Tonotopic and non-auditory organization of the mouse dorsal inferior colliculus revealed by two-photon imaging
- Erasmus MC, University Medical Center Rotterdam, Netherlands
Figures
Figure 1 with 1 supplement
Co-expression between GCaMP6s and GAD67 in the two transgenic mouse lines.
(A) Single optical section of an IC brain slice from a GP4.3 mouse immunolabeled for GAD67 (magenta) and GCaMP6s (green). (B) Distribution of GCaMP+ and GAD67+ cells within the 40 µm brain slice in A…
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Figure 1—source data 1
CellCounter source data XML files generated by Cell counter plug-in in FIJI containing the actual counted datapoints of each cell type, with text files explaning the different cell types being counted.
- https://doi.org/10.7554/eLife.49091.004
Figure 1—figure supplement 1
Expression of parvalbumin and calretinin in GCaMP+ neurons in the two transgenic mouse lines.
(A) Single optical section of an IC brain slice from a GP4.3 mouse immunolabeled for GCaMP6s (green), parvalbumin (PV; red) and calretinin (CR; blue). (B) Distribution of GCaMP+ cells within the 40 …
Figure 2 with 1 supplement
Calcium imaging of dorsal inferior colliculus in awake mice.
(A) Illustration of the cranial window construct imaged with a numerical aperture (NA) 0.95 water-immersion objective. (B) Top-down view of cranial window, showing optically exposed inferior …
Figure 2—figure supplement 1
Reduction of scanner noise by sinusoidal galvo scanning.
(A) Sound spectra measured with a calibration microphone (Model 7016, ACO Pacific) with a sensitivity of −49.1 dBV @ 1 Pa and 40 dB signal amplification, positioned where the right pinna of a mouse …
Figure 3 with 2 supplements
Different sound evoked responses and frequency response areas of representative example cells.
(A) An onset fluorescence response to a 1 s pure tone. Gray vertical lines indicate onset and offset of sound stimulus. Colored and gray traces are average and individual trial fluorescence changes, …
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Figure 3—source data 1
Fluorescence kinetics source data CSV file containing fluorescence kinetics of ROIs, genotype of animal and type of FRA.
- https://doi.org/10.7554/eLife.49091.010
Figure 3—figure supplement 1
Kinetics of fluorescence responses.
(A) Example of the average fluorescence response of a cell with an onset-type response to sound (gray trace). Single exponential fits were performed on the onset (0–1000 ms; solid black curve) and …
Figure 3—figure supplement 2
Another example FRA from a GP4.3 mouse, showing onset response and broad tuning.
Vertical scale bar indicates 1 Fb.
Figure 4
Proportion of response classes and relationship with cell size.
(A–B) Proportions of response classes in GP4.3 (A) and GAD2;Ai96 animals (B). (C) Bee swarm plot and mean ±s.d. for cell size for ROIs of different non-mixed response classes, separated by …
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Figure 4—source data 1
IC ROI Area source data CSV file containing fluorescence area and estimated diameter of ROI, genotype of animal and type of FRA.
- https://doi.org/10.7554/eLife.49091.012
Figure 5 with 3 supplements
Relationship between GCaMP6s fluorescence and spikes in IC neurons.
(A) Imaging of a GCaMP6s+ cell (green) in an awake GP4.3 animal with simultaneous juxtacellular recording with a pipette filled with Alexa 594 (red). (B) Spiking pattern (upper) and fluorescence …
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Figure 5—source data 1
Ground-truth model fitting source data CSV file containing model parameters from ground-truth data fitting and the variance explained, genotype of animal and type of FRA.
- https://doi.org/10.7554/eLife.49091.017
Figure 5—figure supplement 1
Example model fit to a cell with a sustained FRA.
From top to bottom: sound stimulus (purple trace), spiking pattern (black), spike rate (gray histogram, 1 s bins) and fluorescence (black trace) of a cell from a Gad2;Ai96 animal with a sustained …
Figure 5—figure supplement 2
Example model fit to a cell with an inhibited FRA.
