{"paper_id":"2fae67f4-7b33-4356-9567-3c8d30735e80","body_text":"Apelin-13 confers Neuropeptide Y–mediated neuroprotection and preserves learning and allocentric memory in D-glutamic acid-induced excitotoxicity in rats | 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 Apelin-13 confers Neuropeptide Y–mediated neuroprotection and preserves learning and allocentric memory in D-glutamic acid-induced excitotoxicity in rats Kadriye Yagmur Oruc, Aykut Oruc, Ruhat Arslan, Furkan Pasa Diriarin, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6635799/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Jan, 2026 Read the published version in Molecular Neurobiology → Version 1 posted 12 You are reading this latest preprint version Abstract Glutamate-mediated excitotoxicity causes mitochondrial dysfunction, apoptosis, neuronal death. Aim of this study is to investigate whether Apelin-13, together with NPY2 and NPY5 receptors, plays a cooperative role in neuroprotection and in preventing learning and memory impairments under excitotoxic conditions. D-Glutamic acid-induced excitotoxicity was established in 42-male Sprague-Dawley rats (6-8 weeks, 200-250g). Animals were randomly divided into six-groups(n=7); Control (C; 0.9% NaCl, i.p), D-Glutamic Acid (G; 4 mg/kg, i.p), Apelin-13 (A; 300 µg/kg, i.p), D-Glutamic Acid+Apelin-13 (GA), GA+NPY2 receptor(NPY2R) antagonist (GAN2; 1,5 mg/kg, i.p) and GA+NPY5 receptor(NPY5R) antagonist (GAN5; 1,5 mg/kg, i.p). Short-long term memory, learning performance, allocentric-egocentric orientation, locomotor activity were evaluated with Open field (OFT), novel object recognition (NORT), Morris water maze (MWM) tests. In group G, there was an increase in Caspase-3 level(p<0.001), while significant decrease (p<0.001) was observed in Extracellular Signal Regulatory Kinase (ERK1/2) and Protein Kinase B-1(AKT-1) levels. Increased mitochondrial dysfunction indicated neurodegeneration due to excitotoxicity. In MWM an increase latency to the target quadrant (p<0.001), a decrease in the NORT discrimination index (p<0.001) were found. Apelin-13 was observed to have a neuroprotective role by alleviating the damage in GA group. While the protective effect of Apelin-13 was not observed in the presence of NPY2R antagonist; when NPY5R antagonist was applied, more pronounced neuroprotection was detected in GAN5 group compared to GAN2, since NPY2R activity continued. Histochemical staining-scorings showed that protection of Apelin-13 was mediated by NPY2R. Apelin-13 exerts its neuroprotective effects primarily through NPY2R, its modulatory influence via NPY5R appears to be comparatively limited. Excitotoxicity Apelin-13 Neuropeptide Y Allocentric Memory Spatial Memory Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 HIGHLIGHTS • D-Glutamic acid causes excitotoxicity by inhibiting ERK1/2, PI3K/AKT-1, causing oxidative damage and an increase in active caspase-3. • In excitotoxicity, spatial allocentric mapping is disrupted, and an orientation towards adaptive egocentric memory is observed. • Apelin-13 works in coordination with NPY2R and, to a lesser extent, NPY5R against excitotoxic damage. • Apelin-13 provides neuroprotection by strengthening ERK1/2 and PI3K/AKT signaling. • Apelin-13 prevents mitochondrial dysfunction by increasing the antioxidant/oxidant ratio. • Apelin-13 protects allocentric memory and prevents STM-LTM and learning damage. 1. INTRODUCTION Glutamate, the main excitatory neurotransmitter in the central nervous system (CNS), plays a role in neuronal communication, synaptogenesis, learning and memory processes through ionotropic (iGluR) and metabotropic receptors (mGluR). Dysregulation of the glutamatergic system can cause many effects [ 1 ]. Excitotoxicity leads to neuronal loss by acute (excessive)-chronic activation of postsynaptic neurons [ 2 ]. The basal concentration of glutamate in the synaptic cleft varies between 25–600 nM [ 3 , 4 ], and this value cannot activate glutamate receptors [ 2 , 3 , 5 ]. However, reaching basal glutamate concentrations of 2–5 µM causes excitotoxic damage [ 2 , 4 ]. This condition involves NMDAR/AMPAR overactivation, Ca²⁺ dysregulation, and glutamate-glutamine cycle impairment, leading to ROS accumulation, oxidative stress, mitochondrial dysfunction, and apoptosis [ 2 , 6 , 7 ]. These molecular damages cause learning and memory disorders at the macro level [ 2 ]. Today, the protective activity of many endogenous and exogenous molecules against excitotoxic damage is being investigated. Endogenous adipokine and neuropeptide Apelin, produced from adipose tissue, is the ligand of the Apelin receptor (APJR), a G protein-coupled receptor. The 77 amino acid pre-proapelin is cleaved by peptidases and reduced to active apelin isoforms such as 13, 17, and 36 [ 8 – 11 ]. Apelin-13 is the most potent and predominant isoform in plasma and can cross the blood-brain barrier via peripheral pathways [ 12 – 14 ]. The Apelin-13/APJ complex regulates intracellular Ca 2+ by inhibiting NMDAR via extracellular signal-regulated kinase (ERK1/2) and protein kinase B (AKT) signaling. It responds to mitochondrial dysfunction, apoptosis, oxidative stress, learning and memory damage via the same pathways [ 3 , 15 , 16 ]. Neuropeptide Y (NPY) is the most abundant endogenous neuropeptide in the CNS. It regulates neuroprotection, trophic support enhancement, excitotoxicity inhibition, Ca + 2 homeostasis and learning-memory processes. Its receptors are GPCRs and have the subtypes NPY1, -Y2, ​​-Y4, -Y5, -Y6 [ 17 ]. NPY2 receptor (NPY2R) is localized in the presynaptic terminal in the dentate gyrus (DG), Cornu ammonis 1 (CA1), CA3 regions, and NPY5 receptor (NPY5R) is localized postsynaptically in DG, CA1, CA2, CA3 [ 18 ]. NPY prevents excitotoxic damage by regulating glutamate release from the CA1 presynaptic terminal via NPY2R and postsynaptic NPY5R in CA1-CA3 with ERK1/2, PI3K/AKT signaling. Changes in NPY levels have been observed in neurodegenerative diseases [ 4 ]. Additionally, NPY2R enhances consolidation in spatial memory [ 19 ]. Spatial navigation is critical in the hippocampus, which is involved in the learning and memory process. In spatial mapping, hippocampus-dependent allocentric and hippocampus-independent egocentric strategies gain importance in neurodegenerative disease progression. In hippocampal damage, spatial mapping is disrupted and allocentric navigation decreases. Prolonged search time and distance characterize a compensatory shift toward egocentric strategies [ 20 ]. Mitochondrial dysfunction, oxidative stress and faulty protein accumulation leading to excitotoxicity cause learning and memory impairment in hippocampal circuits [ 5 , 21 ]. Although neuroprotective effects of apelin-13 have been demonstrated, the multifactorial nature of the pathophysiological mechanisms makes the specificity of this effect unclear. The colocalization of APJR with NPY2R, NPY5R in hippocampal areas sensitive to excitotoxic damage and their similar protective functions via ERK1/2 and PI3K/AKT signaling pathways have highlighted the idea that Apelin-13 and NPY work in coordination against excitotoxicity [ 10 , 22 , 23 ]. To date, no studies have specifically examined whether Apelin-13 and NPY neuropeptides act in concert to counteract excitotoxic damage. The number of studies modeling D-glutamic acid-mediated excitotoxicity is quite limited [ 24 ]. In this study, we aimed to investigate the protective effects of Apelin-13 in the D-glutamic acid-induced excitotoxicity model and the relationship between NPY2R and NPY5R. 2. METHOD 2.1. Chemicals D-glutamic acid (D-Glutamic acid, Sigma, CAS:6893-26-1, Saint Louis, USA), Apelin-13 (Apelin-13, Cayman Chemicals, Item 13523, Michigan, USA), NPY Y2 receptor antagonist (BIIE 0246 hydrochloride, cat no 7377, Tocris Bioscience, Bristol, UK), NPY Y5 receptor antagonist (L-152.804, catalog no 1382, Tocris Bioscience, Bristol, UK), Ketamine Hydrochloride (HCl) (Ketalar HCl 50 mg/mL, Pfizer, Istanbul, Turkey), Xylazine HCl (Rompun 20 mg/mL, Bayer, Istanbul, Turkey) were used while performing the experiments. 2.2. Animals Forty-two male Sprague Dawley rats, 6–8 weeks old, weighing 200–250 g, belonging to the same generation were obtained from Istanbul University-Cerrahpaşa Nanotechnology and Biotechnology Institute Experimental Medicine Research Laboratory (DETALAB). Ethical approval was obtained from Istanbul University-Cerrahpaşa Animal Experiments Local Ethics Committee (approval number: 2023/11). The animals were housed in 12 hours of light/darkness, 55 ± 10% humidity and 23 ± 1°C. They were fed with standard pellet food and water ad libitum. The cages were taken into the experimental room 1 week before the experiment and cleaned every other day. Rats were randomly assigned to six groups (n = 7); Control (C), Apelin-13 (A), D-Glutamic acid (G), D-Glutamic acid + Apelin-13 (GA), D-Glutamic acid + NPY 2 Receptor Antagonist + Apelin-13 (GAN2), D-Glutamic acid + NPY 5 Receptor Antagonist + Apelin-13 (GAN5). Group C received 1 ml physiological saline (SF) and DMSO on days 1 and 4–10. Group G received 4 mg/kg D-glutamic acid (1 ml, i.p.) on day 1, 1 ml SF on days 4–10. Group A received 1 ml SF on day 1, 300 µg/kg Apelin-13 (1 ml, i.p.) on days 4–10. Group GA received 4 mg/kg D-glutamic acid (1 ml, i.p.) on day 1, 300 µg/kg Apelin-13 (1 ml, i.p.) on days 4–10. GAN2 group received 4 mg/kg D-glutamic acid (1 ml, i.p.) on day 1, 4–10. received 300 µg/kg Apelin-13 + 1,5 mg/kg NPY Y2 receptor antagonist (1 ml, i.p.) on days 1. GAN5 group received 4 mg/kg D-glutamic acid (1 ml, i.p.) on day 1, 300 µg/kg Apelin-13 + 1,5 mg/kg NPY Y5 receptor antagonist (1 ml, i.p.) on days 4–10. 3 days were waited after the first injection for the excitotoxicity model to develop. All chemicals were dissolved in dimethylsulfoxide (DMSO) (Fig. 1 ). D-Glutamic acid accumulates in tissues in rats because it is not deaminated. It can pass through the blood-brain barrier [ 25 ]. D-amino acid oxidase (DAO) and D-aspartate oxidase (DDO) are stereospecific enzymes that metabolize D-amino acids. In particular, DDO reduces flavin adenine dinucleotide (FAD) by converting acidic D-amino acids such as D-aspartate, D-glutamate and NMDA into imino acids [ 26 ]. Reoxidation of FAD in the presence of oxygen causes the production of hydrogen peroxide (H₂O₂), triggering oxidative stress, mitochondrial dysfunction, proinflammatory cytokine activation and cell death [ 27 ]. D-AA accumulation induces inflammation and cell death via H₂O₂ and NF-κB activation [ 28 ]. While determining the i.p. dose of D-glutamic acid, a dose that could induce convulsions but posed minimal risk of mortality was taken into consideration [ 29 ]. Studies in the literature support the D-glutamic acid-mediated excitotoxicity model [ 6 , 30 ]. 2.3. Learning and Memory Tests A 70x70 cm opaque gray colored open-topped cardboard box was used in a room illuminated with constant light in OFT and NORT. Tests evaluated with ANY-Maze Version 7.42 software (AnyMaze, Stoelting Co., Illinois, USA). The experiments were recorded with a camera (Canon PowerShot, SX740 HS, Tokyo, Japan). 2.3.1. Open Field Test Rats were placed in the center of the test box and moved freely for 10 minutes. In the recorded videos, average speed and total distance, number of rearings, number of defecations, time spent in the center and on the edge were evaluated. 2.3.2. Novel Object Recognition Test In order to evaluate short-term memory (STM), rats were given 5 min to explore their surroundings during habituation period. In familiarization, identical green objects (A1, A2) measuring 11x7 cm (lengthxdiameter) were placed diagonally in the box. The rat was placed in the middle of the objects and contact was expected for at least 20 s for 10 min. After 60 min, a new object (B) measuring 11 cm was placed in place of the old A2. The rat touching the object with its nose was considered as “contact”. Discrimination index (DI), average speed and distance, total examination time and number of A1-A2 and A1-B were evaluated. 2.3.3. Morris Water Maze Test MWM was applied to evaluate long-term memory (LTM). The pool consisting of a white Plexiglas pool (150x60 cm, diameterxdepth) and a transparent Plexiglas platform (10x28 cm, diameterxlength) was filled to 30 cm, painted black with non-toxic paint, and heated to 26 ± 1°C. Blue-green shapes were placed on the walls for allocentric memory, and black shapes were placed on the pool perimeter for egocentric memory. Rats were released into the pool from different points in 4 consecutive trials for 5 days [ 31 ], each trial lasted 60 seconds, and the inter-trial interval was determined as 15 seconds. The escape platform was removed on the probe day, and the rats were released into the pool from the northeast, the only direction from which they were not thrown, and swam for 60 seconds. The time to reach the target quadrant, speed, latency, average swimming speed, and distance were evaluated. 2.4. Sacrification Before anesthesia, rats were weighed and given a cocktail of ketamine 50 mg/kg and xylazine HCl 10 mg/kg via the i.p. route. Following the loss of reflexes, intracardiac blood was collected. Serum samples were labeled and stored at -20°C. Cervical dislocation was performed in accordance with ethical guidelines. The brain was dissected and bilateral hippocampus and prefrontal cortex (PFC) were removed. Half of the hippocampus and PFC were stored at -20°C for biochemical analyses, while the remainder was stored in 4% formaldehyde for histological imaging. 2.5. Biochemical Analyses Conducted at Istanbul University-Cerrahpasa, Cerrahpasa Medical Faculty Biochemistry Research Laboratory. Malondialdehyde (MDA), advanced oxidation protein products (AOPP), total thiol (T-SH), dityrosine (DT), kaynurenine (KYN), advanced glycation end products (AGE) and total protein levels were analyzed in plasma, hippocampus and PFC. The brain tissue was rinsed with phosphate buffer saline (PBS, pH 7.4) at + 4°C, weighed and homogenized with PBS at a ratio of 1:9. The homogenates were centrifuged and the supernatants were labeled and stored. 2.5.1. Advanced Glycation End Products, Dityrosine, Kaynurenine According to the procedure in the literature, 150 µL of sample was diluted 1:50 with PBS. It was read in a spectrofluorometer at excitation/emission wavelengths of 325/440 nm for AGE, 330/415 nm for DT, and 365/480 nm for KYN [ 32 ]. 2.5.2. Advanced Oxidation Protein Products 20 µL of sample and 200 µL of citric acid were added to the microplate, waited for 2 min, and 10 µL of potassium iodide was pipetted. The samples were measured spectrophotometrically at a wavelength of 340 nm and the AOPP concentration was determined according to the procedure in the literature [ 33 ]. 2.5.3. Malondialdehyde According to the procedure in the literature [ 34 ], 2 ml of the reagent prepared with 15% TCA, 0.375% TBA and 0.25 mol/L HCl was added to 1 ml of sample and kept in a water bath at 95°C for 15 minutes. The formed precipitate was centrifuged at 1000 RPM for 10 minutes. The absorbance of the supernatant was measured at 532 nm in a spectrophotometer and the MDA concentration was calculated using the molar extinction coefficient of 1.56x105 M-1 cm-1. 2.5.4. Total Thiol According to the method in the literature [ 35 ], 20 µL of sample, 400 µL of Tris buffer (pH 8.2) and 20 µL of dithionitrobenzoic acid (5,5-dithio-bis-(2-nitrobenzoic acid), Cat no: 22582, ThermoFisher Scienfic, USA) were added to the microplate. The absorbance values ​​of the samples were analyzed against the reagent blank in a spectrophotometer at 412 nm and the extinction coefficient and T-SH concentrations were calculated. 2.5.5. Total Protein To determine the total protein amount in tissue and plasma, 194 µL of bicinchoninic acid (BCA) was diluted 1:50 with 4% copper sulfate. 194 µL of BCA and 6 µL of sample were pipetted. Standards were prepared with albumin and added to standard wells. Phosphate buffer was added to blank wells and incubated in the dark for 30 min. Absorbance value was read by spectrophotometric method at 562 nm wavelength. Calibration curve was drawn for standards and samples to calculate the results. 2.6. Enzyme-Linked ImmunoSorbent Assay Experiments were conducted in Istanbul University-Cerrahpasa, Cerrahpasa Faculty of Medicine Biochemistry Research Laboratory and Haliç University Faculty of Medicine Physiology Research Laboratory. Rat extracellular regulated protein kinases 1;2 (ERK1;2) ELISA Kit (Cat no: E1479Ra, BT LAB, China), Rat RAC-alpha serine; threonine-protein kinase (AKT1) ELISA Kit (Cat no: E2548Ra, BT LAB, China) and Rat CASP3 (Caspase 3) ELISA Kit (Cat no: ELK1528, ELK Biotechnology, Denver, USA) kits were purchased from the manufacturers. Standards were prepared by serial dilution at a 1:2 ratio. 25x wash buffer was diluted to 1x. 50 µL of standard was added to the standard wells, 40 µL of sample and 10 µL of biotinylated antibody were added to the sample wells. 50 µL of streptavidin-Horse Radish Peroxidase (HRP) was added and incubated at 37°C for 60 min. It was washed 5 times with 300 µL of wash buffer, 50 µL of substrate solutions A and B were added and incubated at 37°C for 10 min. Stop solution was added and measurement was made at 450 nm and the standard curve was created by regression analysis. 2.7. Histological Analyses Tissue embedding, sectioning and staining were performed in the Histology Research Laboratory of the Faculty of Medicine of Istanbul Altınbaş University, and histological examinations were performed in the Histology Research Laboratory of the Faculty of Medicine of Istanbul University-Cerrahpaşa. All examinations were performed in the hippocampus CA1-CA3 and PFC. Sections stained with Hematoxylin&Eosin (H&E, ABCAM, ab245880, UK) and Cresyl Violet were examined morphologically under 10x, 20x and 40x magnification with a light microscope (Olympus BX61, Japan) integrated camera (Olympus DP72, Japan). By modifying the method of Singh et al. (2018), 5 randomly selected fields from the 40x magnified sections were scored blindly by two researchers. Neurons were histologically evaluated on a scale of 0–5 according to their morphological features (0: undamaged, 5: severely damaged) and statistical analysis was performed [ 36 ]. 2.8. Statistical Analysis Statistical analyses were performed using GraphPad Prism (v10.2.0, Boston, MA, USA). Normality and homogeneity were assessed using Shapiro-Wilk and Levene tests. Since the data were found to be normal and homogeneous, One-Way ANOVA and post-hoc Tukey test were used in ELISA and biochemical analyses. Results are presented as mean ± standard deviation (SD). Repeated-One-Way ANOVA was applied for MWM training days and One-Way ANOVA was applied for probe day. One-Way ANOVA and post-hoc Tukey test were used in OFT and NORT tests. p < 0.05 was considered statistically significant. 