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. 2022 Feb 25;16:836116. doi: 10.3389/fncel.2022.836116

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Copyright © 2022 Szlaga, Sambak, Trenk, Gugula, Singleton, Drwiega, Blasiak, Ma, Gundlach and Blasiak.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Localization, morphology and neuropeptide content of type I and II NI neurons. (A) Reconstruction (relative to bregma, lower right corner), of the localization of type I (blue circles) and type II (pink circles) NI neurons recorded during patch clamp experiments. (B) Post-recording immunofluorescent identification of NI neurons: image of RLN3+ NI neuron (green, white arrowhead); biocytin (red, white arrowhead) and merge. Scale bar: 20 μm. (C) Image of CCK+ NI neuron (yellow, white arrowhead); biocytin (red, white arrowhead) and merge. Scale bar: 20 μm. (D,E) Electrophysiological types of RLN3+ and CCK+ NI neurons, respectively. (F) 2D view of representative dendritic traces of type I (top) and type II (bottom) neurons, drawn with colors representing the number of dendrites intersecting with Sholl spheres. Scale bar: 100 μm. (G) Sholl analysis and comparison of the dendritic trees of type I and II NI neurons; despite a significant interaction between neuron type and distance from soma, significant differences, indicated by asterisks, were found only at 30 and 330 μm from soma. Significance indicated by two-way RM ANOVA and Uncorrected Fisher’s LSD post-hoc test: *p < 0.05, **p < 0.01. (H) Topological parameters of type I and II NI neurons (total dendritic length, maximal branch order, number of primary dendrites, branches, bifurcations and dendritic tips). (I) Images of a representative, biocytin-filled NI RLN3+ neuron (left panel, cell body indicated with a white arrowhead), with thin, tuft-like distal dendrites pointing toward the 4th ventricle (right panel; empty arrowheads indicate exemplary dendritic spines; white dotted line with arrows indicates the extent of tanycytes, T). Boxed areas in the middle panel represent the area surrounding the cell body (left panel) and the tuft-like distal dendrites (right panel). 4V, 4th ventricle; Bar, Barrington’s nucleus; Cb, cerebellum; CCK, cholecystokinin; DR, dorsal raphe nucleus; DTg, dorsal tegmental nucleus; mlf, medial longitudinal fasciculus; NI, nucleus incertus; NIc, nucleus incertus pars compacta; RLN3, relaxin-3; RtTg, reticulotegmental nucleus of the pons; T, tanycytes.