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. 2021 Sep 9;13(1):309–337.e3. doi: 10.1016/j.jcmgh.2021.08.016

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© 2022 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

3D reconstruction of intraganglionic connectivity and synaptic sites. (A1) Intraganglionic connectivity was confirmed based on (A1) confocal imaging and (A2) 3D reconstruction using Imaris (Bitplane, Belfast, United Kingdom). The ganglion shown is from colon area in Figure 2D4. Local cell–cell contact is shown for 3 neurons (arrows). Cell #1 has close contacts (orange balls) with 4 cells. Cell #2 has 4 close contacts (yellow balls), and cell #3 has close contacts on 3 neurons with multiple sites on at least 1 (orange balls). Each of these cells expresses calbindin and ChAT, suggesting that they are IPANs. Immunolabeling with the presynaptic marker synaptophysin, confirms the presence of en passant synapses (B3 and B4). (B1) CGRPA neurons have prominent varicosities and (B2) variable numbers of synaptophysin contacts (B3 and B4) on both their somas and axons. (B1–B4) Images are from the proximal colon area D4 (Figure 2). Scale bars: 50 μm (A1 and A2), and 25 μm (B1–B4).