3. RESULTS Co-activation of NPY2R and NPY5R is required for the full neuroprotective effect of Apelin-13 under excitotoxic conditions Active Caspase-3/Protein (ng/mL) In the hippocampus and PFC, when compared to the C group, a very significant increase (p < 0.001) was found in the Active Caspase-3 levels in the G group. When compared to the G and GA, GAN2 and GAN5 groups, a very significant decrease (p < 0.001) was found in all groups. When compared to the A group, a very significant increase (p < 0.001) was seen in GA, GAN2, GAN5. When compared to the GA group, a very significant increase (p < 0.001) was found in GAN2, GAN5. There was a very significant decrease (p < 0.001***) in GAN5 compared to GAN2 (Shown in Table 1 , Fig. 2 G). Table 1 Active Caspase-3/Protein, ERK1/2 /Protein, AKT-1/Protein levels. Results are given as Mean ± SD. C G A GA GAN2 GAN5 Active Caspase-3/Protein(ng/mL) Hippocampus 0,17 ± 0.03 1,73 ± 0.03 a*** 0,16 ± 0.03 0,74 ± 0.04 b,c*** 1,61 ± 0.06 b,c,d*** 0,97 ± 0.02 b,c,d,e*** PFC 0,27 ± 0.05 1,92 ± 0.04 a*** 0,25 ± 0.03 0,57 ± 0.04 b,c*** 1,80 ± 0.06 b,c,d*** 0,77 ± 0.02 b,c,d,e*** ERK1/2 /Protein(ng/L) Hippocampus 994,4 ± 6.2 471 ± 27.4 a*** 1775 ± 17.9 a*** 1419 ± 6.7 b,c*** 785,1 ± 18.9 b,c,d*** 1014 ± 7.8 b,c,d,e*** PFC 677,1 ± 4.5 450 ± 26.2 a*** 1667 ± 29.6 a*** 1229 ± 30.6 b,c*** 587,4 ± 10.1 b,c,d*** 914 ± 7.8 b,c,d,e*** AKT-1/Protein(ng/L) Hippocampus 1846 ± 66.4 1068 ± 23 a*** 2531 ± 31 a*** 2112 ± 131.1 b,c*** 1263 ± 53 b,c,d*** 1964 ± 58 b,c,e***,d** PFC 1681 ± 12 829,6 ± 60.1 a*** 2307 ± 70 a*** 1850 ± 47 b,c*** 1373 ± 59 b,c,d*** 1749 ± 74 b,c,e***,d* C→G, A, a; G→GA, GAN2, GAN5, b; A→GA, GAN2, GAN5, c; GA→GAN2, GAN5, d; GAN2→GAN5, e; (p < 0.05*, p < 0.01**, p < 0.001***) Extracellular Regulated Kinase 1/2 / Protein (ng/L) In hippocampus and PFC, when compared with group C, a very significant decrease (p < 0.001) in phosphorylated ERK1/2 levels was detected in G and a very significant increase (p < 0.001) in A. A very significant increase (p < 0.001) was detected in GA, GAN2 and GAN5 compared to G. When group A was compared with GA, GAN2, GAN5, very significant decreases (p < 0.001) were observed in all groups. When group GA was compared with GAN2, GAN5, a very significant decrease (p < 0.001) was observed in both groups. A very significant increase (p < 0.001) was observed in GAN5 compared to GAN2 (Shown in Table 1 , Fig. 2 H). AKT-1/Protein (ng/L) In hippocampus and PFC, phosphorylated AKT-1 levels were found to be very significant decrease (p < 0.001) in G and very significant increase (p < 0.001) in A compared to group C. When G was compared with GA, GAN2, GAN5, very significant increases (p < 0.001) were seen in all groups. When A was compared with group GA, GAN2, GAN5, very significant decrease (p < 0.001) was seen in all groups. When compared with group GA, GAN2, GAN5, very significant decrease (p < 0.001) was seen in GAN2 in both hippocampal and cortical tissues, while a significant decrease (p < 0.01) was seen in GAN5 in hippocampus and a slightly significant decrease (p < 0.05) in GAN5 in PFC. When compared with group GAN2, a statistically very significant increase (p < 0.001) was seen in GAN5 (Shown in Table 1 , Fig. 2 J). Excitotoxicity-induced oxidative imbalance is reversed by Apelin-13 treatment Advanced Glycation End Products Hippocampal and serum AGE/Protein µg/ml levels showed a very significant increase in G compared to C (p < 0.001***), and a slightly significant increase in PFC (p < 0.05*). In the hippocampus, there was a very significant decrease in A compared to G (p < 0.001***). There was a slightly significant increase in GAN2 compared to GA (p < 0.05*). Serum AGE/Protein levels showed a slightly significant increase in GA compared to A (p < 0.05*). There was a very significant decrease in GAN5 compared to GAN2 (p < 0.001***). In the hippocampus, PFC and serum, there was a very significant decrease in GA, GAN5 compared to G (p < 0.001***), while a significant decrease in GAN2 (p < 0.01**) was observed. A very significant increase in GAN2 compared to A was found in the PFC and serum (p < 0.001***), and a significant increase in the hippocampus (p < 0.01**) (Fig. 2 A). Advanced Oxidation Protein Products Hippocampal AOPP/Protein levels showed a very significant decrease (p < 0.001***) in GA compared to G. A slightly significant increase (p < 0.05*) in GA and a very significant increase (p < 0.001***) in GAN5 compared to GA. A very significant increase (p < 0.001***) was found in GAN5 compared to GA. PFC AOPP/Protein levels showed a very significant decrease (p < 0.001***) in GA and GAN5 compared to G, while a slightly significant decrease (p < 0.05*) was found in GAN2. Serum AOPP/Protein levels showed a significant decrease (p < 0.01**) in GA compared to G and a significant increase (p < 0.01**) in GAN2. A very significant decrease (p < 0.001***) was seen in GAN5 compared to GAN2. A highly significant increase (p < 0.001***) was observed in G compared to C in hippocampus, PFC and serum. A highly significant increase (p < 0.001***) was observed in GAN2 compared to A. There was a highly significant increase (p < 0.001***) in GAN2 compared to GA in hippocampus and serum (Fig. 2 B). Dityrosine Hippocampal DT/Protein levels showed a slightly significant decrease (p < 0.05*) in GAN2 compared to G. GAN2 showed a very significant (p < 0.001***) and GAN5 showed a slightly significant increase (p < 0.05*) compared to A. PFC DT/Protein levels showed a very significant decrease (p < 0.001***) in GAN2 compared to G. A slightly significant increase (p < 0.05*) was found in GAN2 compared to A. Serum DT/Protein levels showed a very significant increase (p < 0.001***) in GAN2 and GAN5 compared to A. GAN5 showed a significantly increased (p < 0.01**) compared to GA. In hippocampus, PFC and serum, G showed a very significant increase (p < 0.001***) compared to C. A very significant decrease (p < 0.001***) was observed in GA and GAN5 compared to G. A very significant increase (p < 0.001***) was detected in GAN2 compared to GA in the hippocampus and serum. A slightly significant decrease (p < 0.05*) was found in GAN5 compared to GAN2 in the hippocampus, and a very significant decrease (p < 0.001***) was found in the serum (Fig. 2 C). Kaynurenine When hippocampal KYN/Protein levels were examined, a significant increase was detected in GAN2, GAN5 compared to A (p < 0.001***, p < 0.01**, respectively). A very significant increase (p < 0.001***) was observed in GAN2, GAN5 compared to GA. A slightly significant decrease (p < 0.05*) was found in the GAN2 group in the PFC. A very significant increase (p < 0.001***) was found in GAN2 compared to A. There was a very significant increase (p < 0.001***) in GAN2 compared to GA. Serum KYN/Protein levels were recorded in GA, GAN2, GAN5 compared to A (p < 0.05*,p < 0.001***,p < 0.001***). GAN2 showed a very significant increase (p < 0.001***) compared to GA. There was a significant increase in G compared to C in hippocampus, PFC and serum (p < 0.001***,p < 0.01**,p < 0.001***, respectively). GA and GAN5 showed a very significant decrease (p < 0.001***) compared to G. GAN5 showed a significant decrease (p < 0.001***,p < 0.01**,p < 0.001***) compared to GAN2 (Fig. 2 D). Malondialdehyde When hippocampal MDA/Protein levels were examined, a significant increase (p < 0.01**) was found in GA compared to A. In serum MDA/Protein levels, a significant decrease (p < 0.01**) in GAN2 and a very significant decrease (p < 0.001***) in GAN5 were observed compared to G. There was a very significant increase (p < 0.001***) in G in the hippocampus and serum compared to C. There was a very significant decrease (p < 0.001***) in GA, GAN2, GAN5 compared to G. A very significant increase (p < 0.001***) was observed in GAN2, GAN5 compared to A group. GAN2 showed a very significant increase (p < 0.001***) compared to GA (Fig. 2 E). Total Thiol In T-SH levels, a very significant decrease (p < 0.001***) was detected in G compared to C in hippocampus, PFC and serum. A very significant increase (p < 0.001***) was detected in GA, GAN5 compared to G. A very significant decrease (p < 0.001***) was observed in GA, GAN2, GAN5 compared to A. A slightly significant decrease (p < 0.05*) was observed in GAN2 hippocampus and a significant decrease (p < 0.01**) in PFC compared to GA. A very significant increase (p < 0.001***) was observed in GAN5 in hippocampus and a significant increase (p < 0.01**) in PFC compared to GAN2. In serum T-SH/Protein levels, a very significant increase (p < 0.001***) was observed in GAN5 compared to G group. Compared to A, a significant decrease in GA (p < 0.01**) and a very significant decrease in GAN2 (p < 0.001***) were observed (Fig. 2 F). Anxiety-like behaviors observed due to excitotoxicity were suppressed by Apelin-13, while locomotor performance remained constant Open Field Test When the time spent in the center was considered, a very significant decrease (p < 0.001***) was detected in G compared to group C. A significant increase (p < 0.001***) was observed in GA, GAN5, and a significant increase (p < 0.01**) in GAN2 compared to group G. A very significant decrease (p < 0.001***) was observed in GAN2, GAN5 compared to group A, a very significant decrease (p < 0.001***) in GAN2 compared to group GA, and a slightly significant decrease (p < 0.05*) in GAN5. A very significant increase (p < 0.001***) was found in GAN5 compared to GAN2. Since the time spent on the periphery is inversely proportional to the time spent in the center, significant differences were observed to follow a similar trend. There was no statistically significant difference between the groups when the average total distance (m) and speed (m/sec) were examined. A very significant increase (p < 0.001***) was found in G in the number of defecations compared to group C. No difference was observed in the comparisons of other groups. A very significant statistical decrease (p < 0.001***) was detected in G group compared to C group in terms of rearing numbers, and a slightly significant decrease (p < 0.05*) was detected in GAN2 group. A slightly significant increase (p < 0.01**) was observed in GA and GAN5 compared to G group (Fig. 3 B). Apelin-13 provided its cognitive protection on short-term memory via NPY2 and NPY5 receptors NORT Familiarization Compared to group C, there was a slightly significant decrease in the Average Total Distance in G (p < 0.05*) and a very significant decrease in the Average Speed ​​(p < 0.001***). There was no statistically significant difference between the groups in the Exploration Number of A1-A2 Objects. When the Average exploration Time of A1-A2 Objects was considered, there was a significant decrease in G compared to C (p < 0.05*). There was a slightly significant decrease in GAN5 compared to A (p < 0.05*) (Fig. 3 A). NORT Test Period During the NORT test process, a significant decrease (p < 0.05*) was observed in G compared to C in the average speed (m/sec) data. No significant difference was observed when the other groups were compared. When the Average Review Number of A1-B Objects was examined, a very significant decrease (p < 0.001***) was observed in G compared to C group, and a very significant increase (p < 0.001***) was observed in A. In GA, GAN5 groups, a significant increase (p < 0.01**) was observed in the tendency towards B object compared to A1. No significance was found in G and GAN2 groups. A significant increase (p < 0.01**; p < 0.05*) was observed in the tendency towards B object in G, GA groups. When the Average Review Time (sec) of A1-B Objects was considered, a significant increase (p < 0.01**) was observed in the time spent with B object in C and A, and a slightly significant increase (p < 0.05*) was observed in GA. In the comparison between the groups in the orientation towards the B object, a very significant decrease in G compared to C (p < 0.001***), a significant increase in GAN5 compared to G (p < 0.01**), and a significant decrease in GAN2 compared to A (p < 0.01**) were observed. When DI was examined, a significant decrease in G compared to C (p < 0.01**). A significant increase in GA compared to G (p < 0.01**), and a slightly significant increase in GAN5 (p < 0.05*) were observed. When compared to A and GA groups, a significant decrease in GAN2 (p < 0.01**) was observed. A slightly significant increase in GAN5 compared to GAN2 (p < 0.05*) was found (Fig. 3 C). While egocentric strategies were dominant in excitotoxic groups, Apelin-13 treatment enhanced allocentric navigation, which supports long-term memory MWM-Training In the Total Distance Travelled (cm) data, a significant decrease (p < 0.01**) was observed in all groups on Day 4 compared to Day 1 and a very significant decrease (p < 0.001***) on Day 5. Similarly, a significant decrease was observed in the Day 5 data compared to Day 2 (p < 0.001***) and Day 3 (p < 0.05*). In the total time (sec) data, a very significant decrease (p < 0.001***) was observed in all groups on Days 3, 4, and 5 compared to Day 1. A significant decrease was found on Days 3, 4, and 5 compared to Day 2. A very significant decrease (p < 0.001***) was observed in all groups on Day 5 compared to Day 3. When the average swimming speed (m/sec) data of the rats were compared, no statistically significant difference was observed. This indicates that their locomotor activity was normal. When Escape latency (sec) data were examined, a slightly significant decrease (p < 0.05*) was found on Day 2 compared to Day 1 in all groups, and a very significant difference (p < 0.001***) was found on Days 3, 4, and 5. A very significant decrease (p < 0.001***) was found on Days 3, 4, and 5 compared to Day 2, a significant decrease (p < 0.001***) was found on Day 4 compared to Day 3, a significant decrease (p < 0.01**) was found on Day 5 (Fig. 4 A). Thigmotaxis and random search strategy were seen in all groups on Day 1. Direct search strategy was reported in group C on Day 5, indirect search strategy in groups A and GA, and directed search strategy in group GAN5. Random search was present in groups G and GAN2 on Day 5 (Fig. 4 C). MWM-Probe Day In the data of Total Distance traveled (cm), Escape latency (Sec) and Average Swimming Speed ​​(m/sec), there was a very significant increase (p < 0.001***) in G compared to C. A very significant decrease (p < 0.001***) was seen in GA, GAN2, GAN5 compared to G group. A very significant increase (p < 0.001***) was found in GA, GAN2, GAN5 compared to A. A very significant increase (p < 0.001***) was found in GAN2 compared to GA. A significant increase (p < 0.01**) was found in total distance traveled in GAN5 compared to GA. In the data of Total Distance traveled and Escape latency, a very significant decrease (p < 0.001***) was detected in GAN5 compared to GAN2, and a significant decrease (p < 0.01**) was detected in Average Swimming Speed ​​in GAN5. When the number of entries into the target quadrant and the average time spent in the target quadrant (sec) data were examined, a very significant decrease (p < 0.001***) was observed in G compared to C. A very significant increase (p < 0.001***) was found in GA, GAN5 compared to G. A very significant decrease (p < 0.001***) was recorded in GA, GAN2, GAN5 compared to A. There was a very significant decrease (p < 0.001***) in GAN2 compared to GA. A statistically significant increase (p < 0.001***) was recorded in GAN5 compared to GAN2 (Fig. 4 B). During the 60-second probe test, it was observed that there was an increase in the target quadrant entry marks in the C, A, GA, GAN5 groups, as well as an increase in the search tendency in the target quadrant. Random search continued in the G and GAN2 groups (Fig. 4 D). NPY2R activity enables Apelin-13 to protect neuronal structure under excitotoxic stress Histology scoring and imaging In the H&E scoring of the hippocampal CA1 and CA3 areas, a very significant increase (p < 0.001***) was observed in G compared to C. A very significant decrease (p < 0.001***) was observed in GAN2 and GAN5 compared to G. A very significant increase (p < 0.001***) was observed in GAN2 compared to A. A very significant increase (p < 0.001***) was observed in GAN2 compared to GA. A very significant decrease (p < 0.001***) was observed in GAN5 compared to GAN2. In the Cresyl Violet staining scoring of PFC 2nd, 3rd, 4th, 5th layers, G group showed a very significant increase (p < 0.001***) compared to C. A very significant decrease (p < 0.001***) was found in GA, GAN5 compared to G. A very significant increase (p < 0.001***) was seen in GAN2, GAN5 compared to A. A very significant increase (p < 0.001***) was found in GAN2 compared to GA. A very significant decrease (p < 0.001***) was found in GAN5 compared to GAN2 (Fig. 3 D). When hippocampal CA1 and CA3 molecular layer (MT), polymorphic layer (PHT) and pyramidal layers (PT) were examined, healthy (s) light-colored neurons were seen in parasagittal sections in groups C and A (Fig. 5 A, 5 B). In group G neurons, pericellular halo (ph) indicating increased edema in PT and neurons with dark, shrunken, reduced cytoplasm and pyknotic nucleus (pn) indicating apoptotic cells were seen (Fig. 5 C). Tissue loss indicating vacuolization (v) was observed in the same area. In the GA group, a decrease was observed in dark-colored pn and ph neurons (Fig. 5 D). In the GAN2 group, ph, pn and vacuolization were observed with similar density to the G group (Fig. 5 E). In the GAN5 group, healthy neurons with similar density to the GA group and low density of ph and pn compared to GAN2 were observed (Fig. 5 F). Among the PFC molecular (I), external granular (II), external pyramidal (III), internal granular (IV), internal pyramidal (V) and polymorphic (VI) layers, layers II, III, IV and V were examined. In parasagittal sections of groups C and A, homogeneously distributed, normocytic neurons with intact vesicular nuclei were seen (Fig. 6 A, 6 B). In group G, pn, ph, v were observed (Fig. 6 C). In group GA, diffusely distributed small amounts of pn, ph and moderately healthy neurons were seen (Fig. 6 D). In group GAN2, diffuse pn, ph, v, ac and sparse healthy neurons were observed in layers II, III and IV (Fig. 6 E). In group GAN5, small amounts of pn, ph, ac and multiple healthy neurons were observed (Fig. 6 F). 4. DISCUSSION In our study, an excitotoxicity model was established in male rats using D-glutamic acid, and the effectiveness of exogenous Apelin-13 on neuroprotection and learning-memory was investigated in relation to its potential mediation through NPY2R and NPY5R pathways. Caspase-3 is an active proteolytic enzyme that plays a role in the executive pathway of the apoptotic process [ 37 ]. Caspase-3 activation and DNA fragmentation occur in excitotoxicity. Glutamate excitotoxicity was induced in hippocampal neurons and active caspase-3 levels increased [ 38 , 39 ]. In our study, the highly significant increase (p < 0.001) in hippocampal and PFC tissue active caspase-3 levels in group G compared to group C was supported by D-glutamic acid-mediated excitotoxicity. In excitotoxicity and subarachnoid hemorrhage in rats, apoptosis was suppressed by apelin-13 treatment as compared to injury groups due to decreased active caspase-3 levels [ 40 , 41 ]. In Alzheimer's (AD), excitotoxicity and ischemic stroke models, NPY2R activation, and to a lesser extent NPY5R activation, was observed to reduce active caspase-3 levels in CA3, DG, and subgranular regions, suppress glutamate release, and increase AKT phosphorylation. Neurodegeneration was limited and cellular integrity was preserved [ 42 – 44 ]. In line with literature data, our study emphasizes that when NPY2R antagonists are used, the neuroprotective effects of Apelin-13 are largely insufficient and that while Apelin-13 exhibits its biological properties, NPY2R is primarily involved and NPY5R is limited. In patients with AD associated with excitotoxicity, the PI3K/AKT signaling pathway is suppressed as a result of the destruction of AKT by increased caspase-3 activation [ 45 ]. In our study, excitotoxicity increased caspase-3 activation and suppressed phosphorylated ERK1/2 and AKT-1 signaling as a result of ROS increase and mitochondrial dysfunction in the G group. Opposite changes were observed in the GA group due to the neuroprotective ability of Apelin-13. ERK1-ERK2, known as the pro-survival downstream signaling molecule, is a protein-serine/threonine kinase that participates in the Ras-Raf-MEK-ERK signaling cascade and regulates processes such as cell adhesion, migration, survival, and transcription [ 46 ]. Excessive NMDAR activation in excitotoxicity has been shown to directly inhibit ERK1/2 phosphorylation [ 47 ]. In neuronal death due to excitotoxic damage, ERK1/2 and AKT phosphorylation has been shown to be suppressed, and exogenous Apelin-13 treatment has been shown to increase IP3, PKC, MEK1/2, ERK1/2, and AKT phosphorylation, and to reduce excitotoxic damage by inhibiting NMDAR and calpain [ 48 – 50 ]. In our study, D-Glutamic acid inhibited the ERK1/2 signaling pathway and reduced neuronal survival, while Apelin-13 played a neuroprotective role by increasing NMDAR inhibition and ERK1/2 activation. Neuronal apelinergic signaling causes intracellular Ca 2+ release and ERK1/2 activation via endoplasmic reticulum (ER)-IP3 receptors [ 51 ]. It was indicated that the basal ERK1/2 phosphorylation in our group C may be due to endogenous apelinergic signaling and that the increase in phosphorylation in group A is due to exogenous Apelin-13, which enhances the endogenous apelin signal. In addition to ERK1/2 pathway inhibition in glutamate excitotoxicity, augmentation of pro-apoptotic processes and decrease in AKT phosphorylation levels in ER stress are also observed. NPY suppressed the apoptotic process as a treatment agent in this process by increasing the phosphorylation of ERK1/2 and AKT signaling pathways mediated by NPY2R [ 52 ]. In our study, Apelin-13 in the excitotoxicity model; It inhibited active caspase-3 by increasing ERK1/2 and AKT-1 activation, suppressed apoptosis and exhibited this primarily with NPY2R and to a limited extent with NPY5R. Apelin-13 cannot prevent excitotoxic damage on its own in the presence of NPY2R and NPY5R antagonists. AKT, a serine and threonine kinase, is involved in the proliferation, differentiation and survival mechanism. AKT dysfunction is associated with pathology in neurodegenerative diseases, cancer, cardiovascular diseases, inflammatory and autoimmune disorders [ 53 ]. In excitotoxicity, decrease in phosphorylated-AKT and increase in neuronal death have been reported in hippocampal CA1-CA3 areas [ 54 ]. In addition to the suppression of ERK1/2 and AKT phosphorylation due to the increase in active caspase-3 mentioned above, excessive ROS production and Cytochrome c release also occur. Apelin-13 blocks apoptosis and excitotoxic death via APJ/Gi-Gq [ 55 ]. In our study, Apelin-13 increased AKT-1 signaling, while AKT-1 inhibited apoptosis by preserving mitochondrial integrity and played a protective role in excitotoxic damage. NPY selectively inhibits glutamatergic transmission in pyramidal cells by inhibiting voltage-gated Ca 2+ channel (VGCC) through NPY2Rs expressed in high concentrations in the presynaptic terminals of mossy fibers in CA3 and Schaffer collaterals in CA1, and plays a neuroprotective role against excitotoxicity [ 56 – 60 ]. In the Parkinson Disease (PD) model, NPY2R agonists have been shown to have protective effects against excitotoxicity by increasing the activation of both ERK1/2 and PI3K/AKT pathways in glutamatergic cortical afferent fibers, and high NPY2R gene expression slows down the progression of Huntington disease and plays a protective role against cell death [ 61 – 63 ]. In a rat excitotoxicity model, it was reported that NPY2R agonist provided protection against neuronal cell death in DG, CA1 and CA3 pyramidal cell layers, while NPY5R agonist prevented cell damage in DG and CA3 but had no neuroprotective role in CA1 pyramidal cells [ 64 ]. Another study reported that these protective results were lost by using selective NPY2R antagonist (BIIE0246) in DG, CA1 and CA3 pyramidal cells [ 65 – 67 ]. In neurodegenerative pathologies where presynaptic intracellular Ca 2+ levels are increased, NPY2R activation suppresses glutamate release by VGCC blockade and reduces excitotoxic damage by modulating ERK1/2, PI3K/AKT survival signaling pathways. In our study, the contribution of NPY2R to the protective effects of