Midbrain dopaminergic inputs gate amygdala intercalated cell clusters by distinct and cooperative mechanisms in male mice
- Hertie Institute for Clinical Brain Research, Germany
- Centre for Integrative Neuroscience, Germany
- Department of Neurobiology, Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, Germany
- Department of Pharmacology, Medical University of Innsbruck, Austria
Figures
Figure 1 with 5 supplements
Dopaminergic fibers from VTA/SNC targeting dm- and vm-ITC clusters in the amygdala co-label with vGAT and vGluT1/2.
(A–B) Maximum intensity projection confocal images illustrating ChR2-YFP+ axons (green) originating from tyrosine hydroxylase (TH)-positive dopaminergic neurons (TH, red) in the ventral tegmental …
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Figure 1—source data 1
Data Figure 1D.
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Specific expression of ChR2-YFP in dopaminergic midbrain neurons.
(A) Experimental strategy: Targeting of dopaminergic midbrain neurons by injecting DAT-Cre mice with AAV-DIO-ChR2-YFP. Subsequent examination of injection and projection sites using tyrosine hydroxyl…
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Figure 1—figure supplement 1—source data 1
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig1-figsupp1-data1-v1.xlsx
Figure 1—figure supplement 2
Labeling of TH-positive axons in the amygdala.
(A) Fluorescence microscopic overview image of the amygdala showing ChR2-YFP+ axons distributed in central amygdala (CeA), basal amygdala (BA), intercalated cell (ITC) clusters, and the …
Figure 1—figure supplement 3
Example of 3D analysis of axonal volumes in ITC clusters.
Left: Image volume showing FoxP2-labeled nuclei (magenta) of neurons in the dorsomedial-intercalated cell (dm-ITC) cluster and midbrain dopaminergic axons expressing ChR2-YFP (green). Right: Surface …
Figure 1—figure supplement 4
Quantitative analysis TH-staining reveals stronger labeling in dm- versus vm-ITC clusters.
(A) Representative images of tyrosine hydroxylase (TH) and FoxP2 immunoreactivity (IR) from consecutive amygdala sections of C57BL/6J adult male mice. FoxP2-IR, associated primarily with nuclei of …
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Figure 1—figure supplement 4—source data 1
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig1-figsupp4-data1-v1.xlsx
Figure 1—figure supplement 5
Dm-ITC and vm-ITC clusters receive differential inputs from VTA and SNC.
(A) Fluorescence microscopic overview image of the dopaminergic midbrain showing neurons transduced with ChR2-YFP (green) and ChR2-mCherry (red) in ventral tegmental area (VTA) and substantia nigra …
Figure 2 with 4 supplements
Stimulation of midbrain dopaminergic fibers evokes mainly GABAergic PSCs in dm- and vm-ITCs.
(A) Fluorescence (left) and infrared differential interference contrast image of an amygdala slice with patch recording pipette in the dorsomedial-intercalated cell (dm-ITC) cluster (right). …
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Figure 2—source data 1
Data Figure 2C, E-F.
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig2-data1-v1.xlsx
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Figure 2—source data 2
Data Figure 2C, H-I.
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig2-data2-v1.xlsx
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Figure 2—source data 3
Data Figure 2C and Figure 2—figure supplement 2B-C.
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig2-data3-v1.xlsx
Figure 2—figure supplement 1
Overview of ChR2-YFP injection and expression sites in the midbrain for co-release experiments.
Brain atlas overlays of sites showing distribution of ChR2-YFP-labeled neuronal cell bodies in the dopaminergic midbrain for all animals in which fast postsynaptic currents (PSCs) were recorded. (A) …
Figure 2—figure supplement 2
Fast PSCs onto CeA neurons are mainly glutamatergic.
(A) Example traces of mixed postsynaptic currents (PSCs) evoked by dopaminergic fiber stimulation recorded from a central amygdala (CeA) neuron at different holding potentials (−70, 0, and 40 mV). …
Figure 2—figure supplement 3
Locations of neurons receiving co-release and no co-release from DA midbrain afferents.
