VTA-projecting cerebellar neurons mediate stress-dependent depression-like behaviors
- Center for Functional Connectomics, Brain Science Institute, Korea Institute of Science and Technology (KIST), Republic of Korea
- Division of Bio-Medical Science and Technology, KIST School, Korea University of Science and Technology (UST), Republic of Korea
Figures
Figure 1 with 2 supplements
c-Fos expression in the DCN is increased by the chronic stress application.
(A) Schematic diagram of the RS (2 hr/day) application for 2 weeks, followed by 1 day each for the OFT, TST, and FST before fixation, or by fixation for c-Fos staining. (B–D) Immobility time in the …
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Figure 1—source data 1
p Values and statistical tests related to Figure 1.
- https://cdn.elifesciences.org/articles/72981/elife-72981-fig1-data1-v2.xlsx
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Figure 1—source data 2
Effect sizes for behavior results related to Figure 1.
- https://cdn.elifesciences.org/articles/72981/elife-72981-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
Conditioning of mice for the RS protocol.
(A) Examples of movement traces from the CTR and RS groups during 30 min of the OFT. (B) Measurement of the time spent in the center zone or the peripheral zone in the OFT. Data are presented as …
Figure 1—figure supplement 2
Cell types for c-Fos-positive DCN neurons in the DN following 14 days RS.
Images of DN-containing cerebellar slices co-stained for NeuN (A), GAD67 (B), or GFAP (C) and c-Fos with DAPI. Most of c-Fos-positive cells are NeuN-positive cells, as seen in (A). Yellow arrowheads …
Figure 2 with 2 supplements
Manipulation of crus I PC activity improves depression-like behaviors.
(A) Top: a schematic of AAV-sSyn-FLEX-hM3Dq-2AGFP injection into crus I of PCP2-Cre mice. Bottom: confocal image of a whole cerebellar slice showing the expression of Gq-2AGFP. Scale bar: 500 μm. (B)…
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Figure 2—source data 1
p Values and statistical tests related to Figure 2.
- https://cdn.elifesciences.org/articles/72981/elife-72981-fig2-data1-v2.xlsx
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Figure 2—source data 2
Effect sizes for behavior results related to Figure 2.
- https://cdn.elifesciences.org/articles/72981/elife-72981-fig2-data2-v2.xlsx
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Figure 2—source data 3
Numbers of mice for each sex used in each behavioral test related to Figure 2.
- https://cdn.elifesciences.org/articles/72981/elife-72981-fig2-data3-v2.xlsx
Figure 2—figure supplement 1
Similar effects of unilateral and bilateral induction of crus I PC activity on the development of depression-like behaviors.
(A, B, C) Effects of unilateral excitation of crus I PC activity in PCP2-Cre;ICR mice in the TST (A), the FST (B) and the OFT (C). (D, E, F) Effects of bilateral excitation of crus I PC activity in …
Figure 2—figure supplement 2
Changes in body weights of mice used for Figure 2.
Comparisons of body weight changes in mice used for Figure 2G–I (A) and Figure 2J–L (B). *** < 0.001, one-way ANOVA followed by the Fisher’s LSD post hoc test. Data are shown as the mean ± SEM.
Figure 3 with 1 supplement
DCN neurons in the DN structurally project to the VTA.
(A–C) Left: schematic drawing of AAV-sSyn-GFP injection into the right side of the FN (A), the IPN (B), and the DN (C). Note that iontophoretic injection was used, to achieve confined injection of …
Figure 3—figure supplement 1
Anatomical connectivity from each subregion of the DCN to the VTA.
(A–C) Screening of GFP-positive axons in the whole VTA (bottom) followed by an iontophoretic injection of AAV-sSyn-GFP into the FN (A), the IPN (B), or the DN (C). Scale bar: 500 μm.
Figure 4 with 1 supplement
The DCN neurons in the DN make functional synaptic connections to the VTA.
(A) Left: schematic drawing of the injection of AAV-sSyn-ChR2-GFP or AAV-sSyn-ChR2-2AdT into the DN, for whole-cell patch clamp recording from VTA neurons. Right: confocal images of ChR2-2AdT …
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Figure 4—source data 1
p Values and statistical tests related to Figure 4.
- https://cdn.elifesciences.org/articles/72981/elife-72981-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Functional synaptic connections of the DCN-VTA circuit.