Apelin-13 became the focus compared to NPY5R. NPY2R's autoinhibitory function by working through the negative feedback mechanism in the presynaptic terminal leads to its functional dominance. The open field test is a behavioral test that provides data on the locomotor activities and general emotional state of the animal [ 68 ]. In the chronic social defeat stress test, Apelin-13 treatment in rats did not affect the total movement distance in OFT findings and did not cause a stress-drug interaction [ 69 ]. In the depression model, NPY2R expression levels in the PFC of rats decreased and depressive behaviors were observed [ 70 ]. In our study, the fact that the average total distance (sec) and average speed (m/sec) were equivalent to the C group in all groups supported the absence of a locomotor problem. The increase in thigmotaxis and defecation, suppression of exploratory behavior, motivational decrease and reluctance to environmental stimuli in the G and GAN2 groups reflect the emotional disorder caused by glutamate excitotoxicity. The absence of anxious behaviors in the C and A groups shows that apelin-13 does not cause behavioral changes in healthy individuals, but has the potential to improve behavioral disorders due to injury. NPY2R antagonism worsened the behavioral profile, indicating that NPY2R plays a key role in the treatment with apelin-13. MWM assesses spatial memory, learning and LTM. Spatial reference memory, which is based on the re-exploration of stable environmental conditions, enables learning and remembering the location of objects, while developing different search strategies by synchronizing egocentric and allocentric navigation [ 71 , 72 ]. Spatial reference memory transmits the animal's location from the hippocampus to the PFC through anatomical connections, enabling optimization and selection of the appropriate route to the target [ 72 ]. Damage to regions or connections in the hippocampus-PFC significantly impairs memory-learning by impairing spatial memory and mapping [ 73 – 75 ]. Total distance traveled data, which is an indicator of spatial memory and learning in MWM [ 76 , 77 ], showed that complex routes were preferred in the G and GAN2 groups, which had D-glutamic acid-induced excitotoxic damage during the training period, and spatial memory performance was impaired. The therapeutic effects of Apelin-13 on learning and memory were limited with the use of NPY2RA. The successful performance in the GAN5 group, where the effect of NPY5RA was observed, compared to GAN2, further reveals the importance of NPY2R in the treatment with apelin-13. The short-direct route choices of the A and GA groups emphasize that Apelin-13 protects spatial reference memory performance in excitotoxic damage. Hippocampal lesions in striatal regions support the formation of compensatory egocentric search strategies [ 78 , 79 ]. The fact that the G, GAN2 group largely preferred non-spatial egocentric search patterns such as thigmotaxis, scanning and randomized, which do not involve hippocampal-dependent learning, has proven the impairment of allocentric memory mapping. In glutamate excitotoxicity, the conversion process from STM to LTM was impaired, and allocentric memory was disabled, leading to an orientation towards adaptive egocentric memory. The selection of goal-directed direct, focal and indirect search patterns in the C, A, GA and GAN5 groups, and the successful maintenance of hippocampal learning, allocentric spatial mapping and LTM, confirmed the protective function of Apelin-13 mediated by NPY2R/NPY5R. The impairment of hippocampal integrity and spatial memory performance has been reported to have negative effects on total distance and escape latency in MWM training and probe performance [ 80 ]. Histological analyses also showed that significant neuronal damage in CA1, CA3 and PFC disrupted the formation of spatial mapping on a cell-based basis in G and GAN2 groups, while the preservation of cell-tissue integrity in GAN5 and GA groups reinforced the importance of apelin-13 treatment and primarily NPY2R and, to a lesser extent, NPY5R in this treatment. In neurotoxicity models, while an increase in escape latency and total distance travelled, a decrease in the time spent in the target quadrant and the number of entries were observed in the damage group in MWM training and probe sessions, it was reported that Apelin-13 improved these [ 40 , 81 ]. Parallel results were obtained in the total time, swimming speed and escape latency during the training period. During the probe period, the time spent in the target quadrant and the number of entries decreased, and the total distance travelled and escape latency increased in the G, GAN2 group due to neurotoxic damage. NORT is used to assess recognition memory and STM and is associated with the PFC and hippocampus [ 82 ]. NORT results are affected by hippocampal and cortical lesions [ 83 ]. Excitotoxicity leads to neuronal structural changes and functional abnormalities in the hippocampus and cortex [ 6 ]. It has been reported that the impairments in memory performance observed in NORT in the depression model were improved by Apelin-13 treatment, that the recognition memory performance of non-stressed rats was not affected, and that the protective effects of PI3K/AKT and ERK1/2 blockers mediated by Apelin-13 were suppressed [ 16 ]. While a significant decrease was observed in the DI of rats exposed to stress, it was reported that DI increased with Apelin-13 treatment, memory impairment improved, but there was no difference in total object exploration times [ 82 ]. In the PD model, the damage group could not distinguish between new and old objects, while Apelin-13 administration increased DI [ 81 ]. It increases the recall of object recognition memory with exogenous NPY receptor activation [ 84 ]. Apelin-13 and D-Glutamic acid application did not disrupt locomotor activity when the average speed was at the same levels in familiarization, but the increased exploration motivation and environmental curiosity with apelin-13 application decreased in the G group, supporting the suppression of acquiring and processing new information. While the C, A, GA groups exploring the new object during the test period showed healthy STM function, the allocation of equal time to new and old objects in the G, GAN2 and GAN5 groups showed a decrease in STM performance, and impaired consolidation and memory separation. The negative DI seen in the G and GAN2 groups indicates that significant memory damage occurs in an excitotoxic environment, while GAN5 group shows a positive DI close to GA, supporting the protective role of Apelin-13 in cognitive function through NPY2R activation. Oxidative stress resulting from excessive ROS production contributes to neurodegenerative diseases through oxidative damage. Excessive ROS induce DNA methylation and trigger neurotoxicity by increasing oxidants such as AGE, AOPP, KYN, DT, MDA, and decreasing the levels of antioxidant molecules such as T-SH [ 30 , 85 , 86 ]. AGEs promote the accumulation of protease-resistant cross-linked proteins as a result of non-enzymatic reactions between reducing sugars and proteins, lipids, and nucleic acids. Progressive AGE accumulation is also seen in hepatocytes and connective tissue in neurodegenerative pathologies [ 87 – 94 ]. In PD, AGE accumulation occurs in the substantia nigra and induces apoptosis by binding to its receptor RAGE and activating ERK1/2 [ 95 ]. AGE-RAGE activation also induces Ca 2+ signaling in the astrocyte membrane, and contributes to neurodegeneration by increasing vesicular glutamate transporter (VGLUT)-mediated glutamate release by astrocytes [ 96 – 98 ]. Cell survival decreases due to the inhibition of ERK1/2 signaling due to increased ROS in cytotoxicity [ 99 ]. AOPP, which is structurally similar to AGE, is a sensitive marker indicating oxidative stress damage in proteins associated with neurodegenerative diseases [ 100 , 101 ]. AOPPs induce ROS formation and oxidant/antioxidant ratio imbalance [ 102 ]. Some ROS products react with tyrosine residues to produce tyrosine radicals that form intra/intermolecular cross-links in proteins, thus increasing DT formation [ 103 ]. High DT levels have been shown in hippocampal-cortical tissues and cerebrospinal fluid (CSF) amyloid plaque proteins in AD and in the striatal area in PD [ 104 ]. In excitotoxicity, nitric oxide (NO) and superoxide (O2 - ) interact with exaggerated NMDAR activation to release toxic peroxynitrite (ONOO - ) [ 105 ]. ONOO - increases DT accumulation-mediated oxidative damage by causing protein oxidation [ 106 ]. The kynurenine pathway is linked to neurodegenerative pathogenesis, as KYN metabolites play a role in excitotoxic transmission, oxidative stress, neurotransmitter uptake, and amyloid aggregation. KYN is the primary catabolic pathway of tryptophan and affects glutamatergic activity via iGluR, mGluR or VGLUTs. It has been reported that high doses of KYN contribute to neuronal damage by producing reactive free radicals via the glutamatergic pathway [ 107 ]. It has been reported that KYN metabolites accumulate in serum and CSF in AD in parallel with excitotoxicity [ 108 ]. KYN can activate NMDARs by generating sudden action potentials in CA1 pyramidal neurons [ 109 ]. MDA, which is formed as a result of free radical and lipid reactions, causes DNA damage by altering cell membrane structure [ 110 ]. Increased lipid peroxidation, membrane permeability and ionic balance are disrupted, paving the way for neurodegenerative mechanisms [ 111 ]. Antioxidants and agents that reduce ROS production have been reported to reduce MDA levels and oxidative stress [ 112 ]. It has been reported that caspase activation accompanied by an increase in ROS triggers the apoptotic process, suppresses ERK1/2 and AKT-1 signaling pathway activation, and neuronal death occurs [ 113 ]. T-SH shows the content of sulfhydryl bonds with antioxidative properties. The sulfhydryl group (-SH) is found especially in cysteine ​​amino acid [ 114 ]. Oxidative stress causes the formation of disulfide bonds (R-S-S-R) in -SHs in the thiol group (R-SH), oxidation of proteins and damaged folding [ 115 ]. Oxidative stress may increase with disulfidation of cysteines in the Cystine/glutamate antiporter (xCT) structure in the astrocytic membrane, causing glutamate accumulation in the extrasynaptic area [ 116 , 117 ]. It has been suggested that cortical glutathione, T-SH, catalase activity decreases and MDA levels increase in excitotoxicity and oxidative stress is induced [ 118 ]. In nephrotoxicity, Apelin-13 reduced oxidative stress and toxicity by increasing tissue T-SH levels [ 119 ]. The decrease in T-SH levels in the G and GAN2 groups suggests that the sulfhydryl groups due to increased oxidant parameters in these groups are transformed into disulfide bridges. In our study, the increase in oxidant AGE, AOPP, DT, KYN, MDA levels in the tissue and serum of the G group and the inhibition of ERK1/2, AKT, the disruption of the antioxidant/oxidant ratio and the increase in tissue active caspase-3 levels create a predisposing environment for neuronal apoptosis in excitotoxicity. Apelin-13 decreases oxidant parameters in the A and GA groups compared to the G group, while increasing T-SH, indicating that it provides an antioxidative effect. The antagonists used in the GAN2-GAN5 groups reveal that Apelin-13 provides its neuroprotective effects together with NPY2-NPY5 receptors. In neurodegeneration, while ROS production is neutralized, the antioxidant system is compromised and sufficient clearance cannot be provided. The neurodegeneration process accelerates with the development of mitochondrial dysfunction. As a result, it has been reported that cognitive functions such as attention, decision-making, learning and memory are also impaired [ 120 ]. In neurodegenerative models, excessive production or supplementation of antioxidant enzymes increases the consolidation and recall capacity of spatial learning-memory. At the same time, there is a decrease in memory function in young animals exposed to oxidative stress [ 121 ]. It has been reported that resveratrol suppresses mitochondrial damage and apoptosis by reducing free radical formation in mice, increases neuronal superoxide dismutase concentration and reduces MDA levels, and improves learning and memory in MWM test results [ 122 ]. The relationship between increased MDA, AOPP, KYN and oxidative stress levels and neurocognitive, learning, memory and cognitive disorders has been reported with decreases in learning tests [ 108 , 123 , 125 ]. In neurodegeneration, darkly stained pyknotic nuclei (pn) indicating apoptotic nuclei and neurons with disrupted membrane integrity can be observed. Pericellular halo (ph) indicating severe neuronal edema in the extracellular area, and white, round, foamy vacuolization indicating toxicity and oxidative stress in the intracellular area are observed [ 126 ]. In G and GAN2, in cells with preserved membrane integrity, the presence of pn and ph as well as caspase-3 increase showed that the apoptotic process was operating [ 127 ]. In the PD model, neuronal damage accompanying early LTP decrease in the CA1 area was seen in histological analyses, and it was reported that apelin-13 improved this [ 128 ]. In line with our findings and literature studies, the increase in antioxidant T-SH levels in the GA group showed that Apelin-13 supported the activation of the antioxidant defense system, provided protection against oxidative stress and mitochondrial dysfunction, and reduced ROS formation. The fact that apelin-13 works synergistically with NPY2R and NPY5R is also supported by biochemical parameters. MWM, NORT and OFT results also coincide with the changes in oxidative stress parameters. 5. CONCLUSION Our results indicate that Apelin-13 is an effective option in preventing/suppressing excitotoxic damage underlying neurodegenerative pathophysiological mechanisms. Apelin-13 performs this role by binding to its own receptors. The fact that NPY receptors in the hippocampal area are colocalized with APJs and use similar intracellular pathways with Apelin-13 proves that it has cofactor effects in preventing/suppressing excitotoxic damage. The relationships of Apelin-13 with other NPY receptors need to be investigated and revealed in more detail at the in-vitro and in-vivo molecular level. Declarations ACKNOWLEDGEMENTS All videos recorded for Open Field Test, Novel Object Recognition test and Morris Water Maze test were analyzed using ANY-Maze Version 7.42 (AnyMaze, Stoelting Co., Illinois, USA) software. We would like to thank Istanbul University-Cerrahpaşa Scientific Research Projects Coordination Unit for their financial support. Funding Declaration Our study was supported by Istanbul University-Cerrahpaşa Scientific Research Projects Coordination Unit (Project Number: TDK-2023-37312). Data Availability Data will be available upon request. Author Contribution Conceptualization: Kadriye Yagmur Oruc , Hakki Oktay Seymen Methodology: Kadriye Yagmur Oruc Formal analysis and investigation: Kadriye Yagmur Oruc, Aykut Oruc, Ruhat Arslan, Furkan Pasa Diriarin, Murat Mengi, Gamze Tanriverdi, Karolin Yanar, Mediha Ozeren Eser, Gokhan Agturk, Ali Ihsan Sonkurt, Berkay Guler Writing - original draft preparation: Kadriye Yagmur Oruc , Hakki Oktay Seymen Writing - review and editing: Kadriye Yagmur Oruc Supervision: Hakki Oktay Seymen References Martami F, Holton KF (2023) Targeting Glutamate Neurotoxicity through Dietary Manipulation: Potential Treatment for Migraine. Nutrients Sep 12;15 (18):3952. https://doi.org/10.3390/nu15183952. Armada-Moreira A, Gomes JI, Pina CC, et al. (2020) Going the Extra (Synaptic) Mile: Excitotoxicity as the Road Toward Neurodegenerative Diseases. Front Cell Neurosci 14:90. https://doi.org/10.3389/fncel.2020.00090. Herman MA, Jahr CE (2007) Extracellular glutamate concentration in hippocampal slice. J Neurosci 27(36):9736-41. https://doi.org/10.1523/JNEUROSCI.3009-07.2007. Mark LP, Prost RW, Ulmer JL, Smith MM, Daniels DL, Strottmann JM, Brown WD, Hacein-Bey L (2001) Pictorial review of glutamate excitotoxicity: fundamental concepts for neuroimaging. AJNR Am J Neuroradiol 22(10):1813-24. Clements JD, Lester RA, Tong G, Jahr CE, Westbrook GL (1992) The time course of glutamate in the synaptic cleft. Science 258(5087):1498-501. https://doi.org/10.1126/science.1359647. Dong XX, Wang Y, Qin ZH (2009) Molecular mechanisms of excitotoxicity and their relevance to pathogenesis of neurodegenerative diseases. Acta Pharmacol Sin 30(4):379-87. https://doi.org/10.1038/aps.2009.24. Fontana IC, Souza DG, Souza DO, Gee A, Zimmer ER, Bongarzone S (2023) A Medicinal Chemistry Perspective on Excitatory Amino Acid Transporter 2 Dysfunction in Neurodegenerative Diseases. J Med Chem 66(4):2330-2346. https://doi.org/10.1021/acs.jmedchem.2c01572. O'Dowd BF, Heiber M, Chan A, Heng HH, Tsui LC, Kennedy JL, Shi X, Petronis A, George SR, Nguyen T (1993) A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11. Gene. 136(1-2):355-60. https://doi.org/10.1016/0378-1119(93)90495-o. Tatemoto K, Hosoya M, Habata Y, Fujii R, Kakegawa T, Zou MX, Kawamata Y, Fukusumi S, Hinuma S, Kitada C, Kurokawa T, Onda H, Fujino M (1998) Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor. Biochem Biophys Res Commun 251(2):471-6. https://doi.org/10.1006/bbrc.1998.9489. Ivanov MN, Stoyanov DS, Pavlov SP, Tonchev AB (2022) Distribution, Function, and Expression of the Apelinergic System in the Healthy and Diseased Mammalian Brain. Genes (Basel) 13(11):2172. https://doi.org/10.3390/genes13112172. Cirillo P, Ziviello F, Pellegrino G, Conte S, Cimmino G, Giaquinto A, Pacifico F, Leonardi A, Golino P, Trimarco B (2015) The adipokine apelin-13 induces expression of prothrombotic tissue factor. Thromb Haemost Feb;113(2):363-72. https://doi.org/10.1160/TH14-05-0451. Li A, Zhao Q, Chen L, Li Z (2023) Apelin/APJ system: an emerging therapeutic target for neurological diseases. Mol Biol Rep 50(2):1639-1653. https://doi.org/10.1007/s11033-022-08075-9. Wan T, Fu M, Jiang Y, Jiang W, Li P, Zhou S (2022) Research Progress on Mechanism of Neuroprotective Roles of Apelin-13 in Prevention and Treatment of Alzheimer's Disease. Neurochem Res 47(2):205-217. https://doi.org/10.1007/s11064-021-03448-1. Chen P, Wang Y, Chen L, Song N, Xie J (2020) Apelin-13 Protects Dopaminergic Neurons against Rotenone-Induced Neurotoxicity through the AMPK/mTOR/ULK-1 Mediated Autophagy Activation. Int J Mol Sci 21(21):8376. https://doi.org/10.3390/ijms21218376. Lv SY, Chen WD, Wang YD (2020) The Apelin/APJ System in Psychosis and Neuropathy. Front Pharmacol 11:320. https://doi.org/10.3389/fphar.2020.00320. Li E, Deng H, Wang B, Fu W, You Y, Tian S (2016) Apelin-13 exerts antidepressant-like and recognition memory improving activities in stressed rats. Eur Neuropsychopharmacol 26(3):420-30. https://doi.org/10.1016/j.euroneuro.2016.01.007. Li C, Wu X, Liu S, Zhao Y, Zhu J, Liu K (2019) Roles of Neuropeptide Y in Neurodegenerative and Neuroimmune Diseases. Front Neurosci 13:869. https://doi.org/10.3389/fnins.2019.00869. Tanaka M, Yamada S, Watanabe Y (2021) The Role of Neuropeptide Y in the Nucleus Accumbens. Int J Mol Sci 22(14):7287. https://doi.org/10.3390/ijms22147287. Méndez-Couz M, Manahan-Vaughan D, Silva AP, González-Pardo H, Arias JL, Conejo NM (2021) Metaplastic contribution of neuropeptide Y receptors to spatial memory acquisition. Behav Brain Res 396:112864. https://doi.org/10.1016/j.bbr.2020.112864. Curdt N, Schmitt FW, Bouter C, Iseni T, Weile HC, Altunok B, Beindorff N, Bayer TA, Cooke MB, Bouter Y (2022) Search strategy analysis of Tg4-42 Alzheimer Mice in the Morris Water Maze reveals early spatial navigation deficits. Sci Rep 12(1):5451. https://doi.org/10.1038/s41598-022-09270-1. Keimasi M, Salehifard K, Keimasi M, Amirsadri M, Esfahani NMJ, Moradmand M, Esmaeili F, Mofid MR (2023) Alleviation of cognitive deficits in a rat model of glutamate-induced excitotoxicity, using an N-type voltage-gated calcium channel ligand, extracted from Agelena labyrinthica crude venom. Front Mol Neurosci 16:1123343. https://doi.org/10.3389/fnmol.2023.1123343. Behl T, Madaan P, Sehgal A, Singh S, Makeen HA, Albratty M, Alhazmi HA, Meraya AM, Bungau S (2022) Demystifying the Neuroprotective Role of Neuropeptides in Parkinson's Disease: A Newfangled and Eloquent Therapeutic Perspective. Int J Mol Sci 23(9):4565. https://doi.org/10.3390/ijms23094565. Zheng Y, Zhang L, Xie J, Shi L (2021) The Emerging Role of Neuropeptides in Parkinson's Disease. Front Aging Neurosci 13:646726. https://doi.org/10.3389/fnagi.2021.646726. Oruc A, Oruc KY, Yanar K, Mengi M, Caglar A, Kurt BO, Altan M, Sonmez OF, Cakatay U, Uzun H, Simsek G (2024) The Role of Glycogen Synthase Kinase-3β in the Zinc-Mediated Neuroprotective Effect of Metformin in Rats with Glutamate Neurotoxicity. Biol Trace Elem Res 202(1):233-245. https://doi.org/10.1007/s12011-023-03667-3. Arauz-Contreras J, Feria-Velasco A (1984) Monosodium-L-glutamate-induced convulsions--I. Differences in seizure pattern and duration of effect as a function of age in rats. Gen Pharmacol 15(5):391-5. https://doi.org/10.1016/0306-3623(84)90036-3. Katane M, Homma H (2010) D-aspartate oxidase: the sole catabolic enzyme acting on free D-aspartate in mammals. Chem Biodivers 7(6):1435-49. https://doi.org/10.1002/cbdv.200900250. Sacchi S, Cappelletti P, Murtas G (2018) Biochemical Properties of Human D-amino Acid Oxidase Variants and Their Potential Significance in Pathologies. Front Mol Biosci 5:55. https://doi.org/10.3389/fmolb.2018.00055. Yap SH, Lee CS, Furusho A, Ishii C, Shaharudin S, Zulhaimi NS, Kamarulzaman A, Kamaruzzaman