(A) Distribution of recorded dorsomedial (dm)- and ventromedial-intercalated cells (vm-ITCs), as well as central amygdala (CeA) neurons, at different bregma levels color-coded by co-release type. (B)…
Figure 2—figure supplement 4
Fast PSCs from midbrain dopaminergic fibers onto dm-ITCs are monosynaptic.
(A) Example traces of postsynaptic currents (PSCs) evoked by dopaminergic fiber stimulation recorded from a dorsomedial-intercalated cell (dm-ITC) at different holding potentials (−70, 0, and 40 …
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Figure 2—figure supplement 4—source data 1
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig2-figsupp4-data1-v1.xlsx
Figure 3 with 2 supplements
DA application or phasic stimulation of midbrain inputs hyperpolarizes a fraction of dm-ITCs.
(A, D) Representative traces of dorsomedial-intercalated cells (dm-ITCs) recorded at resting membrane potential in current clamp mode from young (postnatal day 20–28) and adult (8–10 weeks) mice. …
Figure 3—figure supplement 1
30 Hz stimulation hyperpolarizes dm-ITCs in the presence of a GABAB receptor blocker.
Left: Representative traces of dorsomedial-intercalated cells (dm-ITCs) recorded in current clamp mode from adult mice. Trains of 10 pulses at 30 Hz optogenetic stimulation of dopaminergic (DA) …
Figure 3—figure supplement 2
Distribution of dm-ITCs according to their response to DA and 30 Hz stimulation.
(A, B) Distribution of recorded dorsomedial-intercalated cells (dm-ITCs) at different bregma levels in young (A) and adult mice (B) color-coded according to their response to dopamine (DA) …
Figure 4 with 3 supplements
Phasic stimulation of midbrain dopaminergic fibers alters sIPSC amplitude in dm-ITCs in a DA-R-dependent manner.
(A, C, E) Top: Experimental protocol. Spontaneous inhibitory postsynaptic current (sIPSC) frequency and amplitude were assessed before and after optogenetic stimulation of midbrain dopaminergic …
Figure 4—figure supplement 1
Overview of ChR2-YFP injection and expression sites in the midbrain for phasic and tonic stimulation experiments.
Brain atlas overlays of sites showing distribution of ChR2-YFP-labeled neuronal cell bodies for experiments related to Figure 4 and its supplements. (A) Injection sites for 30 Hz stimulation onto …
Figure 4—figure supplement 2
Phasic stimulation of midbrain dopaminergic fibers does not affect sEPSCs in dm-ITCs.
(A) Top: Experimental protocol. Spontaneous excitatory postsynaptic currents (sEPSCs) were assessed in dorsomedial-intercalated cells (dm-ITCs) before and after phasic stimulation of dopaminergic …
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Figure 4—figure supplement 2—source data 1
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig4-figsupp2-data1-v1.xlsx
Figure 4—figure supplement 3
Phasic stimulation of midbrain dopaminergic fibers alters sIPSC amplitude in vm-ITCs.
(A) Top: Experimental protocol. sIPSCs were assessed in ventromedial-intercalated cells (vm-ITCs) before and after phasic stimulation of dopaminergic fibers (30 Hz, 10 pulses, 10 sweeps). Left: …
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Figure 4—figure supplement 3—source data 1
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig4-figsupp3-data1-v1.xlsx
Figure 5 with 1 supplement
DA via DRD1 mediates presynaptic depression between ITC clusters.
(A) Confocal images of the amygdala showing ChR2-YFP-expressing neurons in the dorsomedial-intercalated cell (dm-ITC) cluster and their efferents targeting the ventromedial-intercalated cell (vm-ITC)…
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Figure 5—source data 1
Data Figure 5B.
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig5-data1-v1.xlsx
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Figure 5—source data 2
Data Figure 5C.
