Copper loading affects rat neurobehaviour by impairing mitochondria-associated endoplasmic reticulum membranes in hippocampal neurons

Copper loading affects rat neurobehaviour by impairing mitochondria-associated endoplasmic reticulum membranes in hippocampal neurons | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Copper loading affects rat neurobehaviour by impairing mitochondria-associated endoplasmic reticulum membranes in hippocampal neurons Zhengzhe Sun, Shan Jin, Xiang Fang, Wenming Yang, Huaizhen Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6094956/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Sep, 2025 Read the published version in Behavioral and Brain Functions → Version 1 posted 6 You are reading this latest preprint version Abstract Background: To observe the effects of copper sulfate (CuSO4) -induced copper loading on neurobehaviour, mitochondria-associated endoplasmic reticulum membranes (MAMs) and related regulatory proteins in the hippocampal CA1 region of Sprague–Dawley (SD) rats. Methods: Forty SD male rats were randomly divided into control and copper loading groups of 20 rats each. The control group rats were fed with normal feed and water; rats in the copper loading group were fed high copper feed (containing 1g/kg of CuSO4) and CuSO4 deionized water (concentration of 0.185%). After 12 weeks of rearing, the morris water maze (MWM) task and novel object recognition (NOR) test were conducted to compare the neurobehavioral characteristics of the two groups of rats. Morphological changes of neuronal MAMs in the hippocampal CA1 region of copper-loaded rats were observed using a transmission electron microscope (TEM) and immunofluorescence double-labelling techniques. Western-blot analysis was used to detect the expression of MAMs proteins VDAC1, IP3R, GRP75 and Mfn2. Results: The results revealed that rats in the copper-loading group had significantly prolonged escape latency and reduced number of platform crossings in the MWM task (p < 0.01). The percentage of novel objects explored (also known as the Discrimination Ratio, DR) and the discrimination index (DI) were significantly reduced in the NOR test (p < 0.01). In addition, electron microscopy shows increased disruption of neuronal endoplasmic reticulum (ER)-mitochondrion coupling in the hippocampal CA1 region of rats in the copper-loading group (p < 0.05), and the percentage of MAMs in mitochondrial circumference decreased (p < 0.05), the colocalization coefficients between the ER and mitochondria was significantly reduced (p < 0.05). Moreover, the protein expression levels of VDAC1, IP3R, and GRP75 in rat hippocampal tissue were detected to be significantly increased (p < 0.01), while the protein expression level of Mfn2 was significantly decreased (p < 0.01). Conclusions: In this study, it is speculated that the neurobehavioral changes in rats may be related to the increased expression levels of the MAMs proteins VDAC1, IP3R, and GRP75, the reduced expression level of Mfn2, and the disruption of the structural integrity of MAMs in the hippocampal CA1 region of rats caused by copper loading. copper loading neurobehavior MAMs Wilson's disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Copper metabolism disorders can cause various neurological diseases, among which Wilson's disease (WD) is the most common. The copper loading present in the WD can cause neurological damage, exhibiting a variety of neurobehavioural impairments 1-2 . Among them, cognitive impairment has a hidden onset but the risk is relatively high, which has a significant impact on the long-term quality of life of patients, and its severity is positively correlated with the duration of the disease, whereas cognitive deficits of the patients can be significantly improved after decoppering chelation treatment 3 . Copper is highly oxidatively toxic, and the neurological damage caused by copper loading is closely related to free radical production 4 . Copper loading can induce free radical overproduction, leading to mitochondrial and DNA damage, energy metabolism disorders in the body, and cytotoxic effects 5 . In particular, mitochondrial damage is the initial target site for copper-mediated neuronal injury and an important contributor to the development of several neurodegenerative diseases 6-7 . The changes in mitochondrial function are closely related to cognitive impairment 8 , but MAMs play an important role in cognitive related diseases by regulating processes such as mitochondrial fusion, fission, and transport, which affect mitochondrial function 9 . MAMs are enriched with various junctional proteins, among which GRP75, an important protein at the interface of MAMs, affects Ca 2+ signaling and maintains mitochondrial calcium homeostasis by binding to IP3R and VDAC1 to form the IP3R-GRP75-VDAC1 protein complex 10-11 . Mfn2 is localized in the mitochondrial outer membrane and ER, enriched in MAMs, and mainly functions as a physical linkage to maintain the structural stability of MAMs 12 . The combined action of IP3R, GRP75, VDAC1, and Mfn2 is of great significance for stabilizing the structure and function of MAMs 13 . Based on the important role of mitochondria-MAMs interactions in maintaining cognitive function, and the close relationship between MAMs junction proteins and the maintenance of MAMs structure and function, it is speculated that structural and functional changes of MAMs involving multiple connectivity proteins may be an important part of the development of high copper-induced cognitive impairment 14 . In this study, we intend to investigate the possible mechanism of MAMs and related proteins involved in high copper-induced abnormal neurobehaviour by observing the effects of copper loading on neurobehavior, structural homeostasis of MAMs, and expression of functionally related proteins in rats. 2. Materials and Methods 2.1 Ethical Statement The rat experiments in this study were conducted at the Animal Experiment Centre of Anhui University of Traditional Chinese Medicine. All animal experiments were conducted in accordance with the National Institutes of Health's "Guidelines for the Care and Use of Laboratory Animals" (NIH Publication No. 8023, revised in 1978). In addition, this experiment has been reviewed and approved by the Animal Welfare Ethics Committee of Anhui University of Traditional Chinese Medicine (Ethics Approval Code: AHUCM-rats-2024036). 2.2 Experimental animals Forty SPF-grade male SD rats [Production License No. SCXK (Liao) 2020-0001, aged 8 weeks, weighing 220-250 g)] were purchased from Huaxing Laboratory Animal Farm, Huiji District, Zhengzhou City, China. After one week of adaptive feeding in the laboratory, 40 rats were randomly divided into two groups (n=20 each):(a) control group, and (b) copper loading group. According to established protocols 15-16 , the rats in group (b) were given high copper feed (containing 1g/kg of CuSO 4 ) and CuSO 4 deionized water (concentration of 0.185%), while group (a) was given saline and standard normal feed as a control for a total of 12 weeks. During this period, the general condition of the rats was examined at regular intervals. All rats were housed in the same room with a humidity of 40-70% and a temperature of 18-22°C, alternating with a 12-hour light/dark cycle. They were allowed to feed and drink water freely. The specific experimental protocol process is shown in Figure 1 . 2.3 Behavior examination We conducted behavioral examinations on rats, including the MWM task and NOR test. The specific timeline for behavioral testing is shown in Figure 2. 2.3.1 The MWM task The MWM task was conducted at the Animal Experiment Center of Anhui University of Traditional Chinese Medicine. 4 days before the end of the modeling, the rats were trained for 4 days, 4 times a day, with intervals of more than 15 minutes between each session 17 . The pool is divided into four quadrants, with a circular platform placed in the center. The rats were placed into the water from four quadrants in turn, and the time they took to climb up to the platform was recorded, known as the ”escape latency” 18 . The test time was limited to 60s, and those exceeding it were counted as 60s. When the rats did not reach the platform within 60 s, the experimenter guided them to the platform. After all animals had completed 4 days of training, the platform was removed from the pool for the space exploration experiment. The rat was released into the water from any random quadrant, and its movement trajectory was tracked for 60s. Two consecutive tests were performed to record the number of times the rat crossed the original platform position 19 . 