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The demyelinating agent cuprizone induces a male-specific reduction in binge eating in 2 the binge-prone C57BL/6NJ strain 3 4 Abstract Binge eating (BE) is a heritable symptom of eating disorders with an unknown genetic etiology. 32 Rodent models for BE of palatable food permit the study of genetic and biological mechanisms. 33 We previously used genetic mapping and transcriptome analysis to map a coding mutation in 34 Cyfip2 associated with increased BE in the BE-prone C57BL/6NJ substrain compared to the BE-35 resistant C57BL/6J substrain. The increase in BE in C57BL/6NJ mice was associated with a 36 decrease in transcription of genes enriched for myelination in the striatum. Here, we tested the 37 hypothesis that decreasing myelin levels with the demyelinating agent cuprizone would enhance 38 BE. Mice were treated with a 0.3% cuprizone home cage diet for two weeks. Following a three-39 week recovery period, mice were trained for BE in an intermittent, limited access procedure. 40 Cuprizone induced similar weight loss in both substrains and sexes that recovered within 48 h 41 after removal of the cuprizone diet. Surprisingly, cuprizone reduced BE in male but not female 42 C57BL/6NJ mice while having no effect in C57BL/6J mice. Cuprizone also reduced myelin basic 43 protein (MBP) at seven weeks post-cuprizone removal while having no effect on myelin-44 associated glycoprotein (MAG) at this time point. C57BL/6N mice also showed less MBP than 45 C57BL/6J mice. There were no statistical interactions of Treatment with Sex on MBP levels, 46 indicating that differences in MBP are unlikely to account for sex differences in BE. To summarize, 47 cuprizone induced an unexpected, male-specific reduction in BE which could indicate sex-specific 48 biological mechanisms that depend on genetic background. 49 50 KEY WORDS: binge eating disorder, demyelination, sex differences, reduced complexity cross, 51 QTL, sex as a biological variable 52 Binge eating (BE) is operationally defined as the consumption of a relatively large amount 54 of food over a short period of time, and is accompanied by feelings of loss of control (Amianto, 55 Ottone, Abbate Daga, & Fassino, 2015) . Repeated BE that occurs at least once per week for at 56 least three months is termed binge eating disorder (BED). BE has a lifetime prevalence of 57 approximately 4.5% (Hudson, Hiripi, Pope, & Kessler, 2007) and is associated with a number of 58 comorbid behavioral, mood, and physical disorders such as substance abuse, depression, 59 obesity, and chronic pain (Bulik & Reichborn-Kjennerud, 2003; Citrome, 2017; Hudson et al., 60 2007) . Although there is no sex difference in the lifetime prevalence of BE, women are nearly 61 twice as likely as men to progress to the more severe BED (3.5% vs. 2.0% respectively (Hudson 62 et al., 2007) . However, the etiology of this sex difference is not known. amygdala-hippocampus complex in AN, and greater frontal cortical response to food stimuli in 69 BED (Geliebter et al., 2006) . Another important neuroanatomical change common to multiple 70 types of ED involves changes in myelin which is the lipid-rich, membranous insulation sheath that 71 extends from oligodendrocytes to the axons and is involved in axon maintenance (via 72 oligodendrocyte signaling) and function, including rapid salutatory conduction of action potentials. 73 BN is associated with a reduction in fractional anisotropy, a brain imaging measure of white matter 74 integrity, in the corona radiata and corpus callosum ( For MAG detection, blots were incubated in a 1:5,000 dilution of anti-MAG antibody (EMD 194 Millipore, Burlington, MA, USA, #MAB1567) in 5% BSA in TBST overnight at 4C, then a 1:10,000 195 dilution of peroxidase conjugated donkey anti mouse antibody (Jackson Immunoresearch, West 196 Grove, PA, USA, #715-035-151). For MBP detection, blots were incubated in a 1:10,000 dilution 197 of anti-MBP antibody (EDM Millipore, #MAB 386) in 5% BSA in TBST overnight at 4C, then a 198 1:10,000 dilution of peroxidase conjugated donkey anti rat antibody (Jackson Immunoresearch 199 #712-035-153) for one hour. After probing, all blots were imaged using Clarity ECL (BioRad, #170-200 5061) and a c300 imager (Dublin, CA, USA). 201 Blots were then stripped (Thermo Scientific, 46430) at 55°C for 30 min, reblocked with 5% 202 BSA in TBST, incubated in 1:50,000 Beta-actin antibody (Sigma Aldrich, St, Louis, MO, USA, 203 #A2228) in 5% BSA in TBST for 1 h, and then a 1:10,000 dilution of peroxidase conjugated donkey 204 anti mouse antibody for 1 h. Blots were then reimaged for beta actin. Densitometry was conducted 205 using ImageJ, and each lane was normalized to its respective densitometry value for beta actin. 206 Each lane was then normalized to the average wild-type value of that particular blot and then 207 finally, data were combined across blots and statistical analysis was run in R as described below. 