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig5-data2-v1.xlsx
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Figure 5—source data 3
Data Figure 5E-F.
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig5-data3-v1.xlsx
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Figure 5—source data 4
Data Figure 5H–I.
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig5-data4-v1.xlsx
Figure 5—figure supplement 1
An l-ITC→dm-ITC inhibitory pathway that is modulated by DA via a presynaptic mechanism.
(A) Experimental scheme. AAV-DIO-ChR2-YFP was injected into the amygdala of FoxP2-Cre mice to specifically infect the lateral intercalated cell (l-ITC) cluster, and optically evoked postsynaptic …
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Figure 5—figure supplement 1—source data 1
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig5-figsupp1-data1-v1.xlsx
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Figure 5—figure supplement 1—source data 2
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig5-figsupp1-data2-v1.xlsx
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Figure 5—figure supplement 1—source data 3
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig5-figsupp1-data3-v1.xlsx
Figure 6 with 3 supplements
Early extinction enhances GABA release from midbrain terminals and DA-mediated depression of the dm-ITC→vm-ITC pathway.
(A) Experimental scheme: To investigate the VTA/SNC→dm-ITC pathway, ChR2 was transduced in dopaminergic midbrain neurons of DAT-Cre mice. To investigate dopamine (DA) modulation of the dm-ITC→vm-ITC …
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Figure 6—source data 1
Data Figure 6C–D.
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig6-data1-v1.xlsx
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Figure 6—source data 2
Data Figure 6F.
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig6-data2-v1.xlsx
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Figure 6—source data 3
Data Figure 6G.
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig6-data3-v1.xlsx
Figure 6—figure supplement 1
Behavioral data from DAT-Cre mice used for ex vivo recordings and viral injection sites.
(A) Experimental strategy for targeting of dopaminergic midbrain neurons by injecting DAT-Cre mice with AAV-DIO-ChR2-YFP. (B) Freezing levels for behavioral paradigms shown in Figure 6A in virally …
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Figure 6—figure supplement 1—source data 1
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig6-figsupp1-data1-v1.xlsx
Figure 6—figure supplement 2
Behavioral data from mice trained with a long extinction protocol.
Another group of DAT-Cre mice was subjected to extinction training with 25 conditioned stimulus (CS) presentations on days 2 and 3, and extinction recall by four CS presentations on day 4 (n=10 …
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Figure 6—figure supplement 2—source data 1
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig6-figsupp2-data1-v1.xlsx
Figure 6—figure supplement 3
Behavioral data from FoxP2-Cre mice used for ex vivo recordings.
(A) Experimental strategy for targeting of dorsomedial-intercalated cell (dm-ITC) clusters by injecting FoxP2-Cre mice with AAV-DIO-ChR2-YFP. (B) Freezing levels of virally transduced FoxP2-Cre mice …
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Figure 6—figure supplement 3—source data 1
- https://cdn.elifesciences.org/articles/63708/elife-63708-fig6-figsupp3-data1-v1.xlsx
Figure 7
Summary scheme of results.