(A) A trace of EPSCs (top) evoked by 50 Hz photostimulation at a holding potential of −70 mV, and Ih currents (bottom) recorded in the same VTA neuron by applying voltage steps varying from −70 mV …
Figure 5 with 1 supplement
Corticonuclear and nucleocortical connections between crus I and VTA-projecting DCN neurons in the DN.
(A and B) Representative images of a midbrain slice (left) and a cerebellar slice (right) in (A). In the left panel, the area within the white dotted square is magnified in (B), and dotted lines in …
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Figure 5—source data 1
p Values and statistical tests related to Figure 5.
- https://cdn.elifesciences.org/articles/72981/elife-72981-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Nucleocortical connections of the VTA-projecting DCN neurons.
(A) Screening of GFP-positive collateral axons of VTA-projecting DCN neurons in the whole cerebellar cortex, following double injections into the DCN and the VTA, as shown at the top. The …
Figure 6 with 1 supplement
Specific inhibition of VTA-projecting DCN neurons in the DN ameliorates depression-like behaviors.
(A) Diagram of combined AAV injection. Bilateral injections of AAV-sSyn-tdT and AAV-sSyn-FLEX-hM4Di(Gi)–2AGFP was carried out into the DCN and a mixture of AAV-sSyn-mTBFP and retroAAV-CAG-Cre was …
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Figure 6—source data 1
p Values and statistical tests related to Figure 6.
- https://cdn.elifesciences.org/articles/72981/elife-72981-fig6-data1-v2.xlsx
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Figure 6—source data 2
Effect sizes for behavior results related to Figure 6.
- https://cdn.elifesciences.org/articles/72981/elife-72981-fig6-data2-v2.xlsx
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Figure 6—source data 3
Numbers of mice for each sex used in each behavioral test related to Figure 6.
- https://cdn.elifesciences.org/articles/72981/elife-72981-fig6-data3-v2.xlsx
Figure 6—figure supplement 1
Changes in body weights of mice used for Figure 6.
Comparisons of body weight changes in mice used for Figure 6B–D (A) and Figure 6E–G (B). ** < 0.01, *** < 0.001, one-way ANOVA followed by the Fisher’s LSD post hoc test. Data are shown as the mean …
Figure 7 with 2 supplements
Excitation of VTA-projecting DCN neurons in the DN is sufficient to trigger depression-like behaviors in the absence of stress.
(A and E) Diagram of the experimental time course to check the effects of the excitation of VTA-projecting DCN neurons alone on depression-like behaviors. The OFT, TST, and FST were performed after …
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Figure 7—source data 1
p Values and statistical tests related to Figure 7.
- https://cdn.elifesciences.org/articles/72981/elife-72981-fig7-data1-v2.xlsx
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Figure 7—source data 2
Effect sizes for behavior results related to Figure 7.
- https://cdn.elifesciences.org/articles/72981/elife-72981-fig7-data2-v2.xlsx
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Figure 7—source data 3
Numbers of mice for each sex used in each behavioral test related to Figure 7.
- https://cdn.elifesciences.org/articles/72981/elife-72981-fig7-data3-v2.xlsx
Figure 7—figure supplement 1
No additive effects of chemogenetic excitation on RS-dependent depression-like behaviors.
(A) Diagram of combined AAV injection. Bilateral injections of AAV-sSyn-tdT and AAV-sSyn-FLEX-hM3Dq(Gq)–2AGFP was carried out into the DCN and a mixture of AAV-sSyn-mTBFP and retroAAV-CAG-Cre was …
Figure 7—figure supplement 2
Changes in body weights of mice used for Figure 7.
Comparisons of body weight changes in mice used for Figure 7—figure supplement 1B–D (A), Figure 7—figure supplement 1E–G (B), Figure 7B–D (C) and Figure 7F–J and Figure 7—figure supplement 1H (D). …
Figure 8
Diagram summarizing the results of this study.
In this study, we demonstrated that the neural circuit from crus I of the cerebellar cortex to the DN and then to the VTA is functionally connected (left), and that the neuronal circuit composed of …
Videos
Video 1
TST of the CTR group.
Video 2
TST of the RS group.
Video 3
FST of the CTR group.
Video 4
FST of the RS group.
Tables
Table 1
Pearson correlation coefficient analysis between TST and FST, TST and OFT, and FST and TST.
