Strikingly different neurotransmitter release strategies in dopaminergic subclasses
- Centre for Developmental Neurobiology, Institute of Psychiatry, Psychology and Neuroscience (IoPPN), King's College London, United Kingdom
Figures
Figure 1
Labelling putative presynaptic structures in individual olfactory bulb (OB) dopaminergic (DA) neurons.
(A) Log2-normalised expression of Th (tyrosine hydroxylase), Syp (Synaptophysin), and Slc32a1 (vGAT) mRNA in DA neurons from single-cell RNA sequencing data (Brann et al., 2020). (B) Example confocal images of endogenous immunostaining for synaptophysin (green) and TH (magenta) on the left, and vGAT (blue) and TH (magenta) on the right. Both images were taken in the glomerular layer of the OB. Yellow arrowheads point to small clusters where TH and synaptophysin or TH and vGAT co-localise. Scalebars: 5 μm (main) and 0.5 μm (inset) for the images on the left, 4 μm (main) and 1 μm (inset) for the images on the right. (C) Strategy to label putative presynaptic release sites in individual DA neurons. (D) Example confocal image of a successfully labelled DA cell. Inset 1 reveals the TH+ DA identity of the neuron (cyan) and inset 2 highlights the Syn-mRuby puncta (magenta, black). Scalebars: 5 μm. (E) Example confocal image of a GFP+ (green), Syn-mRuby+ (magenta, black) neuronal process co-stained with vGAT (orange, black). Yellow arrowheads indicate examples where Syn-mRuby and vGAT puncta co-localise. The last panel shows the orthogonal views of the bottom punctum. Scalebars: 1 μm.
Figure 2
Anaxonic dopaminergic (DA) neurons have dendritic neurotransmitter release sites.
(A) Example confocal image of a TRIM46-negative anaxonic DA neuron. Blue arrowheads point to examples of other TRIM46+ AISs (orange, black) in the same region which do not co-localise with this neuron’s GFP signal. Scalebars: 5 µm. (B) Snapshot of the same neuron in (A) showing Synaptophysin-mRuby puncta (magenta, black) on the dendrites. Yellow inset highlights a region of the neuron with multiple mRuby+ puncta within the GFP+ (green) processes (yellow arrows). Scalebars: 5 µm and 0.5 µm. (C) Example snapshots from three-dimensional (3D) dendritic reconstructions (green, GFP) and presynaptic puncta detection (magenta, Syn-mRuby) of anaxonic DA neurons. Note: these are not full dendritic reconstructions, but example dendrites. Dotted white circle represents the soma location. Scalebars: 5 µm.
Figure 3 with 2 supplements
Axon-bearing dopaminergic (DA) neurons have release sites on their intermittently myelinated axons, but not on their dendrites.
(A) Stitching of individual confocal stacks processed for maximum intensity projections of olfactory bulb (OB) DA neurons co-stained with tyrosine hydroxylase (TH) (green) and myelin basic protein (MBP, magenta). Yellow arrowheads point to myelinated parts of the axon, blue arrowheads show unmyelinated areas. Scalebar: 10 µm. (B) Example confocal images of a distal DA axon stained with GFP (green), TH (cyan), MBP (orange), and synaptophysin-mRuby (magenta, black). Yellow inset highlights the location of the presynaptic bouton. Yellow arrowheads point to co-localised regions, blue arrowheads show non-co-localisation. Scalebars: 2 µm and 1 µm. (C) Confocal image of an axon-bearing TRIM46+ DA neuron. Yellow arrowheads show co-localised staining for GFP (green) and TRIM46 (orange, black). Scalebars: 2 µm. (D) Soma area of axon-bearing and anaxonic DA neurons. Each dot shows one cell; lines show mean ± SEM; n=11 axon-bearing cells and n=9 anaxonic neurons from N=5 mice; unpaired t-test with Welch’s correction; ****, p<0.0001. (E) Snapshot of the same axon-bearing DA neuron shown in (C), co-stained with GFP (green) and synaptophysin-mRuby (magenta, black). Blue arrows show dendritic segments lacking mRuby label, despite the presence of clear mRuby+ puncta in neighbouring processes from a different GFP+ cell. Scalebars: 2 µm. (F) Dendritic puncta density in axon-bearing and anaxonic DA neurons. All conventions as in D; n=11 axon-bearing cells and n=9 anaxonic neurons from N=5 mice; Mann-Whitney test; ***, p=0.0001.
