Journal: eLife

Article Title: Strikingly different neurotransmitter release strategies in dopaminergic subclasses

doi: 10.7554/eLife.105271

Figure Lengend Snippet: ( A ) Log 2 -normalised expression of Th (tyrosine hydroxylase) , Syp (Synaptophysin), and Slc32a1 (vGAT) mRNA in DA neurons from single-cell RNA sequencing data . ( 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.

Article Snippet: For co-localisation analysis, the 3D centroid coordinates for the synaptophysin-mRuby puncta detected in Imaris were exported and analysed using a custom-written script in MATLAB.

Techniques: Expressing, RNA Sequencing, Immunostaining, Staining

Journal: eLife

Article Title: Strikingly different neurotransmitter release strategies in dopaminergic subclasses

doi: 10.7554/eLife.105271

Figure Lengend Snippet: ( 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.

Article Snippet: For co-localisation analysis, the 3D centroid coordinates for the synaptophysin-mRuby puncta detected in Imaris were exported and analysed using a custom-written script in MATLAB.

Techniques:

Journal: eLife

Article Title: Strikingly different neurotransmitter release strategies in dopaminergic subclasses

doi: 10.7554/eLife.105271

Figure Lengend Snippet: ( 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.

Article Snippet: For co-localisation analysis, the 3D centroid coordinates for the synaptophysin-mRuby puncta detected in Imaris were exported and analysed using a custom-written script in MATLAB.

Techniques: Staining, MANN-WHITNEY

Journal: eLife

Article Title: Strikingly different neurotransmitter release strategies in dopaminergic subclasses

doi: 10.7554/eLife.105271

Figure Lengend Snippet: 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.

Article Snippet: For co-localisation analysis, the 3D centroid coordinates for the synaptophysin-mRuby puncta detected in Imaris were exported and analysed using a custom-written script in MATLAB.

Techniques: Staining

Journal: eLife

Article Title: Strikingly different neurotransmitter release strategies in dopaminergic subclasses

doi: 10.7554/eLife.105271

Figure Lengend Snippet: ( 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 . Yellow arrowheads point to examples of detected puncta. Scalebars: 5 μm.

Article Snippet: For co-localisation analysis, the 3D centroid coordinates for the synaptophysin-mRuby puncta detected in Imaris were exported and analysed using a custom-written script in MATLAB.

Techniques: Expressing