A serial multiplex immunogold labeling method for identifying peptidergic neurons in connectomes
- Max-Planck-Institute for Developmental Biology, Germany
Figures
Figure 1
Development of the siGOLD method.
(A) Schematic flowchart of the siGOLD labeling approach from high-pressure freezing and freeze substitution (HPF-FS) to tracing and 3D reconstruction. Ni, nickel grid, Cu, copper grid. (B) SEM …
Figure 2
Immunolabeling with neuropeptide antibodes on Epon sections.
(A) Representative micrographs with immunogold labeled axons for the neuropeptide antibodies indicated. For the FVa neuropeptide antibody, five adjacent sections are shown (FVa Layer 1–5). (B, C) …
Figure 3 with 1 supplement
Identification of peptidergic neurites by siGOLD in a 72 hpf Platynereis specimen (HT9-5).
(A, C) Anterior view of EM cross-section through the VNC near the first commissure. Dorsal side of larva is to the top. Strongly labeled neurites were analyzed across the whole VNC region. Control …
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Figure 3—source data 1
Tabulated counts of all gold particles in each traced neurite in HT9-5.
(Sheet 1) Detailed gold counts throughout all layers analyzed from the HT9-5 specimen. (Sheet 2) Tabulated data shown in Figure 3B,D. (Sheet 3) DCV counts and gold counts in selected neurites.
- https://doi.org/10.7554/eLife.11147.007
Figure 3—figure supplement 1
Number of DCVs in selected neurite profiles along 100 layers.
Number of DCVs scored along 100 layers in selected neurites in the HT9-5 dataset. The gold counts for the corresponding profiles are shown in Figure 3—source data 1.
Figure 4 with 1 supplement
Whole-mount IF of Platynereis larvae with antibodies raised against neuropeptides labels distinct subsets of neuronal tracks in the VNC.
(A) Ventral view of Platynereis VNC, stained with an anti-acetylated tubulin antibody. This is the registered average VNC generated from scans of 36 larvae. (B–L) Whole-mount IF of Platynereis …
Figure 4—figure supplement 1
Morpholino-mediated knockdown of proneuropeptides followed by whole-mount IF indicates antibody specificities.
Whole-mount IF of 72 hpf uninjected control Platynereis larvae and larvae micro-injected with translation-blocking morpholinos (MOs) targeting different neuropeptide precursor genes. FVa-knockdown …
Figure 5
Axonal arrangements in the VNC detected by whole-body IF spatially match those detected by siGOLD.
(A, B) Ventral overview of individual registered full-body IF with antibodies raised against 11 different neuropeptides (colors). Image is cropped to show only the VNC in the first segment. White …
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Figure 5—source data 1
Acetylated tubulin reference signal used for image registration.
The reference is based on the registration and averaging of the acetylated tubulin signal from 36 whole-body confocal stacks. The file is a mult-image TIFF stack and can be opened e.g. with Fiji or ImageJ.
- https://doi.org/10.7554/eLife.11147.012
Figure 6
siGOLD labeling in a whole-body serial EM dataset (HT9-4).
(A) Schematic of the HT9-4 specimen showing the position of the VNC (grey), ventral view. The entire larva was fully sectioned and imaged. Colored lines indicate the position of sections that were …
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Figure 6—source data 1
Complete layer statistics of the sections and images of HT9-4.
The table is updated from (Randel et al., 2015) with the information on all immunoEM layers.
- https://doi.org/10.7554/eLife.11147.015
Figure 7
Position of siGOLD-labeled neurites in a cross-section of the VNC in HT9-4.
(A) TEM image of a VNC cross-section with the segmented profiles of peptidergic neurites identified by siGOLD. Different colors represent different neuropeptide-antibody labeling. (B) Segmented …
Figure 8
siGOLD labeling and whole-body neuron reconstructions in HT9-4.
(A, G) Full body IF labeling of FVa and PDF-positive cells, ventral view and anterior view, respectively. Note that (A) is 72 hpf and (G) is 48 hpf. Arrowheads point to neuron cell-bodies that were …
Figure 9
siGOLD labeling and whole-body neuron reconstructions in HT9-4.
(A, C) Full body IF labeling of RGWa and MIP-positive cells in 72 hpf larvae, anterior views. Arrowheads point to neurons that were traced in EM reconstructions. (B, D) Traced neurons, identified …
Figure 10 with 2 supplements
Reconstruction of a siGOLD-labeled peptidergic circuit.
(A, B) SEM image of the nuchal organs (arrowhead) in the dorsal-posterior head of a 72 hpf Platynereis larva, dorsal view. Boxed area is shown enlarged in (B). Arrowheads point at motile cilia above …
Figure 10—figure supplement 1
Morphology of SNnuch and INarc neurons.