Figure 3—figure supplement 1
Example confocal images of dopaminergic axons from different mice.
Neurons are stained with GFP (green), tyrosine hydroxylase (TH) (blue), myelin basic protein (MBP) (orange), and Synaptophysin-mRuby (black). Yellow arrows show co-localisation between the different channels; blue arrows show lack of co-localisation. Yellow inset highlights the location of the presynaptic bouton. Images at the bottom are zoomed-in snapshots from the images at the top, and arrows point at the synaptic puncta. Scalebars for axon #2: 2 μm and 1 μm; for axon #3, 10 μm and 5 μm; for axon #4, 1 μm and 0.5 μm; and for axon #5, 2 μm and 1 μm.
Figure 3—figure supplement 2
Axon-bearing DA neurons have larger somas than anaxonic cells, with comparable analysed dendrite lengths and a single outlier showing dramatically high over-expressed levels of syn-mRuby puncta in the soma and dendrites.
(A) Example confocal images showing soma size measurements in axon-bearing and anaxonic dopaminergic (DA) neurons. Axon-bearing (top) and anaxonic (bottom) cells labelled with GFP (green) and TRIM46 (orange). Yellow arrowheads point to the TRIM46-positive segment, indicating that the neuron has an axon. Red lines highlight the soma of the two neurons. Scalebars: 3 μm. (B) Maximum length of traced dendrites for axon-bearing (blue) and anaxonic (magenta) DA neurons. Each dot represents one cell, lines show mean ± SEM, n=11 axon-bearing neurons and n=9 anaxonic cells from N=4 mice, Welch’s t-test, p=0.63, n.s.=non-significant. (C, D) Dendritic mRuby puncta in a strongly over-expressing axon-bearing neuron. (C) Example confocal image with GFP (green) and TRIM46 (orange) labelling, revealing the axon-bearing identity of the neuron. Yellow arrowheads point to the GFP+/TRIM46+ co-localised zone. Scalebars: 2 mm. (D) Example confocal images showing synaptophysin-mRuby label (magenta, black) in the dendrites of the axon-bearing neuron from (C) (green). The levels of synaptophysin-mRuby look dramatically higher and with a less defined puncta profile than in all anaxonic DA cells – note levels of somatic expression compared to Figure 2B. Yellow arrowheads point to examples of detected puncta. Scalebars: 5 μm.
Figure 4 with 2 supplements
Anaxonic dopaminergic (DA) neurons are capable of self-inhibition.
(A) Log2-normalised expression of Th, Drd1, Drd2, Drd3, Drd4, and Drd5 mRNA in olfactory bulb (OB) DA cells. (B) Example trace of an action potential fired by a putative anaxonic DA neuron (left) and its monophasic phase-plane plot profile (right). Note the prolonged repolarisation due to Cs-based internal solution. (C) Example traces of an auto-evoked inhibition (AEI) response recorded before (magenta) and after (grey) the application of gabazine. The subtraction is shown in the orange inset trace. (D) Schematic showing the potential involvement in the AEI response of neighbouring GABAergic neurons activated by dopamine released from the patched DA cell. (E) Example traces of an AEI response before (purple) and after (green) applying D1-like and D2-like receptor blockers (SR 95531 hydrobromide and sulpiride, each at 10 µM). (F) AEI charge before (purple) and after (green) applying dopamine receptor antagonists; n=6 cells from N=4 mice; paired t-test, p=0.21, n.s.=non-significant. (G) Schematic showing the potential involvement in the AEI response of neighbouring GABAergic neurons activated via gap junctions. (H) Example trace of an AEI response in the presence of the gap junction blocker carbenoxolone at 100 µM. (I) AEI charge in the presence of carbenoxolone at 100 µM. Each dot shows one cell; lines show mean ± SEM; n=9 cells from N=4 mice. (J) Schematic showing the potential involvement in the AEI response of neighbouring GABAergic neurons activated by depolarising GABA released from the patched cell. (K) Example trace of an AEI response in the presence of the NKCC1 blocker bumetanide at 20 µM. (L) AEI charge in the presence of bumetanide at 20 µM. All conventions as in F; n=5 cells from N=3 mice.
