Received: 5 October 2020 | Revised: 27 April 2021 | Accepted: 1 May 2021 DOI: 10.1002/jnr.24856 RESEARCH ARTICLE The synaptic blocker botulinum toxin A decreases the density and complexity of oligodendrocyte precursor cells in the adult mouse hippocampus Irene Chacon-De-La-Rocha1 | Gemma L. Fryatt2 | Andrea D. Rivera1,3 | Laura Restani4 | Matteo Caleo4,5 | Diego Gomez-Nicola2 | Arthur M. Butt1 1 Institute of Biomedical and Biomolecular Sciences, School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, UK Centre for Biological Sciences, University of Southampton, Southampton, UK 2 Department of Neuroscience, Institute of Human Anatomy, University of Padua, Padua, Italy 3 4 National Research Council, Neuroscience Institute, Pisa, Italy Department of Biomedical Sciences, University of Padua, Padua, Italy 5 Correspondence Diego Gomez-Nicola, Centre for Biological Sciences, University of Southampton, Southampton, UK. Email: d.gomez-nicola@soton.ac.uk Arthur M. Butt, Institute of Biomedical and Biomolecular Sciences, School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, UK. Email: Arthur.butt@port.ac.uk Funding information BBSRC, Grant/Award Number: BB/ M029379/1; MRC, Grant/Award Number: MR/P025811/1; Alzheimer's Research UK, Grant/Award Number: PG2014B-2; University of Portsmouth; CNR – Joint Laboratories Abstract Oligodendrocyte progenitor cells (OPCs) are responsible for generating oligodendrocytes, the myelinating cells of the CNS. Life-long myelination is promoted by neuronal activity and is essential for neural network plasticity and learning. OPCs are known to contact synapses and it is proposed that neuronal synaptic activity in turn regulates their behavior. To examine this in the adult, we performed unilateral injection of the synaptic blocker botulinum neurotoxin A (BoNT/A) into the hippocampus of adult mice. We confirm BoNT/A cleaves SNAP-25 in the CA1 are of the hippocampus, which has been proven to block neurotransmission. Notably, BoNT/A significantly decreased OPC density and caused their shrinkage, as determined by immunolabeling for the OPC marker NG2. Furthermore, BoNT/A resulted in an overall decrease in the number of OPC processes, as well as a decrease in their lengths and branching frequency. These data indicate that synaptic activity is important for maintaining adult OPC numbers and cellular integrity, which is relevant to pathophysiological scenarios characterized by dysregulation of synaptic activity, such as age-related cognitive decline, Multiple Sclerosis and Alzheimer's disease. KEYWORDS BoNT/A, botulinum toxin A, hippocampus, mouse, oligodendrocyte progenitor cell, RRID:AB_143165, RRID:AB_2313606, RRID:AB_2340613, RRID:AB_11213678, RRID:SCR_002798, RRID:SCR_003070, RRID:SCR_016788, RRID:SCR_017348, SNAP-25, SNARE, Synapse Abbreviations: AD, Alzheimer's disease; BoNT/A, botulinum neurotoxin A; Cspg4, chondroitin sulfate proteoglycan 4 (NG2); EM, electron microscopy; FOV, field of view; GABA, gamma aminobutyric acid; h, hours; min, minutes; MS, multiple sclerosis; NG2, neuron-glia 2 (Cspg4); OPCs, oligodendrocyte progenitor cells; PBS, phosphate-buffered saline; SD, standard deviation; SNAP, synaptosomal-associated protein; SNARE, soluble N-ethylmaleimide-sensitive factor-attachment protein receptors; TTX, tetrodotoxin. Irene Chacon-De-La-Rocha and Gemma L. Fryatt contributed equally to the paper. Edited by Stephen Crocker and Cristina Ghiani. Reviewed by Jack Antel and David Martinelli. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Journal of Neuroscience Research published by Wiley Periodicals LLC. J Neurosci Res. 2021;00:1–12. wileyonlinelibrary.com/journal/jnr | 1 2 | CHACON-DE-LA-ROCHA Et AL. 1 | I NTRO D U C TI O N Oligodendrocyte progenitor cells (OPCs) are a significant population of cells in the adult brain with the fundamental function of lifelong generation of oligodendrocytes, which is required to myelinate new connections formed in response to new life experiences and to replace myelin lost through natural “wear and tear” and disease (Hill et al., 2018; Young et al., 2013; Zawadzka et al., 2010). OPCs are identified by their expression of the proteoglycan NG2 (Cspg4) (Ong & Levine, 1999; Stallcup, 1981) and have a complex morphology, extending processes to contact neuronal synapses and nodes of Ranvier, sensing neuronal glumatergic and GABAergic activity Significance Oligodendrocyte progenitor cells (OPCs) are responsible for life-long myelination, which is essential for higher cognitive function. OPCs are renowned for sensing neuronal synaptic activity, which regulates adaptive myelination and underpins learning and cognitive function. This study shows that silencing synaptic activity induces OPC atrophy in the hippocampus and has important implications for the loss of cognitive function associated with aging and neuropathology. (Bergles et al., 2000; Butt et al., 1999; Hamilton et al., 2010; Kukley et al., 2007; Lin & Bergles, 2004; Ziskin et al., 2007). Several lines of evidence indicate neuronal synaptic activity regulates OPC prolifer- scientific purposes, and approved by the Italian Ministry of Health ation and differentiation (Fannon et al., 2015; Hamilton et al., 2017; (protocol # 346/2013-B). All surgical procedures were performed Mangin et al., 2012; Zonouzi et al., 2015). Optogenetic studies have under deep anesthesia and all efforts were made to ameliorate suf- shown that neuronal activity stimulates OPC proliferation and dif- fering of animals. ferentiation in the cerebral cortex (Gibson et al., 2014). Furthermore, motor learning has been shown to drive OPC differentiation, and failure to generate new oligodendrocytes impairs myelination of 2.2 | Animals and tissues newly formed neuronal connections and learning ability (McKenzie et al., 2014; Xiao et al., 2016). Hence, it is proposed that decreased C57BL/6N mice were bred and group housed in the animal facility of synaptic activity may result in disruption of OPC regenerative capac- the CNR Neuroscience Institute in Pisa (Italy). Mice were housed in ity in neurodegenerative diseases, such as Multiple Sclerosis (MS) groups of 4 to 10, under a 12 hr light/dark cycle at 21°C, with food and Alzheimer's disease (AD) (Chacon-De-La-Rocha et al., 2020; and water ad libitum, and experimental groups contained a spread Rivera et al., 2016; Vanzulli et al., 2020). of sexes. Eight mice aged 11–12 weeks were anesthetized by intra- The synaptic protein