SB, Mita M, Leong KH, Hamase K, Rajasuriar R (2022) Plasma d-amino acids are associated with markers of immune activation and organ dysfunction in people with HIV. AIDS 36(7):911-921. https://doi.org/10.1097/QAD.0000000000003207. Bhagavan HN, Coursin DB, Stewart CN (1971) Monosodium glutamate induces convulsive disorders in rats. Nature 232(5308):275-6. https://doi.org/10.1038/232275a0. Oruç KY, Ağtürk G, Oruç A, Yanar K, Seymen HO (2025) Protective effect of Apelin-13 on D-glutamic acid-induced excitotoxicity in SH-SY5Y cell line: An in-vitro study. Neuropeptides 109:102483. https://doi.org/10.1016/j.npep.2024.102483. Vorhees CV, Williams MT (2006) Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc 1(2):848-58. https://doi.org/10.1038/nprot.2006.116. Sadowska-Bartosz I, Galiniak S, Bartosz G, Rachel M (2014) Oxidative modification of proteins in pediatric cystic fibrosis with bacterial infections. Oxid Med Cell Longev 2014:389629. https://doi.org/10.1155/2014/389629. Hanasand M, Omdal R, Norheim KB, Gøransson LG, Brede C, Jonsson G (2012) Improved detection of advanced oxidation protein products in plasma. Clin Chim Acta 413(9-10):901-6. https://doi.org/10.1016/j.cca.2012.01.038. Buege JA, Aust SD (1978) Microsomal lipid peroxidation. Methods Enzymol 52:302-10. https://doi.org/10.1016/s0076-6879(78)52032-6. Sedlak J, Lindsay RH (1968) Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal Biochem 25(1):192-205. https://doi.org/10.1016/0003-2697(68)90092-4. Singh N, Vijayanti S, Saha L, Bhatia A, Banerjee D, Chakrabarti A (2018) Neuroprotective effect of Nrf2 activator dimethyl fumarate, on the hippocampal neurons in chemical kindling model in rat. Epilepsy Res 143:98-104. https://doi.org/10.1016/j.eplepsyres.2018.02.011. Eskandari E, Eaves CJ (2022) Paradoxical roles of caspase-3 in regulating cell survival, proliferation, and tumorigenesis. J Cell Biol 221(6):e202201159. https://doi.org/10.1083/jcb.202201159. Brecht S, Gelderblom M, Srinivasan A, Mielke K, Dityateva G, Herdegen T (2001) Caspase-3 activation and DNA fragmentation in primary hippocampal neurons following glutamate excitotoxicity. Brain Res Mol Brain Res 94(1-2):25-34. https://doi.org/10.1016/s0006-8993(01)02767-6. Hazzaa SM, Abdelaziz SAM, Abd Eldaim MA, Abdel-Daim MM, Elgarawany GE (2020) Neuroprotective Potential of Allium sativum against Monosodium Glutamate-Induced Excitotoxicity: Impact on Short-Term Memory, Gliosis, and Oxidative Stress. Nutrients 12(4):1028. https://doi.org/10.3390/nu12041028. Mohseni F, Garmabi B, Khaksari M (2021) Apelin-13 attenuates spatial memory impairment by anti-oxidative, anti-apoptosis, and anti-inflammatory mechanism against ethanol neurotoxicity in the neonatal rat hippocampus. Neuropeptides 87:102130. https://doi.org/10.1016/j.npep.2021.102130. Shen X, Yuan G, Li B, Cao C, Cao D, Wu J, Li X, Li H, Shen H, Wang Z, Chen G (2022) Apelin-13 attenuates early brain injury through inhibiting inflammation and apoptosis in rats after experimental subarachnoid hemorrhage. Mol Biol Rep 49(3):2107-2118. https://doi.org/10.1007/s11033-021-07028-y. Spencer B, Potkar R, Metcalf J, Thrin I, Adame A, Rockenstein E, Masliah E (2016) Systemic Central Nervous System (CNS)-targeted Delivery of Neuropeptide Y (NPY) Reduces Neurodegeneration and Increases Neural Precursor Cell Proliferation in a Mouse Model of Alzheimer Disease. J Biol Chem 291(4):1905-1920. https://doi.org/10.1074/jbc.M115.678185. Smiałowska M, Domin H, Zieba B, Koźniewska E, Michalik R, Piotrowski P, Kajta M (2009) Neuroprotective effects of neuropeptide Y-Y2 and Y5 receptor agonists in vitro and in vivo. Neuropeptides 43(3):235-49. https://doi.org/10.1016/j.npep.2009.02.002. Domin H, Przykaza Ł, Jantas D, Kozniewska E, Boguszewski PM, Śmiałowska M (2017) Neuropeptide Y Y2 and Y5 receptors as promising targets for neuroprotection in primary neurons exposed to oxygen-glucose deprivation and in transient focal cerebral ischemia in rats. Neuroscience 344:305-325. https://doi.org/10.1016/j.neuroscience.2016.12.040. Khezri MR, Ghasemnejad-Berenji M, Moloodsouri D (2023) The PI3K/AKT Signaling Pathway and Caspase-3 in Alzheimer's Disease: Which One Is the Beginner? J Alzheimers Dis 92(2):391-393. https://doi.org/10.3233/JAD-221157. Roskoski R Jr (2012) ERK1/2 MAP kinases: structure, function, and regulation. Pharmacol Res 66(2):105-43. https://doi.org/10.1016/j.phrs.2012.04.005. Soriano FX, Hardingham GE (2007) Compartmentalized NMDA receptor signalling to survival and death. J Physiol 584(Pt 2):381-7. https://doi.org/10.1113/jphysiol.2007.138875. Cook DR, Gleichman AJ, Cross SA, Doshi S, Ho W, Jordan-Sciutto KL, Lynch DR, Kolson DL (2011) NMDA receptor modulation by the neuropeptide apelin: implications for excitotoxic injury. J Neurochem 118(6):1113-23. https://doi.org/10.1111/j.1471-4159.2011.07383.x. Ishimaru Y, Sumino A, Kajioka D, Shibagaki F, Yamamuro A, Yoshioka Y, Maeda S (2017) Apelin protects against NMDA-induced retinal neuronal death via an APJ receptor by activating Akt and ERK1/2, and suppressing TNF-α expression in mice. J Pharmacol Sci 133(1):34-41. https://doi.org/10.1016/j.jphs.2016.12.002. Zeng Z, Li H, You M, Rong R, Xia X (2022) Dephosphorylation of ERK1/2 and DRP1 S585 regulates mitochondrial dynamics in glutamate toxicity of retinal neurons in vitro. Exp Eye Res 225:109271. https://doi.org/10.1016/j.exer.2022.109271. Gutkind JS (2000) Regulation of mitogen-activated protein kinase signaling networks by G protein-coupled receptors. Sci STKE 2000(40):re1. https://doi.org/10.1126/stke.2000.40.re1. Palanivel V, Gupta V, Mirshahvaladi SSO, Sharma S, Gupta V, Chitranshi N, Mirzaei M, Graham SL, Basavarajappa D (2022) Neuroprotective Effects of Neuropeptide Y on Human Neuroblastoma SH-SY5Y Cells in Glutamate Excitotoxicity and ER Stress Conditions. Cells 11(22):3665. https://doi.org/10.3390/cells11223665. Manning BD, Toker A (2017) AKT/PKB Signaling: Navigating the Network. Cell 169(3):381-405. https://doi.org/10.1016/j.cell.2017.04.001. Zhou XM, Liu CY, Liu YY, Ma QY, Zhao X, Jiang YM, Li XJ, Chen JX (2021) Xiaoyaosan Alleviates Hippocampal Glutamate-Induced Toxicity in the CUMS Rats via NR2B and PI3K/Akt Signaling Pathway. Front Pharmacol 12:586788. https://doi.org/10.3389/fphar.2021.586788. Zeng XJ, Yu SP, Zhang L, Wei L (2010) Neuroprotective effect of the endogenous neural peptide apelin in cultured mouse cortical neurons. Exp Cell Res 316(11):1773-83. https://doi.org/10.1016/j.yexcr.2010.02.005. Jacques D, Dumont Y, Fournier A, Quirion R (1997) Characterization of neuropeptide Y receptor subtypes in the normal human brain, including the hypothalamus. Neuroscience 79(1):129-48. https://doi.org/10.1016/s0306-4522(96)00639-2. Stanić D, Brumovsky P, Fetissov S, Shuster S, Herzog H, Hökfelt T (2006) Characterization of neuropeptide Y2 receptor protein expression in the mouse brain. I. Distribution in cell bodies and nerve terminals. J Comp Neurol 499(3):357-90. https://doi.org/10.1002/cne.21046. Schlicker E, Kathmann M (2008) Presynaptic neuropeptide receptors. Handb Exp Pharmacol (184):409-34. https://doi.org/10.1007/978-3-540-74805-2_13. Silva AP, Carvalho AP, Carvalho CM, Malva JO (2001) Modulation of intracellular calcium changes and glutamate release by neuropeptide Y1 and Y2 receptors in the rat hippocampus: differential effects in CA1, CA3 and dentate gyrus. J Neurochem 79(2):286-96. https://doi.org/10.1046/j.1471-4159.2001.00560.x. Silva AP, Xapelli S, Grouzmann E, Cavadas C (2005) The putative neuroprotective role of neuropeptide Y in the central nervous system. Curr Drug Targets CNS Neurol Disord 4(4):331-47. https://doi.org/10.2174/1568007054546153. Fatoba O, Kloster E, Reick C, Saft C, Gold R, Epplen JT, Arning L, Ellrichmann G (2018) Activation of NPY-Y2 receptors ameliorates disease pathology in the R6/2 mouse and PC12 cell models of Huntington's disease. Exp Neurol 302:112-128. https://doi.org/10.1016/j.expneurol.2018.01.001. Decressac M, Pain S, Chabeauti PY, Frangeul L, Thiriet N, Herzog H, Vergote J, Chalon S, Jaber M, Gaillard A (2012) Neuroprotection by neuropeptide Y in cell and animal models of Parkinson's disease. Neurobiol Aging 33(9):2125-37. https://doi.org/10.1016/j.neurobiolaging.2011.06.018. Kloster E, Saft C, Akkad DA, Epplen JT, Arning L (2014) Association of age at onset in Huntington disease with functional promoter variations in NPY and NPY2R. J Mol Med (Berl) 92(2):177-84. https://doi.org/10.1007/s00109-013-1092-3. Silva AP, Pinheiro PS, Carvalho AP, Carvalho CM, Jakobsen B, Zimmer J, Malva JO (2003) Activation of neuropeptide Y receptors is neuroprotective against excitotoxicity in organotypic hippocampal slice cultures. FASEB J 17(9):1118-20. https://doi.org/10.1096/fj.02-0885fje. Xapelli S, Bernardino L, Ferreira R, Grade S, Silva AP, Salgado JR, Cavadas C, Grouzmann E, Poulsen FR, Jakobsen B, Oliveira CR, Zimmer J, Malva JO (2008) Interaction between neuropeptide Y (NPY) and brain-derived neurotrophic factor in NPY-mediated neuroprotection against excitotoxicity: a role for microglia. Eur J Neurosci 27(8):2089-102. https://doi.org/10.1111/j.1460-9568.2008.06172.x. Domin H (2021) Neuropeptide Y Y2 and Y5 receptors as potential targets for neuroprotective and antidepressant therapies: Evidence from preclinical studies. Prog Neuropsychopharmacol Biol Psychiatry 111:110349. https://doi.org/10.1016/j.pnpbp.2021.110349. Xapelli S, Silva AP, Ferreira R, Malva JO (2007) Neuropeptide Y can rescue neurons from cell death following the application of an excitotoxic insult with kainate in rat organotypic hippocampal slice cultures. Peptides 28(2):288-94. https://doi.org/10.1016/j.peptides.2006.09.031. Kraeuter AK, Guest PC, Sarnyai Z (2019) The Open Field Test for Measuring Locomotor Activity and Anxiety-Like Behavior. Methods Mol Biol 1916:99-103. https://doi.org/10.1007/978-1-4939-8994-2_9. Tian SW, Xu F, Gui SJ (2018) Apelin-13 reverses memory impairment and depression-like behavior in chronic social defeat stressed rats. Peptides 108:1-6. https://doi.org/10.1016/j.peptides.2018.08.009. Wang W, Xu T, Chen X, Dong K, Du C, Sun J, Shi C, Li X, Yang Y, Li H, Xu ZD (2019) NPY Receptor 2 Mediates NPY Antidepressant Effect in the mPFC of LPS Rat by Suppressing NLRP3 Signaling Pathway. Mediators Inflamm 2019:7898095. https://doi.org/10.1155/2019/7898095. Villarreal-Silva EE, González-Navarro AR, Salazar-Ybarra RA, Quiroga-García O, Cruz-Elizondo MAJ, García-García A, Rodríguez-Rocha H, Morales-Gómez JA, Quiroga-Garza A, Elizondo-Omaña RE, de León ÁRM, Guzmán-López S (2022) Aged rats learn Morris Water maze using non-spatial search strategies evidenced by a parameter-based algorithm. Transl Neurosci 13(1):134-144. https://doi.org/10.1515/tnsci-2022-0221. Negrón-Oyarzo I, Espinosa N, Aguilar-Rivera M, Fuenzalida M, Aboitiz F, Fuentealba P (2018) Coordinated prefrontal-hippocampal activity and navigation strategy-related prefrontal firing during spatial memory formation. Proc Natl Acad Sci U S A 115(27):7123-7128. https://doi.org/10.1073/pnas.1720117115. Jin H, Yang C, Jiang C, Li L, Pan M, Li D, Han X, Ding J (2022) Evaluation of Neurotoxicity in BALB/c Mice following Chronic Exposure to Polystyrene Microplastics. Environ Health Perspect 130(10):107002. https://doi.org/10.1289/EHP10255. Kutlu MG, Gould TJ (2016) Effects of drugs of abuse on hippocampal plasticity and hippocampus-dependent learning and memory: contributions to development and maintenance of addiction. Learn Mem 23(10):515-33. https://doi.org/10.1101/lm.042192.116. Kim EJ, Pellman B, Kim JJ (2015) Stress effects on the hippocampus: a critical review. Learn Mem 22(9):411-6. https://doi.org/10.1101/lm.037291.114. Vorhees CV, Williams MT (2006) Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc 1(2):848-58. https://doi.org/10.1038/nprot.2006.116. Gallagher M, Burwell R, Burchinal M (1993) Severity of spatial learning impairment in aging: development of a learning index for performance in the Morris water maze. Behav Neurosci 107(4):618-26. https://doi.org/10.1037//0735-7044.107.4.618. Hernández-Mercado K, Zepeda A (2022) Morris Water Maze and Contextual Fear Conditioning Tasks to Evaluate Cognitive Functions Associated With Adult Hippocampal Neurogenesis. Front Neurosci 15:782947. https://doi.org/10.3389/fnins.2021.782947. Geerts JP, Chersi F, Stachenfeld KL, Burgess N (2020) A general model of hippocampal and dorsal striatal learning and decision making. Proc Natl Acad Sci U S A 117(49):31427-31437. https://doi.org/10.1073/pnas.2007981117. Inostroza M, Cid E, Brotons-Mas J, Gal B, Aivar P, Uzcategui YG, Sandi C, Menendez de la Prida L (2011) Hippocampal-dependent spatial memory in the water maze is preserved in an experimental model of temporal lobe epilepsy in rats. PLoS One 6(7):e22372. https://doi.org/10.1371/journal.pone.0022372. Haghparast E, Esmaeili-Mahani S, Abbasnejad M, Sheibani V (2018) Apelin-13 ameliorates cognitive impairments in 6-hydroxydopamine-induced substantia nigra lesion in rats. Neuropeptides 68:28-35. https://doi.org/10.1016/j.npep.2018.01.001. Shen P, Yue Q, Fu W, Tian SW, You Y (2019) Apelin-13 ameliorates chronic water-immersion restraint stress-induced memory performance deficit through upregulation of BDNF in rats. Neurosci Lett 696:151-155. https://doi.org/10.1016/j.neulet.2018.11.051. Antunes M, Biala G (2012) The novel object recognition memory: neurobiology, test procedure, and its modifications. Cogn Process 13(2):93-110. https://doi.org/10.1007/s10339-011-0430-z. Kornhuber J, Zoicas I (2017) Neuropeptide Y prolongs non-social memory and differentially affects acquisition, consolidation, and retrieval of non-social and social memory in male mice. Sci Rep 7(1):6821. https://doi.org/10.1038/s41598-017-07273-x. Osredkar J, Gosar D, Maček J, Kumer K, Fabjan T, Finderle P, Šterpin S, Zupan M, Jekovec Vrhovšek M (2019) Urinary Markers of Oxidative Stress in Children with Autism Spectrum Disorder (ASD). Antioxidants (Basel) 8(6):187. https://doi.org/10.3390/antiox8060187. Zgutka K, Tkacz M, Tomasiak P, Tarnowski M (2023) A Role for Advanced Glycation End Products in Molecular Ageing. Int J Mol Sci 24(12):9881. https://doi.org/10.3390/ijms24129881. Prasad C, Davis KE, Imrhan V, Juma S, Vijayagopal P (2017) Advanced Glycation End Products and Risks for Chronic Diseases: Intervening Through Lifestyle Modification. Am J Lifestyle Med 13(4):384-404. https://doi.org/10.1177/1559827617708991. Kothandan D, Singh DS, Yerrakula G, D B, N P, Santhana Sophia B V, A R, Ramya Vg S, S K, M J (2024) Advanced Glycation End Products-Induced Alzheimer's Disease and Its Novel Therapeutic Approaches: A Comprehensive Review. Cureus 16(5):e61373. https://doi.org/10.7759/cureus.61373. Du H, Ma Y, Wang X, Zhang Y, Zhu L, Shi S, Pan S, Liu Z (2023) Advanced glycation end products induce skeletal muscle atrophy and insulin resistance via activating ROS-mediated ER stress PERK/FOXO1 signaling. Am J Physiol Endocrinol Metab 324(3):E279-E287. https://doi.org/10.1152/ajpendo.00218.2022. Wan L, Bai X, Zhou Q, Chen C, Wang H, Liu T, Xue J, Wei C, Xie L (2022) The advanced glycation end-products (AGEs)/ROS/NLRP3 inflammasome axis contributes to delayed diabetic corneal wound healing and nerve regeneration. Int J Biol Sci 18(2):809-825. https://doi.org/10.7150/ijbs.63219. Deng S, He R, Yue Z, Li B, Li F, Xiao Q, Wang X, Li Y, Chen R, Rong S (2024) Association of Advanced Glycation End Products with Cognitive Function: HealthyDance Study. J Alzheimers Dis 100(2):551-562. https://doi.org/10.3233/JAD-240296. Peppa M, Uribarri J, Vlassara H (2008) Aging and glycoxidant stress. Hormones (Athens) 7(2):123-32. https://doi.org/10.1007/BF03401503. Oleniuc M, Secara I, Onofriescu M, Hogas S, Voroneanu L, Siriopol D, Covic A (2011) Consequences of Advanced Glycation End Products Accumulation in Chronic Kidney Disease and Clinical Usefulness of Their Assessment Using a Non-invasive Technique - Skin Autofluorescence. Maedica (Bucur) 6(4):298-307. Wu B, Yu L, Hu P, Lu Y, Li J, Wei Y, He R (2018) GRP78 protects CHO cells from ribosylation. Biochim Biophys Acta Mol Cell Res 1865(4):629-637. https://doi.org/10.1016/j.bbamcr.2018.02.001. Bayarsaikhan E, Bayarsaikhan D, Lee J, Son M, Oh S, Moon J, Park HJ, Roshini A, Kim SU, Song BJ, Jo SM, Byun K, Lee B (2016) Microglial AGE-albumin is critical for neuronal death in Parkinson's disease: a possible implication for theranostics. Int J Nanomedicine 10 Spec Iss(Spec Iss):281-92. https://doi.org/10.2147/IJN.S95077. Malarkey EB, Parpura V (2008) Mechanisms of glutamate release from astrocytes. Neurochem Int 52(1-2):142-54. https://doi.org/10.1016/j.neuint.2007.06.005. Kamynina A, Esteras N, Koroev DO, Angelova PR, Volpina OM, Abramov AY (2021) Activation of RAGE leads to the release of glutamate from astrocytes and stimulates calcium signal in neurons. J Cell Physiol 236(9):6496-6506. https://doi.org/10.1002/jcp.30324. Koerich S, Parreira GM, de Almeida DL, Vieira RP, de Oliveira ACP (2023) Receptors for Advanced Glycation End Products (RAGE): Promising Targets Aiming at the Treatment of Neurodegenerative Conditions. Curr Neuropharmacol 21(2):219-234. https://doi.org/10.2174/1570159X20666220922153903. Lamichhane S, Bastola T, Pariyar R, Lee ES, Lee HS, Lee DH, Seo J (2017) ROS Production and ERK Activity Are Involved in the Effects of d-β-Hydroxybutyrate and Metformin in a Glucose Deficient Condition. Int J Mol Sci 18(3):674. https://doi.org/10.3390/ijms18030674. Alderman CJ, Shah S, Foreman JC, Chain BM, Katz DR (2002) The role of advanced oxidation protein products in regulation of dendritic cell function. Free Radic Biol Med 32(5):377-85. https://doi.org/10.1016/s0891-5849(01)00735-3. Yilmazer UT, Pehlivan B, Guney S, Yar-Saglam AS, Balabanli B, Kaltalioglu K, Coskun-Cevher S (2024) The combined effect of morin and hesperidin on memory ability and oxidative/nitrosative stress in a streptozotocin-induced rat model of Alzheimer's disease. Behav Brain Res 471:115131. https://doi.org/10.1016/j.bbr.2024.115131. Li L, Renier G (2006) Activation of nicotinamide adenine dinucleotide phosphate (reduced form) oxidase by advanced glycation end products links oxidative stress to altered retinal vascular endothelial growth factor expression. Metabolism 55(11):1516-23. https://doi.org/10.1016/j.metabol.2006.06.022. Heijnis WH, Dekker HL, de Koning LJ, Wierenga PA, Westphal AH, de Koster CG, Gruppen H, van Berkel WJ (2011) Identification of the peroxidase-generated intermolecular dityrosine cross-link in bovine α-lactalbumin. J Agric Food Chem 59(1):444-9. https://doi.org/10.1021/jf104298y. Al-Hilaly YK, Williams TL, Stewart-Parker M, Ford L, Skaria E, Cole M, Bucher WG, Morris KL, Sada AA, Thorpe JR, Serpell LC (2013) A central role for dityrosine crosslinking of Amyloid-β in Alzheimer's disease. Acta Neuropathol Commun 1:83. https://doi.org/10.1186/2051-5960-1-83. Wang J, Swanson RA (2020) Superoxide and Non-ionotropic Signaling in Neuronal Excitotoxicity. Front Neurosci 4:861. https://doi.org/10.3389/fnins.2020.00861. Pennathur S, Jackson-Lewis V, Przedborski S, Heinecke JW (1999) Mass spectrometric quantification of 3-nitrotyrosine, ortho-tyrosine, and o,o'-dityrosine in brain tissue of 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine-treated mice, a model of oxidative stress in Parkinson's disease. J Biol Chem 274(49):34621-8. https://doi.org/10.1074/jbc.274.49.34621. Pathak S, Nadar R, Kim S, Liu K, Govindarajulu M, Cook P, Watts Alexander CS, Dhanasekaran M, Moore T (2024) The Influence of Kynurenine Metabolites on Neurodegenerative Pathologies. Int J Mol Sci 25(2):853. https://doi.org/10.3390/ijms25020853. Sorgdrager FJH, Vermeiren Y, Van Faassen M, van der Ley C, Nollen EAA, Kema IP, De Deyn PP (2019) Age- and disease-specific changes of the kynurenine pathway in Parkinson's and Alzheimer's disease. J Neurochem 151(5):656-668. https://doi.org/10.1111/jnc.14843. Ganong AH, Cotman CW (1986) Kynurenic acid and quinolinic acid act at N-methyl-D-aspartate receptors in the rat hippocampus. J Pharmacol Exp Ther 236(1):293-9. Dharmajaya R, Sari DK (2022) Malondialdehyde value as radical oxidative marker and endogenous antioxidant value analysis in brain tumor. Ann Med Surg (Lond) 77:103231. https://doi.org/10.1016/j.amsu.2021.103231. Peña-Bautista C, Vento M, Baquero M, Cháfer-Pericás C (2019) Lipid peroxidation in neurodegeneration. Clin Chim Acta 497:178-188. https://doi.org/10.1016/j.cca.2019.07.037. Haro Girón S, Monserrat Sanz J, Ortega MA, Garcia-Montero C, Fraile-Martínez O, Gómez-Lahoz AM, Boaru DL, de Leon-Oliva D, Guijarro LG, Atienza-Perez M, Diaz D, Lopez-Dolado E, Álvarez-Mon M (2023) Prognostic Value of Malondialdehyde (MDA) in the Temporal Progression of Chronic Spinal Cord Injury. J Pers Med 13(4):626. https://doi.org/10.3390/jpm13040626. Li J, Zhou Q, Yang T, Li Y, Zhang Y, Wang J, Jiao Z (2018) SGK1 inhibits PM2.5-induced apoptosis and oxidative stress in human lung alveolar epithelial A549 cells. Biochem Biophys Res Commun 496(4):1291-1295. https://doi.org/10.1016/j.bbrc.2018.02.002. Ajsuvakova OP, Tinkov AA, Aschner M, Rocha JBT, Michalke B, Skalnaya MG, Skalny AV, Butnariu M, Dadar M, Sarac I, Aaseth J, Bjørklund G (2020) Sulfhydryl groups as targets of mercury toxicity. Coord Chem Rev 417:213343. https://doi.org/10.1016/j.ccr.2020.213343. Eroglu N, Sahin G, Yesil S, Fettah A, Yildiz YT, Erel O (2023) Thiol disulfide homeostasis in ionizing radiation and chemotherapeutic drug exposure. North Clin Istanb 10(1):53-58. https://doi.org/10.14744/nci.2021.59913. Pham TK, Buczek WA, Mead RJ, Shaw PJ, Collins MO (2021) Proteomic Approaches to Study Cysteine Oxidation: Applications in Neurodegenerative Diseases. Front Mol Neurosci 14:678837. https://doi.org/10.3389/fnmol.2021.678837. Kazama M, Kato Y, Kakita A, Noguchi N, Urano Y, Masui K, Niida-Kawaguchi M, Yamamoto T, Watabe K, Kitagawa K, Shibata N (2020) Astrocytes release glutamate via cystine/glutamate antiporter upregulated in response to increased oxidative stress related to sporadic amyotrophic lateral sclerosis. Neuropathology 40(6):587-598. https://doi.org/10.1111/neup.12716. Shivasharan BD, Nagakannan P, Thippeswamy BS, Veerapur VP (2013) Protective Effect of Calendula officinalis L. Flowers Against Monosodium Glutamate Induced Oxidative Stress and Excitotoxic Brain Damage in Rats. Indian J Clin Biochem 28(3):292-8. https://doi.org/10.1007/s12291-012-0256-1. Topcu A, Saral S, Mercantepe T, Akyildiz K, Tumkaya L, Yilmaz A (2023) The effects of apelin-13 against cisplatin-induced nephrotoxicity in rats. Drug Chem Toxicol 46(1):77-87. https://doi.org/10.1080/01480545.2021.2011309. Singh P, Barman B, Thakur MK (2022) Oxidative stress-mediated memory impairment during aging and its therapeutic intervention by natural bioactive compounds. Front Aging Neurosci 14:944697. https://doi.org/10.3389/fnagi.2022.944697. Fukui K, Omoi NO, Hayasaka T, Shinnkai T, Suzuki S, Abe K, Urano S (2002) Cognitive impairment of rats caused by oxidative stress and aging, and its prevention by vitamin E. Ann N Y Acad Sci 959:275-84. https://doi.org/10.1111/j.1749-6632.2002.tb02099.x. Wang B, Yang Q, Sun YY, Xing YF, Wang YB, Lu XT, Bai WW, Liu XQ, Zhao YX (2014) Resveratrol-enhanced autophagic flux ameliorates myocardial oxidative stress injury in diabetic mice. J Cell Mol Med 18(8):1599-611. https://doi.org/10.1111/jcmm.12312. Negahdar H, Hosseini SR, Parsian H, Kheirkhah F, Mosapour A, Khafri S, Haghighi AH (2015) Homocysteine, trace elements and oxidant/antioxidant status in mild cognitively impaired elderly persons: a cross-sectional study. Rom J Intern Med 53(4):336-42. https://doi.org/10.1515/rjim-2015-0043. Solvang SH, Nordrehaug JE, Tell GS, Nygård O, McCann A, Ueland PM, Midttun Ø, Meyer K, Vedeler CA, Aarsland D, Refsum H, Smith AD, Giil LM (2019) The kynurenine pathway and cognitive performance in community-dwelling older adults. The Hordaland Health Study. Brain Behav Immun 75:155-162. https://doi.org/10.1016/j.bbi.2018.10.003. Logan S, Royce GH, Owen D, Farley J, Ranjo-Bishop M, Sonntag WE, Deepa SS (2019) Accelerated decline in cognition in a mouse model of increased oxidative stress. Geroscience 41(5):591-607. https://doi.org/10.1007/s11357-019-00105-y. Mandour DA, Bendary MA, Alsemeh AE (2021) Histological and imunohistochemical alterations of hippocampus and prefrontal cortex in a rat model of Alzheimer like-disease with a preferential role of the flavonoid \"hesperidin\". J Mol Histol 52(5):1043-1065. https://doi.org/10.1007/s10735-021-09998-6. Fricker M, Tolkovsky AM, Borutaite V, Coleman M, Brown GC (2018) Neuronal Cell Death. Physiol Rev 98(2):813-880. https://doi.org/10.1152/physrev.00011.2017. Esmaeili-Mahani S, Haghparast E, Nezhadi A, Abbasnejad M, Sheibani V (2020) Apelin-13 prevents hippocampal synaptic plasticity impairment in Parkinsonism rats. J Chem Neuroanat 111:101884. https://doi.org/10.1016/j.jchemneu.2020.101884. