A novel class of inferior colliculus principal neurons labeled in vasoactive intestinal peptide-Cre mice
- University of Michigan, United States
- Northeast Ohio Medical University, United States
Figures
Figure 1
VIP neurons are distributed across multiple subdivisions of the IC.
Photomicrographs of transverse sections through the IC ranging from caudal (left-most) to rostral (right-most). VIP-expressing cells (labeled with tdTomato) are shown in magenta, and GAD67 staining …
Figure 2
VIP neurons are glutamatergic and represent 3.5% of neurons in the ICc.
(A) Confocal z-stack projections showing IC VIP neurons (magenta, left), GAD67 staining (cyan, middle), and an overlay (right). White arrowheads mark VIP neurons, yellow arrows GABAergic cell …
Figure 3
VIP neurons have sustained firing patterns and moderate membrane properties.
(A) VIP neurons exhibited a regular, sustained firing pattern in response to depolarizing current steps, while hyperpolarizing current steps elicited minimal voltage sag (A1). As the amplitude of …
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Figure 3—source data 1
Intrinsic physiology of VIP neurons and from non-targeted recordings in mouse IC.
- https://doi.org/10.7554/eLife.43770.009
Figure 4
Intrinsic physiology of VIP neurons in the ICc varies along the tonotopic axis.
(A) A 2D coordinate system was fit to every IC slice a VIP neuron was recorded from. The medial-lateral axis runs from the midline (zero) to the lateral edge (max) of the slice, the dorsal-ventral …
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Figure 4—source data 1
Intrinsic physiology of VIP neurons matched to their location in the ICc.
- https://doi.org/10.7554/eLife.43770.011
Figure 5
VIP neurons in the ICc are a class of stellate cells and most VIP neurons have dendritic spines.
(A, B) Maximum-intensity projections of confocal z-stacks showing streptavidin-Alexa Fluor-stained VIP neurons from the ICc. Insets: enlarged views of dendritic segments show dendritic spines. (C) …
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Figure 5—source data 1
Morphometric analysis of VIP neurons in the ICc.
- https://doi.org/10.7554/eLife.43770.013
Figure 6
VIP neurons project to multiple local and long-range targets.
(A) Photomicrograph of an AAV deposit site in the right IC. AAV-infected, VIP-expressing cells are labeled with eGFP (yellow), while all VIP-expressing cells are labeled with tdTomato (magenta). …
Figure 7
VIP neurons in the ICc receive excitatory and inhibitory synaptic input from the contralateral IC.
(A) Experimental setup. An AAV encoding Chronos-GFP was injected into the right IC. Three weeks later, light-evoked postsynaptic potentials were recorded from VIP neurons in the left ICc. (B) Image …
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Figure 7—source data 1
EPSP and IPSP analysis of commissural inputs to VIP neurons.
- https://doi.org/10.7554/eLife.43770.016
Figure 8
VIP neurons in the ICc receive direct synaptic input from the DCN and feedforward inhibition driven by DCN afferents.
(A) Experimental setup. An AAV encoding Chronos-GFP was injected into the right DCN. For every experiment, the injection site and Chronos-GFP expression were confirmed through GFP fluorescence. …
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Figure 8—source data 1
Analysis of EPSPs from DCN inputs in VIP neurons and non-targeted recordings.
- https://doi.org/10.7554/eLife.43770.018
Figure 9
Summary of inputs and projection targets of VIP neurons in mouse IC.
(A) Summary of the major projection targets of VIP neurons identified by axonal tract tracing: MGB (medial geniculate body), SC (superior colliculus), contralateral IC, PAG (periaqueductal gray), …
Tables
Table 1
VIP neurons are glutamatergic.
Across three mice, an average of 1.3% of tdTomato+ neurons were labeled with an antibody against GAD67.
| Animal | Slice # | # tdTomato+ | # GAD67+ | # co-labeled | % tdTomato+co-labeled |
|---|---|---|---|---|---|
| P58 female, #1 | 1 (caudal) | 210 | 184 | 3 | 1.4 |
| 2 (middle) | 172 | 65 | 2 | 1.2 | |
| Total | 382 | 249 | 5 | 1.3 | |
| P58 male | 1 (caudal) | 151 | 152 | 2 | 1.3 |
| 2 (middle) | 46 | 212 | 2 | 4.3 | |
| Total | 197 | 364 | 4 | 2.0 | |
| P58 female, #2 | 1 (caudal) | 161 | 137 | 0 | 0.0 |
| 2 (middle) | 53 | 187 | 1 | 1.9 | |
| Total | 214 | 324 | 1 | 0.5 | |
| Grand total | 793 | 937 | 10 | 1.3 | |
| Average across three mice (mean ± SD) | 1.3 ± 0.8% | ||||
Table 2
VIP neurons represent 3.5% of ICc neurons, 1.5% of IC shell neurons, and are present at a higher density in the caudal ICc and IC shell.