2.3.2 The NOR test After completing the MWM task, all rats were carried out a NOR test. This experiment uses an open wooden box device with a size of 60cm × 60cm × 60cm, placed in a quiet, weakly lit room 20 . We modified the testing technique based on similar research by Bekci et al 21 . This experiment is divided into three stages: adaptation, familiarity, and testing 22 . During the adaptation phase (3 days in total), the rats were placed in an empty test box and each rat was allowed to freely explore the box for 5 minutes to acclimatize to the environment. The familiarity phase was entered on the forth day by placing two identical rectangular boxes (A1 and A2) of the same height and hardness, odorless, non-smooth, and unable to be moved by rats at will in the center of two opposing areas. Subsequently, the rats were placed into the test box with their backs toward the objects and the distance from the tip of their noses to the two objects was equal, so that they were familiarised with the objects A1 and A2, and were observed for 5 min. On the fifth day, the testing phase was started. The familiar object A2 was replaced with the new object B (a cylindrical box), and the rats were placed into the test box with their backs toward the objects and the tips of their noses at equal distances from both objects. The movement tracks of rats were recorded and analyzed by connecting the camera with Any-Maze animal tracking system software. The situation of rats detecting objects within 10 minutes was recorded and used for behavioral analysis. The time spent exploring object A1 was denoted as F, the time spent exploring new object B was denoted as N, and the total time spent exploring two objects was denoted as F+N. The percentage of rats exploring the new object [DR = N ÷ (N+F) × 100%] and discrimination index [DI = (N-F) ÷ (N+F) × 100%] were calculated 23 . The observation box and 2 objects were thoroughly cleaned with 75% alcohol after each rat experiment to eliminate odors. Collection of hippocampal tissue samples After 24 hours of behavioral testing, all rats were transferred to the sampling room and anesthetized using 1% sodium pentobarbital (20 mg/kg) by intraperitoneal injection. After full anesthesia, 6 rats in each group were randomly selected and decapitated, and the brain tissue was stripped off on ice. The hippocampus was rapidly separated, and the tissue of the hippocampal CA1 region was dissected out and weighed for recording. It was stored in a centrifuge tube containing 4% paraformaldehyde solution at -80°C for Western-blot detection 24 . The remaining rats were fixed on the operating table, and the chest hair was shaved. Tissue scissors were used to dissect the chest wall tissue along the edge of the diaphragm from below the xiphoid process, and vascular clamps were fliped up to fix the anterior chest wall and fully expose the heart and aortic root. Ophthalmic scissors were used to carefully open the pericardium of rats, and the perfusion needle was inserted from the apex of the heart and slowly pushed to the ascending aorta. After the silk thread was secured, the right atrial appendage was clipped open, rapidly infused with physiological saline, and waited for the fluid to flow out of the right atrial appendage and become clear 25-26 . 6 rats were taken from each group and perfused with glutaraldehyde solution instead. Successful perfusion was indicated when the rats' tails trembled and limbs turned white and hard. Rats were executed by decapitation and bluntly dissected of bilateral hippocampal CA1 region tissues on ice, trimmed appropriately, and immersed in centrifuge tubes filled with glutaraldehyde solution stored at 4°C protected from light for transmission electron microscopy 27 . 6 rats were taken from each group for the remaining rats and were perfused with 4% paraformaldehyde solution. After the perfusion was completed, the rats were decapitated, and the tissues of the bilateral hippocampal CA1 region were stripped and stored in 4% paraformaldehyde solution at 4°C for immunofluorescence double-labeling detection 28 . Three sections per animal were analyzed, all within the dorsal CA1 mid-segment (AP -3.3 to -3.8 mm from bregma). 2.4 Transmission electron microscope After successive fixation in 2.5% glutaraldehyde and 1% osmium tetroxide, the hippocampal tissue was dehydrated twice in various graded series of acetone for 10 minutes each time before soaking and embedding. Thereafter, ultrathin (50-75nm) sections were cut with a diamond knife (diatome, ultra45°) on an ultramicrotome (Leica, emuc7), and then stained with lead citrate for 10 minutes. Finally, the sections were observed on a 200 kV transmission electron microscope (FEI, TalosF200S). 2.5 Immunofluorescence double-labeling detection Fixed with 4% paraformaldehyde and embedded in paraffin, routine paraffin sectioning is performed. Hippocampus slices were soaked in xylene I and xylene II for dewaxing, hydrated, and high-pressure repaired with 0.01M sodium citrate buffer solution for 15 minutes. After natural cooling, they were washed with 0.02M PBS for 3 minutes x 3 times. After that, they were preincubated in 5% BSA in phosphate buffer solution for 30 min, followed by dropwise addition of PDI and COX IV primary antibody (1:100; Abcam, Shanghai, China) and overnight incubation at 4 ° C, and incubation of secondary antibody (1:100; Abcam, Shanghai, China) for 1 hour 29 . Finally, the slices were mounted with mounting medium (containing DAPI; Beyotime, Shanghai, China) and photographed with a circular fluorescence microscope (Nikon, Tokyo, Japan). 2.6 Western-blot analysis Proteins were extracted from hippocampal tissues using RIPA lysis buffer (1:100; Beyotim, Shanghai, China). The protein solution was added to 5×reduced protein loading buffer (Solarbio, Beijing, China) in the ratio of 4:1, denatured in a boiling water for 15 minutes, and stored in a refrigerator at -20°C for spare use. Equal amounts of proteins were separated using an SDS-PAGE gel preparation kit (SangonBiotech, Shanghai, China). Electrophoresis was performed on SDS-polyacrylamide gels with 25 μg of total protein per lane. After electrophoresis, the proteins were transferred to a PVDF membrane. Then, the membrane was incubated with the corresponding primary antibodies (Mfn21:1000, VDAC11:1000, IP3R1:1000, GRP751:1000, Shanghai, China) overnight at 4°C, and the secondary antibody (1:5000, SignalwayAntibody, Nanjing, China) was incubated at room temperature for 30 min. Finally, the bands were developed by applying ECL luminescent solution (Millipore, Shanghai, China) and analysed using ImageJ software to calculate the grey values of the bands. The assay results were normalized to the relative protein expression using β-actin as an internal reference protein. 2.7 Statistical Analysis Quantitative data were represented as`x±s and analyzed using SPSS 26.0 software (International Business Machines Corporation, Armonk, New York, USA). The quantitative data comparison between the two groups was conducted using a t-test, and the qualitative data using a chi-square test. Differences between multiple groups were tested using two-way ANOVA and Tukey's post hoc test. P <0.05 was considered statistically significant. 3. Results 3.1 General conditions of the two groups of rats The rats in the control group were in a good mental state, with normal activities and faster weight gain. After one week of modeling, the rats in the copper loading group showed depression, reduced activity, and decreased glossiness of their fur. The rats showed significant weight loss and hair loss in the 3rd week of modelling. Starting from the 10th week of modeling, some rats showed marked emaciation, decreased activity, and sparse hair. Throughout the experimental period, there were no deaths in the control rats, while two deaths in the copper loading group. 3.2 Effect of copper loading on neurobehaviour in rats 3.2.1 The MWM task Two groups of rats underwent MWM tasks after modeling, and the results are shown in Figures 3 and 4 . Compared with the control group, the escape latency of rats in the loading group was significantly prolonged, while the number of crossing platforms was significantly reduced ( P <0.01). 3.2.2 The NOR test Both groups of rats underwent the NOR test. The results showed that compared with the control group, the copper-loaded group of rats exhibited a significant impairment in the recognition and memory of new objects, which can be demonstrated by the significant decrease in the percentage of DR and DI in the copper-loaded group rats ( P <0.01) (Figure 5) . 3.3 The effect of copper loading on the ultrastructure of MAMs in rat hippocampal tissue The changes in the ultrastructure of MAMs observed under transmission electron microscopy are shown in Figure 6. In the hippocampal neurons of the control group rats, regular mitochondria and ER were observed, with intact mitochondrial cristae and tightly packed MAMs structures. The mitochondria and ER of hippocampal neurons in the copper loading group were highly swollen, structurally dilated, and the mitochondrial cristae structure was blurred, broken, and disappeared. In addition, the structure of MAMs in the copper loading group was sparsed, and the percentage of coupling length to mitochondrial circumference was significantly reduced ( P <0.05) 3.4 Effects of copper loading on mitochondrial ER co-localization Immunofluorescence double-labeling techniques were used to observe the co-localization of ER and mitochondria, and ImageJ software was used to analyze their co-localization coefficients. Compared with the control group, the number of MAMs in the hippocampal CA1 region of the copper-loaded group rats was significantly reduced, and the co-localization coefficients of ER and mitochondria were significantly decreased ( P <0.05), indicating a weakened interaction between ER and mitochondria ( Figure 7, Figure 8 ). 