SNAP-25 is necessary for synaptic vesicle peritoneal injection of Avertin (20 ml/kg, 2,2,2-tribromoethanol so- fusion and its cleavage by botulinum neurotoxins (BoNTs) blocks neu- lution; Sigma-Aldrich, Cat. #T48402) and mounted in a stereotaxic rotransmitter release and synaptic signaling (Caleo & Restani, 2018). frame (David Kopf Instruments, Tujunga, CA), for injection of BoNT/A Developmental myelination is inhibited by ablation of neuronal (n = 4 mice) or sterile vehicle (n = 4 mice). The isolation and purifi- SNAP-25 either by BoNT/A in cell culture (Wake et al., 2011), or cation of BoNT/A was performed in house, as previously described genetically in vivo (Korrell et al., 2019), but the effects of synaptic (Schiavo & Montecucco, 1995; Shone & Tranter, 1995), and its activ- activity on adult OPCs remain unclear. In the present study, we ity assayed and characterized in the hippocampal model (Antonucci investigated the effects of prolonged synaptic silencing on adult et al., 2008; Caleo et al., 2012) (Figure 1). In brief, BoNT/A was iso- OPCs, by local injection of botulinum neurotoxin A (BoNT/A) into lated from cultures of Clostridium botulinum and purified as 150 kDa the mouse hippocampal CA1 region, which has been demonstrated di-chain neurotoxin, as described by (Shone & Tranter, 1995) and to produce a sustained blockade of synaptic transmission via cleav- (Schiavo & Montecucco, 1995). After purification, toxin was dialyzed age of the synaptic protein SNAP-25 (Antonucci et al., 2008; Caleo in 10 mM TrisCl, 150 mM NaCl, pH 6.8 and stored at −80°C until & Restani, 2018; Caleo et al., 2012). Our results demonstrate that use. For hippocampal injections, stock of toxin (50 nM) was thawed the synaptic blocker BoNT/A decreases the density and complexity on ice, diluted to working concentration (1nM, in 2% rat serum albu- of OPCs, leading to overall cellular atrophy. These results indicate min, PBS) and kept on ice until briefly warming to hand temperature synaptic activity is important in the long-term maintenance of the immediately prior to brain injections. For hippocampal injections, a population of OPCs, with relevance for understanding pathological stereotaxically guided injection of BoNT/A (0.2 µl of a 1 nM solution) process leading to progressive demyelination. or vehicle (2% rat serum albumin in PBS) was made into the dorsal hippocampus using fine glass micropipettes (at the coordinates, in 2 | M ATE R I A L S A N D M E TH O DS 2.1 | Ethics statement mm with respect to the Bregma, A–P −2.0, M–L 1.5, H 1.7 below the dura), as previously characterized (Antonucci et al., 2008; Caleo et al., 2012). The experiments were designed in compliance with ARRIVE guidelines and no mice were excluded from analyses. In all injected animals, recovery was uneventful and no overt behavioral All procedures were performed in compliance with the EU Council abnormalities were observed. Two weeks after injections, mice were Directive 2010/63/EU on the protection of animals used for deeply anesthetized with chloral hydrate and perfused through the | CHACON-DE-LA-ROCHA Et AL. 3 F I G U R E 1 BoNT/A cleaves SNAP-25 in the hippocampus. Representative fluorescence micrographs of coronal sections of the adult mouse hippocampus illustrating the CA1 area following stereotaxical injection of BoNT/A or vehicle. Sections are immunolabeled with an antibody specific for BoNT/A-truncated SNAP-25 (red), which has been thoroughly characterized in previous studies (Antonucci et al., 2008; Caleo et al., 2012), and counterstained with Hoechst nuclear dye. Scale bar = 100 µm; images representative of two sections each from four animals heart with freshly prepared 4% paraformaldehyde in 0.1 M phos- blocking solution (Alexa Fluor 488-AffiniPure Goat Anti-Rabbit IgG phate buffer (PB), and the brains processed for immunohistochem- (H+L), Thermo Fisher Scientific Cat# A-11008, RRID:AB_143165), or istry (see below). biotinylated secondary antibody, diluted at 1:200 in blocking solution (Biotinylated Goat Anti-Rabbit IgG (H+L), Vector Laboratories 2.3 | Immunohistochemistry Cat# BA-1000, RRID:AB_2313606). Finally, sections were washed three times with PBS before being mounted on glass slides and covered with mounting medium and glass coverslips ready for imaging. Brains were post-fixed for 1 hr at 4°C and brain sections (50 μm thick) were cut with a freezing microtome and stored at 4°C until use in cryoprotectant solution containing 25% sucrose and 3.5% 2.4 | Imaging and analysis glycerol in 0.05 M PBS (all chemicals from Sigma-Aldrich, unless otherwise stated), at pH 7.4, prior to immunostaining (for details of Immunofluorescence images were captured using a Zeiss Axiovert antibodies, see Table 1). To detect SNAP-25 cleavage, we used a rab- LSM 710 VIS40S confocal microscope and maintaining the acqui- bit polyclonal antibody specific for BoNT/A-truncated SNAP-25 (gift sition parameters constant to allow comparison between samples from Prof Ornella Rossetto, University of Padua); antibody raised within the same experiment. Acquisition of images for cell counts against the immunizing sequence CKADSNKTRIDEANQ (aa 184- was performed with ×20 objective. Images for OPC reconstruction 197), affinity purified and previously characterized with full controls were taken using ×100 objective and capturing z-stacks formed by in our previous studies (Antonucci et al., 2008; Caleo et al., 2012). 80–100 single plains with an interval of 0.3 µm. All analyses were Sections were blocked with 10% donkey serum in PBS containing performed by a person who was blind to experimental conditions 0.25% Triton X-100 and then incubated overnight at room tempera- and no exclusion criteria were applied. OPC cell counts were per- ture with the anti-cleaved SNAP-25 antibody (1:500 dilution). On the formed manually on images using a constant field of view (FOV) of following day, sections were incubated with anti-rabbit Rhodamine 200 µm × 200 µm centered on the CA1 area, and data expressed as Red X, 1:500 (Jackson ImmunoResearch Labs Cat# 711-295-152, OPC density per mm2; data from two sections per mouse were aver- RRID:AB_2340613), washed in PBS, counterstained with Hoechst aged, to provide a final n = 4 mice per treatment group, on which 33342 (Thermo Fisher Scientific Cat#62249). For NG2 immunostain- statistics were performed. To determine the process domains (cell ing, sections were blocked with 0.5% bovine serum albumin (BSA) coverage) of