Midbrain dopaminergic inputs gate amygdala intercalated cell clusters by distinct and cooperative mechanisms in male mice

  1. Ayla Aksoy-Aksel
  2. Andrea Gall
  3. Anna Seewald
  4. Francesco Ferraguti
  5. Ingrid Ehrlich  Is a corresponding author
  1. Hertie Institute for Clinical Brain Research, Germany
  2. Centre for Integrative Neuroscience, Germany
  3. Department of Neurobiology, Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, Germany
  4. Department of Pharmacology, Medical University of Innsbruck, Austria
7 figures, 1 table and 1 additional file

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 …

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…

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 …

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). …

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 …

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 GABAreceptor 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 …

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: …

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)…

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 …

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 …

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 …

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 …

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 …

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
DesignationSource or
reference
IdentifiersAdditional
information
Strain, strain background
(Mus musculus)
Male C57BL/6J miceCharles River or EnvigoStock#: 000664,
RRID:IMSR_JAX:000664
Genetic reagent
(Mus musculus)
Male heterozygousB6.SJL-Slc6a3tm1.1(cre)Bkmn/J miceThe Jackson LaboratoryStock#: 006660,
RRID:IMSR_JAX:006660
Maintained on C57BL/6J background
Genetic reagent
(Mus musculus)
Male heterozygousB6.Cg-Foxp2tm1.1(cre)Rpa/J miceThe Jackson LaboratoryStock#: 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.10905RRID:IMSR_RBRC03674Maintained on
C57BL/6J background
Strain, strain background
(adeno-associated virus)
AAV2/1.EF1a.DIO.hChR2(H134R).eYFPAddgene /
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
AntibodyAnti-GFP (goat polyclonal)GeneTexCat# GTX26673, RRID:AB_371426IF(1:500)
AntibodyAnti-GFP (rabbit polyclonal)InvitrogenCat# A11122, RRID:AB_221569IF(1:750 or 1:1000)
AntibodyAnti-FoxP2 (rabbit polyclonal)AbcamCat# AB16046, RRID:AB_2107107IF(1:1000)
AntibodyAnti-FoxP2 (mouse monoclonal)Merck MilliporeCat# MABE415, RRID:AB_2721039IF(1:1000)
AntibodyAnti-FoxP2 (sheep polyclonal)R and D SystemsCat# AF5647, RRID:AB_2107133IF(1:200),
IHC(1:200)
AntibodyAnti-TH (rabbit polyclonal)MilliporeCat# AB152, RRID:AB_390204IF(1:1000),
IHC
(1:4000)
AntibodyAnti-TH (mouse monoclonal)Merck
Millipore
Cat# MAB318, RRID:AB_2313764IF(1:1000)
AntibodyAnti-Bassoon (mouse monoclonal)AbcamCat# AB82958, RRID:AB_1860018IF(1:500)
AntibodyAnti-VGAT (guinea pig polyclonal)Synaptic SystemsCat# 131 004, RRID:AB_887873IF(1:500)
AntibodyAnti-VGLUT1 (guinea pig polyclonal)Chemicon/
Millipore
Cat# AB5905, RRID:AB_2301751IF(1:3000)
AntibodyAnti-VGLUT2 (guinea pig monoclonal)Chemicon/
Millipore
Cat# AB5907, RRID:AB_2301731IF(1:5000)
AntibodyAnti-Goat Alexa 488 (donkey polyclonal)Invitrogen/Life TechnologiesCat# A-11055, RRID:AB_2534102IF(1:1000)
AntibodyAnti-Rabbit Alexa 555 (donkey polyclonal)Invitrogen/Life TechnologiesCat# A-31572, RRID:AB_162543IF(1:1000)
AntibodyAnti-Mouse Alexa 555 (goat polyclonal)Invitrogen/Life TechnologiesCat# A-21424, RRID:AB_141780IF(1:1000)
AntibodyAnti-Rabbit Alexa 647 (donkey polyclonal)Invitrogen/Life TechnologiesCat# A-31573, RRID:AB_2536183IF(1:1000)
AntibodyAnti-Mouse Alexa 633 (goat polyclonal)Invitrogen/Life TechnologiesCat# A-21052, RRID:AB_2535719IF(1:1000)
AntibodyAnti-Rabbit Alexa 633 (goat polyclonal)Invitrogen/Life TechnologiesCat# A-21071, RRID:AB_2535732IF(1:1000)
AntibodyAnti-Rabbit Alexa 488 (donkey polyclonal)Life TechnologiesCat# A21206, RRID:AB_2535792IF(1:1000)
AntibodyAnti-Goat Cy5 (donkey polyclonal)Jackson Immuno-researchCat# 705-175-147, RRID:AB_2340415IF(1:400)
AntibodyAnti-Guinea pig DyLight 405 (donkey polyclonal)Jackson Immuno-researchCat# 706-475-148, RRID:AB_2340470IF(1:500)
AntibodyAnti-rabbit (goat polyclonal)Vector LaboratoriesCat# BA-1000, RRID:AB_2313606IHC(1:500)
AntibodyAnti-goat/sheep (horse polyclonal)Vector LaboratoriesCat# BA-9500, RRID:AB_2336123IHC(1:500)
Chemical compound, drugTetrodotoxin citrate (TTX)Alomone LabsCat# T-550
Chemical compound, drug4-amino pyridine (4-AP)Sigma AldrichCat# 275875
Chemical compound, drugPicrotoxin (PTX)Sigma AldrichCat# P1675
Chemical compound, drug6,7-dinitro quinoxaline-2,3-dione (DNQX) disodium saltTocris BioscienceCat# 2312
Chemical compound, drugDL-2-Amino-5-phosphono pentanoic acid sodium salt (DL-AP5)BioTrendCat# BN0086
Chemical compound, drugDopamine hydrochlorideSigma AldrichCat# H8502
Chemical compound, drugDihydrexidine hydrochloride (DH)Tocris BioscienceCat# 0884
Chemical compound, drugQuinpirole hydrochlorideSigma AldrichCat# Q102
Chemical compound, drugCGP55845 hydrochlorideTocris BioscienceCat# 1248
Chemical compound, drugSCH23390 hydrochlorideTocris BioscienceCat# 0925
Chemical compound, drugSulpirideTocris BioscienceCat# 0895
Chemical compound, drugL-745870 trihydro- chlorideTocris BioscienceCat# 1002
Software, algorithmSPSSIBMRRID:SCR_002865
Software, algorithmIgor ProWaveMetricsRRID:SCR_000325
Software, algorithmNeuroMaticNeuroMaticRRID:SCR_004186
Software, algorithmImageJNIHRRID:SCR_003070
Software, algorithmFijiFijiRRID:SCR_002285
Software, algorithmpClamp 10Molecular DevicesRRID:SCR_011323
Software, algorithmZEN 2009 SoftwareZeissRRID:SCR_013672
Software, algorithmHuygens Suite 19.04Scientific Volume ImagingRRID:SCR_014237
Software, algorithmImaris Software, versions 7.6.1 and 9.7.0Oxford Instruments, BitplaneRRID:SCR_007370
OtherNeuroTrace stain 435/455Thermo Fisher ScientificCat# N21479
OtherVectastain Elite ABC-Peroxidase kitVector LaboratoriesCat# PK-7100, RRID:AB_2336827
OtherCY3-StreptavidinDianova/
Jackson Immuno-Research
Cat# 016-160-084, RRID:AB_2337244
OtherPacific Blue-StreptavidinThermo Fisher ScientificCat# S11222

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