3.5 The effect of copper loading on the expression of functionally related proteins in MAMs The expression levels of functionally related proteins of MAMs in rats are shown in Figure 9. The results showed that the protein expression levels of VDAC1, IP3R, and GRP75 were significantly increased and the protein expression level of Mfn2 was significantly decreased in the copper-loaded group compared with the control group ( P <0.01). 4. Discussion Copper is one of the essential trace elements for the body to maintain normal physiological functions 30 . Excessive deposition of copper in the body may result in "direct" damage to the conformational changes of functional active molecules; on the other hand, it can induce the generation of reactive oxygen species and reactive nitrogen species, causing "indirect" damage. The "indirect" damage can activate intracellular early response genes that are extremely sensitive to oxidative stress, ultimately causing DNA damage, lipid peroxidation, and altered thiol homeostasis 31 . Changes in the mechanism of copper homeostasis can lead to impaired Cytox function, causing abnormal mitochondrial respiration, and oxidative stress on intracellular organelles such as mitochondria 32 . Sustained oxidative stress may induce mitochondrial permeability transition and a decrease in its oxidative potential, and trigger a series of cascading reactions that ultimately result in neuronal damage. Therefore, mitochondria are considered an early and sensitive key to copper-induced oxygen stress in neuronal cells 33 . In addition, mitochondria are the main site of energy metabolism, and neuronal cells associated with cognition are highly polarized cells whose large energy demands are mainly met by mitochondria. Normal mitochondrial function is crucial for neuroprotection and repair of cognition-related brain regions. However, mitochondrial dysfunction can also lead to the production of excessive free radicals and the imbalance of calcium ion concentration, resulting in neuronal dysfunction 34 . Mitochondrial dysfunction is directly or indirectly associated with cognitive impairment in various diseases 35 . Mitochondria communicate with other organelles inside the cell through the outer membrane, and play an important role in cognitive impairment-related diseases through mitochondrial self-metabolism and intracellular signaling pathways. There is a co-localized structure between mitochondria and ER, and this tightly contacted subcellular structure is called MAMs. MAMs act as a physical and biochemical bridge between mitochondria and ER, are critical for ER stress and unfolded protein responses, autophagy, inflammatory mediator signal transduction, and widely influence normal intracellular life activities 36 . Structural and functional changes in MAMs may cause disturbances in calcium homeostasis, abnormal cholesterol and phospholipid metabolism, which may affect mitochondrial function and participate in the development of cognitive impairment 37 . Reactive oxygen species, as the intersection of high copper-induced oxidative stress response and autophagy cascade, serve as a bridge and link between pathogenic factors and the organism's defense response 38 . When the body perceives the threat of oxidative stress posed by excess reactive oxygen species, it will regulate the structure of MAMs and stabilize the mitochondrial function through various MAMs constituent proteins, so as to achieve the role of repairing the damage and maintaining cellular functional homeostasis 39 . Among a variety of MAMs constituent proteins, Mfn2 is a MAMs-associated mitochondrial fusion protein, localized on the surface of the ER, which can form homodimers or heterodimers with Mfn1 at the mitochondrial outer membrane and plays an important role in regulating mitochondrial morphology 40 . IP3R is a Ca 2+ release channel located on the ER membrane, which allows Ca 2+ to be released from the ER lumen into the cytoplasm 41 . VDAC1 is a voltage-dependent Ca 2+ uptake channel located on the outer mitochondrial membrane, which is involved in the regulation of Ca 2+ concentration in mitochondria and is an important channel protein for maintaining intracellular calcium homeostasis 42 . GRP75 plays an important bridging role in intracellular Ca 2+ transport. GRP75 promotes the binding of mitochondria to the ER, and acts as a molecular chaperone linking IP3R and VDAC1 to form the VDAC1-GRP75-IP3R complex, which can mediate Ca 2+ transport from the ER to mitochondria, and regulate the calcium homeostasis of mitochondria 43 . Under pathological conditions, dysfunction of the VDAC1-GRP75-IP3R complex can result in excessive calcium ion concentrations in mitochondria, leading to alterations in mitochondrial membrane permeability, opening of mitochondrial permeability transition pores (MPTP), causing changes in mitochondrial osmolality, and rupture of the outer membrane, leading to mitochondrial morphology and functional disorders 44 . The MAMs proteins IP3R, GRP75, VDAC1, and Mfn2 participate in regulating MAMs function from multiple perspectives to maintain mitochondrial morphology and membrane integrity, affecting cognitive function. The copper-loaded rat model can better simulate the pathological process of copper deposition in vivo and has been widely used in studies related to WD 45-46 . In this study, MWM and NOR were used for neurobehavioural assessment of rats. MWM includes localization navigation experiments and spatial probe tests, which can reflect the learning and memory abilities of rats toward platform spatial positions 47 . NOR can reflect the non spatial recognition memory ability of animals 48 . The combination of MWM and NOR methods can effectively evaluate abnormal neurobehaviour in copper-loaded rats. In this study, copper-loaded rats had significantly prolonged escape latency and reduced number of platform crossings, and the percentage of DR and the DI were significantly reduced. It suggests that copper loading can impair the abilities of learning, spatial exploration, and non-spatial recognition memory in rats, causing neurobehavioural dysfunction. In addition, this study found that copper loading can disrupt the microstructure of rat MAMs: Under transmission electron microscopy, neuronal mitochondria and ER in the hippocampal CA1 region of the copper-loaded group rats were highly swollen, structurally dilated, and the mitochondrial cristae structure was blurred, broken, and disappeared. Besides, the number of MAMs was significantly reduced, and the percentage of coupling length to mitochondrial circumference was significantly decreased. ER and mitochondrial co-localization coefficients were significantly reduced in immunofluorescence double-labeling assays. Moreover, copper loading can induce differential expression of structural and functional proteins related to MAMs in the hippocampal CA1 region, with significantly increased expression levels of the Ca 2+ channel proteins VDAC1, IP3R, and GRP75, and significantly decreased expression levels of mitochondrial outer membrane fusion protein Mfn2. We have to acknowledge that our study focused on the ultrastructure and function of MAMs as sensitive indicators of ER-mitochondria disruption, future work employing ER-specific staining may provide complementary insights. 5. Conclusions In this study, we found that copper loading can cause abnormal expression of MAMs homeostasis-related proteins IP3R1, GRP75, VDAC1, and Mfn2. It is speculated that high copper-induced neurobehavioural impairments may be related to the structural changes and related protein expression changes in MAMs, and the specific mechanism is worthy of further in-depth study. However, whether cognitive dysfunction can be improved by regulating the expression of IP3R, GRP75, VDAC1, and Mfn2 proteins to maintain the MAMs homeostasis, needs to be further investigated in the future. Declarations Disclosure statement All authors report no relevant conflicts of interest/financial disclosures. Informed Consent Statement Not applicable. Availability of data and materials The datasets for this article are public. Ethics approval statement This study has been reviewed and approved by the Animal Welfare Ethics Committee of Anhui University of Traditional Chinese Medicine (Ethics Approval Code: AHUCM-rats-2024036). Author’s contribution ZZ S, S J, X F:drafting/revision of the manuscript for content, including medical writing for content WM Y, HZ C: major role in the acquisition of data The final draft was read and approved by all the authors. Acknowledgments We sincerely thank all editors and reviewers for their help with our paper. Consent for publication All authors read and approve of the final manuscript for publication. Funding This research was supported by The General program of Anhui Natural Science Foundation (2208085MH271), The General Program of the National Natural Science Foundation of China (81973825), Anhui Province Traditional Chinese Medicine Inheritance and Innovation Research Project (2024CCCX093), Anhui Province Higher Education Science Research Major Project (2024AH040153) and Anhui Province Higher Education Science Research Key Project (2024AH051016). Abbreviations MAMs: Mitochondria-associated endoplasmic reticulum membranes CuSO 4 : Copper sulfate SD: Sprague–Dawley MWM: Morris water maze NOR: Novel object recognition TEM: Transmission electron microscope DR: Discrimination Ratio, DI: Discrimination index ER: Edoplasmic reticulum WD: Wilson's disease MPTP: Mitochondrial permeability transition pores References Schilsky M L, Roberts E A, Bronstein J M, et al. 