OPCs, chromogenic NG2 immunostained sections for 1-2h, washed three times in PBS, and incubated overnight in the were examined on an Olympus dotSlide digital slide scanning sys- antibody solution which comprised of the primary antibody rabbit tem based on a BX51 microscope stand with an integrated scan- anti-NG2, 1:500 (Millipore Cat# AB5320, RRID:AB_11213678), di- ning stage and Olympus CC12 color camera, and the cell coverage luted in blocking solution containing 0.25% Triton-X. Tissues were of OPCs was measured using ImageJ by drawing a line around the then washed three times in PBS and incubated for 1–2h with ap- entire cell process field and the area within the line was measured propriate fluorochrome secondary antibody, diluted at 1:500 in using ImageJ (RRID:SCR_003070), and data were expressed as µm2; 4 | TA B L E 1 List of antibodies used Antibody Supplier information RRID Immunogen Dilution Cleaved SNAP-25 Custom made by Prof Ornella Rossetto (University of Padua) From Prof Ornella Rossetto (University of Padua) Immunizing sequence: CKADSNKTRIDEANQ (aa 1 84-197) 1:500 RRID:AB_11213678 Immunoaffinity purified NG2 Chondroitin Sulfate Proteoglycan from rat 1:500 RRID:AB_143165 IgG (H+L), anti-IgG (H+L) 1:500 RRID:AB_2313606 IgG (H+L), anti-IgG (H+L) 1:200 RRID:AB_2340613 Rabbit IgG (H+L) 1:500 See Antonucci et al. (2008), for testing and full controls (Figure 1b–d) Rabbit polyclonal (affinity purified) Anti-NG2 Chondroitin Sulfate Proteoglycan antibody Millipore Rabbit polyclonal Cat# AB5320 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 Thermo Fisher Scientific Goat Anti-Rabbit IgG Antibody (H+L), Biotinylated Vector Laboratories Goat polyclonal Cat# A-11008 Goat polyclonal Cat# BA-1000 Rhodamine Red-X-AffiniPure Donkey Anti-Rabbit IgG (H+L) (min X Bov, Ck, Gt,GP, Sy Hms, Hrs, Hu, Ms, Rat, Shp Sr Prot) antibody Jackson ImmunoResearch Labs Donkey polyclonal Cat# 711-295-152 CHACON-DE-LA-ROCHA Et AL. | CHACON-DE-LA-ROCHA Et AL. 5 data from two sections per mouse were averaged, to provide a final two-way ANOVA with Sidak's post hoc multiple comparisons test. n = 4 mice per treatment group, on which statistics were performed. Statistics, actual p values, when significant, and number of samples For detailed morphological analysis of single OPCs, cells were drawn are reported in the text. Data were analyzed by a person who was using Neurolucida 360 (MBF Bioscience, RRID:SCR_016788), and blind to experimental conditions and no exclusion criteria were ap- their morphology was analyzed using Neurolucida 360 explorer plied. Statistical significance in the figures is indicated as: *p ≤ 0.05; (MBF Bioscience, RRID:SCR_017348) for measurements of the **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001, ns = p > 0.05. number of processes per cell, process lengths, number of process terminals (end points), and number of nodes (branch points); three cells were drawn from the center of the CA1 field in one section per mouse (12 cells from 4 mice per treatment group), and data were averaged in each mouse, to provide a final n = 4 mice per treatment group, on which statistics were performed. Sholl analysis was performed on the same cells, using a 5µm interval between Sholl shells 3 | R E S U LT S 3.1 | The synaptic blocker BoNT/A causes a reduction in OPC numerical density and size in the CA1 region of the hippocampus (n = 12 cells from fours mice in each experimental group). The long-term effects of synaptic silencing on adult OPCs have not 2.5 | Statistical analysis been previously studied. We addressed this by injecting adult mice into the left hippocampus with BoNT/A (1 nM solution, 0.2 µl), which causes persistent blockade of hippocampal synaptic activity for up All data are represented as mean ± SD. Data sets were analyzed statis- to 120 days (Antonucci et al., 2008; Caleo et al., 2012). The hip- tically by Graphpad Prism 6.0 software (Graphpad, San Diego, USA, pocampus was analyzed 2 weeks post-injection and we confirmed RRID:SCR_002798). Statistical differences between two parametric that BoNT/A mediates long-term SNAP-25 cleavage in the CA1 samples were measured by the unpaired two tailed Student's t test, area of inoculated side (Figure 1), which has been detailed compre- and statistical differences of multiple parameters were assessed by hensively in earlier studies, by immunohistochemistry and western F I G U R E 2 Effect of synaptic silencing with BoNT/A on adult hippocampal OPC in vivo. Analysis of OPCs in hippocampus following injection with BoNT/A to silence synaptic activity, or vehicle in controls. (a–c) Immunofluorescence labeling for NG2 (green) and counterstain with Hoechst nuclear dye (blue), illustrating the overall distribution of OPCs in the CA1 area of the hippocampus following injection of vehicle (a) or BoNT/A (b), together with box–whisker plots of the numerical density of OPCs in the CA1 (c); scale bars = 50 µm; data were averaged from two sections per mouse to provide a mean ± SEM from n = 4 mice per treatment group; p = 0.0267, unpaired t test. (d-f) High magnification of individual chromogenic NG2 immunolabeled OPCs in the CA1 area of the hippocampus illustrating their process domains (broken lines) following injection of vehicle (d) or BoNT/A (e), together with box–whisker plots of OPC cell domains (f); scale bars = 20 µm; data were averaged from two sections per mouse (37 cells for vehicle, 43 cells for BoNT/A), to provide a mean ± SEM from n = 4 animals per treatment group; p =0.0003, unpaired t test 6 | CHACON-DE-LA-ROCHA Et AL. blot (Antonucci et al., 2008; Caleo et al., 2012). Next, we used im- group). The results indicate a primary effect of BoNT/A on cellular munolabeling for NG2 to identify OPCs (Butt et al., 1999), and we branching and this was examined further by Sholl analysis (Figure 4). focused on the CA1 area as OPCs had been previously shown to The data indicate that following synaptic silencing the most marked form synapses with neurones and respond to synaptic signaling in changes in OPC processes were within 20–30 μm of the cell body, this area (Bergles et al., 2000; Lin & Bergles, 2004). Confocal images with significant decreases in the number of nodes, or branch points demonstrate an evident decrease in NG2 immunostaining 14 days (Figure 4b; two-way ANOVA followed by Sidak's multiple compari- after BoNT/A injection (Figure 2a), compared to controls injected sons tests, F(13, 308) = 90.49, p < 