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 22 Jan, 2026 Read the published version in Molecular Neurobiology → Version 1 posted Editorial decision: Revision requested 20 Jun, 2025 Reviews received at journal 19 Jun, 2025 Reviews received at journal 18 Jun, 2025 Reviews received at journal 16 Jun, 2025 Reviewers agreed at journal 11 Jun, 2025 Reviewers agreed at journal 10 Jun, 2025 Reviewers agreed at journal 10 Jun, 2025 Reviewers agreed at journal 02 Jun, 2025 Reviewers invited by journal 30 May, 2025 Editor assigned by journal 23 May, 2025 Submission checks completed at journal 23 May, 2025 First submitted to journal 10 May, 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. 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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-6635799\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":464816090,\"identity\":\"220f3c79-549e-435e-a8ec-0d8f9da9b594\",\"order_by\":0,\"name\":\"Kadriye Yagmur 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Active Caspase-3/Protein \\u003cstrong\\u003e(G)\\u003c/strong\\u003e, ERK1/2/Protein \\u003cstrong\\u003e(H)\\u003c/strong\\u003e, AKT-1/Protein \\u003cstrong\\u003e(J)\\u003c/strong\\u003ehippocampus and prefrontal cortex levels. 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(h) Healty neuron, (ph) Pericellular halo, (pn) Pyknotic nuclei, (v) vacuolization, (ab) apoptotic body, MT (Molecular layer), PHT (Pyramidal cell layer), PT (Polymorphic layer).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6635799/v1/30434f6d8874169e5beca29e.png\"},{\"id\":83780394,\"identity\":\"a054a233-39a9-4b52-b703-08b9957103d0\",\"added_by\":\"auto\",\"created_at\":\"2025-06-02 15:17:53\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1369484,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePrefrontal Cortex Cresyl Violet staining C group \\u003cstrong\\u003e(A)\\u003c/strong\\u003e, A Group \\u003cstrong\\u003e(B)\\u003c/strong\\u003e, G Group \\u003cstrong\\u003e(C)\\u003c/strong\\u003e, GA Group \\u003cstrong\\u003e(D)\\u003c/strong\\u003e,\\u003cstrong\\u003e \\u003c/strong\\u003eGAN2 Group \\u003cstrong\\u003e(E)\\u003c/strong\\u003e,\\u003cstrong\\u003e \\u003c/strong\\u003eGAN5 Group \\u003cstrong\\u003e(F)\\u003c/strong\\u003e. (h) Healty neuron, (ph) Pericellular halo, (pn) Pyknotic nuclei, (v) vacuolization, (ab) apoptotic body. Molecular (I), external granular (II), external pyramidal (III), internal granular (IV), internal pyramidal (V) and polymorphic (VI) layers.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6635799/v1/0d6819beec7bc8fe561b57c7.png\"},{\"id\":101151798,\"identity\":\"467ef0fb-ca5b-4576-8456-300c32db868b\",\"added_by\":\"auto\",\"created_at\":\"2026-01-26 16:05:45\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":6404758,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6635799/v1/bb6190d8-04f3-459f-b157-c7d8bf872ab4.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Apelin-13 confers Neuropeptide Y–mediated neuroprotection and preserves learning and allocentric memory in D-glutamic acid-induced excitotoxicity in rats\",\"fulltext\":[{\"header\":\"HIGHLIGHTS\",\"content\":\"\\u003cp\\u003e• D-Glutamic acid causes excitotoxicity by inhibiting ERK1/2, PI3K/AKT-1, causing oxidative damage and an increase in active caspase-3.\\u003c/p\\u003e\\n\\u003cp\\u003e• In excitotoxicity, spatial allocentric mapping is disrupted, and an orientation towards adaptive egocentric memory is observed.\\u003c/p\\u003e\\n\\u003cp\\u003e• Apelin-13 works in coordination with NPY2R and, to a lesser extent, NPY5R against excitotoxic damage.\\u003c/p\\u003e\\n\\u003cp\\u003e• Apelin-13 provides neuroprotection by strengthening ERK1/2 and PI3K/AKT signaling.\\u003c/p\\u003e\\n\\u003cp\\u003e• Apelin-13 prevents mitochondrial dysfunction by increasing the antioxidant/oxidant ratio.\\u003c/p\\u003e\\n\\u003cp\\u003e• Apelin-13 protects allocentric memory and prevents STM-LTM and learning damage.\\u003c/p\\u003e\"},{\"header\":\"1. INTRODUCTION\",\"content\":\"\\u003cp\\u003eGlutamate, the main excitatory neurotransmitter in the central nervous system (CNS), plays a role in neuronal communication, synaptogenesis, learning and memory processes through ionotropic (iGluR) and metabotropic receptors (mGluR). Dysregulation of the glutamatergic system can cause many effects [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. Excitotoxicity leads to neuronal loss by acute (excessive)-chronic activation of postsynaptic neurons [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. The basal concentration of glutamate in the synaptic cleft varies between 25\\u0026ndash;600 nM [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e], and this value cannot activate glutamate receptors [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. However, reaching basal glutamate concentrations of 2\\u0026ndash;5 \\u0026micro;M causes excitotoxic damage [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. This condition involves NMDAR/AMPAR overactivation, Ca\\u0026sup2;⁺ dysregulation, and glutamate-glutamine cycle impairment, leading to ROS accumulation, oxidative stress, mitochondrial dysfunction, and apoptosis [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. These molecular damages cause learning and memory disorders at the macro level [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eToday, the protective activity of many endogenous and exogenous molecules against excitotoxic damage is being investigated. Endogenous adipokine and neuropeptide Apelin, produced from adipose tissue, is the ligand of the Apelin receptor (APJR), a G protein-coupled receptor. The 77 amino acid pre-proapelin is cleaved by peptidases and reduced to active apelin isoforms such as 13, 17, and 36 [\\u003cspan additionalcitationids=\\\"CR9 CR10\\\" citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. Apelin-13 is the most potent and predominant isoform in plasma and can cross the blood-brain barrier via peripheral pathways [\\u003cspan additionalcitationids=\\\"CR13\\\" citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. The Apelin-13/APJ complex regulates intracellular Ca\\u003csup\\u003e2+\\u003c/sup\\u003e by inhibiting NMDAR via extracellular signal-regulated kinase (ERK1/2) and protein kinase B (AKT) signaling. It responds to mitochondrial dysfunction, apoptosis, oxidative stress, learning and memory damage via the same pathways [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eNeuropeptide Y (NPY) is the most abundant endogenous neuropeptide in the CNS. It regulates neuroprotection, trophic support enhancement, excitotoxicity inhibition, Ca\\u003csup\\u003e+\\u0026thinsp;2\\u003c/sup\\u003e homeostasis and learning-memory processes. Its receptors are GPCRs and have the subtypes NPY1, -Y2, ​​-Y4, -Y5, -Y6 [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. NPY2 receptor (NPY2R) is localized in the presynaptic terminal in the dentate gyrus (DG), Cornu ammonis 1 (CA1), CA3 regions, and NPY5 receptor (NPY5R) is localized postsynaptically in DG, CA1, CA2, CA3 [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. NPY prevents excitotoxic damage by regulating glutamate release from the CA1 presynaptic terminal via NPY2R and postsynaptic NPY5R in CA1-CA3 with ERK1/2, PI3K/AKT signaling. Changes in NPY levels have been observed in neurodegenerative diseases [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. Additionally, NPY2R enhances consolidation in spatial memory [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eSpatial navigation is critical in the hippocampus, which is involved in the learning and memory process. In spatial mapping, hippocampus-dependent allocentric and hippocampus-independent egocentric strategies gain importance in neurodegenerative disease progression. In hippocampal damage, spatial mapping is disrupted and allocentric navigation decreases. Prolonged search time and distance characterize a compensatory shift toward egocentric strategies [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. Mitochondrial dysfunction, oxidative stress and faulty protein accumulation leading to excitotoxicity cause learning and memory impairment in hippocampal circuits [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eAlthough neuroprotective effects of apelin-13 have been demonstrated, the multifactorial nature of the pathophysiological mechanisms makes the specificity of this effect unclear. The colocalization of APJR with NPY2R, NPY5R in hippocampal areas sensitive to excitotoxic damage and their similar protective functions via ERK1/2 and PI3K/AKT signaling pathways have highlighted the idea that Apelin-13 and NPY work in coordination against excitotoxicity [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eTo date, no studies have specifically examined whether Apelin-13 and NPY neuropeptides act in concert to counteract excitotoxic damage. The number of studies modeling D-glutamic acid-mediated excitotoxicity is quite limited [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. In this study, we aimed to investigate the protective effects of Apelin-13 in the D-glutamic acid-induced excitotoxicity model and the relationship between NPY2R and NPY5R.\\u003c/p\\u003e\"},{\"header\":\"2. METHOD\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1. Chemicals\\u003c/h2\\u003e \\u003cp\\u003eD-glutamic acid (D-Glutamic acid, Sigma, CAS:6893-26-1, Saint Louis, USA), Apelin-13 (Apelin-13, Cayman Chemicals, Item 13523, Michigan, USA), NPY Y2 receptor antagonist (BIIE 0246 hydrochloride, cat no 7377, Tocris Bioscience, Bristol, UK), NPY Y5 receptor antagonist (L-152.804, catalog no 1382, Tocris Bioscience, Bristol, UK), Ketamine Hydrochloride (HCl) (Ketalar HCl 50 mg/mL, Pfizer, Istanbul, Turkey), Xylazine HCl (Rompun 20 mg/mL, Bayer, Istanbul, Turkey) were used while performing the experiments.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2. Animals\\u003c/h2\\u003e \\u003cp\\u003eForty-two male Sprague Dawley rats, 6\\u0026ndash;8 weeks old, weighing 200\\u0026ndash;250 g, belonging to the same generation were obtained from Istanbul University-Cerrahpaşa Nanotechnology and Biotechnology Institute Experimental Medicine Research Laboratory (DETALAB). Ethical approval was obtained from Istanbul University-Cerrahpaşa Animal Experiments Local Ethics Committee (approval number: 2023/11). The animals were housed in 12 hours of light/darkness, 55\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;10% humidity and 23\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1\\u0026deg;C. They were fed with standard pellet food and water ad libitum. The cages were taken into the experimental room 1 week before the experiment and cleaned every other day. Rats were randomly assigned to six groups (n\\u0026thinsp;=\\u0026thinsp;7); Control (C), Apelin-13 (A), D-Glutamic acid (G), D-Glutamic acid\\u0026thinsp;+\\u0026thinsp;Apelin-13 (GA), D-Glutamic acid\\u0026thinsp;+\\u0026thinsp;NPY 2 Receptor Antagonist\\u0026thinsp;+\\u0026thinsp;Apelin-13 (GAN2), D-Glutamic acid\\u0026thinsp;+\\u0026thinsp;NPY 5 Receptor Antagonist\\u0026thinsp;+\\u0026thinsp;Apelin-13 (GAN5).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eGroup C received 1 ml physiological saline (SF) and DMSO on days 1 and 4\\u0026ndash;10. Group G received 4 mg/kg D-glutamic acid (1 ml, i.p.) on day 1, 1 ml SF on days 4\\u0026ndash;10. Group A received 1 ml SF on day 1, 300 \\u0026micro;g/kg Apelin-13 (1 ml, i.p.) on days 4\\u0026ndash;10. Group GA received 4 mg/kg D-glutamic acid (1 ml, i.p.) on day 1, 300 \\u0026micro;g/kg Apelin-13 (1 ml, i.p.) on days 4\\u0026ndash;10. GAN2 group received 4 mg/kg D-glutamic acid (1 ml, i.p.) on day 1, 4\\u0026ndash;10. received 300 \\u0026micro;g/kg Apelin-13\\u0026thinsp;+\\u0026thinsp;1,5 mg/kg NPY Y2 receptor antagonist (1 ml, i.p.) on days 1. GAN5 group received 4 mg/kg D-glutamic acid (1 ml, i.p.) on day 1, 300 \\u0026micro;g/kg Apelin-13\\u0026thinsp;+\\u0026thinsp;1,5 mg/kg NPY Y5 receptor antagonist (1 ml, i.p.) on days 4\\u0026ndash;10. 3 days were waited after the first injection for the excitotoxicity model to develop. All chemicals were dissolved in dimethylsulfoxide (DMSO) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eD-Glutamic acid accumulates in tissues in rats because it is not deaminated. It can pass through the blood-brain barrier [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. D-amino acid oxidase (DAO) and D-aspartate oxidase (DDO) are stereospecific enzymes that metabolize D-amino acids. In particular, DDO reduces flavin adenine dinucleotide (FAD) by converting acidic D-amino acids such as D-aspartate, D-glutamate and NMDA into imino acids [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. Reoxidation of FAD in the presence of oxygen causes the production of hydrogen peroxide (H₂O₂), triggering oxidative stress, mitochondrial dysfunction, proinflammatory cytokine activation and cell death [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. D-AA accumulation induces inflammation and cell death via H₂O₂ and NF-κB activation [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e]. While determining the i.p. dose of D-glutamic acid, a dose that could induce convulsions but posed minimal risk of mortality was taken into consideration [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. Studies in the literature support the D-glutamic acid-mediated excitotoxicity model [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3. Learning and Memory Tests\\u003c/h2\\u003e \\u003cp\\u003eA 70x70 cm opaque gray colored open-topped cardboard box was used in a room illuminated with constant light in OFT and NORT. Tests evaluated with ANY-Maze Version 7.42 software (AnyMaze, Stoelting Co., Illinois, USA). The experiments were recorded with a camera (Canon PowerShot, SX740 HS, Tokyo, Japan).\\u003c/p\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.1. Open Field Test\\u003c/h2\\u003e \\u003cp\\u003eRats were placed in the center of the test box and moved freely for 10 minutes. In the recorded videos, average speed and total distance, number of rearings, number of defecations, time spent in the center and on the edge were evaluated.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.2. Novel Object Recognition Test\\u003c/h2\\u003e \\u003cp\\u003eIn order to evaluate short-term memory (STM), rats were given 5 min to explore their surroundings during habituation period. In familiarization, identical green objects (A1, A2) measuring 11x7 cm (lengthxdiameter) were placed diagonally in the box. The rat was placed in the middle of the objects and contact was expected for at least 20 s for 10 min. After 60 min, a new object (B) measuring 11 cm was placed in place of the old A2. The rat touching the object with its nose was considered as \\u0026ldquo;contact\\u0026rdquo;. Discrimination index (DI), average speed and distance, total examination time and number of A1-A2 and A1-B were evaluated.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.3. Morris Water Maze Test\\u003c/h2\\u003e \\u003cp\\u003eMWM was applied to evaluate long-term memory (LTM). The pool consisting of a white Plexiglas pool (150x60 cm, diameterxdepth) and a transparent Plexiglas platform (10x28 cm, diameterxlength) was filled to 30 cm, painted black with non-toxic paint, and heated to 26\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1\\u0026deg;C. Blue-green shapes were placed on the walls for allocentric memory, and black shapes were placed on the pool perimeter for egocentric memory. Rats were released into the pool from different points in 4 consecutive trials for 5 days [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e], each trial lasted 60 seconds, and the inter-trial interval was determined as 15 seconds. The escape platform was removed on the probe day, and the rats were released into the pool from the northeast, the only direction from which they were not thrown, and swam for 60 seconds. The time to reach the target quadrant, speed, latency, average swimming speed, and distance were evaluated.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4. Sacrification\\u003c/h2\\u003e \\u003cp\\u003eBefore anesthesia, rats were weighed and given a cocktail of ketamine 50 mg/kg and xylazine HCl 10 mg/kg via the i.p. route. Following the loss of reflexes, intracardiac blood was collected. Serum samples were labeled and stored at -20\\u0026deg;C. Cervical dislocation was performed in accordance with ethical guidelines. The brain was dissected and bilateral hippocampus and prefrontal cortex (PFC) were removed. Half of the hippocampus and PFC were stored at -20\\u0026deg;C for biochemical analyses, while the remainder was stored in 4% formaldehyde for histological imaging.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.5. Biochemical Analyses\\u003c/h2\\u003e \\u003cp\\u003eConducted at Istanbul University-Cerrahpasa, Cerrahpasa Medical Faculty Biochemistry Research Laboratory. Malondialdehyde (MDA), advanced oxidation protein products (AOPP), total thiol (T-SH), dityrosine (DT), kaynurenine (KYN), advanced glycation end products (AGE) and total protein levels were analyzed in plasma, hippocampus and PFC. The brain tissue was rinsed with phosphate buffer saline (PBS, pH 7.4) at +\\u0026thinsp;4\\u0026deg;C, weighed and homogenized with PBS at a ratio of 1:9. The homogenates were centrifuged and the supernatants were labeled and stored.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.5.1. Advanced Glycation End Products, Dityrosine, Kaynurenine\\u003c/h2\\u003e \\u003cp\\u003eAccording to the procedure in the literature, 150 \\u0026micro;L of sample was diluted 1:50 with PBS. It was read in a spectrofluorometer at excitation/emission wavelengths of 325/440 nm for AGE, 330/415 nm for DT, and 365/480 nm for KYN [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.5.2. Advanced Oxidation Protein Products\\u003c/h2\\u003e \\u003cp\\u003e20 \\u0026micro;L of sample and 200 \\u0026micro;L of citric acid were added to the microplate, waited for 2 min, and 10 \\u0026micro;L of potassium iodide was pipetted. The samples were measured spectrophotometrically at a wavelength of 340 nm and the AOPP concentration was determined according to the procedure in the literature [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.5.3. Malondialdehyde\\u003c/h2\\u003e \\u003cp\\u003eAccording to the procedure in the literature [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e], 2 ml of the reagent prepared with 15% TCA, 0.375% TBA and 0.25 mol/L HCl was added to 1 ml of sample and kept in a water bath at 95\\u0026deg;C for 15 minutes. The formed precipitate was centrifuged at 1000 RPM for 10 minutes. The absorbance of the supernatant was measured at 532 nm in a spectrophotometer and the MDA concentration was calculated using the molar extinction coefficient of 1.56x105 M-1 cm-1.