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Neurotoxic effect of flavonol myricetin in the presence of excess copper[J]. Molecules, 2021, 26(4): 845-852. Witt B, Stiboller M, Raschke S, et al. Characterizing effects of excess copper levels in a human astrocytic cell line with focus on oxidative stress markers[J]. Journal of Trace Elements in Medicine and Biology, 2021, 65: 126711. Baev A Y, Vinokurov A Y, Novikova I N, et al. Interaction of mitochondrial calcium and ROS in neurodegeneration[J]. Cells, 2022, 11(4): 706-712. Sharma C, Kim S, Nam Y, et al. Mitochondrial dysfunction as a driver of cognitive impairment in Alzheimer’s disease[J]. International Journal of Molecular Sciences, 2021, 22(9): 4850. Ji C, Zhang Z, Li Z, et al. Mitochondria‐Associated Endoplasmic Reticulum Membranes: Inextricably Linked with Autophagy Process[J]. Oxidative medicine and cellular longevity, 2022, 2022(1): 7086807. Eysert F, Kinoshita P F, Mary A, et al. Molecular dysfunctions of mitochondria-associated membranes (MAMs) in Alzheimer’s disease[J]. International journal of molecular sciences, 2020, 21(24): 9521. Qiao N, Dai X, Chen J, et al. Single nucleus RNA sequencing reveals cellular and molecular responses to vanadium exposure in duck kidneys[J]. J Hazard Mater, 2024, 480(2): 136492. Resende R, Fernandes T, Pereira A C, et al. Endoplasmic reticulum-mitochondria contacts modulate reactive oxygen species-mediated signaling and oxidative stress in brain disorders: The key role of sigma-1 receptor[J]. Antioxidants & Redox Signaling, 2022, 37(10-12): 758-780. Hu Y, Chen H, Zhang L, et al. The AMPK-MFN2 axis regulates MAM dynamics and autophagy induced by energy stresses[J]. Autophagy, 2021, 17(5): 1142-1156. Erustes A G, D'Eletto M, Guarache G C, et al. Overexpression of α‐synuclein inhibits mitochondrial Ca2+ trafficking between the endoplasmic reticulum and mitochondria through MAMs by altering the GRP75–IP3R interaction[J]. 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Hepatology, 2019, 70(1): 108-126. Zorzo C, Arias J L, Méndez M. Are there sex differences in spatial reference memory in the Morris water maze? A large-sample experimental study[J]. Learning & Behavior, 2024, 52(2): 179-190. Kishi T, Kobayashi K, Sasagawa K, et al. Automated analysis of a novel object recognition test in mice using image processing and machine learning[J]. Behavioural Brain Research, 2024: 115278. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 30 Sep, 2025 Read the published version in Behavioral and Brain Functions → Version 1 posted Editorial decision: Revision requested 22 Apr, 2025 Reviews received at journal 20 Apr, 2025 Reviewers agreed at journal 20 Apr, 2025 Reviewers invited by journal 15 Apr, 2025 Submission checks completed at journal 07 Apr, 2025 First submitted to journal 06 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6094956","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":443396696,"identity":"1a05aeb2-36a1-4401-aedd-0e0b784b2629","order_by":0,"name":"Zhengzhe Sun","email":"","orcid":"","institution":"The Second Affiliated Hospital of Anhui University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhengzhe","middleName":"","lastName":"Sun","suffix":""},{"id":443396697,"identity":"2fbe2a06-94fb-4b28-b9cc-ca75e59896db","order_by":1,"name":"Shan Jin","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shan","middleName":"","lastName":"Jin","suffix":""},{"id":443396698,"identity":"61d222c8-ff92-4470-83fe-278d9b0b8402","order_by":2,"name":"Xiang Fang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYBAC9gYGAwaGCoYECJeNCC08B0BazjAk8JCmhbGNJC0SydukeefZ5dmzn05g+FB2mIF/dgMBLTzHig1nbksu5uHJ3cA449xhBok7B/BrsWfvMXzwcRtzYo8E7wZm3rbDDAYSCQRsYeYxOJA4px6i5S9RWsC2NByGaGEkSgvILzOOHU/sOZO74WDPuXQeiRuEtIBCjKemOrG9/ezGBz/KrOX4ZxDQggIOgMwgQf0oGAWjYBSMAlwAAA/gQDQhPu6gAAAAAElFTkSuQmCC","orcid":"","institution":"The First Affiliated Hospital of Anhui University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Fang","suffix":""},{"id":443396699,"identity":"3288ba66-28ed-4f6f-8fbd-6c84f197eacd","order_by":3,"name":"Wenming Yang","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wenming","middleName":"","lastName":"Yang","suffix":""},{"id":443396702,"identity":"4f9636aa-56f4-46d4-bae7-890235e50414","order_by":4,"name":"Huaizhen Chen","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Huaizhen","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-02-24 08:53:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6094956/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6094956/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12993-025-00277-y","type":"published","date":"2025-09-30T15:56:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80743738,"identity":"9deb719b-c63a-4173-be26-8e7562375d60","added_by":"auto","created_at":"2025-04-16 14:54:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":195494,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic of the experimental design\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6094956/v1/c11f8c8495b8813d51e3b8ce.png"},{"id":80744224,"identity":"2e724c7e-4ceb-4c50-9578-bed136947460","added_by":"auto","created_at":"2025-04-16 15:02:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":41612,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTime–line of the behavior alanalysis.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6094956/v1/587550d2eb16346704d63d54.png"},{"id":80744230,"identity":"1e81bfbc-25c8-4c79-88c9-41f88082adcd","added_by":"auto","created_at":"2025-04-16 15:02:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":66329,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEscape latency time in both groups of rats. \u003c/strong\u003eDuring 1-4 days, the copper loading group significantly prolonged the escape latency of rats compared to the control group. Control: the control group; CuSO\u003csub\u003e4\u003c/sub\u003e: the copper loading group. Data are presented as mean±SD (n=15). **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 versus the control group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6094956/v1/36bf0e6082cada29cbe3cb48.png"},{"id":80743746,"identity":"3f8f4ae9-3e5f-4609-a436-5dc331161178","added_by":"auto","created_at":"2025-04-16 14:54:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":54549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe number of platform crossings in both groups of rats.\u003c/strong\u003e The number of platform crossings was significantly reduced in the copper loading group compared to the control group of rats. Control: the control group; CuSO\u003csub\u003e4\u003c/sub\u003e: the copper loading group. Data are presented as mean±SD (n=15). **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 versus the control group.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6094956/v1/cdb313c42dccd800c1d4b482.png"},{"id":80744225,"identity":"73f9df85-2bd4-4a82-b20e-9021b794f037","added_by":"auto","created_at":"2025-04-16 15:02:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":63416,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the results of the NOR test in two groups of rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth DR and DI values were significantly reduced in the copper loading group compared to the control group rats.Control: the control group; CuSO\u003csub\u003e4\u003c/sub\u003e: the copper loading group. Data are presented as mean±SD (n=15). **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 versus the control group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6094956/v1/e6462e2d3a95f6e7b6ce1290.png"},{"id":80744228,"identity":"47f99dfa-1644-4b90-8e0e-887d6a061ba0","added_by":"auto","created_at":"2025-04-16 15:02:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":517941,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in the ultrastructure of MAMs in hippocampal neurons of two groups of rats.\u003c/strong\u003e Control: the control group; CuSO\u003csub\u003e4\u003c/sub\u003e: the copper loading group. The yellow dashed lines outline the ultrastructural features of the MAMs. Data are presented as mean±SD (n=6). **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 versus the control group.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6094956/v1/0ab155bcae9cb46914962464.png"},{"id":80743764,"identity":"ee27d9a3-bfc9-442e-9ec0-e3e54bdf53f5","added_by":"auto","created_at":"2025-04-16 14:54:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":329448,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of copper loading on the co-localization of ER and mitochondria in the CA1 region of rats (×200). \u003c/strong\u003eAnti-PDI antibody labels the ER as green, anti-COX IV antibody labels mitochondria as red, and DAPI labels the nucleus as blue.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6094956/v1/23487be51ddcbc51910ba364.png"},{"id":80743748,"identity":"85eaf7c2-d029-438a-9b3e-8958ac66649a","added_by":"auto","created_at":"2025-04-16 14:54:09","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":38962,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePearson's correlation coefficient of mitochondria and ER of different groups.