Figure 4—figure supplement 1
Comparison of the decay constant k of the auto-evoked inhibition response before (magenta) and after (green) the application of D1 and D2 receptor antagonists.
Each dot represents one cell, n=6 cells from N=4 mice, Wilcoxon signed-rank test, p=0.84, n.s.=non-significant.
Figure 4—figure supplement 2
The auto-evoked inhibition response charge is not affected by performing three repeats of the protocol or by switching the holding voltage to -50 mV.
(A) Comparison of the analysed auto-evoked inhibition charge after three repeats of the auto-evoked inhibition protocol. Circles represent individual cells, lines connect data from the same neuron; n=5 cells from N=4 mice; repeated-measures one-way ANOVA, p=0.44, n.s.=non-significant. (B) Comparison of the auto-evoked inhibition charge from a holding potential of either –50 mV or –80 mV. All conventions as in (A), n=5 cells from N=3 mice, paired t-test, p=0.96, n.s.=non-significant.
Figure 5 with 1 supplement
Axon-bearing dopaminergic (DA) neurons do not self-inhibit.
(A) Example trace of an action potential fired by a putative axon-bearing DA neuron (left) and its biphasic phase-plane plot profile (right). Arrows point to spike onset kink (left) and initial segment bump (right). Note the prolonged repolarisation due to Cs-based internal solution. (B) Example traces showing tail current responses in an axon-bearing DA neuron before (blue) and after (grey) the application of gabazine. The subtraction is shown in the orange inset trace. (C) Auto-evoked inhibition (AEI) charge in axon-bearing and anaxonic DA neurons. Each dot shows one cell; lines show mean ± SEM; n=9 axon-bearing cells, n=31 anaxonic cells from N=18 mice; Mann-Whitney test; ****, p<0.0001. (D) Example traces showing spontaneous IPSCs in an axon-bearing DA neuron before (blue) and after (grey) the application of gabazine. (E, F) Example traces showing tail current responses in axon-bearing DA neurons before (blue) and after (grey) the application of gabazine using a paired-pulse protocol (E) and prolonged depolarisation (F). Subtractions are shown in the orange inset traces. (G) Example traces showing that the tail current recorded in axon-bearing DA neurons (blue) is blocked by cadmium (ocre). The subtraction is shown in the orange inset trace.
Figure 5—figure supplement 1
Passive membrane properties reflect soma-size differences between axon-bearing and anaxonic DA neurons.
Axon-bearing neurons show no auto-evoked inhibition across multiple stimulation protocols and display cadmium-sensitive tail currents. (A) Input resistance in axon-bearing (blue) and anaxonic (magenta) dopaminergic (DA) neurons. Each dot represents one cell, horizontal lines show the mean ± SEM, n=11 axon-bearing neurons and n=45 anaxonic cells from N=32 mice, ****, p<0.0001, Mann-Whitney test. (B) Membrane capacitance in axon-bearing (blue) and anaxonic (magenta) DA neurons. All conventions as in (A), n=11 axon-bearing neurons and n=45 anaxonic cells from N=32 mice, Mann-Whitney test, n.s.=non-significant. (C) Comparison of auto-evoked inhibition (AEI) charge for single (blue) and paired response (black) in axon-bearing cells. Each dot represents one cell, grey lines connect measurements from the same cell; n=8 cells from N=7 mice; Wilcoxon matched-pairs signed rank test, p=0.06, n.s.=non-significant. (D) Comparison of the AEI charge in axon-bearing cells following either a 10 ms or 100 ms depolarisation step. All conventions as in (C), n=7 cells from N=6 mice; Wilcoxon matched-pairs signed-rank test, p=0.30, n.s.=non-significant. (E) Cadmium-sensitive current in axon-bearing DA neurons, n=3 cells from N=3 mice, all conventions as in (A).