(A) Impact of dopaminergic midbrain inputs onto intercalated cell (ITC) clusters, central amygdala (CeA), and basal amygdala (BLA). Dopaminergic inputs from ventral tegmental area/substantia nigra …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Male C57BL/6J mice | Charles River or Envigo | Stock#: 000664, RRID:IMSR_JAX:000664 | |
| Genetic reagent (Mus musculus) | Male heterozygousB6.SJL-Slc6a3tm1.1(cre)Bkmn/J mice | The Jackson Laboratory | Stock#: 006660, RRID:IMSR_JAX:006660 | Maintained on C57BL/6J background |
| Genetic reagent (Mus musculus) | Male heterozygousB6.Cg-Foxp2tm1.1(cre)Rpa/J mice | The Jackson Laboratory | Stock#: 030541, RRID:IMSR_JAX: 030541 | Maintained on C57BL/6J background |
| Genetic reagent (Mus musculus) | Male heterozygous ICR.Cg-Gad1tm1.1Tam/Rbrc mice | Tamamaki et al., 2003. J Comp Neurol 467(1):60–79. doi:10.1002/cne.10905 | RRID:IMSR_RBRC03674 | Maintained on C57BL/6J background |
| Strain, strain background (adeno-associated virus) | AAV2/1.EF1a.DIO.hChR2(H134R).eYFP | Addgene / U. Penn Vector Core | Cat# 20298-AAV1, RRID:Addgene viral prep # 20298-AAV1 | |
| Strain, strain background (adeno-associated virus) | AAV2/9.EF1a.DIO.hChR2 (H134R).eYFP | Addgene / U. Penn Vector Core | Cat# 20298-AAV9, RRID:Addgene viral prep # 20298-AAV9 | |
| Strain, strain background (adeno-associated virus) | AAV2/1.EF1a.DIO.hChR2 (H134R).mCherry | Addgene/Viral Vector Facility U. Zurich | Cat# 20297-AAV1, RRID:Addgene viral prep # 20297-AAV1 | |
| Antibody | Anti-GFP (goat polyclonal) | GeneTex | Cat# GTX26673, RRID:AB_371426 | IF(1:500) |
| Antibody | Anti-GFP (rabbit polyclonal) | Invitrogen | Cat# A11122, RRID:AB_221569 | IF(1:750 or 1:1000) |
| Antibody | Anti-FoxP2 (rabbit polyclonal) | Abcam | Cat# AB16046, RRID:AB_2107107 | IF(1:1000) |
| Antibody | Anti-FoxP2 (mouse monoclonal) | Merck Millipore | Cat# MABE415, RRID:AB_2721039 | IF(1:1000) |
| Antibody | Anti-FoxP2 (sheep polyclonal) | R and D Systems | Cat# AF5647, RRID:AB_2107133 | IF(1:200), IHC(1:200) |
| Antibody | Anti-TH (rabbit polyclonal) | Millipore | Cat# AB152, RRID:AB_390204 | IF(1:1000), IHC (1:4000) |
| Antibody | Anti-TH (mouse monoclonal) | Merck Millipore | Cat# MAB318, RRID:AB_2313764 | IF(1:1000) |
| Antibody | Anti-Bassoon (mouse monoclonal) | Abcam | Cat# AB82958, RRID:AB_1860018 | IF(1:500) |
| Antibody | Anti-VGAT (guinea pig polyclonal) | Synaptic Systems | Cat# 131 004, RRID:AB_887873 | IF(1:500) |
| Antibody | Anti-VGLUT1 (guinea pig polyclonal) | Chemicon/ Millipore | Cat# AB5905, RRID:AB_2301751 | IF(1:3000) |
| Antibody | Anti-VGLUT2 (guinea pig monoclonal) | Chemicon/ Millipore | Cat# AB5907, RRID:AB_2301731 | IF(1:5000) |
| Antibody | Anti-Goat Alexa 488 (donkey polyclonal) | Invitrogen/Life Technologies | Cat# A-11055, RRID:AB_2534102 | IF(1:1000) |
| Antibody | Anti-Rabbit Alexa 555 (donkey polyclonal) | Invitrogen/Life Technologies | Cat# A-31572, RRID:AB_162543 | IF(1:1000) |
| Antibody | Anti-Mouse Alexa 555 (goat polyclonal) | Invitrogen/Life Technologies | Cat# A-21424, RRID:AB_141780 | IF(1:1000) |