From top to bottom: sound stimulus (purple trace), spiking pattern (black), spike rate (gray histogram, 1 s bins) and fluorescence (black trace) of a cell from a Gad2;Ai96 animal with an inhibited …
Figure 5—figure supplement 3
Example model fit to spontaneous activity of a cell.
From top to bottom: spiking pattern (black), spike rate (gray histogram, 1 s bins) and fluorescence (black trace) of a spontaneously active cell from a GP4.3 animal and the model fit (green). Fit …
Figure 6 with 9 supplements
Tonotopic organization.
(A) Combined spatial distribution of characteristic frequencies (CFs) in the two transgenic lines, aligned to the same top-down image of an exposed left IC. Symbol size represents the size of the …
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Figure 6—source data 1
IC CF Distribution source data CSV file containing 3D coordinate of ROIs, projected distance of ROIs, genotype of animal and type of FRA.
Source code for fit function used for fitting the spatial distribution.
- https://doi.org/10.7554/eLife.49091.027
Figure 6—figure supplement 1
Tonotopic organization of a single GP4.3 animal (Mouse 20976–07; B6CBAF1/J background), registered to widefield image in Figure 6A.
https://doi.org/10.7554/eLife.49091.019
Figure 6—figure supplement 2
Tonotopic organization of a single GP4.3 animal (Mouse 11605–01; C57BL/6J background), registered to widefield image in Figure 6A.
https://doi.org/10.7554/eLife.49091.020
Figure 6—figure supplement 3
Tonotopic organization of a single Gad2;Ai96 animal (Mouse 12156–03), registered to widefield image in Figure 6A.
https://doi.org/10.7554/eLife.49091.021
Figure 6—figure supplement 4
Tonotopic organization of a single Gad2;Ai96 animal (Mouse 12156–04), registered to widefield image in Figure 6A.
Same animal as Figure 9A.
Figure 6—figure supplement 5
Tonotopic organization of a single GP4.3 animal (Mouse 11605–04; C57BL/6J background), registered to widefield image in Figure 6A.
Same animal as Figure 9E.
Figure 6—figure supplement 6
Tonotopic organization of a single Gad2;Ai96 animal (Mouse 14234–01), registered to widefield image in Figure 6A.
https://doi.org/10.7554/eLife.49091.024
Figure 6—figure supplement 7
Relationship between CF and depth of cell.
Scatter plot of CF against depth of each ROI from pia surface. Black: all cells; red: cells within the central strip, r = 525–725 μm. Solid lines represent regression lines for the two data sets.
Figure 6—figure supplement 8
Tonotopic organization of the most superficial cells.
Combined spatial distribution of characteristic frequencies (CFs) for cells within 50 μm of the pia surface in the two transgenic lines, aligned to the same top-down image of an exposed left IC. …
Figure 6—video 1
Video of all sound-responsive.
ROIs plotted in a common coordinate system, color coded according to their characteristic frequencies. The Z-dimension indicates the depth from pia surface. Numbers on the axes are in μm. The same …
Figure 7
Spatial organization of response classes.
Similar proportions of response classes along (A) the presumed tonotopic axis (bin size 50 μm) and (B) the direction orthogonal to the axis (bin size 100 μm). Zero position was taken as the contact …
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Figure 7—source data 1
IC Response Distribution source data CSV file containing 3D coordinate of ROIs, projected distance of ROIs, genotype of animal and type of FRA.
- https://doi.org/10.7554/eLife.49091.030
Figure 8 with 1 supplement
Cells with motion-related responses.
(A) Two example cells that were spontaneously active in the absence of sound presentation. Calcium transients occurred when the animal was moving its paws or showing facial movement (e.g. whisking). …
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Figure 8—source data 1
IC Movement cells source data CSV file containing 3D coordinate of ROIs, projected distance of ROIs, genotype of animal, type of FRA and parameters related to movement-related response.
- https://doi.org/10.7554/eLife.49091.033
Figure 8—figure supplement 1
Evidence that the motion-related responses were not due to motion artefacts in imaging.