The correlation was calculated from data of all mice used in each series of experiments for Figure 2J–L, Figure 6B–D, or Figure 7B–D.
| Figure | Test | Sample numbers | Comparison | Coefficient (r) | p value | Significance |
|---|---|---|---|---|---|---|
| Figure 2J-L | Effects of excitation of crus I PCs on RS | 119 | TST vs FST | 0.464 | 1.17 × 10–7 | *** |
| TST vs OFT | 0.153 | 0.0961 | ||||
| FST vs OFT | 0.0303 | 0.743 | ||||
| Figure 6B-D | Effects of inhibition of VTA-projecting DCN neurons on RS | 116 | TST vs FST | 0.327 | 3.42 × 10–4 | *** |
| TST vs OFT | –0.119 | 0.204 | ||||
| FST vs OFT | 0.0474 | 0.0613 | ||||
| Figure 7B-D | Only excitation of VTA-projecting DCN neurons | 70 | TST vs FST | 0.439 | 1.43 × 10–4 | *** |
| TST vs OFT | –0.0308 | 0.8 | ||||
| FST vs OFT | –0.0143 | 0.907 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL/6 J mice | Orient Bio | N/A | |
| Strain, strain background (Mus musculus) | ICR mice; CrljOri:CD1 | Orient Bio | N/A | |
| Genetic reagent (Mus musculus) | Gad2-IRES-Cre mice; Gad2tm2(cre)Zjh/J | Jackson Laboratories | Stock No: 010802; RRID:IMSR_JAX:010802 | |
| Genetic reagent (Mus musculus) | Pcp2-cre mice; B6.129-Tg(Pcp2-cre)2Mppin/J | Jackson Laboratories | Stock No: 004146; RRID:IMSR_JAX:004146 | |
| Genetic reagent (Mus musculus) | Ai six reporter mice; B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J | Jackson Laboratories | Stock No: 007906; RRID:IMSR_JAX:007906 | |
| Antibody | Mouse monoclonal anti-calbindin | Sigma-Aldrich | Cat#C9848; RRID:AB_476894 | (1:200) |
| Antibody | Rabbit polyclonal anti-TH | Millipore | Cat#AB152; RRID: AB_390204 | (1:400) |
| Antibody | Rabbit monoclonal anti-c-Fos | Cell Signalling Technology | Cat#2250; RRID: AB_2247211 | (1:300) |
| Antibody | Mouse monoclonal anti-NeuN | Millipore | Cat#MAB377; RRID: AB_2298772 | (1:200) |
| Antibody | Mouse monoclonal anti-GAD67 | Millipore | Cat#MAB5406; RRID:AB_2278725 | (1:1000) |
| Antibody | Mouse monoclonal anti-GFAP | Millipore | Cat#MAB360; RRID:11212597 | (1:200) |
| Antibody | Goat anti-mouse IgG(H + L) alexa fluor@647 | Invitrogen | Cat#A21235; RRID: AB_2535804 | (1:200) |
| Antibody | Goat anti-rabbit IgG(H + L) alexa fluor@647 | Invitrogen | Cat#A21245; RRID: AB_141775 | (1:200) |
| Antibody | Rabbit polyclonal anti-OLLAS | Antibodies-online | ABIN1842163 | (1:1000) |
| Chemical compound, drug | Clozapine N-oxide | Hello Bio | Cat#HB1807 | 10 mg/kg, i.p. |
| Chemical compound, drug | Bicuculline methochloride | Tocris Bioscience | Cat#0131 | |
| Chemical compound, drug | Mounting medium | Vector Laboratories | Cat#H-1400 | |
| Chemical compound, drug | Mounting medium with DAPI | Vector Laboratories | Cat#H-1500 | |
| Chemical compound, drug | Prolong diamond antifade mountant | Thermo Fisher Scientific | Cat#P36961 | |
| Chemical compound, drug | Cholera toxin subunit B, Alexa flour 647 conjugate | Thermo Fisher Scientific | Cat#C34778 | |
| Chemical compound, drug | Tetrodotoxin | Tocris Bioscience | Cat#1,069 | |
| Chemical compound, drug | 4-Aminopyridine | Tocris Bioscience | Cat#0940 | |
| Recombinant DNA reagent | pAAV-hSyn-DIO-hM3Dq(Gq)-mCherry | Addgene | Addgene plasmid #44,361 | |