0.0001, n = 12 cells from four with vehicle (Figure 2b). Quantification confirmed OPC numerical animals in each group), as well as the number of process terminals density was decreased following BoNT/A injection compared to (Figure 4c; two-way ANOVA followed by Sidak's multiple compari- controls (Figure 2c; unpaired t test, t = 2.918, df = 6, p = 0.0267; sons tests, F(13, 308) = 98.22, p < 0.0001, n = 12 cells from four data from two sections per mouse were averaged, to provide a final animals in each group), and the lengths of processes (Figure 4d; two- n = 4 mice per treatment group). Moreover, mapping the process do- way ANOVA followed by Sidak's multiple comparisons tests, F(13, mains of individual OPCs (Figure 2d,e) demonstrated these were sig- 308) = 182.1, p < 0.0001, n = 12 cells from four animals in each nificantly reduced following synaptic silencing (Figure 2f; unpaired t group). In addition, we analyzed the processes length in the different test, t = 7.651, df = 6, p = 0.0003; data from 37 cells for vehicle and branch orders, identifying that synaptic silencing primarily caused 43 cells for BoNT/A from two sections per mouse were averaged, shrinkage of the distal branches, and the maximum branch order to provide a final n = 4 mice per treatment group). The data show was decreased to 13 compared to 15 in controls (Figure 4e; two- that the synaptic blocker BoNT/A results in a decline in the overall way ANOVA followed by Sidak's multiple comparisons tests, F(15, number of OPC and those that persist display a marked shrinkage. 352) = 94.80, p < 0.0001, n = 12 cells from four animals in each group). Although the possibility of bias may have been introduced 3.2 | BoNT/A decreases the morphological complexity of OPCs in the Neurolucida and Sholl analyses of a relatively small number of OPCs, it is evident that OPCs were shrunken following BoNT/A, with very little variation, and these measurements are corroborated by the atrophy of OPC process domains based on a much larger num- Adult OPCs respond to pathology by changes in their morphol- ber of cells demonstrated in Figure 2. Together, the morphological ogy (Butt et al., 2002), and in the adult hippocampus they react to analyses show that OPC shrinkage is a key feature following synaptic toxic activation of glutamatergic receptors by extending a greater silencing with BoNT/A and is mainly due to process retraction and an number of short process (Ong & Levine, 1999). We therefore ex- evident decrease in branching. amined OPC morphology in detail using Neurolucida (Figure 3) and Sholl analysis (Figure 4). Overall, OPCs had a characteristic complex morphology in vehicle-injected controls, with on average 12 primary 4 | D I S CU S S I O N processes that extended radially for 50–100 µm from a central cell body (Figure 3a). In comparison, OPCs displayed overall atrophy An important feature of OPCs is that they form synapses with neu- following BoNT/A injection, with an evident decrease in cellular rones and sense neuronal synaptic activity (Bergles et al., 2000). complexity (Figure 3b); the shrinkage of the OPC process field fol- Here, we show that BoNT/A, which silences synaptic activity in the lowing BoNT/A treatment is amplified in the y-plane (upper insets, hippocampus, results in a decrease in OPC numbers and cellular at- Figure 3a,b), and in the x-plane OPCs in controls are seen to have a rophy. Notably, similar changes in OPCs are associated with synaptic dense network of branching processes, which is stunted following dysregulation in AD-like pathology in mice (Chacon-De-La-Rocha synaptic silencing (lower insets, Figure 3a,b). Multiple parameters of et al., 2020; Vanzulli et al., 2020). The results support a role for process complexity were quantified (Figure 3c–e); data were from synaptic signaling in maintaining adult OPC numbers and integrity, three cells drawn from the center of the CA1 field in one section which is relevant to neuropathologies in which neuronal activity is per mouse, which was averaged in each mouse, to provide a final altered, such as AD, MS, and traumatic injury, as well as age-related n = 4 mice per treatment group. There were significant decreases cognitive decline. in the number of processes extending from the cell body follow- BoNT/A is a member of the family of botulinum neurotoxins ing BoNT/A injection, ranging from 2 to 12, compared to 8–25 in (BoNTs A-G) that inhibit neurotransmission by blocking vesicu- controls (Figure 3c; unpaired t test, p = 0.0448, t = 2.528, df = 6, lar neurotransmitter release (Caleo & Restani, 2018). BoNTs enter n = 4 animals in each group), together with significant decreases in presynaptic terminals mainly via activity-dependent synaptic endo- the length of processes from each cell (Figure 3d; unpaired t test, cytosis and cleave proteins of the SNARE complex, which is neces- p = 0.0116, t = 3.582, df = 6, n = 4 animals in each group), as well as sary for synaptic vesicle fusion (Verderio et al., 2006). It has been the number of process terminals, or end points (Figure 3e; unpaired demonstrated that infusion of BoNT/A into the mouse hippocam- t test, p = 0.0280, t = 2.881, df = 6, n = 4 animals in each group), pus cleaves SNAP-25 and prevents neuronal synaptic activity in the and the number of branch points along processes (nodes) (Figure 3f; CA1 area (Antonucci et al., 2008). It is possible that BoNT/A cleaves unpaired t test, p = 0.0277, t = 2.891, df = 6, n = 4 animals in each SNAP-25 in OPCs, but in the hippocampus SNAP-25 is concentrated CHACON-DE-LA-ROCHA Et AL. | 7 F I G U R E 3 Effects of synaptic silencing with BoNT/A on OPC morphology in the adult hippocampus. Confocal images of hippocampus sections were immunolabeled for NG2 following injection with BoNT/A to silence synaptic activity, or vehicle in controls. (a, b) Confocal images were analyzed using Neurolucida 360 (MBF Bioscience), to generate 3D images (main panels in a, b); insets illustrate the cell in the x- and y-planes. (c–f) Box–whisker plots of the number of processes per cell (c), process lengths (d), number of process terminals, or end points (e), and the number of branch points, or nodes (f). Data were from three cells drawn from the center of the CA1 field in one section per mouse, which was averaged in each mouse, to provide a mean ± SEM from n = 4 animals per treatment group; p values as indicated, from unpaired t tests 8 | CHACON-DE-LA-ROCHA Et AL. F I G U R E 4 Sholl