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.5.4. Total Thiol\\u003c/h2\\u003e \\u003cp\\u003eAccording to the method in the literature [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e], 20 \\u0026micro;L of sample, 400 \\u0026micro;L of Tris buffer (pH 8.2) and 20 \\u0026micro;L of dithionitrobenzoic acid (5,5-dithio-bis-(2-nitrobenzoic acid), Cat no: 22582, ThermoFisher Scienfic, USA) were added to the microplate. The absorbance values ​​of the samples were analyzed against the reagent blank in a spectrophotometer at 412 nm and the extinction coefficient and T-SH concentrations were calculated.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.5.5. Total Protein\\u003c/h2\\u003e \\u003cp\\u003eTo determine the total protein amount in tissue and plasma, 194 \\u0026micro;L of bicinchoninic acid (BCA) was diluted 1:50 with 4% copper sulfate. 194 \\u0026micro;L of BCA and 6 \\u0026micro;L of sample were pipetted. Standards were prepared with albumin and added to standard wells. Phosphate buffer was added to blank wells and incubated in the dark for 30 min. Absorbance value was read by spectrophotometric method at 562 nm wavelength. Calibration curve was drawn for standards and samples to calculate the results.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.6. Enzyme-Linked ImmunoSorbent Assay\\u003c/h2\\u003e \\u003cp\\u003eExperiments were conducted in Istanbul University-Cerrahpasa, Cerrahpasa Faculty of Medicine Biochemistry Research Laboratory and Hali\\u0026ccedil; University Faculty of Medicine Physiology Research Laboratory. Rat extracellular regulated protein kinases 1;2 (ERK1;2) ELISA Kit (Cat no: E1479Ra, BT LAB, China), Rat RAC-alpha serine; threonine-protein kinase (AKT1) ELISA Kit (Cat no: E2548Ra, BT LAB, China) and Rat CASP3 (Caspase 3) ELISA Kit (Cat no: ELK1528, ELK Biotechnology, Denver, USA) kits were purchased from the manufacturers. Standards were prepared by serial dilution at a 1:2 ratio. 25x wash buffer was diluted to 1x. 50 \\u0026micro;L of standard was added to the standard wells, 40 \\u0026micro;L of sample and 10 \\u0026micro;L of biotinylated antibody were added to the sample wells. 50 \\u0026micro;L of streptavidin-Horse Radish Peroxidase (HRP) was added and incubated at 37\\u0026deg;C for 60 min. It was washed 5 times with 300 \\u0026micro;L of wash buffer, 50 \\u0026micro;L of substrate solutions A and B were added and incubated at 37\\u0026deg;C for 10 min. Stop solution was added and measurement was made at 450 nm and the standard curve was created by regression analysis.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.7. Histological Analyses\\u003c/h2\\u003e \\u003cp\\u003eTissue embedding, sectioning and staining were performed in the Histology Research Laboratory of the Faculty of Medicine of Istanbul Altınbaş University, and histological examinations were performed in the Histology Research Laboratory of the Faculty of Medicine of Istanbul University-Cerrahpaşa. All examinations were performed in the hippocampus CA1-CA3 and PFC. Sections stained with Hematoxylin\\u0026amp;Eosin (H\\u0026amp;E, ABCAM, ab245880, UK) and Cresyl Violet were examined morphologically under 10x, 20x and 40x magnification with a light microscope (Olympus BX61, Japan) integrated camera (Olympus DP72, Japan). By modifying the method of Singh et al. (2018), 5 randomly selected fields from the 40x magnified sections were scored blindly by two researchers. Neurons were histologically evaluated on a scale of 0\\u0026ndash;5 according to their morphological features (0: undamaged, 5: severely damaged) and statistical analysis was performed [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.8. Statistical Analysis\\u003c/h2\\u003e \\u003cp\\u003eStatistical analyses were performed using GraphPad Prism (v10.2.0, Boston, MA, USA). Normality and homogeneity were assessed using Shapiro-Wilk and Levene tests. Since the data were found to be normal and homogeneous, One-Way ANOVA and post-hoc Tukey test were used in ELISA and biochemical analyses. Results are presented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation (SD). Repeated-One-Way ANOVA was applied for MWM training days and One-Way ANOVA was applied for probe day. One-Way ANOVA and post-hoc Tukey test were used in OFT and NORT tests. p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 was considered statistically significant.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. RESULTS\",\"content\":\"\\u003cp\\u003e \\u003cul\\u003e \\u003cli\\u003e \\u003cp\\u003e \\u003cb\\u003eCo-activation of NPY2R and NPY5R is required for the full neuroprotective effect of Apelin-13 under excitotoxic conditions\\u003c/b\\u003e \\u003c/p\\u003e \\u003c/li\\u003e \\u003c/ul\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eActive Caspase-3/Protein (ng/mL)\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn the hippocampus and PFC, when compared to the C group, a very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) was found in the Active Caspase-3 levels in the G group. When compared to the G and GA, GAN2 and GAN5 groups, a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) was found in all groups. When compared to the A group, a very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) was seen in GA, GAN2, GAN5. When compared to the GA group, a very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) was found in GAN2, GAN5. There was a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) in GAN5 compared to GAN2 (Shown in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eG).\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eActive Caspase-3/Protein, ERK1/2 /Protein, AKT-1/Protein levels. Results are given as Mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"7\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eC\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eG\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eA\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eGA\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eGAN2\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eGAN5\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"7\\\" nameend=\\\"c7\\\" namest=\\\"c1\\\"\\u003e \\u003cp\\u003eActive Caspase-3/Protein(ng/mL)\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eHippocampus\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0,17\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.03\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1,73\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.03\\u003csup\\u003ea***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0,16\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.03\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0,74\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.04\\u003csup\\u003eb,c***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1,61\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.06\\u003csup\\u003eb,c,d***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0,97\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.02\\u003csup\\u003eb,c,d,e***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003ePFC\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0,27\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.05\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1,92\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.04\\u003csup\\u003ea***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0,25\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.03\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0,57\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.04\\u003csup\\u003eb,c***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1,80\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.06\\u003csup\\u003eb,c,d***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0,77\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.02\\u003csup\\u003eb,c,d,e***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"7\\\" nameend=\\\"c7\\\" namest=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eERK1/2 /Protein(ng/L)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eHippocampus\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e994,4\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;6.2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e471\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;27.4\\u003csup\\u003ea***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1775\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;17.9\\u003csup\\u003ea***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1419\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;6.7\\u003csup\\u003eb,c***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e785,1\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;18.9\\u003csup\\u003eb,c,d***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e1014\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;7.8\\u003csup\\u003eb,c,d,e***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003ePFC\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e677,1\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e450\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;26.2\\u003csup\\u003ea***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1667\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;29.6\\u003csup\\u003ea***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1229\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;30.6\\u003csup\\u003eb,c***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e587,4\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;10.1\\u003csup\\u003eb,c,d***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e914\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;7.8\\u003csup\\u003eb,c,d,e***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"7\\\" nameend=\\\"c7\\\" namest=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eAKT-1/Protein(ng/L)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eHippocampus\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1846\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;66.4\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1068\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;23\\u003csup\\u003ea***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e2531\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;31\\u003csup\\u003ea***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2112\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;131.1\\u003csup\\u003eb,c***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1263\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;53\\u003csup\\u003eb,c,d***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e1964\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;58\\u003csup\\u003eb,c,e***,d**\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003ePFC\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1681\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;12\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e829,6\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;60.1\\u003csup\\u003ea***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e2307\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;70\\u003csup\\u003ea***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1850\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;47\\u003csup\\u003eb,c***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1373\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;59\\u003csup\\u003eb,c,d***\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e1749\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;74\\u003csup\\u003eb,c,e***,d*\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eC\\u0026rarr;G, A, a; G\\u0026rarr;GA, GAN2, GAN5, b; A\\u0026rarr;GA, GAN2, GAN5, c; GA\\u0026rarr;GAN2, GAN5, d; GAN2\\u0026rarr;GAN5, e; (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***)\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eExtracellular Regulated Kinase 1/2 / Protein (ng/L)\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn hippocampus and PFC, when compared with group C, a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) in phosphorylated ERK1/2 levels was detected in G and a very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) in A. A very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) was detected in GA, GAN2 and GAN5 compared to G. When group A was compared with GA, GAN2, GAN5, very significant decreases (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) were observed in all groups. When group GA was compared with GAN2, GAN5, a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) was observed in both groups. A very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) was observed in GAN5 compared to GAN2 (Shown in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eH).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eAKT-1/Protein (ng/L)\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn hippocampus and PFC, phosphorylated AKT-1 levels were found to be very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) in G and very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) in A compared to group C. When G was compared with GA, GAN2, GAN5, very significant increases (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) were seen in all groups. When A was compared with group GA, GAN2, GAN5, very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) was seen in all groups. When compared with group GA, GAN2, GAN5, very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) was seen in GAN2 in both hippocampal and cortical tissues, while a significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01) was seen in GAN5 in hippocampus and a slightly significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) in GAN5 in PFC. When compared with group GAN2, a statistically very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) was seen in GAN5 (Shown in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eJ).\\u003c/p\\u003e \\u003cp\\u003e \\u003cul\\u003e \\u003cli\\u003e \\u003cp\\u003e \\u003cb\\u003eExcitotoxicity-induced oxidative imbalance is reversed by Apelin-13 treatment\\u003c/b\\u003e \\u003c/p\\u003e \\u003c/li\\u003e \\u003c/ul\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eAdvanced Glycation End Products\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eHippocampal and serum AGE/Protein \\u0026micro;g/ml levels showed a very significant increase in G compared to C (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***), and a slightly significant increase in PFC (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*). In the hippocampus, there was a very significant decrease in A compared to G (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***). There was a slightly significant increase in GAN2 compared to GA (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*). Serum AGE/Protein levels showed a slightly significant increase in GA compared to A (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*). There was a very significant decrease in GAN5 compared to GAN2 (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***). In the hippocampus, PFC and serum, there was a very significant decrease in GA, GAN5 compared to G (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***), while a significant decrease in GAN2 (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) was observed. A very significant increase in GAN2 compared to A was found in the PFC and serum (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***), and a significant increase in the hippocampus (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eAdvanced Oxidation Protein Products\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eHippocampal AOPP/Protein levels showed a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) in GA compared to G. A slightly significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*) in GA and a very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) in GAN5 compared to GA. A very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was found in GAN5 compared to GA. PFC AOPP/Protein levels showed a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) in GA and GAN5 compared to G, while a slightly significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*) was found in GAN2. Serum AOPP/Protein levels showed a significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) in GA compared to G and a significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) in GAN2. A very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was seen in GAN5 compared to GAN2. A highly significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in G compared to C in hippocampus, PFC and serum. A highly significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in GAN2 compared to A. There was a highly significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) in GAN2 compared to GA in hippocampus and serum (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eDityrosine\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eHippocampal DT/Protein levels showed a slightly significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*) in GAN2 compared to G. GAN2 showed a very significant (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) and GAN5 showed a slightly significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*) compared to A. PFC DT/Protein levels showed a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) in GAN2 compared to G. A slightly significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*) was found in GAN2 compared to A. Serum DT/Protein levels showed a very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) in GAN2 and GAN5 compared to A. GAN5 showed a significantly increased (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) compared to GA. In hippocampus, PFC and serum, G showed a very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) compared to C. A very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in GA and GAN5 compared to G. A very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was detected in GAN2 compared to GA in the hippocampus and serum. A slightly significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*) was found in GAN5 compared to GAN2 in the hippocampus, and a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was found in the serum (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eKaynurenine\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eWhen hippocampal KYN/Protein levels were examined, a significant increase was detected in GAN2, GAN5 compared to A (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**, respectively). A very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in GAN2, GAN5 compared to GA. A slightly significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*) was found in the GAN2 group in the PFC. A very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was found in GAN2 compared to A. There was a very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) in GAN2 compared to GA. Serum KYN/Protein levels were recorded in GA, GAN2, GAN5 compared to A (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*,p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***,p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***). GAN2 showed a very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) compared to GA. There was a significant increase in G compared to C in hippocampus, PFC and serum (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***,p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**,p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***, respectively). GA and GAN5 showed a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) compared to G. GAN5 showed a significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***,p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**,p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) compared to GAN2 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eMalondialdehyde\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eWhen hippocampal MDA/Protein levels were examined, a significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) was found in GA compared to A. In serum MDA/Protein levels, a significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) in GAN2 and a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) in GAN5 were observed compared to G. There was a very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) in G in the hippocampus and serum compared to C. There was a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) in GA, GAN2, GAN5 compared to G. A very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in GAN2, GAN5 compared to A group. GAN2 showed a very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) compared to GA (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eE).