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A cross-modality enhancement of defensive flight via parvalbumin neurons in zona incerta

  1. Xiyue Wang
  2. Xiaolin Chou
  3. Bo Peng
  4. Li Shen
  5. Junxiang J Huang
  6. Li I Zhang  Is a corresponding author
  7. Huizhong W Tao  Is a corresponding author
  1. University of Southern California, United States
Research Article
Cite this article as: eLife 2019;8:e42728 doi: 10.7554/eLife.42728
4 figures, 1 table and 2 additional files

Figures

Figure 1 with 2 supplements
Tactile stimulation enhances sound-induced flight response via SSp.

(A) Left, illustration of the head-fixed animal behavioral paradigm. Right, plots of running speed under noise presentation without (black) and with (red) concurrent whisker stimulation for an example animal. Red line marks the duration of noise/whisker stimulation. (B) Summary of peak noise-induced running speed in the absence and presence of whisker stimulation. **p=0.0011, two-sided paired t-test, n = 7 animals. (C) Summary of total travel distance. **p=0.0072, two-sided paired t-test, n = 7 animals. (D) Left, illustration of the experimental paradigm: SSp was silenced with infusion of muscimol (red) as shown in the confocal image (upper left, scale: 500 µm). Right, plots of speed without (black) and with (red) whisker stimulation for an example animal. (E) Summary of peak speed in the absence and presence of whisker stimulation. ‘n.s.', not significant, two-sided paired t-test, n = 5 animals. (F) Summary of total travel distance. ‘n.s.', not significant, two-sided paired t-test, n = 5 animals. Open symbols represent mean ± s.d. for all panels.

https://doi.org/10.7554/eLife.42728.002
Figure 1—figure supplement 1
Control experiments and analysis of individual animals.

(A) Illustration of the auditory and whisker stimulation paradigm. Right, photograph of the whisker stimulation detail. (B) Summary of baseline locomotion speed without (contr) and with (whisk) whisker stimulation. ‘n.s.', non-significant, two-sided paired t-test, n = 6 animals. Error bars represent mean ± s.d. (C) Normalized peak speed (averaged by trials) for each individual animal without (gray) and with (black) whisker stimulation. ***p<0.001, *p<0.05, two-sided unpaired t-test. Error bars represent mean ± s.e.m. (D) Normalized total travel distance for each animal. ***p<0.001, **p<0.01, *p<0.05, two-sided unpaired t-test. Error bars represent mean ± s.e.m.

https://doi.org/10.7554/eLife.42728.003
Figure 1—figure supplement 2
A flght test in freely moving mice.

(A) Illustration of the two-chamber flight test. (B) Summary of average speed for flight crossing the channel before and after whisker trimming. Data points for the same animal are connected with a line. **p<0.01, two-sided paired t-test, n = 7 animals. Error bars represent mean ± s.d.

https://doi.org/10.7554/eLife.42728.004
Figure 2 with 1 supplement
The SSp-ZIv projection mediates the tactile enhancement of sound-induced flight.

(A) Illustration of the injection paradigm. (B) Anterogradely labeled axon terminals in rostral (left), dorsal and ventral (middle), as well as caudal (right) sectors of ZI. Scale bar, 200 µm. Blue shows Nissl staining; red shows PV+ neuron or axon distribution. (C) Left, illustration of the experimental paradigm: optic fibers were implanted to stimulate ChR2-expressing SSp axons in ZI. Right, plots of speed without (black) and with (blue) LED stimulation for an example animal. (D) Summary of peak noise-induced speed in the absence and presence of LED stimulation of SSp-ZI terminals. **p=0.003195, two-sided paired t-test, n = 7 animals. (E) Summary of the travel distance. *p=0.01854, two-sided paired t-test, n = 7 animals. (F) Left, ZIv was silenced with muscimol (red) as shown in the confocal image (lower, scale: 500 µm). Right, plots of speed without (black) and with (red) whisker stimulation for an example animal. (G) Summary of peak speed in the absence and presence of whisker stimulation. ‘n.s.”, not significant, two-sided paired t-test, n = 5 animals. (H) Summary of total travel distance. ‘n.s.”, not significant, two-sided paired t-test, n = 5 animals. (I) Experimental paradigm for slice recording. (J) Average LED-evoked EPSC in an example ZIv PV+ neuron before and after (lower) perfusion of CNQX. Arrow points to the onset of LED light. Recording was made in the presence of TTX and 4-AP. Scale: 25 pA, 25 ms. (K) Amplitudes of LED-evoked EPSCs of 8 responding neurons out of 10 recorded ZIv PV+ cells. Bars represent s.d. for all panels.

https://doi.org/10.7554/eLife.42728.006
Figure 2—figure supplement 1
Analysis of individual animals and control experiment of LED stimulation alone.

(A) Normalized peak speed for each animal without (gray) and with (black) LED light stimulation. *p<0.05, two-sided unpaired t-test. Error bars represent mean ± s.e.m. (B) Normalized total travel distance for each animal. **p<0.01, *p<0.05, two-sided unpaired t-test. (C) Baseline locomotion speed without (OFF) and with (ON) LED light stimulation. ‘n.s.', non-significant, two-sided paired t-test, n = 7 animals.

https://doi.org/10.7554/eLife.42728.007
Figure 3 with 3 supplements
PV+ neurons in ZIv mediate the tactile enhancement of flight behavior.