Vertical or horizontal blurring was due to subpixel image registration. (A) Single image frame for GCaMP fluorescence from experiment in Figure 8, i-iii corresponds to frames marked in Figure 8A. (B)…
Figure 9 with 7 supplements
Comparison of tonotopic organization with histological data and literature.
(A) Epifluorescence image of the IC in a horizontal brain section stained for GAD67. This brain slice was from a Gad2;Ai96 mouse after two-photon imaging. Black straight line indicates the minimum …
Figure 9—figure supplement 1
GAD67 staining in the IC of a series of consecutive 40 μm horizontal brain slices from the same Gad2;Ai96 animal as in Figure 9A (animal: 12156–04), displayed from dorsal (top-left) to ventral (right-bottom) showing that the fainter GAD67 staining highlighted in Figure 9A is contiguous with the well-stained neurochemical modules in more ventral slices (arrowheads and numbers).
Same sections as supplements 2 & 3.
Figure 9—figure supplement 2
Calretinin staining in the IC of a series of consecutive 40 μm horizontal brain slices from the same Gad2;Ai96 animal as in Figure 9A (animal: 12156–04), displayed from dorsal to ventral (left to right, top to bottom).
Same sections as supplements 1 and 3.
Figure 9—figure supplement 3
GFP staining of GCaMP6s in the IC for a series of consecutive 40 μm horizontal brain slices from the same Gad2;Ai96 animal as in Figure 9A (animal: 12156–04), displayed from dorsal to ventral (left to right, top to bottom).
Same sections as supplements 1 and 2.
Figure 9—figure supplement 4
GAD67 staining in the IC of a series of consecutive 40 μm horizontal brain slices from the same GP4.3 animal as in Figure 9E (animal: 11605–04; C57BL/6J background), displayed from dorsal (top-left) to ventral (right-bottom), showing that the fainter GAD67 staining highlighted in Figure 9E is contiguous with the well-stained neurochemical modules in more ventral slices (arrowheads and numbers).
Same sections as supplements 5 and 6. Figure 9—figure supplement 5.
Figure 9—figure supplement 5
Calretinin staining in the IC of a series of consecutive 40 μm horizontal brain slices from the same GP4.3 animal as in Figure 9E (animal: 11605–04; C57BL/6J background), displayed from dorsal to ventral (left to right, top to bottom).
Same sections as supplements 4 and 6.
Figure 9—figure supplement 6
GFP staining of GCaMP6s in the inferior colliculus for the series of consecutive 40 μm horizontal brain slices from the same GP4.3 animal as in Figure 9E (animal: 11605–04; C57BL/6J background), displayed from dorsal to ventral (left to right, top to bottom).
Same sections as supplements 4 and 5.
Figure 9—video 1
Video showing the 3D reconstruction of the most dorsal aspect of the left and right inferior colliculi in animal 12156–04 from 40 μm serial sections.
GAD67-dense modules are marked in both ICs. The same sections used for reconstruction are shown in Figure 9A and Figure 9—figure supplements 1–3.
Figure 10
Example motion-sensitive cells with dendritic arbor extending into GAD67-dense modules.
(A) Maximum projection of GCaMP staining showing dendritic arbor of cell three in Figure 9D2, overlaid with a single optical section of GAD67 staining showing a module (m; at same focus as Figure 9D2…
Author response image 1
Histogram of the fraction of significant responses showing the largest response class (dominant class) in the FRA of all responsive ROIs.
Author response image 2
Defining medial and lateral subregions for analysis.
Author response image 3
Polynomial fit to characteristic frequencies of medial cells.
Author response image 4
Polynomial fit to characteristic frequencies of lateral cells.
Author response image 5
Polynomial fit to characteristic frequencies (CF) of cells with >= 8 kHz CF.
Author response image 6
Highly correlated movement in facial and whisker pad areas.