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eTotal Thiol\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn T-SH levels, a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was detected in G compared to C in hippocampus, PFC and serum. A very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was detected in GA, GAN5 compared to G. A very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in GA, GAN2, GAN5 compared to A. A slightly significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*) was observed in GAN2 hippocampus and a significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) in PFC compared to GA. A very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in GAN5 in hippocampus and a significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) in PFC compared to GAN2. In serum T-SH/Protein levels, a very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in GAN5 compared to G group. Compared to A, a significant decrease in GA (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) and a very significant decrease in GAN2 (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) were observed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eF).\\u003c/p\\u003e \\u003cp\\u003e \\u003cul\\u003e \\u003cli\\u003e \\u003cp\\u003e \\u003cb\\u003eAnxiety-like behaviors observed due to excitotoxicity were suppressed by Apelin-13, while locomotor performance remained constant\\u003c/b\\u003e \\u003c/p\\u003e \\u003c/li\\u003e \\u003c/ul\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eOpen Field Test\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eWhen the time spent in the center was considered, a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was detected in G compared to group C. A significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in GA, GAN5, and a significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) in GAN2 compared to group G. A very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in GAN2, GAN5 compared to group A, a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) in GAN2 compared to group GA, and a slightly significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*) in GAN5. A very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was found in GAN5 compared to GAN2. Since the time spent on the periphery is inversely proportional to the time spent in the center, significant differences were observed to follow a similar trend. There was no statistically significant difference between the groups when the average total distance (m) and speed (m/sec) were examined. A very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was found in G in the number of defecations compared to group C. No difference was observed in the comparisons of other groups. A very significant statistical decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was detected in G group compared to C group in terms of rearing numbers, and a slightly significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*) was detected in GAN2 group. A slightly significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) was observed in GA and GAN5 compared to G group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB).\\u003c/p\\u003e \\u003cp\\u003e \\u003cul\\u003e \\u003cli\\u003e \\u003cp\\u003e \\u003cb\\u003eApelin-13 provided its cognitive protection on short-term memory via NPY2 and NPY5 receptors\\u003c/b\\u003e \\u003c/p\\u003e \\u003c/li\\u003e \\u003c/ul\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eNORT Familiarization\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eCompared to group C, there was a slightly significant decrease in the Average Total Distance in G (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*) and a very significant decrease in the Average Speed ​​(p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***). There was no statistically significant difference between the groups in the Exploration Number of A1-A2 Objects. When the Average exploration Time of A1-A2 Objects was considered, there was a significant decrease in G compared to C (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*). There was a slightly significant decrease in GAN5 compared to A (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eNORT Test Period\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eDuring the NORT test process, a significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*) was observed in G compared to C in the average speed (m/sec) data. No significant difference was observed when the other groups were compared. When the Average Review Number of A1-B Objects was examined, a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in G compared to C group, and a very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in A. In GA, GAN5 groups, a significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) was observed in the tendency towards B object compared to A1. No significance was found in G and GAN2 groups. A significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**; p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*) was observed in the tendency towards B object in G, GA groups. When the Average Review Time (sec) of A1-B Objects was considered, a significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) was observed in the time spent with B object in C and A, and a slightly significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*) was observed in GA. In the comparison between the groups in the orientation towards the B object, a very significant decrease in G compared to C (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***), a significant increase in GAN5 compared to G (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**), and a significant decrease in GAN2 compared to A (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) were observed. When DI was examined, a significant decrease in G compared to C (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**). A significant increase in GA compared to G (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**), and a slightly significant increase in GAN5 (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*) were observed. When compared to A and GA groups, a significant decrease in GAN2 (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) was observed. A slightly significant increase in GAN5 compared to GAN2 (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*) was found (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cul\\u003e \\u003cli\\u003e \\u003cp\\u003e \\u003cb\\u003eWhile egocentric strategies were dominant in excitotoxic groups, Apelin-13 treatment enhanced allocentric navigation, which supports long-term memory\\u003c/b\\u003e \\u003c/p\\u003e \\u003c/li\\u003e \\u003c/ul\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eMWM-Training\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn the Total Distance Travelled (cm) data, a significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) was observed in all groups on Day 4 compared to Day 1 and a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) on Day 5. Similarly, a significant decrease was observed in the Day 5 data compared to Day 2 (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) and Day 3 (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*). In the total time (sec) data, a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in all groups on Days 3, 4, and 5 compared to Day 1. A significant decrease was found on Days 3, 4, and 5 compared to Day 2. A very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in all groups on Day 5 compared to Day 3. When the average swimming speed (m/sec) data of the rats were compared, no statistically significant difference was observed. This indicates that their locomotor activity was normal. When Escape latency (sec) data were examined, a slightly significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05*) was found on Day 2 compared to Day 1 in all groups, and a very significant difference (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was found on Days 3, 4, and 5. A very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was found on Days 3, 4, and 5 compared to Day 2, a significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was found on Day 4 compared to Day 3, a significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) was found on Day 5 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). Thigmotaxis and random search strategy were seen in all groups on Day 1. Direct search strategy was reported in group C on Day 5, indirect search strategy in groups A and GA, and directed search strategy in group GAN5. Random search was present in groups G and GAN2 on Day 5 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eMWM-Probe Day\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn the data of Total Distance traveled (cm), Escape latency (Sec) and Average Swimming Speed ​​(m/sec), there was a very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) in G compared to C. A very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was seen in GA, GAN2, GAN5 compared to G group. A very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was found in GA, GAN2, GAN5 compared to A. A very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was found in GAN2 compared to GA. A significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) was found in total distance traveled in GAN5 compared to GA. In the data of Total Distance traveled and Escape latency, a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was detected in GAN5 compared to GAN2, and a significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01**) was detected in Average Swimming Speed ​​in GAN5. When the number of entries into the target quadrant and the average time spent in the target quadrant (sec) data were examined, a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in G compared to C. A very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was found in GA, GAN5 compared to G. A very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was recorded in GA, GAN2, GAN5 compared to A. There was a very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) in GAN2 compared to GA. A statistically significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was recorded in GAN5 compared to GAN2 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB). During the 60-second probe test, it was observed that there was an increase in the target quadrant entry marks in the C, A, GA, GAN5 groups, as well as an increase in the search tendency in the target quadrant. Random search continued in the G and GAN2 groups (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD).\\u003c/p\\u003e \\u003cp\\u003e \\u003cul\\u003e \\u003cli\\u003e \\u003cp\\u003e \\u003cb\\u003eNPY2R activity enables Apelin-13 to protect neuronal structure under excitotoxic stress\\u003c/b\\u003e \\u003c/p\\u003e \\u003c/li\\u003e \\u003c/ul\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eHistology scoring and imaging\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn the H\\u0026amp;E scoring of the hippocampal CA1 and CA3 areas, a very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in G compared to C. A very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in GAN2 and GAN5 compared to G. A very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in GAN2 compared to A. A very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in GAN2 compared to GA. A very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was observed in GAN5 compared to GAN2. In the Cresyl Violet staining scoring of PFC 2nd, 3rd, 4th, 5th layers, G group showed a very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) compared to C. A very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was found in GA, GAN5 compared to G. A very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was seen in GAN2, GAN5 compared to A. A very significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was found in GAN2 compared to GA. A very significant decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001***) was found in GAN5 compared to GAN2 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eWhen hippocampal CA1 and CA3 molecular layer (MT), polymorphic layer (PHT) and pyramidal layers (PT) were examined, healthy (s) light-colored neurons were seen in parasagittal sections in groups C and A (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA, \\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB). In group G neurons, pericellular halo (ph) indicating increased edema in PT and neurons with dark, shrunken, reduced cytoplasm and pyknotic nucleus (pn) indicating apoptotic cells were seen (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC). Tissue loss indicating vacuolization (v) was observed in the same area. In the GA group, a decrease was observed in dark-colored pn and ph neurons (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD). In the GAN2 group, ph, pn and vacuolization were observed with similar density to the G group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE). In the GAN5 group, healthy neurons with similar density to the GA group and low density of ph and pn compared to GAN2 were observed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eF).\\u003c/p\\u003e \\u003cp\\u003eAmong the PFC molecular (I), external granular (II), external pyramidal (III), internal granular (IV), internal pyramidal (V) and polymorphic (VI) layers, layers II, III, IV and V were examined. In parasagittal sections of groups C and A, homogeneously distributed, normocytic neurons with intact vesicular nuclei were seen (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA, \\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB). In group G, pn, ph, v were observed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eC). In group GA, diffusely distributed small amounts of pn, ph and moderately healthy neurons were seen (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eD). In group GAN2, diffuse pn, ph, v, ac and sparse healthy neurons were observed in layers II, III and IV (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eE). In group GAN5, small amounts of pn, ph, ac and multiple healthy neurons were observed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eF).\\u003c/p\\u003e\"},{\"header\":\"4. DISCUSSION\",\"content\":\"\\u003cp\\u003eIn our study, an excitotoxicity model was established in male rats using D-glutamic acid, and the effectiveness of exogenous Apelin-13 on neuroprotection and learning-memory was investigated in relation to its potential mediation through NPY2R and NPY5R pathways.\\u003c/p\\u003e \\u003cp\\u003eCaspase-3 is an active proteolytic enzyme that plays a role in the executive pathway of the apoptotic process [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e]. Caspase-3 activation and DNA fragmentation occur in excitotoxicity. Glutamate excitotoxicity was induced in hippocampal neurons and active caspase-3 levels increased [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e]. In our study, the highly significant increase (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) in hippocampal and PFC tissue active caspase-3 levels in group G compared to group C was supported by D-glutamic acid-mediated excitotoxicity. In excitotoxicity and subarachnoid hemorrhage in rats, apoptosis was suppressed by apelin-13 treatment as compared to injury groups due to decreased active caspase-3 levels [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e]. In Alzheimer's (AD), excitotoxicity and ischemic stroke models, NPY2R activation, and to a lesser extent NPY5R activation, was observed to reduce active caspase-3 levels in CA3, DG, and subgranular regions, suppress glutamate release, and increase AKT phosphorylation. Neurodegeneration was limited and cellular integrity was preserved [\\u003cspan additionalcitationids=\\\"CR43\\\" citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e]. In line with literature data, our study emphasizes that when NPY2R antagonists are used, the neuroprotective effects of Apelin-13 are largely insufficient and that while Apelin-13 exhibits its biological properties, NPY2R is primarily involved and NPY5R is limited. In patients with AD associated with excitotoxicity, the PI3K/AKT signaling pathway is suppressed as a result of the destruction of AKT by increased caspase-3 activation [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e]. In our study, excitotoxicity increased caspase-3 activation and suppressed phosphorylated ERK1/2 and AKT-1 signaling as a result of ROS increase and mitochondrial dysfunction in the G group. Opposite changes were observed in the GA group due to the neuroprotective ability of Apelin-13.\\u003c/p\\u003e \\u003cp\\u003eERK1-ERK2, known as the pro-survival downstream signaling molecule, is a protein-serine/threonine kinase that participates in the Ras-Raf-MEK-ERK signaling cascade and regulates processes such as cell adhesion, migration, survival, and transcription [\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e]. Excessive NMDAR activation in excitotoxicity has been shown to directly inhibit ERK1/2 phosphorylation [\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e]. In neuronal death due to excitotoxic damage, ERK1/2 and AKT phosphorylation has been shown to be suppressed, and exogenous Apelin-13 treatment has been shown to increase IP3, PKC, MEK1/2, ERK1/2, and AKT phosphorylation, and to reduce excitotoxic damage by inhibiting NMDAR and calpain [\\u003cspan additionalcitationids=\\\"CR49\\\" citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e]. In our study, D-Glutamic acid inhibited the ERK1/2 signaling pathway and reduced neuronal survival, while Apelin-13 played a neuroprotective role by increasing NMDAR inhibition and ERK1/2 activation. Neuronal apelinergic signaling causes intracellular Ca\\u003csup\\u003e2+\\u003c/sup\\u003e release and ERK1/2 activation via endoplasmic reticulum (ER)-IP3 receptors [\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e]. It was indicated that the basal ERK1/2 phosphorylation in our group C may be due to endogenous apelinergic signaling and that the increase in phosphorylation in group A is due to exogenous Apelin-13, which enhances the endogenous apelin signal. In addition to ERK1/2 pathway inhibition in glutamate excitotoxicity, augmentation of pro-apoptotic processes and decrease in AKT phosphorylation levels in ER stress are also observed. NPY suppressed the apoptotic process as a treatment agent in this process by increasing the phosphorylation of ERK1/2 and AKT signaling pathways mediated by NPY2R [\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e]. In our study, Apelin-13 in the excitotoxicity model; It inhibited active caspase-3 by increasing ERK1/2 and AKT-1 activation, suppressed apoptosis and exhibited this primarily with NPY2R and to a limited extent with NPY5R. Apelin-13 cannot prevent excitotoxic damage on its own in the presence of NPY2R and NPY5R antagonists.\\u003c/p\\u003e \\u003cp\\u003eAKT, a serine and threonine kinase, is involved in the proliferation, differentiation and survival mechanism. AKT dysfunction is associated with pathology in neurodegenerative diseases, cancer, cardiovascular diseases, inflammatory and autoimmune disorders [\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e]. In excitotoxicity, decrease in phosphorylated-AKT and increase in neuronal death have been reported in hippocampal CA1-CA3 areas [\\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e]. In addition to the suppression of ERK1/2 and AKT phosphorylation due to the increase in active caspase-3 mentioned above, excessive ROS production and Cytochrome c release also occur. Apelin-13 blocks apoptosis and excitotoxic death via APJ/Gi-Gq [\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e]. In our study, Apelin-13 increased AKT-1 signaling, while AKT-1 inhibited apoptosis by preserving mitochondrial integrity and played a protective role in excitotoxic damage.\\u003c/p\\u003e \\u003cp\\u003eNPY selectively inhibits glutamatergic transmission in pyramidal cells by inhibiting voltage-gated Ca\\u003csup\\u003e2+\\u003c/sup\\u003e channel (VGCC) through NPY2Rs expressed in high concentrations in the presynaptic terminals of mossy fibers in CA3 and Schaffer collaterals in CA1, and plays a neuroprotective role against excitotoxicity [\\u003cspan additionalcitationids=\\\"CR57 CR58 CR59\\\" citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e60\\u003c/span\\u003e]. In the Parkinson Disease (PD) model, NPY2R agonists have been shown to have protective effects against excitotoxicity by increasing the activation of both ERK1/2 and PI3K/AKT pathways in glutamatergic cortical afferent fibers, and high NPY2R gene expression slows down the progression of Huntington disease and plays a protective role against cell death [\\u003cspan additionalcitationids=\\\"CR62\\\" citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e61\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR63\\\" class=\\\"CitationRef\\\"\\u003e63\\u003c/span\\u003e]. In a rat excitotoxicity model, it was reported that NPY2R agonist provided protection against neuronal cell death in DG, CA1 and CA3 pyramidal cell layers, while NPY5R agonist prevented cell damage in DG and CA3 but had no neuroprotective role in CA1 pyramidal cells [\\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e64\\u003c/span\\u003e]. Another study reported that these protective results were lost by using selective NPY2R antagonist (BIIE0246) in DG, CA1 and CA3 pyramidal cells [\\u003cspan additionalcitationids=\\\"CR66\\\" citationid=\\\"CR65\\\" class=\\\"CitationRef\\\"\\u003e65\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR67\\\" class=\\\"CitationRef\\\"\\u003e67\\u003c/span\\u003e]. In neurodegenerative pathologies where presynaptic intracellular Ca\\u003csup\\u003e2+\\u003c/sup\\u003e levels are increased, NPY2R activation suppresses glutamate release by VGCC blockade and reduces excitotoxic damage by modulating ERK1/2, PI3K/AKT survival signaling pathways. In our study, the contribution of NPY2R to the protective effects of Apelin-13 became the focus compared to NPY5R. NPY2R's autoinhibitory function by working through the negative feedback mechanism in the presynaptic terminal leads to its functional dominance.