208 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/865600 doi: bioRxiv preprint 9 209 210 We analyzed food consumption in R (https://www.r-project.org/) using mixed model 212 ANOVAs with Genotype, Sex, and Treatment as independent variables, and Day as a repeated 213 measure. When the data were separated by Sex, mixed model ANOVAs were employed with 214 Genotype and Treatment as independent variables, and Day as a repeated measure. Bonferroni-215 adjusted post hoc pairwise unpaired t-tests were used to determine whether group differences 216 were significant on individual days. Slope analyses were conducted as previously described (R. We employed a regimen consisting of one week of habituation to the colony, two weeks 223 of the cuprizone diet, three weeks of recovery from cuprizone, and four weeks of training for BE 224 of PF (Fig.1) . A single cuprizone-assigned male B6J mouse died early on during the study. 225 Therefore, the results are presented for 63 mice instead of 64 mice. In examining the sex-226 combined dataset, we found significantly lower body weight in cuprizone-treated mice of both the 227 B6J and B6NJ strains on D8-D14 of cuprizone treatment ( Fig.2A) . Remarkably, within 24 h after 228 the cuprizone diet was replaced with standard laboratory chow (D15), mice regained nearly all of 229 their body weight and did not differ significantly from control mice ( Fig.2A) . 230 When considering the female and male datasets separately, females showed significant 231 weight loss earlier on at D4 and D5 (Fig.2B) and both sexes showed a significant reduction in 232 body weight from D8-D14, after which there was recovery of weight loss within 24 following 233 removal of the cuprizone diet and replacement with normal home cage chow (Fig.2B,C) . 234 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/865600 doi: bioRxiv preprint Cuprizone treatment reduces BE and compulsive-like eating in male but not female mice 235 of the BE-prone B6NJ substrain 236 Consistent with our previous report (Kirkpatrick et al., 2017) , B6NJ mice showed greater 237 overall PF consumption compared to B6J mice (Fig.3 ) -a behavior that we previously showed 238 was associated with an enrichment of downregulated genes involved in myelination in the striatum 239 (Kirkpatrick et al., 2017) . However, contrary to our hypothesis that the demyelinating agent 240 cuprizone would increase BE, prior treatment with cuprizone in the BE-prone B6NJ substrain 241 actually decreased the amount of PF intake and decreased the slope of escalation in PF intake 242 intake across days (Fig.3A,B) . Despite the lack of effect of cuprizone on the amount of PF intake 246 in B6J mice, cuprizone induced a small but significant non-zero slope in escalation of PF intake 247 across days (Fig.3B ). In examining compulsive-like PF intake in the light/dark conflict test on D23, 248 as expected, the BE-prone B6NJ strain showed a significant increase in PF intake relative to the 249 BE-resistant B6J strain; however, there was no effect of cuprizone or interaction with substrain in 250 the sex-combined dataset (Fig.3C) . 251 In examining females only, there was no significant effect of the cuprizone diet on PF 252 consumption or compulsive-like PF intake (Fig.3D ,F). However, there was a small but significant 253 increase in the slope of escalation of PF intake in cuprizone-treated B6J females due to the uptick 254 in PF intake on the final day of BE training (D18; Fig.3D,E) . Furthermore, the escalation in 255 cuprizone-treated B6J females was significantly greater than control B6J females (#p = 0.045; 256 In examining males only, cuprizone-treated B6NJ mice showed a robust reduction in PF 258 intake relative to their control B6NJ counterparts (Fig.3G) . Additionally, cuprizone treatment 259 eliminated the slope in escalation of PF intake (Fig.3H ) and significantly reduced compulsive-like 260 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/865600 doi: bioRxiv preprint 11 PF intake in B6NJ males in the light/dark conflict test (Fig.3I) . Thus, the cuprizone-induced 261 reduction of PF intake in the sex-combined dataset was completely accounted for by male B6NJ 262 In examining locomotor activity on D23 in the light side of the light/dark test for compulsive-264 like eating, there was a main effect of Genotype (F1,55 = 4.72; p = 0.034) and Sex (F1,55 = 3.97; 265 p = 0.051) but no effect of Treatment [F(1,55) = 0.03; p = 0.86) and no interactions (ps > 0.54) 266 (data not shown). In examining time spent in the light-paired side, there was also no effect of We did not observe any evidence for a potential confounding influence of cuprizone 273 treatment on locomotor activity that could explain differences in PF intake. Specifically, in 274 considering the sex-combined dataset for D1 locomotor activity during initial preference for the 275 PF-paired side (5 weeks after the beginning of cuprizone treatment or i.e., three weeks after the 276 termination of cuprizone treatment), there was no effect of Substrain or interaction with Treatment 277 We previously reported a BE-induced downregulation of a gene network enriched for 291 myelinationan effect that was driven by the BE-prone B6NJ substrain (Kirkpatrick et al., 2017) . 