Left, two different analysis regions-of-interest (ROIs). Right: Changes in intensity at the facial ROI (Y-axis, as analyzed in the manuscript) is highly correlated with that in the whisker pad …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | B6;129S6-Gt(ROSA)26Sortm96(CAG-GCaMP6s)Hze/J | The Jackson Laboratory; originally reported in Madisen et al. (2015) | IMSR Cat# JAX:024106, RRID:IMSR_JAX:024106 | Designated as ‘Ai96’ in this manuscript. Maintained in heterozygous state by backcrossing to C57BL/6J. |
| Genetic reagent (Mus musculus) | STOCK Gad2tm2(cre)Zjh/J | The Jackson Laboratory; originally reported in Taniguchi et al. (2011) | IMSR Cat# JAX:010802, RRID:IMSR_JAX:010802 | Designated as ‘Gad2-IRES-Cre’ in this manuscript. Maintained in homozygous state after > 10 generations of backcrossing to C57BL/6J, and re-backcrossed to C57BL/6J every 4–5 generations. |
| Genetic reagent (Mus musculus) | C57BL/6J-Tg(Thy1-GCaMP6s)GP4.3Dkim/J | The Jackson Laboratory; originally reported in Dana et al. (2014) | IMSR Cat# JAX:024275, RRID:IMSR_JAX:024275 | Designated as ‘GP4.3’ in this manuscript. Maintained in heterozygous state by backcrossing to C57BL/6J, or crossed with CBA/JRj to obtain mice with B6CBAF1/J background. |
| Strain, strain background (Mus musculus) | JAX C57BL/6J | Charles Rivers Laboratories | IMSR Cat# JAX:000664, RRID:IMSR_JAX:000664 | |
| Strain, strain background (Mus musculus) | CBA/JRj | Janvier Labs | MGI Cat# 6157506, RRID:MGI:6157506 | |
| antibody | chicken anti-GFP (Chicken polyclonal) | Aves | Aves Labs Cat# GFP-1020, RRID:AB_10000240 | IF(1:1000) |
| Antibody | mouse anti-Gad67 (Mouse monoclonal) | Millipore | Millipore Cat# MAB5406, RRID:AB_2278725 | IF(1:1000) |
| Antibody | rabbit anti-NeuN (Rabbit polyclonal) | Millipore | Millipore Cat# ABN78, RRID:AB_10807945 | IF(1:1000) |
| Antibody | mouse anti-parvalbumin (Mouse monoclonal) | Swant | Swant Cat# 235, RRID:AB_10000343 | IF(1:7000) |
| Antibody | rabbit anti-calretinin (Rabbit polyclonal) | Swant | Swant Cat# 7699/4, RRID:AB_2313763 | IF(1:5000) |
| Antibody | Alexa Fluor 488-conjugated Donkey anti-chicken antibody (Donkey polyclonal) | Jackson ImmunoResearch Labs | Cat# 703-545-155, RRID:AB_2340375 | IF(1:200) |
| Antibody | Alexa Fluor 594-conjugated Donkey anti-mouse antibody (Donkey polyclonal) | Jackson ImmunoResearch Labs | Cat# 715-585-150, RRID:AB_2340854 | IF(1:200) |
| Antibody | Alexa Fluor 647-conjugated Donkey anti-rabbit antibody (Donkey polyclonal) | Jackson ImmunoResearch Labs | Cat# 711-605-152, RRID:AB_2492288 | IF(1:200) |
| Software, algorithm | IGOR Pro | Wavemetrics | RRID:SCR_000325 | Analysis of calcium imaging and electrophysiology data |
| Software, algorithm | pClamp | Molecular Devices | RRID:SCR_011323 | Signal digitization. software, algorithm |
| MATLAB | MathWorks | RRID:SCR_001622 | Stimulus generation and presentation | |
| Software, algorithm | LabView | National Instruments | RRID:SCR_014325 | Control of microscope and other hardware |
| Software, algorithm | Fiji | http://fiji.sc | RRID:SCR_002285 | Processing and analysis of images from immunohistochemistry |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.49091.044