Author response image 1
Traces of an AEI response recorded before (magenta) and after (green) the application of carbenoxolone (n=1 cell from N=1 mouse).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL/6J mice | Charles River | Strain code 027; RRID:MGI:3028467 | |
| Strain, strain background (M. musculus) | DAT-Cre, B6.SJL-Slc6a3tm1.1(cre)Bkmn/J | The Jackson Laboratory | Jax stock 006660; RRID:IMSR_JAX:006660 | |
| Strain, strain background (M. musculus) | VGAT-Cre, Slc32a1tm2(cre)Lowl/J | The Jackson Laboratory | Jax stock 016962; RRID:IMSR_JAX:016962 | |
| Strain, strain background (M. musculus) | Flex-tdTomato, B6.Cg–Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J | The Jackson Laboratory | Jax stock 007909; RRID:IMSR_JAX:007909 | |
| Antibody | Mouse monoclonal Anti-Tyrosine Hydroxylase | Merck | Catalogue number MAB318; RRID:AB_2201528 | Used at 1:500 1:5000 1:10,000 1:50,000 |
| Antibody | Chicken polyclonal Anti-GFP | Abcam | Catalogue number ab13970; RRID:AB_300798 | Used at 1:1000 |
| Antibody | Guinea pig polyclonal Anti-TRIM46 | Synaptic Systems | Catalogue number 377005; RRID:AB_2721101 | Used at 1:500 |
| Antibody | Rabbit polyclonal Anti-vGAT | Synaptic Systems | Catalogue number 131002; RRID:AB_887871 | Used at 1:1000 |
| Antibody | Rat monoclonal Anti-mCherry (16D7) | Thermo Fisher Scientific | Catalogue number M11217; RRID:AB_2536611 | Used at 1:500 |
| Antibody | Rabbit polyclonal Anti-MBP | Thermo Fisher Scientific | Catalogue number PA5-78397; RRID:AB_2736178 | Used at 1:1000 |
| Antibody | Rat monoclonal Anti-MBP (clone 12) | Merck | Catalogue number MAB386; RRID:AB_94975 | Used at 1:500 |
| Antibody | Guinea pig polyclonal Anti-Synaptophysin1 | Synaptic Systems | Catalogue number 101 004; RRID:AB_1210382 | Used at 1:500 |
| Antibody | Alexa Fluor 488, Goat Anti-Chicken IgY (H+L) secondary antibody | Invitrogen | Catalogue number A-11039; RRID:AB_2534096 | Used at 1:1000 |
| Antibody | Alexa Fluor 488, F(ab’)2-Goat Anti-Mouse IgG (H+L) secondary antibody | Invitrogen | Catalogue number A-11017; RRID:AB_ 2534084 | Used at 1:1000 |
| Antibody | Alexa Fluor 488, Goat Anti-Guinea Pig IgG (H+L) secondary antibody | Invitrogen | Catalogue number A-11073; RRID:AB_ 2534117 | Used at 1:1000 |
| Antibody | Alexa Fluor 647, Goat Anti-Mouse IgG (H+L) secondary antibody | Invitrogen | Catalogue number A-21235; RRID:AB_ 2535804 | Used at 1:1000 |
| Antibody | Alexa Fluor 488, Goat Anti-Rabbit IgG (H+L) secondary antibody | Invitrogen | Catalogue number A-11008; RRID:AB_ 143165 | Used at 1:1000 |
| Antibody | Alexa Fluor 546, Goat Anti-Rabbit IgG (H+L) secondary antibody | Invitrogen | Catalogue number A-11010; RRID:AB_2534077 | Used at 1:1000 |
| Antibody | Alexa Fluor 594, Goat Anti-Rat IgG (H+L) secondary antibody | Invitrogen | Catalogue number A-11007; RRID:AB_10561522 | Used at 1:1000 |