(A, D) Four examples of SNnuch sensory neurons with the presynaptic (red) and postsynaptic sites shown. (E, H) Morphology of the four INarc interneurons with the presynaptic (red) and postsynaptic …
Figure 10—figure supplement 2
Number of synaptic inputs from SNnuch cells to all postsynaptic targets.
All postsynaptic targets of the SNnuch neurons are listed.
Videos
Video 1
Neuronal arrangements in the ventral nerve cord detected by whole-body IF and image registration.
Ventral view of 72 hpf Platynereis. VNC (grey) of average whole-body reference template stained with acetylated tubulin, generated from scans of 36 individuals. Onto this reference scaffold, we …
Video 2
3D view of the 72 hpf HT9-4 larva showing fully-traced peptidergic neurons identified by siGOLD.
Ventral view of the 72 hpf Platynereis siGOLD-tagged peptidergic neurons are shown in the following colors: FVa (orange), RGWa (yellow), LEUC (brown), FVRIa (lavender), RYa (forest green), LUQ …
Video 3
Reconstruction of the nuchal organ.
Reconstruction of the nuchal organ from the dorsal head of the 72 hpf Platynereis larva dataset HT9-4. The nuchal organ consists of three multiciliated cells and 16 sensory cells which project a …
Video 4
Reconstruction of the nuchal organ circuit.
The nuchal organ sensory cells (SNnuch, red) connect to two pairs of INarc interneurons (orange). The adult eye photoreceptors (dark blue) are shown for reference. Several other neurons are shown in …
Tables
Table 1
List of antibodies used
| NP precursor name | Abbreviation | Antigen |
|---|---|---|
| FMRFamide | FMRFa | (C)FMRFa |
| RYamide | RYa | (C)VFRYa |
| Myoinhibitory peptide/Allatostatin B | MIP | (C)AWNKNSMRVWa or (C)VWa |
| RGWamide | RGWa | (C)RGWa or (C)GWa |
| Proenkephalin | PENK | (C)YGDLSFSNSNYa |
| Luqin | LUQ | (C)WRPQGRFa |
| Allatotropin | ATO | (C)GFRTGAYDRFSHGFa |
| Pigment dispersing factor | (C)NPGTLDAVLDMPDLMSLa | |
| Leucokinin | LEUC | (C)KFTPWAa |
| FVamide | FVa | (C)AHRFVa or (C)FVa |
| FVRIamide | FVRIa | (C)FVRIa |
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The full name and abbreviation of neuropeptide precursors that contain the neuropeptides used for immunoEM. The FMRFa, RYa, MIP short and long, RGWa, FVa short and long, and FVRIa antibodies have been described previously (Conzelmann and Jékely, 2012; Conzelmann et al., 2011; Conzelmann et al., 2013a; Jékely et al., 2008). All 11 neuropeptides are amidated (a). A Cys (C) was added to the N-terminus of each peptide to allow coupling during immunization and affinity purification. All antibodies were generated in rabbits. For FVa long, a rat antibody was also generated.
Table 2
Number of gold particles in SNnuch neurons in three layers labeled with the PDF antibody.
| Layer | Layer | ||||||
|---|---|---|---|---|---|---|---|
| SNnuch | 703 | 723 | 787 | SNnuch | 703 | 723 | 787 |
| r1 | 0 | 2 | 0 | l1 | 0 | 0 | 0 |
| r2 | 0 | 0 | 0 | l2 | 1 | 0 | 4 |
| r3 | 0 | 0 | 0 | l3 | 0 | 0 | 0 |
| r4 | 0 | 0 | 7 | l4 | 0 | 0 | 0 |
| r5 | 0 | 0 | 0 | l5 | 0 | 0 | 0 |
| r6 | 2 | 0 | 0 | l6 | 0 | 3 | 10 |
| r7 | 0 | 0 | 0 | l7 | 0 | 0 | 2 |
| r8 | 0 | 1 | 2 | l8 | 1 | 0 | 3 |
| r9 | 0 | 0 | 0 | l9 | 0 | 0 | 0 |
| r10 | 0 | 0 | 0 | l10 | 0 | 0 | 0 |
| r11 | 0 | 0 | 0 | l11 | 0 | 0 | 0 |
| r12 | 0 | 3 | 0 | l12 | 6 | 1 | 0 |
| r13 | 1 | 3 | 2 | l13 | 9 | 9 | 6 |
| r14 | 0 | 0 | 0 | l14 | 18 | 0 | 0 |
| r15-r20 | 0 | 0 | 0 | l15 | 0 | 0 | 0 |
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Number of gold particles in SNnuch neurons identified in three different immunogold layers labeled with the PDF antibody in all SNnuch sensory neurons.