(A) Left, experimental paradigm. Right, Plots of speed without (black) and with (blue) LED stimulation for an example animal. Blue line marks the duration of noise/LED stimulation. (B) Summary of peak noise-induced speed in the absence and presence of LED stimulation of ZIv PV+ neurons. ***p=0.0009, two-sided paired t-test, n = 6 animals. (C) Summary of total travel distance. **p=0.0042, two-sided paired t-test, n = 6 animals. (D) Left, experimental paradigm. Right, plots of speed without (black) and with (green) LED stimulation for an example animal. Green line marks the duration of noise/LED stimulation. (E) Summary of peak noise-induced speed in the absence and presence of LED inhibition. ***p=0.0004, two-sided paired t-test, n = 5 animals. (F) Summary of total travel distance. *p=0.0136, two-sided paired t-test, n = 5 animals. (G) Left, expressing DREADDi in ZIv PV+ neurons. Right, plots of speed without (black) and with (red) whisker stimulation for an example animal. (H) Summary of peak noise-induced speed in the absence and presence of whisker stimulation with ZIv PV+ neurons inhibited by CNO. ‘n.s.”, not significant, two-sided paired t-test, n = 8 animals. (I) Summary of total travel distance. ‘n.s.”, not significant, two-sided paired t-test, n = 8 animals. Open symbols represent mean ± s.d. (J) Upper, optrode recording in the head-fixed animal. Lower, raster plot of an example ZIv PV+ neuron to 20 Hz LED stimulation in seven trials. Scale: 50 ms. (K) Peri-stimulus spike time histogram for an example PV+ neuron in response to whisker (red), noise (yellow) and whisker plus noise (black) stimulation. Bin size = 100 ms. (L) Summary of evoked firing rates of recorded PV+ neurons (within the stimulation window). ***p<0.0001, one-way ANOVA with post hoc test, n = 22 cells.

https://doi.org/10.7554/eLife.42728.009
Figure 3—figure supplement 1
Tests of efficacies of ChR2 and ArchT stimulation and analysis of individual animals.

(A) Confocal images showing the expression of ChR2 (green) and tdTomato (red) in ZIv in a PV-Cre::Ai14 animal. White arrows point to cells showing colocalization of ChR2 and tdTomato. Scale: 50 µm. (B) Upper, blue light induced spiking in an example ChR2-expressing neuron. Each blue dot represents a blue light pulse. Scale: 20 mV, 100 ms. Lower, summary of probability of spiking in response to 10 pulses (at 20 Hz) of blue light stimulation (n = 5 neurons). (C) Upper, green light (500 ms duration) induced hyperpolarization in an example ArchT-expressing neuron. Lower, summary of maximum level of hyperpolarization induced by light for 4 recorded neurons. (D) Normalized peak speed for each animal without (gray) and with (black) blue LED light stimulation for the ChR2 group. **p<0.01, *p<0.05, two-sided unpaired t-test. Error bars represent mean ± s.e.m. (E) Normalized total travel distance for each animal without (gray) and with (black) blue LED light stimulation for the ChR2 group. **p<0.01, *p<0.05, two-sided unpaired t-test. Error bars represent mean ± s.e.m. (F) Normalized peak speed for each animal without (gray) and with (black) green LED light stimulation for the ArchT group, **p<0.01, *p<0.05, two-sided unpaired t-test. Error bars represent mean ± s.e.m. (G) Normalized total travel distance for each animal without (gray) and with (black) green LED light stimulation for the ArchT group. *p<0.05, two-sided unpaired t-test. Error bars represent mean ± s.e.m.

https://doi.org/10.7554/eLife.42728.010
Figure 3—figure supplement 2
Control experiments of LED stimulation alone.

(A, B) Summary of average locomotion speed without and with LED simulation alone for the ChR2 (A, n = 5 animals) and ArchT (B, n = 5 animals) group. ‘n.s.', non-significant, two-sided paired t-test. Error bars represent mean ± s.d.

https://doi.org/10.7554/eLife.42728.011
Figure 3—figure supplement 3
Test of efficacy of chemogenetic silencing.

(A) Current-clamp recording traces for an example hM4D(Gi)-expressing neuron in response to a series of current injections (500 ms duration) with amplitudes ranging from 0 to 120 pA with a step of 20 pA, before (left) and after (middle) CNO perfusion and after washing out CNO (right). Scale: 20 mV. Red color labels the trace at the maximum level of current injection. (B) Summary of minimum amplitude of current injection needed to induce spiking before and after CNO infusion and after washing out CNO. (C) Summary of number of spikes induced by current injection at the same level (which was the threshold level after CNO application) before and after CNO application (n = 6 neurons).

https://doi.org/10.7554/eLife.42728.012
Figure 4 with 2 supplements
The projection of ZIv PV+ neurons to POm enhances sound-induced flight.

