software, algorithm | 2 P microscope control software | MCS, Sutter Instruments, Novato CA | ||
| Software, algorithm | MATLAB | Mathworks, Natick MA | ||
| Software, algorithm | Doric Neuroscience Studio | Doric Lenses, Quebec, Quebec Canada |
Table 1
AAV-injected mice.
| AAV-injected mice | # mice (vessels) for 2-photon imaging | # mice for chronic electrophysiology | Immunohistochemistry | Fiber photometry |
|---|---|---|---|---|
| C57BL/6J: AAV5-CMV-TurboRFP-WPRE-rBG (UNC Vector Core) | 12 mice (56 vessels) | Four mice | Three mice | |
| C57BL/6J: AAV8-hSYN-HA-hM3D(Gq)-mCherry (Addgene #50474-AAV8) | Seven mice (37 vessels) | Three mice | Three mice | |
| C57BL/6J: AAV5-hSYN-HA-hM4D(Gi)- mCherry (UNC Vector Core) | Seven mice (45 vessels) | Three mice | Two mice | |
| C57BL/6J: AAV5-CaMKIIa-hM3D(Gq)- mCherry (Addgene # 50476-AAV5) | Six mice (41 vessels) | Four mice | Two mice | |
| C57BL/6J: AAV5-CaMKIIa-hM4D(Gi)- mCherry (UNC Vector Core) | Seven mice (38 vessels) | Five mice | Two mice | |
| B6.129-Nos1tm1(cre)Mgmj/J: AAV8-DIO-hM3D(Gq)-mCherry in nNOS-cre mice (Addgene #44361-AAV8) | Eight mice (52 vessels) | Four mice | Two mice | |
| B6.129-Nos1tm1(cre)Mgmj/J: AAV8-DIO-hM4D(Gi)-mCherry in nNOS-cre mice (Addgene #44362-AAV8) | Nine mice (49 vessels) | Five mice | Two mice | |
| B6;FVB-Tg(Aldh1l1-cre/ERT2)1Khakh/J: AAV8-DIO-hM4D(Gi)-mCherry in astrocyte-cre mice (Addgene #44362-AAV8) | Five mice (24 vessels) | Three mice | Two mice | |
| B6;FVB-Tg(Aldh1l1-cre/ERT2)1Khakh/J: AAV8-DIO-hM4D(Gi)-mCherry in astrocyte -cre mice (Addgene #44362-AAV8) | Seven mice (31 vessels) | Four mice | Two mice | |
| C57BL/6J: AAV9-CaMKII-GCaMP6s (Addgene) | Five mice | |||
| C57BL/6J: AAV8-syn-GCaMP-WPRE (Vigene) | Four mice | |||
| B6.129-Nos1tm1(cre)Mgmj/J: AAV9-CaMKII-GCaMP6s (Addgene) | Nine mice | |||
| C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ (Jackson Labs) | Three mice |
Table 2
Infused mice.
| Infused mice | # mice (vessels) for two-photon imaging | # mice for chronic Electrophysiology |
|---|---|---|
| C57BL/6J: Muscimol (10 mM) | Nine mice (26 vessels) | Three mice |
| C57BL/6J: L-NAME (1 mM) | Six mice (36 vessels) | Five mice |
| C57BL/6J: Substance P (1 µM) | Seven mice (30 vessels) | Four mice |
| C57BL/6J: CP-99994 (8 mM) | Seven mice (28 vessels) | Four mice |
| C57BL/6J: ML-133 (10 µM) | Five mice (36 vessels) | Four mice |
| C57BL/6J: BaCl2 (100 µM) | Five mice (24 vessels) | Three mice |