analysis of OPC process morphology in the adult hippocampus following synaptic silencing with BoNT/A. (a) Isosurface rendering to illustrate 3D morphology of OPCs (generated with Volocity software, PerkinElmer), obtained following NG2 immunolabeled cells in CA1 area of the hippocampus following injection of BoNT/A (ai) or vehicle (aii). The schematic representation in (aiii) illustrates parameters measured, where the concentric circles (termed Sholl shells) are placed at 5 μm apart, with the cell body in the middle (yellow lines in ai, aii); the points of process branching are termed nodes (blue dots), and the points where the processes intersect the Sholl shells are termed intersections (green dots) (adapted from Sholl (1953) and Rietveld et al. (2015)). (b–e) Graphs of the number of nodes or branch points (b), number of process terminals or end points (c), processes lengths (d), and length of processes of different branch orders (e). Data are mean ± SEM; n = 12 cells from four animals per group (three cells per section, taken from the center of the CA1 field); p values as indicated, from two-way ANOVA followed by Sidak's multiple comparisons test in neuronal terminals and is largely absent from glial processes 2008; Caleo et al., 2012). It is not discounted that BoNT/A could act (Schubert et al., 2011), and transcriptomic data indicate that SNAP- on synaptic transmission by OPCs (Hamilton et al., 2010), or have 25 is barely detectable in OPCs and other glia, compared to neurons other effects on OPCs unrelated to synaptic transmission, but the (Figure S1; (Zhang et al., 2014)). Glia can express SNAP-23 (Feldmann results of the present study are consistent with BoNT/A acting to et al., 2009; Hepp et al., 1999; Schardt et al., 2009; Schubert silence neuronal synapses and cause a decrease in OPC numbers et al., 2011), but BoNT/A acts mainly on SNAP-25 and not SNAP-23 and cell shrinkage, resulting in marked loss of overall coverage of the (Sikorra et al., 2016). In addition, it has been shown that BoNT/A hippocampus by OPCs. has little effect on astrocytes (Rojewska et al., 2018), or microglia Our data demonstrate that BoNT/A treatment resulted in a (Caleo et al., 2012), and the primary effect of BoNT/A is on synaptic decrease in OPC numbers, indicating that OPC self-renewal is signaling, with little evidence of axonal disruption (Antonucci et al., dependent on synaptic signaling. These findings support previous | CHACON-DE-LA-ROCHA Et AL. 9 studies showing OPC proliferation is respectively increased or in the hippocampal model (Antonucci et al., 2008; Caleo et al., decreased when neuronal electrical activity is stimulated (Gibson 2012), which has been explained by the fact that SNAP-25 is ex- et al., 2014) or blocked (Barres & Raff, 1993). The decrease in OPCs pressed only at very low levels in GABAergic synapses (Verderio following BoNT/A indicates their population was not replenished et al., 2004). Furthermore, although OPCs in the hippocampus by self-renewal, although further studies to measure cell prolifer- receive synaptic inputs from GABAergic neurones (Bergles et al., ation are required to confirm this. A novel finding of our study is 2000; Lin & Bergles, 2004), their blockade is reported to increase that BoNT/A caused shrinkage of adult OPC in vivo, with a marked OPC numbers in vitro in cortical slices (Hamilton et al., 2017; retraction of distal processes and decreased branching. A simi- Zonouzi et al., 2015), which is opposite to that observed following lar reduction in OPC process extension and branching has been synaptic silencing. Also, as noted above, OPC disruption following reported in vitro in postnatal cerebellar slices following blockade BoNT/A in vivo is mirrored by blockade of glutamatergic signaling of neuronal electrical activity and glutamate receptors (Fannon in vitro in cerebellar slices (Fannon et al., 2015). Thus, the balance et al., 2015). This is in comparison to the response of OPCs to of evidence indicates excitatory synaptic signaling plays an im- CNS insults, which is usually marked by increased process branch- portant role in maintaining OPC numbers and integrity in the adult ing to give OPCs a fibrous appearance (Butt et al., 2002; Levine hippocampus. et al., 2001). Time-lapse imaging demonstrates that NG2+ OPCs processes are highly motile and respond rapidly to changes in their environment (Hughes et al., 2013). Hence, it is possible that OPC 5 | CO N C LU S I O N S processes are retracted from silent synapses following BoNT/A treatment, resulting in decreased coverage by OPCs, which is This study demonstrates that the synaptic blocker BoNT/A has a comparable to early atrophy of OPCs observed in mouse models major negative impact on adult OPCs, which have the fundamen- of AD that are characterized by synaptic dysregulation (Chacon- tal function of life-long generation of oligodendrocytes. It will be De-La-Rocha et al., 2020; Vanzulli et al., 2020). The overall extent of considerable interest in future studies to determine whether of synaptic disruption in neuropathology is unlikely to be as ex- synaptic blockade has longer term effects on myelination that is tensive as that of the hippocampus following BoNT/A injection, required for the formation of new neuronal connections and learn- and the changes observed in the present study are more likely to ing (McKenzie et al., 2014; Xiao et al., 2016). Notably, disruption reflect those that occur focally within lesions. Indeed, changes in of OPCs has been shown to result in impaired myelination in aging OPC morphology are characteristic of neuropathology in human (Neumann et al., 2019; Rivera et al., 2021), and this is a key factor AD and MS, as well as mouse models of these diseases (Butt & in the age-related decline in cognitive function (Bartzokis, 2004), as Dinsdale, 2005; Dong et al., 2018; Levine & Reynolds, 1999; Li well as the failure of remyelination in chronic MS (Sanai et al., 2016), et al., 2013; Nielsen et al., 2013; Reynolds et al., 2002; Zhang and the accelerated loss of myelin observed in AD (Nasrabady et al., et al., 2019). These data support evidence that synaptic signaling 2018; Vanzulli et al., 2020). In conclusion, disruption of OPCs fol- regulates adult OPC morphology and the maintenance of their lowing synaptic blockade in the hippocampus is relevant to cogni- numbers. tive function and neuropathologies where there is dysregulation of The effects of BoNT/A on hippocampal OPCs may be due neuronal synaptic activity. mainly to blockade of glutamatergic synapses, since BoNT/A has been shown to act preferentially on excitatory synapses (Antonucci et al., 2008; Caleo et al., 2012), which are contacted by OPCs D EC L A R ATI O N O F TR A N S PA R E N C Y (Bergles et al., 2000). Furthermore, neurotransmitters released at synapses or along axons would also activate non-synaptic recep- The authors, reviewers and editors affirm that in accordance to the tors on adult OPCs through what is termed volume transmission policies set by the Journal of Neuroscience Research, this manuscript (Kula et al., 2019; Vélez-Fort et al., 2010; Hamilton et al., 2010), presents an accurate and transparent account of the study being re- which would also be blocked by BoNT/A. Glutamatergic signal- ported and that all critical details describing the methods and results ing has been shown to promote OPC proliferation (Chen et al., are present. 