(A) Illustration of injection paradigm. (B) Confocal images showing GFP expression at the injection site (left; scale: 500 µm) and in major target regions (middle and right; scale: 200 µm). Blue shows Nissl staining. SC, superior colliculus; POm, posterior medial nucleus of thalamus. (C) Left, stimulating ChR2-expressing ZI PV+ neuron axons in SC. Right, plots of speed without (black) and with (blue) LED stimulation for an example animal. (D) Summary of peak noise-induced speed in the absence and presence of LED activation of ZIv-SC axons. ‘n.s.”, not significant, two-sided paired t-test, n = 5 animals. (E) Summary of total travel distance. Two-sided paired t-test, n = 5 animals. (F) Left, stimulating ChR2-expressing ZI PV+ neuron axons in POm. Right, plots of speed without (black) and with (blue) LED stimulation for an example animal. (G) Summary of peak noise-induced speed in the absence and presence of LED activation of ZI-POm axons. *p=0.0198, two-sided paired t-test, n = 5 animals. (H) Summary of total travel distance. **p=0.0034, two-sided paired t-test, n = 5 animals. (I) Left, silencing DREADDi-expressing ZI PV+ neuron axons in POm. Right, plots of speed without (black) and with (red) whisker stimulation after local infusion of CNO in POm for an example animal. (J) Summary of peak noise-induced speed in the absence and presence of whisker stimulation when silencing ZIv-POm axons. ‘n.s.', not significant, two-sided paired t-test, n = 7 animals. (K) Summary of total travel distance. ‘n.s.', not significant, two-sided paired t-test, n = 7 animals. Open symbols represent mean ± s.d. for all panels.

https://doi.org/10.7554/eLife.42728.014
Figure 4—figure supplement 1
Quantification of relative fluorescence density of GFP-labeled processes in different downstream regions of ZIv PV+ neurons (n = 4 animals).

Bar = s.d. Abbreviations: vlPAG, ventrolateral periaqueductal gray; dlPAG, dorsolateral periaqueductal gray; IC, inferior colliculus; MRN, midbrain reticular nucleus; RN, red nucleus; APN, anterior pretectal nucleus; PRN, pontine reticular nucleus; M1, primary motor cortex; S1, primary somatosensory cortex; V1, primary visual cortex; A1, primary auditory cortex; LA, lateral amygdalar nucleus; BLA, basolateral amygdalar nucleus; CEA, central amygdalar nucleus.

https://doi.org/10.7554/eLife.42728.015
Figure 4—figure supplement 2
Analysis of individual animals and control experiment of LED stimulation alone.

(A) Normalized peak speed for each animal without (gray) and with (black) blue LED light stimulation. *p<0.05, two-sided unpaired t-test. Error bars represent mean ± s.e.m. (B), Normalized total travel distance for each animal without (gray) and with (black) blue LED light stimulation. **p<0.01, *p<0.05, two-sided unpaired t-test. Error bars represent mean ± s.e.m. (C), Summary of average locomotion speed without and with LED light stimulation alone. ‘n.s.”, non-significant, two-sided paired t-test, n = 5 animals. Error bars represent mean ± s.d. Supplementary and source data files.

https://doi.org/10.7554/eLife.42728.016

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain (mouse)Pvalb-ires-CreJachson LaboratoryStock No.008069
Strain (mouse)Ai14Jachson LaboratoryStock No.007914
Strain (mouse)C57BL/6JJachson LaboratoryStock No.000664
Recombinant DNA reagentAAV2/1-CamKII-hChR2-eYFP-WPRE-hGhUPenn Vector Core
Recombinant DNA reagentAAV1-CAG-FLEX-eGFP-WPRE-bGHUPenn Vector Core
Recombinant DNA reagentAAV2/1-pEF1α-DIO-hChR2-eYFPUPenn Vector Core
Recombinant DNA reagentAAV1-CAG-FLEX-ArchT-GFPUNC vector Core
Recombinant DNA reagentpAAV-hSyn-hM4D(Gi)-mCherryAddgenePlasmid #50475
Other (stains)NeuroTrace 640/660 Deep-Red Fluorescent Nissl StainThermoFisherN21483IHC 1:500
Chemical compound, drugMuscimolThermoFisherM23400
Chemical compound, drugTetrodotoxinTorcrisCat. No.10781 µM
Chemical compound, drug4-Aminopyridine (4-AP)TorcrisCat. No. 09401 mM
Chemical compound, drugcyanquixaline (CNQX)Sigma-AldrichC23920 µM
Chemical compound, drugclozapine-N-oxide (CNO)TorcrisCat. No. 49361 mg/kg IP; 3 µM local infusion; 5 µM in slice recording
SoftwareOffline SorterPlexonversion 4
SoftwareMATLABMathworksversion R2017a

Additional files

Supplementary file 1

Analysis of temporal profiles of speed traces in different sets of experiments.

Data are presented as mean ± SD. Two-sided paired t-test were performed to compared values between control and manipulation conditions. The type of experiment is shown by the corresponding figure number in main figures.

https://doi.org/10.7554/eLife.42728.018
Transparent reporting form
https://doi.org/10.7554/eLife.42728.019

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