\u003c/strong\u003e n=6. Data are presented as mean±SD (n=6), *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6094956/v1/7597ce444354f4b09a6651f7.png"},{"id":80745120,"identity":"8312e177-0764-4a92-9f56-ab217592aa63","added_by":"auto","created_at":"2025-04-16 15:10:09","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":193698,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of copper loading on the protein expression levels of Mfn2, VDAC1, IP3R, and GRP75 in the hippocampal CA1 region of rats.\u003c/strong\u003e (a) Western-blot assay for \u003ca href=\"https://kns.cnki.net/kns8/Detail?sfield=fn\u0026amp;QueryID=0\u0026amp;CurRec=13\u0026amp;FileName=1023542112.nh\u0026amp;DbName=CDFDTEMP\u0026amp;DbCode=CDFD\" target=\"_blank\"\u003eMfn2\u003c/a\u003e, VDAC1, IP3R and GRP75. (b) \u003ca href=\"https://kns.cnki.net/kns8/Detail?sfield=fn\u0026amp;QueryID=0\u0026amp;CurRec=13\u0026amp;FileName=1023542112.nh\u0026amp;DbName=CDFDTEMP\u0026amp;DbCode=CDFD\" target=\"_blank\"\u003eMfn2\u003c/a\u003e, VDAC1, IP3R and GRP75 MAMs related protein expression levels. Control: the control group; CuSO\u003csub\u003e4\u003c/sub\u003e: the copper loading group. Data are presented as mean±SD (n=6). **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 versus the control group.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6094956/v1/638bfc1ef6010d5ebe9dec92.png"},{"id":92883592,"identity":"a5c9d23e-a66d-4c76-a750-71d2c8476e42","added_by":"auto","created_at":"2025-10-06 16:03:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2384414,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6094956/v1/a231dd13-8f65-47ea-bcb7-f1b3089cc3e5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Copper loading affects rat neurobehaviour by impairing mitochondria-associated endoplasmic reticulum membranes in hippocampal neurons","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCopper metabolism disorders can cause various neurological diseases, among which Wilson's disease (WD) is the most common. The copper loading present in the WD can cause neurological damage, exhibiting a variety of neurobehavioural impairments\u003csup\u003e1-2\u003c/sup\u003e. Among them, cognitive impairment has a hidden onset but the risk is relatively high, which has a significant impact on the long-term quality of life of patients, and its severity is positively correlated with the duration of the disease, whereas cognitive deficits of the patients can be significantly improved after decoppering\u0026nbsp;chelation treatment\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Copper is highly oxidatively toxic, and the neurological damage caused by copper loading is closely related to free radical production\u003csup\u003e4\u003c/sup\u003e. Copper loading can induce free radical overproduction, leading to mitochondrial and DNA damage, energy metabolism disorders in the body, and cytotoxic effects\u003csup\u003e5\u003c/sup\u003e. In particular, mitochondrial damage is the initial target site for copper-mediated neuronal injury and an important contributor to the development of several neurodegenerative diseases\u003csup\u003e6-7\u003c/sup\u003e. The changes in mitochondrial function are closely related to cognitive impairment\u003csup\u003e8\u003c/sup\u003e, but MAMs play an important role in cognitive related diseases by regulating processes such as mitochondrial fusion, fission, and transport, which affect mitochondrial function\u003csup\u003e9\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eMAMs are enriched with various junctional proteins, among which GRP75, an important protein at the interface of MAMs, affects Ca\u003csup\u003e2+\u003c/sup\u003e signaling and maintains mitochondrial calcium homeostasis by binding to IP3R and VDAC1 to form the IP3R-GRP75-VDAC1 protein complex\u003csup\u003e10-11\u003c/sup\u003e. Mfn2 is localized in the mitochondrial outer membrane and ER, enriched in MAMs, and mainly functions as a physical linkage to maintain the structural stability of MAMs\u003csup\u003e12\u003c/sup\u003e. The combined action of IP3R, GRP75, VDAC1, and Mfn2 is of great significance for stabilizing the structure and function of MAMs\u003csup\u003e13\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBased on the important role of mitochondria-MAMs interactions in maintaining cognitive function, and the close relationship between MAMs junction proteins and the maintenance of MAMs structure and function, it is speculated that structural and functional changes of MAMs involving multiple connectivity proteins may be an important part of the development of high copper-induced cognitive impairment\u003csup\u003e14\u003c/sup\u003e. In this study, we intend to investigate the possible mechanism of MAMs and related proteins involved in high copper-induced abnormal neurobehaviour by observing the effects of copper loading on neurobehavior, structural homeostasis of MAMs, and expression of functionally related proteins in rats.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 \u0026nbsp;Ethical Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rat experiments in this study were conducted at the Animal Experiment Centre of Anhui University of Traditional Chinese Medicine. All animal experiments were conducted in accordance with the National Institutes of Health\u0026apos;s \u0026quot;Guidelines for the Care and Use of Laboratory Animals\u0026quot; (NIH Publication No. 8023, revised in 1978).\u003c/p\u003e\n\u003cp\u003eIn addition, this experiment has been reviewed and approved by the Animal Welfare Ethics Committee of Anhui University of Traditional Chinese Medicine (Ethics Approval Code: AHUCM-rats-2024036).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 \u0026nbsp;Experimental animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eForty SPF-grade male SD rats [Production License No. SCXK (Liao) 2020-0001, aged 8 weeks, weighing 220-250 g)] were purchased from Huaxing Laboratory Animal Farm, Huiji District, Zhengzhou City, China. After one week of adaptive feeding in the laboratory, 40 rats were randomly divided into two groups (n=20 each):(a) control group, and (b) copper loading group. According to established protocols\u003csup\u003e15-16\u003c/sup\u003e, the rats in group (b) were given high copper feed (containing 1g/kg of CuSO\u003csub\u003e4\u003c/sub\u003e) and CuSO\u003csub\u003e4\u003c/sub\u003e deionized water (concentration of 0.185%), while group (a) was given saline and standard normal feed as a control for a total of 12 weeks. During this period, the general condition of the rats was examined at regular intervals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll rats were housed in the same room with a humidity of 40-70% and a temperature of 18-22\u0026deg;C, alternating with a 12-hour light/dark cycle. They were allowed to feed and drink water freely. The specific experimental protocol process is shown in\u003cstrong\u003e\u0026nbsp;Figure 1\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 \u0026nbsp;Behavior examination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe conducted behavioral examinations on rats, including the MWM task and NOR test. The specific timeline for behavioral testing is shown in Figure 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.3.1 The MWM task\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MWM task was conducted at the Animal Experiment Center of Anhui University of Traditional Chinese Medicine. 4 days before the end of the modeling, the rats were trained for 4 days, 4 times a day, with intervals of more than 15 minutes between each session\u003csup\u003e17\u003c/sup\u003e. The pool is divided into four quadrants, with a circular platform placed in the center. The rats were placed into the water from four quadrants in turn, and the time they took to climb up to the platform was recorded, known as the \u0026rdquo;escape latency\u0026rdquo;\u003csup\u003e18\u003c/sup\u003e. The test time was limited to 60s, and those exceeding it were counted as 60s. When the rats did not reach the platform within 60 s, the experimenter guided them to the platform.\u003c/p\u003e\n\u003cp\u003eAfter all animals had completed 4 days of training, the platform was removed from the pool for the space exploration experiment. The rat was released into the water from any random quadrant, and its movement trajectory was tracked for 60s. Two consecutive tests were performed to record the number of times the rat crossed the original platform position\u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.3.2 The NOR test\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter completing the MWM task, all rats were carried out a NOR test. This experiment uses an open wooden box device with a size of 60cm \u0026times; 60cm \u0026times; 60cm, placed in a quiet, weakly lit room\u003csup\u003e20\u003c/sup\u003e. We modified the testing technique based on similar research by Bekci et al\u003csup\u003e21\u003c/sup\u003e. This experiment is divided into three stages: adaptation, familiarity, and testing\u003csup\u003e22\u003c/sup\u003e. During the adaptation phase (3 days in total), the rats were placed in an empty test box and each rat was allowed to freely explore the box for 5 minutes to acclimatize to the environment. The familiarity phase was entered on the forth day by placing two identical rectangular boxes (A1 and A2) of the same height and hardness, odorless, non-smooth, and unable to be moved by rats at will in the center of two opposing areas.