2018; Wake et al., 2011), hence blockade of glutamate release by BoNT/A would result in the observed decline in OPC numbers. To AC K N OW L E D G M E N T S confirm this unequivocally will require comprehensive electro- We thank Dr Olivier Raineteau for assistance and use of Neurolucida physiological, immunohistochemical, EM, cell proliferation, and in the INSERM Stem Cell and Brain Research Institute, Univ Lyon, fate-mapping studies. Nonetheless, we demonstrate that BoNT/A France. The research presented in this paper was supported by causes cleavage of SNAP-25 in our model (Figure 1), which is grants from the BBSRC (AB, Grant Number BB/M029379/1), MRC essential for synaptic vesicular release of glutamate (Caleo & (AB, Grant Number MR/P025811/1), Alzheimer's Research UK (DG- Restani, 2018). In contrast, electrophysiological and EM studies N, AB, Grant Number PG2014B-2), University of Portsmouth PhD indicate BoNT/A does not affect GABAergic synapses when used Programme (AB, ICR), and CNR – Joint Laboratories (MC, LR). 10 | CHACON-DE-LA-ROCHA Et AL. C O N FL I C T O F I N T E R E S T Prof Arthur Butt and Dr Andrea Rivera are shareholders and cofounders of the company GliaGenesis. All authors declare no other conflict. AU T H O R C O N T R I B U T I O N S Conceptualization, D.G.-N. and A.M.B.; Methodology, I.C.-D.-L.-R., G.F., and L.R.; Validation, D.G.-N. and A.M.B.; Formal Analysis, I.C.D.-L.-R., G.F., and A.M.B.; Investigation, I.C.-D.-L.-R., G.F., L.R., and A.D.R.; Resources, M.C., D.G.-N., and A.M.B.; Data Curation, D.G.-N. and A.M.B.; Writing – Original Draft, I.C.-D.-L.-R. and A.M.B.; Writing – Review & Editing, L.R., M.C., D.G.-N., and A.M.B.; Visualization, D.G.-N. and A.M.B.; Supervision, D.G.-N. and A.M.B.; Project Administration, D.G.-N. and A.M.B.; Funding Acquisition, D.G.-N. and A.M.B. PEER REVIEW The peer review history for this article is available at https://publo ns.com/publon/10.1002/jnr.24856 DATA AVA I L A B I L I T Y S TAT E M E N T The data that support the findings of this study are available from the corresponding authors upon reasonable request. ORCID Arthur M. Butt https://orcid.org/0000-0001-7579-0746 REFERENCES Antonucci, F., Rossi, C., Gianfranceschi, L., Rossetto, O., & Caleo, M. (2008). Long-distance retrograde effects of botulinum neurotoxin A. Journal of Neuroscience, 28(14), 3689–3696. https://doi.org/10.1523/ JNEUROSCI.0375- 08.2008 Barres, B. A., & Raff, M. C. (1993). Proliferation of oligodendrocyte precursor cells depends on electrical activity in axons. Nature, 361(6409), 258–260. https://doi.org/10.1038/361258a0 Bartzokis, G. (2004). Age-related myelin breakdown: A developmental model of cognitive decline and Alzheimer's disease. Neurobiology of Aging, 25(1), 5–18; author reply 49–62. https://doi.org/10.1016/j. neurobiolaging.2003.03.001 Bergles, D. E., Roberts, J. D. B., Somogyi, P., & Jahr, C. E. (2000). Glutamatergic synapses on oligodendrocyte precursor cells in the hippocampus. Nature, 405(6783), 187–191. https://doi. org/10.1038/35012083 Butt, A. M., & Dinsdale, J. (2005). Fibroblast growth factor 2 induces loss of adult oligodendrocytes and myelin in vivo. Experimental Neurology, 192(1), 125–133. https://doi.org/10.1016/j.expne urol.2004.11.007 Butt, A. M., Duncan, A., Hornby, M. F., Kirvell, S. L., Hunter, A., Levine, J. M., & Berry, M. (1999). Cells expressing the NG2 antigen contact nodes of Ranvier in adult CNS white matter. Glia, 26(1), 84–91. https://doi.org/10.1002/(SICI)1098-1136(19990 3)26:1<84:AIDGLIA9>3.0.CO;2-L Butt, A. M., Kiff, J., Hubbard, P., & Berry, M. (2002). Synantocytes: New functions for novel NG2 expressing glia. Journal of Neurocytology, 31(6/7), 551–565. http://dx.doi.org/10.1023/a:1025751900356 Caleo, M., & Restani, L. (2018). Exploiting botulinum neurotoxins for the study of brain physiology and pathology. Toxins, 10(5), 175. https:// doi.org/10.3390/toxins10050175 Caleo, M., Restani, L., Vannini, E., Siskova, Z., Al-Malki, H., Morgan, R., O'Connor, V., & Perry, V. H. (2012). The role of activity in synaptic degeneration in a protein misfolding disease, prion disease. PLoS One, 7(7), e41182. Chacon-De-La-Rocha, I., Fryatt, G., Rivera, A. D., Verkhratsky, A., Raineteau, O., Gomez-Nicola, D., & Butt, A. M. (2020). Accelerated dystrophy and decay of oligodendrocyte precursor cells in the APP/PS1 model of Alzheimer's-like pathology. Frontiers in Cellular Neuroscience, 14, 575082. https://doi.org/10.3389/ fncel.2020.575082 Chen, T. J., Kula, B., Nagy, B., Barzan, R., Gall, A., Ehrlich, I., & Kukley, M. (2018). In vivo regulation of oligodendrocyte precursor cell proliferation and differentiation by the AMPA-receptor subunit GluA2. Cell Reports, 25(4), 852–861.e7. http://dx.doi.org/10.1016/j. celrep.2018.09.066 Dong, Y.-X., Zhang, H.-Y., Li, H.-Y., Liu, P.-H., Sui, Y. I., & Sun, X.-H. (2018). Association between Alzheimer's disease pathogenesis and early demyelination and oligodendrocyte dysfunction. Neural Regeneration Research, 13(5), 908–914. https://doi.org/10.4103/167 3-5374.232486 Fannon, J., Tarmier, W., & Fulton, D. (2015). Neuronal activity and AMPAtype glutamate receptor activation regulates the morphological development of oligodendrocyte precursor cells. Glia, 63(6), 1021– 1035. https://doi.org/10.1002/glia.22799 Feldmann, A., Winterstein, C., White, R., Trotter, J., & Krämer-Albers, E.