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Nitric Oxide Critically Regulates Purkinje Neuron Dendritic Development Through a Metabotropic Glutamate Receptor Type 1–Mediated Mechanism

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Abstract

Nitric oxide (NO), specifically derived from neuronal nitric oxide synthase (nNOS), is a well-established regulator of synaptic transmission in Purkinje neurons (PNs), governing fundamental processes such as motor learning and coordination. Previous phenotypic analyses showed similar cerebellar structures between neuronal nitric oxide null (nNOS−/−) and wild-type (WT) adult male mice, despite prominent ataxic behavior within nNOS−/− mice. However, a study has yet to characterize PN molecular structure and their excitatory inputs during development in nNOS−/− mice. This study is the first to explore morphological abnormalities within the cerebellum of nNOS−/− mice, using immunohistochemistry and immunoblotting. This study sought to examine PN dendritic morphology and the expression of metabotropic glutamate receptor type 1 (mGluR1), vesicular glutamate transporter type 1 and 2 (vGluT1 and vGluT2), stromal interaction molecule 1 (STIM1), and calpain-1 within PNs of WT and nNOS−/− mice at postnatal day 7 (PD7), 2 weeks (2W), and 7 weeks (7W) of age. Results showed a decrease in PN dendritic branching at PD7 in nNOS−/− cerebella, while aberrant dendritic spine formation was noted in adult ages. Total protein expression of mGluR1 was decreased in nNOS−/− cerebella across development, while vGluT2, STIM1, and calpain-1 were significantly increased. Ex vivo treatment of WT slices with NOS inhibitor L-NAME increased calpain-1 expression, whereas treating nNOS−/− cerebellar slices with NO donor NOC-18 decreased calpain-1. Moreover, mGluR1 agonist DHPG increased calpain-1 in WT, but not in nNOS−/− slices. Together, these results indicate a novel role for nNOS/NO signaling in PN development, particularly by regulating an mGluR1-initiated calcium signaling mechanism.

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Acknowledgments

This work was supported by the Canadian Institutes of Health Research (MOP-133504) awarded to W-Y L. VT, and MJEM were awarded Ontario Graduate Scholarships.

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Fig. S1

Analysis of STIM1 particles within the PN soma. Depicted is a representative image of WT 7W cerebellar tissue stained for STIM1, which is selective for PNs. (A) First, a rectangular region of interest (ROI) is created to encompass the entirety of the PN soma. (B) Next, the ROI is duplicated, and any background staining not associated with PN soma is removed. (C) The Particle Tracker 2D/3D plugin is run using FIJI and identifies the number of STIM1 clusters (visualized by the red circles), and a total number of particles is reported by the plugin. (PPT 908 kb).

Fig. S2

Method of determining number and localization of vGluT2 clusters. (A) Depicted is a representative image of WT 7W CalB staining. (B) The distal border of the PN dendrites (closest to the pial surface) is determined using the freehand line selection tool in FIJI. A straight line is drawn where the proximal dendrites begin. The area in between the white dotted line and the yellow line denotes 100% of the molecular layer. (C) The length of this region is determined, and 20% of the length is calculated, the border of which is represented with a blue line. The area between the blue line and the dotted line denotes the bottom 80% of the molecular layer (where climbing fibers are expected to innervate PNs). (D) These borders determined on the CalB staining are then applied to the respective vGluT2 stain. (E) The Particle Tracker 2D/3D is run, giving the total number of particles detected. Particles detected above the blue line are considered as vGluT2 clusters occupying the upper 20% of the molecular layer, while particles detected between the blue line and white dotted line represents vGluT2 clusters occupying the lower 80% of the molecular layer. (PPT 825 kb).

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Tellios, V., Maksoud, M.J.E., Xiang, YY. et al. Nitric Oxide Critically Regulates Purkinje Neuron Dendritic Development Through a Metabotropic Glutamate Receptor Type 1–Mediated Mechanism. Cerebellum 19, 510–526 (2020). https://doi.org/10.1007/s12311-020-01125-7

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