| Recombinant DNA reagent | pAAV-hSyn-DIO-hM4Di(Gi)-mCherry | Addgene | Addgene plasmid #44,362 | |
| Recombinant DNA reagent | pAAV-CaMKIIa-hChR2(H134R)-EYFP | Addgene | Addgene plasmid #26,969 | |
| Recombinant DNA reagent | AAV-CaMKIIa- GCaMP6f-P2A-nls-dTomato | Addgene | Addgene plasmid #51,087 | |
| Recombinant DNA reagent | pBAD-mTagBFP2 | Addgene | Addgene plasmid #34,632 | |
| Recombinant DNA reagent | ptdTomato-N1 | Clontech | Cat#632,532 | |
| Recombinant DNA reagent | paavCAG-iCre | Addgene | Addgene plasmid #51,904 | |
| Recombinant DNA reagent | paavCAG-sfGFP | Addgene | Will be deposited | |
| Recombinant DNA reagent | paavCAG-pre- mGRASPi- mCerulean | Addgene | Will be deposited | |
| Recombinant DNA reagent | paavCAG-post-mGRASPi-2AdT | Addgene | Addgene plasmid #34,912 | |
| Recombinant DNA reagent | rAAV2-retro helper | Addgene | Addgene plasmid #81,070 | |
| Biological sample (AAV) | AAV-sSyn-GFP | Kim et al., Kim et al., 2015a | ||
| Biological sample (AAV) | AAV-sSyn-tdT | This paper | Further information will be provided by the Lead Contact upon request. | |
| Biological sample (AAV) | AAV-sSyn-mTBFP | This paper | Further information will be provided by the Lead Contact upon request. | |
| Biological sample (AAV) | AAV-sSyn-ChR2-GFP | This paper | Further information will be provided by the Lead Contact upon request. | |
| Biological sample (AAV) | AAV-sSyn-ChR2-2AdT | This paper | Further information will be provided by the Lead Contact upon request. | |
| Biological sample (AAV) | AAV-sSyn-FLEX-GFP | This paper | Further information will be provided by the Lead Contact upon request. | |
| Biological sample (AAV) | AAV-sSyn-FLEX-ChR2-GFP | This paper | Further information will be provided by the Lead Contact upon request. | |
| Biological sample (AAV) | AAV-sSyn-FLEX-hM3Dq(Gq)–2AGFP | This paper | Further information will be provided by the Lead Contact upon request. | |
| Biological sample (AAV) | AAV-sSyn-FLEX-hM4Di(Gi)–2AGFP | This paper | Further information will be provided by the Lead Contact upon request. | |
| Software, algorithm | pClamp 10 | Axon instruments | RRID:SCR_011323 | |
| Software, algorithm | NIS-Element | Nikon | RRID:SCR_014329 | |
| Software, algorithm | Fiji | NIH | RRID:SCR_002285 | |
| Software, algorithm | Ethovision | Noldus | RRID:SCR_000441 | |
| Software, algorithm | Origin | Origin Lab | RRID:SCR_014212 |
Table 2
Numbers of DAPI- and c-Fos-positive cells in each subregion of DCN at different time periods of RS.
| RS duration | DCN sub-region | Control | Stress | RS-Gq-CNO | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| DAPI | c-Fos | N | DAPI | c-Fos | N | DAPI | c-Fos | N | ||
| 3 day | DN | 94.6 ± 8 | 31.4 ± 4.2 | 5 | 97.2 ± 28.5 | 50.8 ± 14.6 | 5 | - | - | - |
| 7 day | DN | 152 ± 15 | 41.8 ± 14.2 | 5 | 152 ± 21.7 | 96.4 ± 20.2 | 5 | - | - | - |
| 10 day | DN | 128.2 ± 18.7 | 32.6 ± 8.7 | 5 | 119.6 ± 20.3 | 67 ± 11.2 | 5 | - | - | - |
| 14 day | DN | 147.6 ± 46.5 | 36.4 ± 12.9 | 8 | 109.3 ± 53.9 | 54.8 ± 22.3 | 8 | 95.8 ± 11.1 | 21.4 ± 10.7 | 5 |
| IPN | 114.3 ± 30.5 | 35.5 ± 9.5 | 8 | 99.6 ± 32 | 47 ± 24 | 8 | - | - | - | |
| FN | 103.8 ± 29 | 35.5 ± 9.9 | 8 | 112.1 ± 33.2 | 47.5 ± 21 | 8 | - | - | - | |