-M. (2009). Comprehensive analysis of expression, subcellular localization, and cognate pairing of SNARE proteins in oligodendrocytes. Journal of Neuroscience Research, 87(8), 1760–1772. https:// doi.org/10.1002/jnr.22020 Gibson, E. M., Purger, D., Mount, C. W., Goldstein, A. K., Lin, G. L., Wood, L. S., Inema, I., Miller, S. E., Bieri, G., Zuchero, J. B., Barres, B. A., Woo, P. J., Vogel, H., & Monje, M. (2014). Neuronal activity promotes oligodendrogenesis and adaptive myelination in the mammalian brain. Science, 344(6183), 1252304. https://doi.org/10.1126/science.1252304 Hamilton, N. B., Clarke, L. E., Arancibia-Carcamo, I. L., Kougioumtzidou, E., Matthey, M., Káradóttir, R., Whiteley, L., Bergersen, L. H., Richardson, W. D., & Attwell, D. (2017). Endogenous GABA controls oligodendrocyte lineage cell number, myelination, and CNS internode length. Glia, 65(2), 309–321. https://doi.org/10.1002/glia.23093 Hamilton, N., Vayro, S., Wigley, R., & Butt, A. M. (2010). Axons and astrocytes release ATP and glutamate to evoke calcium signals in NG2glia. Glia, 58(1), 66–79. https://doi.org/10.1002/glia.20902 Hepp, R., Perraut, M., Chasserot-Golaz, S., Galli, T., Aunis, D., Langley, K., & Grant, N. J. (1999). Cultured glial cells express the SNAP-25 analogue SNAP-23. Glia, 27(2), 181–187. https://doi.org/10.1002/ (SICI)1098-1136(19990 8)27:2<181:AID-GLIA8>3.0.CO;2-9 Hill, R. A., Li, A. M., & Grutzendler, J. (2018). Lifelong cortical myelin plasticity and age-related degeneration in the live mammalian brain. Nature Neuroscience, 21(5), 683–695. https://doi.org/10.1038/s4159 3- 018- 0120-6 Hughes, E. G., Kang, S. H., Fukaya, M., & Bergles, D. E. (2013). Oligodendrocyte progenitors balance growth with self-repulsion to achieve homeostasis in the adult brain. Nature Neuroscience, 16(6), 668–676. https://doi.org/10.1038/nn.3390 Korrell, K. V., Disser, J., Parley, K., Vadisiute, A., Requena-Komuro, M.-C., Fodder, H., Pollart, C., Knott, G., Molnár, Z., & Hoerder-Suabedissen, A. (2019). Differential effect on myelination through abolition of activity-dependent synaptic vesicle release or reduction of overall electrical activity of selected cortical projections in the mouse. Journal of Anatomy, 235(3), 452–467. https://doi.org/10.1111/joa.12974 Kukley, M., Capetillo-Zarate, E., & Dietrich, D. (2007). Vesicular glutamate release from axons in white matter. Nature Neuroscience, 10(3), 311–320. https://doi.org/10.1038/nn1850 CHACON-DE-LA-ROCHA Et AL. Kula, B., Chen, T. J., & Kukley, M. (2019). Glutamatergic signaling between neurons and oligodendrocyte lineage cells: Is it synaptic or non-synaptic? Glia, 67(11), 2071–2091. https://doi.org/10.1002/ glia.23617 Levine, J. M., & Reynolds, R. (1999). Activation and proliferation of endogenous oligodendrocyte precursor cells during ethidium bromideinduced demyelination. Experimental Neurology, 160(2), 333–347. https://doi.org/10.1006/exnr.1999.7224 Levine, J. M., Reynolds, R., & Fawcett, J. W. (2001). The oligodendrocyte precursor cell in health and disease. Trends in Neurosciences, 24(1), 39–47. https://doi.org/10.1016/S0166-2236(00)01691-X Li, W., Tang, Y., Fan, Z., Meng, Y. A., Yang, G., Luo, J., & Ke, Z.-J. (2013). Autophagy is involved in oligodendroglial precursor-mediated clearance of amyloid peptide. Molecular Neurodegeneration, 8, 27. https:// doi.org/10.1186/1750-1326-8-27 Lin, S. C., & Bergles, D. E. (2004). Synaptic signaling between GABAergic interneurons and oligodendrocyte precursor cells in the hippocampus. Nature Neuroscience, 7(1), 24–32. https://doi.org/10.1038/ nn1162 Mangin, J.- M., Li, P., Scafidi, J., & Gallo, V. (2012). Experiencedependent regulation of NG2 progenitors in the developing barrel cortex. Nature Neuroscience, 15(9), 1192–1194. https://doi. org/10.1038/nn.3190 McKenzie, I. A., Ohayon, D., Li, H., Paes de Faria, J., Emery, B., Tohyama, K., & Richardson, W. D. (2014). Motor skill learning requires active central myelination. Science, 346(6207), 318–322. https://doi. org/10.1126/science.1254960 Nasrabady, S. E., Rizvi, B., Goldman, J. E., & Brickman, A. M. (2018). White matter changes in Alzheimer's disease: A focus on myelin and oligodendrocytes. Acta Neuropathologica Communications, 6(1), 22. https://doi.org/10.1186/s40478- 018- 0515-3 Neumann, B., Baror, R., Zhao, C., Segel, M., Dietmann, S., Rawji, K., Foerster, S., McClain, C. R., Chalut, K., van Wijngaarden, P., & Franklin, R. J. M. (2019). Metformin restores CNS remyelination capacity by rejuvenating aged stem cells. Cell Stem Cell. 25(4), 473–485. e8. http://dx.doi.org/10.1016/j.stem.2019.08.015 Nielsen, H. M., Ek, D., Avdic, U., Orbjörn, C.; Hansson, O., Veerhuis, R., Rozemuller, A. J. M., Brun, A., Minthon, L., & Wennström, M. (2013). NG2 cells, a new trail for Alzheimer's disease mechanisms? Acta Neuropathologica Communications, 1(1), 7. https://doi. org/10.1186/2051-5960-1-7 Ong, W. Y., & Levine, J. M. (1999). A light and electron microscopic study of NG2 chondroitin sulfate proteoglycan-positive oligodendrocyte precursor cells in the normal and kainate-lesioned rat hippocampus. Neuroscience, 92(1), 83–95. https://doi.org/10.1016/ S0306- 4522(98)00751-9 Reynolds, R., Dawson, M., Papadopoulos, D., Polito, A., Di Bello, I. C., Pham-Dinh, D., & Levine, J. (2002). The response of NG2-expressing oligodendrocyte progenitors to demyelination in MOG-EAE and MS. Journal of Neurocytology, 31(6–7), 523–536. https://doi. org/10.1023/A:1025747832215 Rietveld L., Stuss D. P., McPhee D., & Delaney K. R. (2015). Genotypespecific effects of Mecp2 loss-of-function on morphology of Layer V pyramidal neurons in heterozygous female Rett syndrome model mice. Frontiers in Cellular Neuroscience, 9. http://dx.doi.org/10.3389/ fncel.2015.00145 Rivera, A. D., Pieropan, F., Chacon-De-La-Rocha, I., Lecca, D., Abbracchio, M. P., Azim, K., & Butt, A. M. (2021). Functional genomic analyses a shift in Gpr17-regulated cellular processes in oligodendrocyte progenitor cells and underlying myelin dysregulation in the aged mouse cerebrum. Aging Cell, 20(4), e13335. http://dx.doi.org/10.1111/acel.13335 Rivera, A., Vanzuli, I., Arellano, J. J., & Butt, A. (2016). Decreased regenerative capacity of oligodendrocyte progenitor cells (NG2-Glia) in the ageing brain: A vicious cycle of synaptic dysfunction, myelin loss and neuronal disruption? Current Alzheimer Research, 13(4), 413–418. https://doi.org/10.2174/1567205013666151116125518 | 11 Rojewska, E., Piotrowska, A., Popiolek-Barczyk, K., & Mika, J. (2018). Botulinum toxin type A-A modulator of spinal neuron-glia interactions under neuropathic pain conditions. Toxins, 10(4), 