\u0026nbsp;Subsequently, the rats were placed into the test box with their backs toward the objects and the distance from the tip of their noses to the two objects was equal, so that they were familiarised with the objects A1 and A2, and were observed for 5 min. On the fifth day, the testing phase was started. The familiar object A2 was replaced with the new object B (a cylindrical box), and the rats were placed into the test box with their backs toward the objects and the tips of their noses at equal distances from both objects.\u0026nbsp;The movement tracks of rats were recorded and analyzed by connecting the camera with Any-Maze animal tracking system software. The situation of rats detecting objects within 10 minutes was recorded and used for behavioral analysis. The time spent exploring object A1 was denoted as F, the time spent exploring new object B was denoted as N, and the total time spent exploring two objects was denoted as F+N. The percentage of rats exploring the new object [DR = N \u0026divide; (N+F) \u0026times; 100%] and discrimination index [DI = (N-F) \u0026divide; (N+F) \u0026times; 100%] were calculated\u003csup\u003e23\u003c/sup\u003e. The observation box and 2 objects were thoroughly cleaned with 75% alcohol after each rat experiment to eliminate odors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollection of hippocampal tissue samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 24 hours of behavioral testing, all rats were transferred to the sampling room and anesthetized using 1% sodium pentobarbital (20 mg/kg) by intraperitoneal injection. After full anesthesia, 6 rats in each group were randomly selected and decapitated, and the brain tissue was stripped off on ice. The hippocampus was rapidly separated, and the tissue of the hippocampal CA1 region was dissected out and weighed for recording. It was stored in a centrifuge tube containing 4% paraformaldehyde solution at -80\u0026deg;C for Western-blot detection\u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe remaining rats were fixed on the operating table, and the chest hair was shaved. Tissue scissors were used to dissect the chest wall tissue along the edge of the diaphragm from below the xiphoid process, and vascular clamps were fliped up to fix the anterior chest wall and fully expose the heart and aortic root. Ophthalmic scissors were used to carefully open the pericardium of rats, and the perfusion needle was inserted from the apex of the heart and slowly pushed to the ascending aorta. After the silk thread was secured, the right atrial appendage was clipped open, rapidly infused with physiological saline, and waited for the fluid to flow out of the right atrial appendage and become clear\u003csup\u003e25-26\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e6 rats were taken from each group and perfused with glutaraldehyde solution instead. Successful perfusion was indicated when the rats\u0026apos; tails trembled and limbs turned white and hard. Rats were executed by decapitation and bluntly dissected of bilateral hippocampal CA1 region tissues on ice, trimmed appropriately, and immersed in centrifuge tubes filled with glutaraldehyde solution stored at 4\u0026deg;C protected from light for transmission electron microscopy\u003csup\u003e27\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e6 rats were taken from each group for the remaining rats and were perfused with 4% paraformaldehyde solution. After the perfusion was completed, the rats were decapitated, and the tissues of the bilateral hippocampal CA1 region were stripped and stored in 4% paraformaldehyde solution at 4\u0026deg;C for immunofluorescence double-labeling detection\u003csup\u003e28\u003c/sup\u003e. Three sections per animal were analyzed, all within the dorsal CA1 mid-segment (AP -3.3 to -3.8 mm from bregma).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 \u0026nbsp;Transmission electron microscope\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter successive fixation in 2.5% glutaraldehyde and 1% osmium tetroxide, the hippocampal tissue was dehydrated twice in various graded series of acetone for 10 minutes each time before soaking and embedding. Thereafter, ultrathin (50-75nm) sections were cut with a diamond knife (diatome, ultra45\u0026deg;) on an ultramicrotome (Leica, emuc7), and then stained with lead citrate for 10 minutes. Finally, the sections were observed on a 200 kV transmission electron microscope (FEI, TalosF200S).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 \u0026nbsp;Immunofluorescence double-labeling detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFixed with 4% paraformaldehyde and embedded in paraffin, routine paraffin sectioning is performed. Hippocampus slices were soaked in xylene I and xylene II for dewaxing, hydrated, and high-pressure repaired with 0.01M sodium citrate buffer solution for 15 minutes. After natural cooling, they were washed with 0.02M PBS for 3 minutes x 3 times. After that, they were preincubated in 5% BSA in phosphate buffer solution for 30 min, followed by dropwise addition of PDI and COX IV primary antibody (1:100; Abcam, Shanghai, China) and overnight incubation at 4 \u0026deg; C, and incubation of secondary antibody (1:100; Abcam, Shanghai, China) for 1 hour\u003csup\u003e29\u003c/sup\u003e. Finally, the slices were mounted with mounting medium (containing DAPI; Beyotime, Shanghai, China) and photographed with a circular fluorescence microscope (Nikon, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 \u0026nbsp;Western-blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteins were extracted from hippocampal tissues using RIPA lysis buffer (1:100; Beyotim, Shanghai, China). The protein solution was added to 5\u0026times;reduced protein loading buffer (Solarbio, Beijing, China) in the ratio of 4:1, denatured in a boiling water for 15 minutes, and stored in a refrigerator at -20\u0026deg;C for spare use. Equal amounts of proteins were separated using an SDS-PAGE gel preparation kit (SangonBiotech, Shanghai, China). Electrophoresis was performed on SDS-polyacrylamide gels with 25 \u0026mu;g of total protein per lane. After electrophoresis, the proteins were transferred to a PVDF membrane. Then, the membrane was incubated with the corresponding primary antibodies (Mfn21:1000, VDAC11:1000, IP3R1:1000, GRP751:1000, Shanghai, China) overnight at 4\u0026deg;C, and the secondary antibody (1:5000, SignalwayAntibody, Nanjing, China) was incubated at room temperature for 30 min. Finally, the bands were developed by applying ECL luminescent solution (Millipore, Shanghai, China) and analysed using ImageJ software to calculate the grey values of the bands. The assay results were normalized to the relative protein expression using \u0026beta;-actin as an internal reference protein.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 \u0026nbsp;Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantitative data were represented as`x\u0026plusmn;s and analyzed using SPSS 26.0 software (International Business Machines Corporation, Armonk, New York, USA). The quantitative data comparison between the two groups was conducted using a t-test, and the qualitative data using a chi-square test. Differences between multiple groups were tested using two-way ANOVA and Tukey\u0026apos;s post hoc test. \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 \u0026nbsp;General conditions of the two groups of rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rats in the control group were in a good mental state, with normal activities and faster weight gain. After one week of modeling, the rats in the copper loading group showed depression, reduced activity, and decreased glossiness of their fur.\u003c/p\u003e\n\u003cp\u003eThe rats showed significant weight loss and hair loss in the 3rd week of modelling.\u003c/p\u003e\n\u003cp\u003eStarting from the 10th week of modeling, some rats showed marked emaciation, decreased activity, and sparse hair. Throughout the experimental period, there were no deaths in the control rats, while two deaths in the copper loading group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 \u0026nbsp;Effect of copper loading on neurobehaviour in rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.2.1 The MWM task\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo groups of rats underwent MWM tasks after modeling, and the results are shown in \u003cstrong\u003eFigures 3 and 4\u003c/strong\u003e. Compared with the control group, the escape latency of rats in the loading group was significantly prolonged, while the number of crossing platforms was significantly reduced (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.2.2 The NOR test\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth groups of rats underwent the NOR test. The results showed that compared with the control group, the copper-loaded group of rats exhibited a significant impairment in the recognition and memory of new objects, which can be demonstrated by the significant decrease in the percentage of DR and DI in the copper-loaded group rats (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01) \u003cstrong\u003e(Figure 5)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 \u0026nbsp;The effect of copper loading on the ultrastructure of MAMs in rat hippocampal tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe changes in the ultrastructure of MAMs observed under transmission electron microscopy are shown in \u003cstrong\u003eFigure 6.