292 Here, we examined differences in the myelin proteins MAG and MBP in B6NJ versus B6J mice 293 and the effect of cuprizone on these protein levels. There were no effect of cuprizone treatment 294 or substrain on MAG levels ( Fig.5A ,B). However, for MBP, cuprizone treatment induced a 295 significant decrease in total MBP and in each of the MBP isoforms compared to control mice 296 The demyelinating agent cuprizone induced comparable weight loss in females and males 303 of both the BE-resistant B6J substrain and the BE-prone B6NJ substrain (Figs.1,2 ), yet it induced 304 robust substrain-and sex-dependent effects on PF intake. In contrast to our hypothesis that 305 administration of the demyelinating agent cuprizone would enhance BE, we observed the 306 opposite result -a robust reduction in BE in male but not female mice of the BE-prone B6NJ strain 307 (Fig.3) . The male-selective decrease in BE in cuprizone-treated B6NJ males could not be 308 explained by confounding effects on locomotor activity (Fig.4) contributes to the high mortality rates in cuprizone doses exceeding 0.3% (Hiremath et al., 1998; 314 Stidworthy et al., 2003) . The mechanism behind cuprizone-induced weight loss is unclear, but 315 could involve copper chelation, given that copper deficiency in both rats (C. G. Taylor, Bettger, & 316 Bray, 1988) and mice (Prohaska, 1983 ) results in reduced body weight. In the present study, we 317 report a similar degree of weight loss in both female and male cuprizone-treated mice of both 318 substrains (Fig. 2) . Thus, it is unlikely that weight loss alone is responsible for the male-specific 319 reduction in BE and compulsive-like eating in the BE-prone B6NJ substrain. Also, food restriction-320 induced weight loss promotes rather than reduces BE in rodents (Consoli, Contarino, Tabarin The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/865600 doi: bioRxiv preprint 14 the cuprizone-treated B6NJ males showed a sharp escalation in PF intake from the first to the 339 second training day, after which they plateaued rather than continuing to escalate like their control 340 B6NJ male counterparts (Fig.3G,H) . It is also possible that cuprizone induced an anhedonic-like 341 state selectively in B6NJ males that caused a reduction in PF intake (Sen et al., 2019) or, e.g., 342 novelty-induced hypophagia due to a depressive-like effect (Dulawa & Hen, 2005) . 343 Here, we showed that two weeks of exposure to the cuprizone diet induced a significant 344 reduction in the level of MBP when assessed at nearly nine weeks after the beginning of cuprizone 345 without affecting MAG at the time of assessment, irrespective of Sex or Substrain (Fig.5) . MBP 346 is a major structural protein produced by oligodendrocytes and Schwan cells and redistributes to 347 the processes to initiate axon ensheathment, compaction, thickening, and adhesion (Han, following the beginning of cuprizone treatment as the process of remyelination ensued (Ludwin & 363 Sternberger, 1984) . It is also possible that there are brain region-specific changes in myelin-364 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/865600 doi: bioRxiv preprint 15 associated proteins and that the striatum shows a different profile than, e.g., the corpus callosum 365 which is the hallmark tissue that is typically employed to assess the degree of cuprizone-induced 366 We observed a decrease in MBP in the BE-prone B6NJ substrain versus BE-resistant B6J 368 substrain that was significant for the14 kDa isoform (Fig.5) and was consistent with our previous 369 observation of an association of BE with the downregulated expression of a set of genes enriched 370 for myelination (Kirkpatrick et al., 2017) . In that study, the enrichment was identified as a function An additional factor that could contribute to sex differences in the effect of cuprizone on 392 BE is weight loss-induced rebound hyperphagia. We are unaware of any studies demonstrating 393 sex differences in eating behavior after weight loss in rodentswith or without cuprizone. 394 However, greater hyperphagia has been reported in females compared to males after 395 hypothalamic lesions in rats (Valenstein, Cox, & Kakolewski, 1969) and Il18 deletion in mice 396 The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/865600 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/865600 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/865600 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/865600 doi: bioRxiv preprint