145. https:// doi.org/10.3390/toxins1004 0145 Sanai, S. A., Saini, V., Benedict, R. H. B., Zivadinov, R., Teter, B. E., Ramanathan, M., & Weinstock-Guttman, B. (2016). Aging and multiple sclerosis. Multiple Sclerosis Journal, 22(6), 717–725. https://doi. org/10.1177/1352458516634871 Schardt, A., Brinkmann, B. G., Mitkovski, M., Sereda, M. W., Werner, H. B., & Nave, K. A. (2009). The SNARE protein SNAP-29 interacts with the GTPase Rab 3A: Implications for membrane trafficking in myelinating glia. Journal of Neuroscience Research, 87(15), 3465–3479. https://doi.org/10.1002/jnr.22005 Schiavo, G., & Montecucco, C. (1995). Tetanus and botulism neurotoxins: Isolation and assay. Methods in Enzymology, 248, 643–652. https:// doi.org/10.1016/0076-6879(95)48041-2 Schubert, V., Bouvier, D., & Volterra, A. (2011). SNARE protein expression in synaptic terminals and astrocytes in the adult hippocampus: A comparative analysis. Glia, 59(10), 1472–1488. https://doi. org/10.1002/glia.21190 Sholl, D. A. (1953). Dendritic organization in the neurons of the visual and motor cortices of the cat. Journal of Anatomy, 87(4), 387–406. Shone, C. C., & Tranter, H. S. (1995). Growth of clostridia and preparation of their neurotoxins. In C. Montecucco (Ed.), Clostridial Neurotoxins, Current Topics in Microbiology and Immunology (Vol. 195, pp. 143– 160). Berlin, Heidelberg: Springer. https://doi.org/10.1007/978-3642-85173-5_7 Sikorra, S., Litschko, C., Müller, C., Thiel, N., Galli, T., Eichner, T., & Binz, T. (2016). Identification and characterization of botulinum neurotoxin A substrate binding pockets and their re-engineering for human SNAP23. Journal of Molecular Biology, 428(2 Pt A), 372–384. https://doi. org/10.1016/j.jmb.2015.10.024 Stallcup, W. B. (1981). The NG2 antigen, a putative lineage marker: Immunofluorescent localization in primary cultures of rat brain. Developmental Biology, 83(1), 154–165. https://doi.org/10.1016/ S0012-1606(81)80018-8 Vanzulli, I., Papanikolaou, M., De-La-Rocha, I. C., Pieropan, F., Rivera, A. D., Gomez-Nicola, D., Verkhratsky, A., Rodríguez, J. J., & Butt, A. M. (2020). Disruption of oligodendrocyte progenitor cells is an early sign of pathology in the triple transgenic mouse model of Alzheimer's disease. Neurobiology of Aging, 94, 130–139. https://doi.org/10.1016/j. neurobiolaging.2020.05.016 Velez-Fort, M., Maldonado, P. P., Butt, A. M., Audinat, E., & Angulo, M. C. (2010). Postnatal switch from synaptic to extrasynaptic transmission between interneurons and NG2 cells. Journal of Neuroscience, 30(20), 6921–6929. https://doi.org/10.1523/JNEUROSCI.0238-10.2010 Verderio, C., Pozzi, D., Pravettoni, E., Inverardi, F., Schenk, U., Coco, S., Proux-Gillardeaux, V., Galli, T., Rossetto, O., Frassoni, C., & Matteoli, M. (2004). SNAP-25 modulation of calcium dynamics underlies differences in GABAergic and glutamatergic responsiveness to depolarization. Neuron, 41(4), 599–610. https://doi.org/10.1016/ S0896-6273(04)00077-7 Verderio, C., Rossetto, O., Grumelli, C., Frassoni, C., Montecucco, C., & Matteoli, M. (2006). Entering neurons: Botulinum toxins and synaptic vesicle recycling. EMBO Reports, 7(10), 995–999. https://doi. org/10.1038/sj.embor.7400796 Wake, H., Lee, P. R., & Fields, R. D. (2011). Control of local protein synthesis and initial events in myelination by action potentials. Science, 333(6049), 1647–1651. https://doi.org/10.1126/science.1206998 Xiao, L., Ohayon, D., McKenzie, I. A., Sinclair-Wilson, A., Wright, J. L., Fudge, A. D., Emery, B., Li, H., & Richardson, W. D. (2016). Rapid production of new oligodendrocytes is required in the earliest stages of motor-skill learning. Nature Neuroscience, 19(9), 1210–1217. https:// doi.org/10.1038/nn.4351 Young, K. M., Psachoulia, K., Tripathi, R. B., Dunn, S.-J., Cossell, L., Attwell, D., Tohyama, K., & Richardson, W. D. (2013). Oligodendrocyte 12 | dynamics in the healthy adult CNS: Evidence for myelin remodeling. Neuron, 77(5), 873–885. https://doi.org/10.1016/j. neuron.2013.01.006 Zawadzka, M., Rivers, L. E., Fancy, S. P. J., Zhao, C., Tripathi, R., Jamen, F., Young, K., Goncharevich, A., Pohl, H., Rizzi, M., Rowitch, D. H., Kessaris, N., Suter, U., Richardson, W. D., & Franklin, R. J. M. (2010). CNS-resident glial progenitor/stem cells produce Schwann cells as well as oligodendrocytes during repair of CNS demyelination. Cell Stem Cell, 6(6), 578–590. https://doi.org/10.1016/j. stem.2010.04.002 Zhang, P., Kishimoto, Y., Grammatikakis, I., Gottimukkala, K., Cutler, R. G., Zhang, S., Abdelmohsen, K., Bohr, V. A., Misra Sen, J., Gorospe, M., & Mattson, M. P. (2019). Senolytic therapy alleviates Aβ-associated oligodendrocyte progenitor cell senescence and cognitive deficits in an Alzheimer's disease model. Nature Neuroscience, 22(5), 719–728. https://doi.org/10.1038/s41593- 019- 0372-9 Zhang, Y., Chen, K., Sloan, S. A., Bennett, M. L., Scholze, A. R., O'Keeffe, S., Phatnani, H. P., Guarnieri, P., Caneda, C., Ruderisch, N., Deng, S., Liddelow, S. A., Zhang, C., Daneman, R., Maniatis, T., Barres, B. A., & Wu, J. Q. (2014). An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. The Journal of Neuroscience, 34(36), 11929–11947. https://doi. org/10.1523/JNEUROSCI.1860-14.2014 Ziskin, J. L., Nishiyama, A., Rubio, M., Fukaya, M., & Bergles, D. E. (2007). Vesicular release of glutamate from unmyelinated axons in white matter. Nature Neuroscience, 10(3), 321–330. https://doi. org/10.1038/nn1854 CHACON-DE-LA-ROCHA Et AL. Zonouzi, M., Scafidi, J., Li, P., McEllin, B., Edwards, J., Dupree, J. L., Harvey, L., Sun, D., Hübner, C. A., Cull-Candy, S. G., Farrant, M., & Gallo, V. (2015). GABAergic regulation of cerebellar NG2 cell development is altered in perinatal white matter injury. Nature Neuroscience, 18(5), 674–682. https://doi.org/10.1038/nn.3990 S U P P O R T I N G I N FO R M AT I O N Additional Supporting Information may be found online in the Supporting Information section. FIGURE S1 Visualization of SNAP-25 mRNA expression in OPCs compared to neurons and other glia, from Transcriptomic data sets generated by Zhang et al., 2014. Adapted from www.brainrnaseq.org Transparent Peer Review Report Transparent Science Questionnaire for Authors How to cite this article: Chacon-De-La-Rocha I, Fryatt GL, Rivera AD, et al. The synaptic blocker botulinum toxin A decreases the density and complexity of oligodendrocyte precursor cells in the adult mouse hippocampus. J Neurosci Res. 2021;00:1–12. https://doi.org/10.1002/jnr.24856