\u0026nbsp;\u003c/strong\u003eIn the hippocampal neurons of the control group rats, regular mitochondria and ER were observed, with intact mitochondrial cristae and tightly packed MAMs structures. The mitochondria and ER of hippocampal neurons in the copper loading group were highly swollen, structurally dilated, and the mitochondrial cristae structure was blurred, broken, and disappeared. In addition, the structure of MAMs in the copper loading group was sparsed, and the percentage of coupling length to mitochondrial circumference was significantly reduced (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 \u0026nbsp;Effects of copper loading on mitochondrial ER co-localization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunofluorescence double-labeling techniques were used to observe the co-localization of ER and mitochondria, and ImageJ software was used to analyze their co-localization coefficients. Compared with the control group, the number of MAMs in the hippocampal CA1 region of the copper-loaded group rats was significantly reduced, and the co-localization coefficients of ER and mitochondria were significantly decreased (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05), indicating a weakened interaction between ER and mitochondria (\u003cstrong\u003eFigure 7, Figure 8\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 \u0026nbsp;The effect of copper loading on the expression of functionally related proteins in MAMs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression levels of functionally related proteins of MAMs in rats are shown in \u003cstrong\u003eFigure 9.\u003c/strong\u003e The results showed that the protein expression levels of VDAC1, IP3R, and GRP75 were significantly increased and the protein expression level of Mfn2 was significantly decreased in the copper-loaded group compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCopper is one of the essential trace elements for the body to maintain normal physiological functions\u003csup\u003e30\u003c/sup\u003e. Excessive deposition of copper in the body may result in \"direct\" damage to the conformational changes of functional active molecules; on the other hand, it can induce the generation of reactive oxygen species and reactive nitrogen species, causing \"indirect\" damage. The \"indirect\" damage can activate intracellular early response genes that are extremely sensitive to oxidative stress, ultimately causing DNA damage, lipid peroxidation, and altered thiol homeostasis\u003csup\u003e31\u003c/sup\u003e. Changes in the mechanism of copper homeostasis can lead to impaired Cytox function, causing abnormal mitochondrial respiration, and oxidative stress on intracellular organelles such as mitochondria\u003csup\u003e32\u003c/sup\u003e. Sustained oxidative stress may induce mitochondrial permeability transition and a decrease in its oxidative potential, and trigger a series of cascading reactions that ultimately result in neuronal damage. Therefore, mitochondria are considered an early and sensitive key to copper-induced oxygen stress in neuronal cells\u003csup\u003e33\u003c/sup\u003e. In addition, mitochondria are the main site of energy metabolism, and neuronal cells associated with cognition are highly polarized cells whose large energy demands are mainly met by mitochondria. Normal mitochondrial function is crucial for neuroprotection and repair of cognition-related brain regions. However, mitochondrial dysfunction can also lead to the production of excessive free radicals and the imbalance of calcium ion concentration, resulting in neuronal dysfunction\u003csup\u003e34\u003c/sup\u003e. Mitochondrial dysfunction is directly or indirectly associated with cognitive impairment in various diseases\u003csup\u003e35\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eMitochondria communicate with other organelles inside the cell through the outer membrane, and play an important role in cognitive impairment-related diseases through mitochondrial self-metabolism and intracellular signaling pathways.\u003c/p\u003e\n\u003cp\u003eThere is a co-localized structure between mitochondria and ER, and this tightly contacted subcellular structure is called MAMs. MAMs act as a physical and biochemical bridge between mitochondria and ER, are critical for ER stress and unfolded protein responses, autophagy, inflammatory mediator signal transduction, and widely influence normal intracellular life activities\u003csup\u003e36\u003c/sup\u003e. Structural and functional changes in MAMs may cause disturbances in calcium homeostasis, abnormal cholesterol and phospholipid metabolism, which may affect mitochondrial function and participate in the development of cognitive impairment\u003csup\u003e37\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eReactive oxygen species, as the intersection of high copper-induced oxidative stress response and autophagy cascade, serve as a bridge and link between pathogenic factors and the organism's defense response\u003csup\u003e38\u003c/sup\u003e. When the body perceives the threat of oxidative stress posed by excess reactive oxygen species, it will regulate the structure of MAMs and stabilize the mitochondrial function through various MAMs constituent proteins, so as to achieve the role of repairing the damage and maintaining cellular functional homeostasis\u003csup\u003e39\u003c/sup\u003e. Among a variety of MAMs constituent proteins, Mfn2 is a MAMs-associated mitochondrial fusion protein, localized on the surface of the ER, which can form homodimers or heterodimers with Mfn1 at the mitochondrial outer membrane and plays an important role in regulating mitochondrial morphology\u003csup\u003e40\u003c/sup\u003e. IP3R is a Ca\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003erelease channel located on the ER membrane, which allows Ca\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003eto be released from the ER lumen into the cytoplasm\u003csup\u003e41\u003c/sup\u003e. VDAC1 is a voltage-dependent Ca\u003csup\u003e2+\u003c/sup\u003e uptake channel located on the outer mitochondrial membrane, which is involved in the regulation of Ca\u003csup\u003e2+\u003c/sup\u003e concentration in mitochondria and is an important channel protein for maintaining intracellular calcium homeostasis\u003csup\u003e42\u003c/sup\u003e. GRP75 plays an important bridging role in intracellular Ca\u003csup\u003e2+\u003c/sup\u003e transport. GRP75 promotes the binding of mitochondria to the ER, and acts as a molecular chaperone linking IP3R and VDAC1 to form the VDAC1-GRP75-IP3R complex, which can mediate Ca\u003csup\u003e2+\u003c/sup\u003e transport from the ER to mitochondria, and regulate the calcium homeostasis of mitochondria\u003csup\u003e43\u003c/sup\u003e. Under pathological conditions, dysfunction of the VDAC1-GRP75-IP3R complex can result in excessive calcium ion concentrations in mitochondria, leading to alterations in mitochondrial membrane permeability, opening of mitochondrial permeability transition pores (MPTP), causing changes in mitochondrial osmolality, and rupture of the outer membrane, leading to mitochondrial morphology and functional disorders\u003csup\u003e44\u003c/sup\u003e.\u0026nbsp;The MAMs proteins IP3R, GRP75, VDAC1, and Mfn2 participate in regulating MAMs function from multiple perspectives to maintain mitochondrial morphology and membrane integrity, affecting cognitive function.\u003c/p\u003e\n\u003cp\u003eThe copper-loaded rat model can better simulate the pathological process of copper deposition in vivo and has been widely used in studies related to WD\u003csup\u003e45-46\u003c/sup\u003e. In this study, MWM and NOR were used for neurobehavioural assessment of rats. MWM includes localization navigation experiments and spatial probe tests, which can reflect the learning and memory abilities of rats toward platform spatial positions\u003csup\u003e47\u003c/sup\u003e. NOR can reflect the non spatial recognition memory ability of animals\u003csup\u003e48\u003c/sup\u003e. The combination of MWM and NOR methods can effectively evaluate abnormal neurobehaviour in copper-loaded rats.\u003c/p\u003e\n\u003cp\u003eIn this study, copper-loaded rats had significantly prolonged escape latency and reduced number of platform crossings, and the percentage of DR and the DI were significantly reduced. It suggests that copper loading can impair the abilities of learning, spatial exploration, and non-spatial recognition memory in rats, causing neurobehavioural dysfunction. In addition, this study found that copper loading can disrupt the microstructure of rat MAMs: Under transmission electron microscopy, neuronal mitochondria and ER in the\u0026nbsp;hippocampal CA1 region\u0026nbsp;of\u0026nbsp;the copper-loaded group rats\u0026nbsp;were highly swollen, structurally dilated, and the mitochondrial cristae structure was blurred, broken, and disappeared.