Table shows results from stereological analysis of the percentage of neurons (NeuN+) in the ICc and IC shell (ICd + IClc) that express tdTomato in VIP-IRES-Cre x Ai14 mice. Values indicate mean ± SEM…
| ICc | ||||
|---|---|---|---|---|
| Coronal slice | P54 male 1 | P54 male 2 | Per slice plane | Grand average |
| Caudal | 3.1 ± 0.9% (12/503) five samples | 8.4 ± 1.2% (26/338) four samples | 5.8 ± 2.7% (38/841) | |
| Middle | 2.4 ± 0.8% (20/741) eight samples | 3.9 ± 0.9% (44/1173) eight samples | 3.2 ± 0.7% (64/1914) | |
| Rostral | 1.9 ± 0.6% (21/929) eight samples | 1.2 ± 0.4% (12/1024) seven samples | 1.5 ± 0.4% (33/1953) | |
| Per mouse | 2.5 ± 0.3% (53/2173) | 4.5 ± 2.1% (82/2535) | 3.5 ± 1.0% (135/4708) | |
| IC shell | ||||
| Coronal slice | P54 male 1 | P54 male 2 | Per slice plane | Grand average |
| Caudal | 2.9 ± 0.8% (35/1092) 10 samples | 1.9 ± 0.8% (9/615) five samples | 2.4 ± 0.5% (44/1707) | |
| Middle | 0.9 ± 0.6% (4/534) six samples | 0.9 ± 0.6% (10/944) eight samples | 0.9 ± 0.0% (14/1478) | |
| Rostral | 1.2 ± 0.4% (10/842) eight samples | 1.2 ± 0.6% (5/569) four samples | 1.2 ± 0.0% (15/1411) | |
| Per mouse | 1.6 ± 0.6% (49/2468) | 1.3 ± 0.3% (24/2128) | 1.5 ± 0.2% (73/4596) | |
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Table 2—source data 1
Percentages of VIP neurons in ICc and IC shell.
- https://doi.org/10.7554/eLife.43770.007
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL/6J | The Jackson Laboratory | JAX:000664 | |
| Genetic reagent (Mus musculus) | VIP-IRES-Cre | The Jackson Laboratory | JAX:010908 | |
| Genetic reagent (Mus musculus) | Ai14 | The Jackson Laboratory | JAX:007914 | |
| Antibody | anti-GAD67 (mouse monoclonal) | Millipore | RRID:AB_2278725 Cat#:MAB5406 | IHC (1:1000) |
| Antibody | anti-NeuN (rabbit polyclonal) | Millipore | RRID:AB_10807945 Cat#:ABN78 | IHC (1:500) |
| Antibody | anti-bNOS (mouse monoclonal) | Sigma-Aldrich | RRID:AB_260754 Cat#:N2280 | IHC (1:1000) |
| Antibody | anti-mouse IgG Alexa Fluor 488 (donkey polyclonal) | ThermoFisher | RRID:AB_141607 Cat#:A-21202 | IHC (1:500) |
| Antibody | anti-rabbit IgG Alexa Fluor 488 (donkey polyclonal) | ThermoFisher | RRID:AB_2535792 Cat#:A-21206 | IHC (1:500) |
| Recombinant DNA reagent | AAV1.Syn.Chronos-GFP.WPRE.bGH | University of Pennsylvania Vector Core/Addgene | Addgene:59170-AAV1 RRID:Addgene_59170 | http://n2t.net/addgene:59170 |
| Recombinant DNA reagent | AAV1.CAG.FLEX.eGFP.WPRE.bGH | University of Pennsylvania Vector Core/Addgene | Addgene:51502-AAV1 RRID:Addgene_51502 | http://n2t.net/addgene:51502 |
| Chemical compound, drug | gabazine | Hello Bio | Cat#:HB0901 | also called SR95531 hydrobromide |
| Chemical compound, drug | strychnine hydrochloride | Sigma-Aldrich | Cat#:S8753 | |
| Chemical compound, drug | D-AP5 | Hello Bio | Cat#:HB0225 | |
| Chemical compound, drug | NBQX disodium salt | Hello Bio | Cat#:HB0443 | |
| Software, algorithm | Igor Pro 7 and 8 | Wavemetrics | RRID:SCR_000325 | |
| Software, algorithm | MATLAB R2018a and R2018b | Mathworks | RRID:SCR_001622 | |
| Software, algorithm | Neurolucida | MBF Bioscience | RRID:SCR_001775 | |
| Software, algorithm | Neurolucida 360 | MBF Bioscience | RRID:SCR_016788 |
Table 3
Stereotaxic coordinates for virus injections. All coordinates are relative to the lambda suture.
| Location | X coordinate (caudal) | Y coordinate (lateral) | Z coordinates (depth) |
|---|---|---|---|
| Right IC penetration 1 (CRACM) | -900 µm | 1000 µm | 2250 - 1500 µm, 250 µm interval |
| Right IC penetration 2 (CRACM) | -900 µm | 1250 µm | 2250 - 1750 µm, 250 µm interval |
| Right IC penetration 1 (axonal tracing) | -900 µm | 1000 µm | 1850 µm, 2000 µm |
| Right DCN | -1325 µm | 2150 µm | 4750 µm, 4550 µm |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.43770.021