| Transfected construct (Adeno-associated virus) | AAV-DJ-hSyn-FLEx-mGFP-T2A-Synaptophysin-mRuby | Stanford Vector Core Facility | Reference GVVC-AAV-100 | Used at ~3 × 1012 GC/ml |
| Chemical compound, drug | DL-2-Amino-5-phosphonopentanoic acid (APV) | Sigma-Aldrich | Catalogue number A5282 | Used at 50 μM |
| Chemical compound, drug | 2,3-Dioxo-6-nitro-7-sulfamoyl-bnzo[f]quinoxaline (NBQX) | Sigma-Aldrich | Catalogue number N183 | Used at 10 μM |
| Chemical compound, drug | Tetrodoxin citrate (TTX) | Alomone and Tocris | Catalogue numbers T-550 and 1069 | Used at 1 μM |
| Chemical compound, drug | SR 95531 hydrobromide (Gabazine) | Tocris | Catalogue number 1262 | Used at 10 μM |
| Chemical compound, drug | SKF 83566 hydrobromide | Tocris | Catalogue number 1586 | Used at 10 μM |
| Chemical compound, drug | Sulpiride | Sigma-Aldrich | Catalogue number S8010 | Used at 10 μM |
| Chemical compound, drug | Cadmium chloride | Sigma-Aldrich | Catalogue number 202908 | Used at 200 μM |
| Chemical compound, drug | Carbenoxolone disodium | Tocris | Catalogue number 3096 | Used at 100 μM |
| Chemical compound, drug | Bumetanide | Tocris | Catalogue number 3108 | Used at 20 μM |
| Software, algorithm | R package Seurat | The R Project for Statistical Computing | RRID:SCR_016341 | |
| Software, algorithm | ImageJ software (Fiji) | NIH; Schindelin et al., 2012 | RRID:SCR_003070 | |
| Software, algorithm | Prism 10 | GraphPad | RRID:SCR_002798 | |
| Software, algorithm | Patchmaster v2x73 | HEKA Elektronik | RRID:SCR_000034 | |
| Software, algorithm | MATLAB R2024b | MathWorks | RRID:SCR_01622 | |
| Software, algorithm | Imaris v9.1.2 | Oxford Instruments | RRID:SCR_007370 | |
| Software, algorithm | ImarisViewer (v10.2.0) | Oxford Instruments | RRID:SCR_027660 | |
| Software, algorithm | Zen 2010 B-2P1 | Zeiss | RRID:SCR_013672 | |
| Software, algorithm | Inkscape v1.3.2 | Inkscape | RRID:SCR_014479 |
Table 1
Primary antibodies.
| Target | Host | Dilution | Supplier | Reference | Identifier |
|---|---|---|---|---|---|
| TH | Mouse | 1:500 1:5000 1:10,000 1:50,000 | Merck | MAB318 | RRID:AB_2201528 |
| GFP | Chicken | 1:1000 | Abcam | ab13970 | RRID:AB_300798 |
| TRIM46 | Guinea pig | 1:500 | Synaptic Systems | 377005 | RRID:AB_2721101 |
| vGAT | Rabbit | 1:1000 | Synaptic Systems | 131002 | RRID:AB_887871 |
| mCherry | Rat | 1:500 | Thermo Fisher | M11217 | RRID:AB_2536611 |
| MBP | Rabbit | 1:1000 | Thermo Fisher | PA5-78397 | RRID:AB_2736178 |
| MBP | Rat | 1:500 | Millipore | MAB386 | RRID:AB_94975 |
| Synaptophysin | Guinea pig | 1:500 | Synaptic Systems | 101004 | RRID:AB_1210382 |
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Strikingly different neurotransmitter release strategies in dopaminergic subclasses
eLife 14:RP105271.
https://doi.org/10.7554/eLife.105271.3