\u0026nbsp;Besides, the number of MAMs was significantly reduced, and the percentage of coupling length to mitochondrial circumference was significantly decreased. ER and mitochondrial co-localization coefficients were significantly reduced in immunofluorescence double-labeling assays.\u0026nbsp;Moreover,\u0026nbsp;copper loading can induce differential expression of structural and functional proteins related to MAMs in the hippocampal CA1 region, with significantly increased expression levels of the Ca\u003csup\u003e2+\u003c/sup\u003e channel proteins VDAC1, IP3R, and GRP75, and significantly decreased expression levels of mitochondrial outer membrane fusion protein Mfn2. We have to acknowledge that our study focused on the ultrastructure and function of MAMs as sensitive indicators of ER-mitochondria disruption, future work employing ER-specific staining may provide complementary insights.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn this study, we found that copper loading can cause abnormal expression of MAMs homeostasis-related proteins IP3R1, GRP75, VDAC1, and Mfn2. It is speculated that high copper-induced neurobehavioural impairments may be related to the structural changes and related protein expression changes in MAMs, and the specific mechanism is worthy of further in-depth study. However, whether cognitive dysfunction can be improved by regulating the expression of IP3R, GRP75, VDAC1, and Mfn2 proteins to maintain the MAMs homeostasis, needs to be further investigated in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDisclosure statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors report no relevant conflicts of interest/financial disclosures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets for this article are public.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has been reviewed and approved by the Animal Welfare Ethics Committee of Anhui University of Traditional Chinese Medicine (Ethics Approval Code: AHUCM-rats-2024036).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor’s contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZZ S, S J, X F:drafting/revision of the manuscript for content, including medical writing for content\u003c/p\u003e\n\u003cp\u003eWM Y, HZ C:\u0026nbsp;major role in the acquisition of data\u003c/p\u003e\n\u003cp\u003eThe final draft was read and approved by all the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank all editors and reviewers for their help with our paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors read and approve of the final manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by The General program of Anhui Natural Science Foundation (2208085MH271),\u0026nbsp;The General Program of the National Natural Science Foundation of China\u0026nbsp;(81973825),\u0026nbsp;Anhui Province Traditional Chinese Medicine Inheritance and Innovation Research Project (2024CCCX093), Anhui Province Higher Education Science Research Major Project (2024AH040153) and Anhui Province Higher Education Science Research Key Project (2024AH051016).\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMAMs: Mitochondria-associated endoplasmic reticulum membranes\u003c/p\u003e\n\u003cp\u003eCuSO\u003csub\u003e4\u003c/sub\u003e: Copper sulfate\u003c/p\u003e\n\u003cp\u003eSD: Sprague\u0026ndash;Dawley\u003c/p\u003e\n\u003cp\u003eMWM: Morris water maze\u003c/p\u003e\n\u003cp\u003eNOR: Novel object recognition\u003c/p\u003e\n\u003cp\u003eTEM: Transmission electron microscope\u003c/p\u003e\n\u003cp\u003eDR: Discrimination Ratio,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDI: Discrimination index\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eER: Edoplasmic reticulum\u003c/p\u003e\n\u003cp\u003eWD: Wilson\u0026apos;s disease\u003c/p\u003e\n\u003cp\u003eMPTP: Mitochondrial permeability transition pores\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSchilsky M L, Roberts E A, Bronstein J M, et al. 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Behavioural Brain Research, 2024: 115278.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"behavioral-and-brain-functions","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"babf","sideBox":"Learn more about [Behavioral and Brain Functions](http://behavioralandbrainfunctions.biomedcentral.com)","snPcode":"12993","submissionUrl":"https://submission.nature.com/new-submission/12993/3","title":"Behavioral and Brain Functions","twitterHandle":"@BBF_Journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"copper loading, neurobehavior, MAMs, Wilson's disease","lastPublishedDoi":"10.21203/rs.3.rs-6094956/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6094956/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: To observe the effects of copper sulfate (CuSO4) -induced copper loading on neurobehaviour, mitochondria-associated endoplasmic reticulum membranes (MAMs) and related regulatory proteins in the hippocampal CA1 region of Sprague–Dawley (SD) rats.\u003c/p\u003e\n\u003cp\u003eMethods: Forty SD male rats were randomly divided into control and copper loading groups of 20 rats each. The control group rats were fed with normal feed and water; rats in the copper loading group were fed high copper feed (containing 1g/kg of CuSO4) and CuSO4 deionized water (concentration of 0.185%). After 12 weeks of rearing, the morris water maze (MWM) task and novel object recognition (NOR) test were conducted to compare the neurobehavioral characteristics of the two groups of rats. Morphological changes of neuronal MAMs in the hippocampal CA1 region of copper-loaded rats were observed using a transmission electron microscope (TEM) and immunofluorescence double-labelling techniques. Western-blot analysis was used to detect the expression of MAMs proteins VDAC1, IP3R, GRP75 and Mfn2.\u003c/p\u003e\n\u003cp\u003eResults: The results revealed that rats in the copper-loading group had significantly prolonged escape latency and reduced number of platform crossings in the MWM task (p \u0026lt; 0.01). The percentage of novel objects explored (also known as the Discrimination Ratio, DR) and the discrimination index (DI) were significantly reduced in the NOR test (p \u0026lt; 0.01). In addition, electron microscopy shows increased disruption of neuronal endoplasmic reticulum (ER)-mitochondrion coupling in the hippocampal CA1 region of rats in the copper-loading group (p \u0026lt; 0.05), and the percentage of MAMs in mitochondrial circumference decreased (p \u0026lt; 0.05), the colocalization coefficients between the ER and mitochondria was significantly reduced (p \u0026lt; 0.05). Moreover, the protein expression levels of VDAC1, IP3R, and GRP75 in rat hippocampal tissue were detected to be significantly increased (p \u0026lt; 0.01), while the protein expression level of Mfn2 was significantly decreased (p \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003eConclusions: In this study, it is speculated that the neurobehavioral changes in rats may be related to the increased expression levels of the MAMs proteins VDAC1, IP3R, and GRP75, the reduced expression level of Mfn2, and the disruption of the structural integrity of MAMs in the hippocampal CA1 region of rats caused by copper loading.\u003c/p\u003e","manuscriptTitle":"Copper loading affects rat neurobehaviour by impairing mitochondria-associated endoplasmic reticulum membranes in hippocampal neurons","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-16 14:54:04","doi":"10.21203/rs.3.rs-6094956/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-22T18:33:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-20T23:36:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"61145234399530426732791194755259880155","date":"2025-04-20T22:41:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-15T15:58:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-07T08:21:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Behavioral and Brain Functions","date":"2025-04-06T16:32:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"behavioral-and-brain-functions","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"babf","sideBox":"Learn more about [Behavioral and Brain Functions](http://behavioralandbrainfunctions.biomedcentral.com)","snPcode":"12993","submissionUrl":"https://submission.nature.com/new-submission/12993/3","title":"Behavioral and Brain Functions","twitterHandle":"@BBF_Journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"025f0d86-d8f9-43e6-a743-d592dcb68986","owner":[],"postedDate":"April 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T15:58:44+00:00","versionOfRecord":{"articleIdentity":"rs-6094956","link":"https://doi.org/10.1186/s12993-025-00277-y","journal":{"identity":"behavioral-and-brain-functions","isVorOnly":false,"title":"Behavioral and Brain Functions"},"publishedOn":"2025-09-30 15:56:54","publishedOnDateReadable":"September 30th, 2025"},"versionCreatedAt":"2025-04-16 14:54:04","video":"","vorDoi":"10.1186/s12993-025-00277-y","vorDoiUrl":"https://doi.org/10.1186/s12993-025-00277-y","workflowStages":[]},"version":"v1","identity":"rs-6094956","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6094956","identity":"rs-6094956","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}