| Antibody | Anti-Rabbit Alexa 647 (donkey polyclonal) | Invitrogen/Life Technologies | Cat# A-31573, RRID:AB_2536183 | IF(1:1000) |
| Antibody | Anti-Mouse Alexa 633 (goat polyclonal) | Invitrogen/Life Technologies | Cat# A-21052, RRID:AB_2535719 | IF(1:1000) |
| Antibody | Anti-Rabbit Alexa 633 (goat polyclonal) | Invitrogen/Life Technologies | Cat# A-21071, RRID:AB_2535732 | IF(1:1000) |
| Antibody | Anti-Rabbit Alexa 488 (donkey polyclonal) | Life Technologies | Cat# A21206, RRID:AB_2535792 | IF(1:1000) |
| Antibody | Anti-Goat Cy5 (donkey polyclonal) | Jackson Immuno-research | Cat# 705-175-147, RRID:AB_2340415 | IF(1:400) |
| Antibody | Anti-Guinea pig DyLight 405 (donkey polyclonal) | Jackson Immuno-research | Cat# 706-475-148, RRID:AB_2340470 | IF(1:500) |
| Antibody | Anti-rabbit (goat polyclonal) | Vector Laboratories | Cat# BA-1000, RRID:AB_2313606 | IHC(1:500) |
| Antibody | Anti-goat/sheep (horse polyclonal) | Vector Laboratories | Cat# BA-9500, RRID:AB_2336123 | IHC(1:500) |
| Chemical compound, drug | Tetrodotoxin citrate (TTX) | Alomone Labs | Cat# T-550 | |
| Chemical compound, drug | 4-amino pyridine (4-AP) | Sigma Aldrich | Cat# 275875 | |
| Chemical compound, drug | Picrotoxin (PTX) | Sigma Aldrich | Cat# P1675 | |
| Chemical compound, drug | 6,7-dinitro quinoxaline-2,3-dione (DNQX) disodium salt | Tocris Bioscience | Cat# 2312 | |
| Chemical compound, drug | DL-2-Amino-5-phosphono pentanoic acid sodium salt (DL-AP5) | BioTrend | Cat# BN0086 | |
| Chemical compound, drug | Dopamine hydrochloride | Sigma Aldrich | Cat# H8502 | |
| Chemical compound, drug | Dihydrexidine hydrochloride (DH) | Tocris Bioscience | Cat# 0884 | |
| Chemical compound, drug | Quinpirole hydrochloride | Sigma Aldrich | Cat# Q102 | |
| Chemical compound, drug | CGP55845 hydrochloride | Tocris Bioscience | Cat# 1248 | |
| Chemical compound, drug | SCH23390 hydrochloride | Tocris Bioscience | Cat# 0925 | |
| Chemical compound, drug | Sulpiride | Tocris Bioscience | Cat# 0895 | |
| Chemical compound, drug | L-745870 trihydro- chloride | Tocris Bioscience | Cat# 1002 | |
| Software, algorithm | SPSS | IBM | RRID:SCR_002865 | |
| Software, algorithm | Igor Pro | WaveMetrics | RRID:SCR_000325 | |
| Software, algorithm | NeuroMatic | NeuroMatic | RRID:SCR_004186 | |
| Software, algorithm | ImageJ | NIH | RRID:SCR_003070 | |
| Software, algorithm | Fiji | Fiji | RRID:SCR_002285 | |
| Software, algorithm | pClamp 10 | Molecular Devices | RRID:SCR_011323 | |
| Software, algorithm | ZEN 2009 Software | Zeiss | RRID:SCR_013672 | |
| Software, algorithm | Huygens Suite 19.04 | Scientific Volume Imaging | RRID:SCR_014237 | |
| Software, algorithm | Imaris Software, versions 7.6.1 and 9.7.0 | Oxford Instruments, Bitplane | RRID:SCR_007370 | |
| Other | NeuroTrace stain 435/455 | Thermo Fisher Scientific | Cat# N21479 | |
| Other | Vectastain Elite ABC-Peroxidase kit | Vector Laboratories | Cat# PK-7100, RRID:AB_2336827 | |
| Other | CY3-Streptavidin | Dianova/ Jackson Immuno-Research | Cat# 016-160-084, RRID:AB_2337244 | |
| Other | Pacific Blue-Streptavidin | Thermo Fisher Scientific | Cat# S11222 |