\\u003c/p\\u003e \\u003cp\\u003eThe open field test is a behavioral test that provides data on the locomotor activities and general emotional state of the animal [\\u003cspan citationid=\\\"CR68\\\" class=\\\"CitationRef\\\"\\u003e68\\u003c/span\\u003e]. In the chronic social defeat stress test, Apelin-13 treatment in rats did not affect the total movement distance in OFT findings and did not cause a stress-drug interaction [\\u003cspan citationid=\\\"CR69\\\" class=\\\"CitationRef\\\"\\u003e69\\u003c/span\\u003e]. In the depression model, NPY2R expression levels in the PFC of rats decreased and depressive behaviors were observed [\\u003cspan citationid=\\\"CR70\\\" class=\\\"CitationRef\\\"\\u003e70\\u003c/span\\u003e]. In our study, the fact that the average total distance (sec) and average speed (m/sec) were equivalent to the C group in all groups supported the absence of a locomotor problem. The increase in thigmotaxis and defecation, suppression of exploratory behavior, motivational decrease and reluctance to environmental stimuli in the G and GAN2 groups reflect the emotional disorder caused by glutamate excitotoxicity. The absence of anxious behaviors in the C and A groups shows that apelin-13 does not cause behavioral changes in healthy individuals, but has the potential to improve behavioral disorders due to injury. NPY2R antagonism worsened the behavioral profile, indicating that NPY2R plays a key role in the treatment with apelin-13.\\u003c/p\\u003e \\u003cp\\u003eMWM assesses spatial memory, learning and LTM. Spatial reference memory, which is based on the re-exploration of stable environmental conditions, enables learning and remembering the location of objects, while developing different search strategies by synchronizing egocentric and allocentric navigation [\\u003cspan citationid=\\\"CR71\\\" class=\\\"CitationRef\\\"\\u003e71\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR72\\\" class=\\\"CitationRef\\\"\\u003e72\\u003c/span\\u003e]. Spatial reference memory transmits the animal's location from the hippocampus to the PFC through anatomical connections, enabling optimization and selection of the appropriate route to the target [\\u003cspan citationid=\\\"CR72\\\" class=\\\"CitationRef\\\"\\u003e72\\u003c/span\\u003e]. Damage to regions or connections in the hippocampus-PFC significantly impairs memory-learning by impairing spatial memory and mapping [\\u003cspan additionalcitationids=\\\"CR74\\\" citationid=\\\"CR73\\\" class=\\\"CitationRef\\\"\\u003e73\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR75\\\" class=\\\"CitationRef\\\"\\u003e75\\u003c/span\\u003e]. Total distance traveled data, which is an indicator of spatial memory and learning in MWM [\\u003cspan citationid=\\\"CR76\\\" class=\\\"CitationRef\\\"\\u003e76\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR77\\\" class=\\\"CitationRef\\\"\\u003e77\\u003c/span\\u003e], showed that complex routes were preferred in the G and GAN2 groups, which had D-glutamic acid-induced excitotoxic damage during the training period, and spatial memory performance was impaired. The therapeutic effects of Apelin-13 on learning and memory were limited with the use of NPY2RA. The successful performance in the GAN5 group, where the effect of NPY5RA was observed, compared to GAN2, further reveals the importance of NPY2R in the treatment with apelin-13. The short-direct route choices of the A and GA groups emphasize that Apelin-13 protects spatial reference memory performance in excitotoxic damage.\\u003c/p\\u003e \\u003cp\\u003eHippocampal lesions in striatal regions support the formation of compensatory egocentric search strategies [\\u003cspan citationid=\\\"CR78\\\" class=\\\"CitationRef\\\"\\u003e78\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR79\\\" class=\\\"CitationRef\\\"\\u003e79\\u003c/span\\u003e]. The fact that the G, GAN2 group largely preferred non-spatial egocentric search patterns such as thigmotaxis, scanning and randomized, which do not involve hippocampal-dependent learning, has proven the impairment of allocentric memory mapping. In glutamate excitotoxicity, the conversion process from STM to LTM was impaired, and allocentric memory was disabled, leading to an orientation towards adaptive egocentric memory. The selection of goal-directed direct, focal and indirect search patterns in the C, A, GA and GAN5 groups, and the successful maintenance of hippocampal learning, allocentric spatial mapping and LTM, confirmed the protective function of Apelin-13 mediated by NPY2R/NPY5R.\\u003c/p\\u003e \\u003cp\\u003eThe impairment of hippocampal integrity and spatial memory performance has been reported to have negative effects on total distance and escape latency in MWM training and probe performance [\\u003cspan citationid=\\\"CR80\\\" class=\\\"CitationRef\\\"\\u003e80\\u003c/span\\u003e]. Histological analyses also showed that significant neuronal damage in CA1, CA3 and PFC disrupted the formation of spatial mapping on a cell-based basis in G and GAN2 groups, while the preservation of cell-tissue integrity in GAN5 and GA groups reinforced the importance of apelin-13 treatment and primarily NPY2R and, to a lesser extent, NPY5R in this treatment. In neurotoxicity models, while an increase in escape latency and total distance travelled, a decrease in the time spent in the target quadrant and the number of entries were observed in the damage group in MWM training and probe sessions, it was reported that Apelin-13 improved these [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR81\\\" class=\\\"CitationRef\\\"\\u003e81\\u003c/span\\u003e]. Parallel results were obtained in the total time, swimming speed and escape latency during the training period. During the probe period, the time spent in the target quadrant and the number of entries decreased, and the total distance travelled and escape latency increased in the G, GAN2 group due to neurotoxic damage.\\u003c/p\\u003e \\u003cp\\u003eNORT is used to assess recognition memory and STM and is associated with the PFC and hippocampus [\\u003cspan citationid=\\\"CR82\\\" class=\\\"CitationRef\\\"\\u003e82\\u003c/span\\u003e]. NORT results are affected by hippocampal and cortical lesions [\\u003cspan citationid=\\\"CR83\\\" class=\\\"CitationRef\\\"\\u003e83\\u003c/span\\u003e]. Excitotoxicity leads to neuronal structural changes and functional abnormalities in the hippocampus and cortex [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. It has been reported that the impairments in memory performance observed in NORT in the depression model were improved by Apelin-13 treatment, that the recognition memory performance of non-stressed rats was not affected, and that the protective effects of PI3K/AKT and ERK1/2 blockers mediated by Apelin-13 were suppressed [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. While a significant decrease was observed in the DI of rats exposed to stress, it was reported that DI increased with Apelin-13 treatment, memory impairment improved, but there was no difference in total object exploration times [\\u003cspan citationid=\\\"CR82\\\" class=\\\"CitationRef\\\"\\u003e82\\u003c/span\\u003e]. In the PD model, the damage group could not distinguish between new and old objects, while Apelin-13 administration increased DI [\\u003cspan citationid=\\\"CR81\\\" class=\\\"CitationRef\\\"\\u003e81\\u003c/span\\u003e]. It increases the recall of object recognition memory with exogenous NPY receptor activation [\\u003cspan citationid=\\\"CR84\\\" class=\\\"CitationRef\\\"\\u003e84\\u003c/span\\u003e]. Apelin-13 and D-Glutamic acid application did not disrupt locomotor activity when the average speed was at the same levels in familiarization, but the increased exploration motivation and environmental curiosity with apelin-13 application decreased in the G group, supporting the suppression of acquiring and processing new information.\\u003c/p\\u003e \\u003cp\\u003eWhile the C, A, GA groups exploring the new object during the test period showed healthy STM function, the allocation of equal time to new and old objects in the G, GAN2 and GAN5 groups showed a decrease in STM performance, and impaired consolidation and memory separation. The negative DI seen in the G and GAN2 groups indicates that significant memory damage occurs in an excitotoxic environment, while GAN5 group shows a positive DI close to GA, supporting the protective role of Apelin-13 in cognitive function through NPY2R activation.\\u003c/p\\u003e \\u003cp\\u003eOxidative stress resulting from excessive ROS production contributes to neurodegenerative diseases through oxidative damage. Excessive ROS induce DNA methylation and trigger neurotoxicity by increasing oxidants such as AGE, AOPP, KYN, DT, MDA, and decreasing the levels of antioxidant molecules such as T-SH [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR85\\\" class=\\\"CitationRef\\\"\\u003e85\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR86\\\" class=\\\"CitationRef\\\"\\u003e86\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eAGEs promote the accumulation of protease-resistant cross-linked proteins as a result of non-enzymatic reactions between reducing sugars and proteins, lipids, and nucleic acids. Progressive AGE accumulation is also seen in hepatocytes and connective tissue in neurodegenerative pathologies [\\u003cspan additionalcitationids=\\\"CR88 CR89 CR90 CR91 CR92 CR93\\\" citationid=\\\"CR87\\\" class=\\\"CitationRef\\\"\\u003e87\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR94\\\" class=\\\"CitationRef\\\"\\u003e94\\u003c/span\\u003e]. In PD, AGE accumulation occurs in the substantia nigra and induces apoptosis by binding to its receptor RAGE and activating ERK1/2 [\\u003cspan citationid=\\\"CR95\\\" class=\\\"CitationRef\\\"\\u003e95\\u003c/span\\u003e]. AGE-RAGE activation also induces Ca\\u003csup\\u003e2+\\u003c/sup\\u003e signaling in the astrocyte membrane, and contributes to neurodegeneration by increasing vesicular glutamate transporter (VGLUT)-mediated glutamate release by astrocytes [\\u003cspan additionalcitationids=\\\"CR97\\\" citationid=\\\"CR96\\\" class=\\\"CitationRef\\\"\\u003e96\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR98\\\" class=\\\"CitationRef\\\"\\u003e98\\u003c/span\\u003e]. Cell survival decreases due to the inhibition of ERK1/2 signaling due to increased ROS in cytotoxicity [\\u003cspan citationid=\\\"CR99\\\" class=\\\"CitationRef\\\"\\u003e99\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eAOPP, which is structurally similar to AGE, is a sensitive marker indicating oxidative stress damage in proteins associated with neurodegenerative diseases [\\u003cspan citationid=\\\"CR100\\\" class=\\\"CitationRef\\\"\\u003e100\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR101\\\" class=\\\"CitationRef\\\"\\u003e101\\u003c/span\\u003e]. AOPPs induce ROS formation and oxidant/antioxidant ratio imbalance [\\u003cspan citationid=\\\"CR102\\\" class=\\\"CitationRef\\\"\\u003e102\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eSome ROS products react with tyrosine residues to produce tyrosine radicals that form intra/intermolecular cross-links in proteins, thus increasing DT formation [\\u003cspan citationid=\\\"CR103\\\" class=\\\"CitationRef\\\"\\u003e103\\u003c/span\\u003e]. High DT levels have been shown in hippocampal-cortical tissues and cerebrospinal fluid (CSF) amyloid plaque proteins in AD and in the striatal area in PD [\\u003cspan citationid=\\\"CR104\\\" class=\\\"CitationRef\\\"\\u003e104\\u003c/span\\u003e]. In excitotoxicity, nitric oxide (NO) and superoxide (O2\\u003csup\\u003e-\\u003c/sup\\u003e) interact with exaggerated NMDAR activation to release toxic peroxynitrite (ONOO\\u003csup\\u003e-\\u003c/sup\\u003e) [\\u003cspan citationid=\\\"CR105\\\" class=\\\"CitationRef\\\"\\u003e105\\u003c/span\\u003e]. ONOO\\u003csup\\u003e-\\u003c/sup\\u003e increases DT accumulation-mediated oxidative damage by causing protein oxidation [\\u003cspan citationid=\\\"CR106\\\" class=\\\"CitationRef\\\"\\u003e106\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eThe kynurenine pathway is linked to neurodegenerative pathogenesis, as KYN metabolites play a role in excitotoxic transmission, oxidative stress, neurotransmitter uptake, and amyloid aggregation. KYN is the primary catabolic pathway of tryptophan and affects glutamatergic activity via iGluR, mGluR or VGLUTs. It has been reported that high doses of KYN contribute to neuronal damage by producing reactive free radicals via the glutamatergic pathway [\\u003cspan citationid=\\\"CR107\\\" class=\\\"CitationRef\\\"\\u003e107\\u003c/span\\u003e]. It has been reported that KYN metabolites accumulate in serum and CSF in AD in parallel with excitotoxicity [\\u003cspan citationid=\\\"CR108\\\" class=\\\"CitationRef\\\"\\u003e108\\u003c/span\\u003e]. KYN can activate NMDARs by generating sudden action potentials in CA1 pyramidal neurons [\\u003cspan citationid=\\\"CR109\\\" class=\\\"CitationRef\\\"\\u003e109\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eMDA, which is formed as a result of free radical and lipid reactions, causes DNA damage by altering cell membrane structure [\\u003cspan citationid=\\\"CR110\\\" class=\\\"CitationRef\\\"\\u003e110\\u003c/span\\u003e]. Increased lipid peroxidation, membrane permeability and ionic balance are disrupted, paving the way for neurodegenerative mechanisms [\\u003cspan citationid=\\\"CR111\\\" class=\\\"CitationRef\\\"\\u003e111\\u003c/span\\u003e]. Antioxidants and agents that reduce ROS production have been reported to reduce MDA levels and oxidative stress [\\u003cspan citationid=\\\"CR112\\\" class=\\\"CitationRef\\\"\\u003e112\\u003c/span\\u003e]. It has been reported that caspase activation accompanied by an increase in ROS triggers the apoptotic process, suppresses ERK1/2 and AKT-1 signaling pathway activation, and neuronal death occurs [\\u003cspan citationid=\\\"CR113\\\" class=\\\"CitationRef\\\"\\u003e113\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eT-SH shows the content of sulfhydryl bonds with antioxidative properties. The sulfhydryl group (-SH) is found especially in cysteine ​​amino acid [\\u003cspan citationid=\\\"CR114\\\" class=\\\"CitationRef\\\"\\u003e114\\u003c/span\\u003e]. Oxidative stress causes the formation of disulfide bonds (R-S-S-R) in -SHs in the thiol group (R-SH), oxidation of proteins and damaged folding [\\u003cspan citationid=\\\"CR115\\\" class=\\\"CitationRef\\\"\\u003e115\\u003c/span\\u003e]. Oxidative stress may increase with disulfidation of cysteines in the Cystine/glutamate antiporter (xCT) structure in the astrocytic membrane, causing glutamate accumulation in the extrasynaptic area [\\u003cspan citationid=\\\"CR116\\\" class=\\\"CitationRef\\\"\\u003e116\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR117\\\" class=\\\"CitationRef\\\"\\u003e117\\u003c/span\\u003e]. It has been suggested that cortical glutathione, T-SH, catalase activity decreases and MDA levels increase in excitotoxicity and oxidative stress is induced [\\u003cspan citationid=\\\"CR118\\\" class=\\\"CitationRef\\\"\\u003e118\\u003c/span\\u003e]. In nephrotoxicity, Apelin-13 reduced oxidative stress and toxicity by increasing tissue T-SH levels [\\u003cspan citationid=\\\"CR119\\\" class=\\\"CitationRef\\\"\\u003e119\\u003c/span\\u003e]. The decrease in T-SH levels in the G and GAN2 groups suggests that the sulfhydryl groups due to increased oxidant parameters in these groups are transformed into disulfide bridges. In our study, the increase in oxidant AGE, AOPP, DT, KYN, MDA levels in the tissue and serum of the G group and the inhibition of ERK1/2, AKT, the disruption of the antioxidant/oxidant ratio and the increase in tissue active caspase-3 levels create a predisposing environment for neuronal apoptosis in excitotoxicity. Apelin-13 decreases oxidant parameters in the A and GA groups compared to the G group, while increasing T-SH, indicating that it provides an antioxidative effect. The antagonists used in the GAN2-GAN5 groups reveal that Apelin-13 provides its neuroprotective effects together with NPY2-NPY5 receptors.\\u003c/p\\u003e \\u003cp\\u003eIn neurodegeneration, while ROS production is neutralized, the antioxidant system is compromised and sufficient clearance cannot be provided. The neurodegeneration process accelerates with the development of mitochondrial dysfunction. As a result, it has been reported that cognitive functions such as attention, decision-making, learning and memory are also impaired [\\u003cspan citationid=\\\"CR120\\\" class=\\\"CitationRef\\\"\\u003e120\\u003c/span\\u003e]. In neurodegenerative models, excessive production or supplementation of antioxidant enzymes increases the consolidation and recall capacity of spatial learning-memory. At the same time, there is a decrease in memory function in young animals exposed to oxidative stress [\\u003cspan citationid=\\\"CR121\\\" class=\\\"CitationRef\\\"\\u003e121\\u003c/span\\u003e]. It has been reported that resveratrol suppresses mitochondrial damage and apoptosis by reducing free radical formation in mice, increases neuronal superoxide dismutase concentration and reduces MDA levels, and improves learning and memory in MWM test results [\\u003cspan citationid=\\\"CR122\\\" class=\\\"CitationRef\\\"\\u003e122\\u003c/span\\u003e]. The relationship between increased MDA, AOPP, KYN and oxidative stress levels and neurocognitive, learning, memory and cognitive disorders has been reported with decreases in learning tests [\\u003cspan citationid=\\\"CR108\\\" class=\\\"CitationRef\\\"\\u003e108\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR123\\\" class=\\\"CitationRef\\\"\\u003e123\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR125\\\" class=\\\"CitationRef\\\"\\u003e125\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eIn neurodegeneration, darkly stained pyknotic nuclei (pn) indicating apoptotic nuclei and neurons with disrupted membrane integrity can be observed. Pericellular halo (ph) indicating severe neuronal edema in the extracellular area, and white, round, foamy vacuolization indicating toxicity and oxidative stress in the intracellular area are observed [\\u003cspan citationid=\\\"CR126\\\" class=\\\"CitationRef\\\"\\u003e126\\u003c/span\\u003e]. In G and GAN2, in cells with preserved membrane integrity, the presence of pn and ph as well as caspase-3 increase showed that the apoptotic process was operating [\\u003cspan citationid=\\\"CR127\\\" class=\\\"CitationRef\\\"\\u003e127\\u003c/span\\u003e]. In the PD model, neuronal damage accompanying early LTP decrease in the CA1 area was seen in histological analyses, and it was reported that apelin-13 improved this [\\u003cspan citationid=\\\"CR128\\\" class=\\\"CitationRef\\\"\\u003e128\\u003c/span\\u003e]. In line with our findings and literature studies, the increase in antioxidant T-SH levels in the GA group showed that Apelin-13 supported the activation of the antioxidant defense system, provided protection against oxidative stress and mitochondrial dysfunction, and reduced ROS formation. The fact that apelin-13 works synergistically with NPY2R and NPY5R is also supported by biochemical parameters. MWM, NORT and OFT results also coincide with the changes in oxidative stress parameters.\\u003c/p\\u003e\"},{\"header\":\"5. CONCLUSION\",\"content\":\"\\u003cp\\u003eOur results indicate that Apelin-13 is an effective option in preventing/suppressing excitotoxic damage underlying neurodegenerative pathophysiological mechanisms. Apelin-13 performs this role by binding to its own receptors. The fact that NPY receptors in the hippocampal area are colocalized with APJs and use similar intracellular pathways with Apelin-13 proves that it has cofactor effects in preventing/suppressing excitotoxic damage. The relationships of Apelin-13 with other NPY receptors need to be investigated and revealed in more detail at the in-vitro and in-vivo molecular level.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eACKNOWLEDGEMENTS\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll videos recorded for Open Field Test, Novel Object Recognition test and Morris Water Maze test were analyzed using ANY-Maze Version 7.42 (AnyMaze, Stoelting Co., Illinois, USA) software. \\u0026nbsp;We would like to thank Istanbul University-Cerrahpaşa Scientific Research Projects Coordination Unit for their financial support.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding Declaration\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eOur study was supported by Istanbul University-Cerrahpaşa Scientific Research Projects Coordination Unit (Project Number: TDK-2023-37312).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData Availability\\u003c/strong\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eData will be available upon request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor Contribution\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConceptualization:\\u0026nbsp;\\u003c/strong\\u003eKadriye Yagmur Oruc\\u003cstrong\\u003e,\\u0026nbsp;\\u003c/strong\\u003eHakki Oktay Seymen\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMethodology:\\u0026nbsp;\\u003c/strong\\u003eKadriye Yagmur Oruc\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFormal analysis and investigation:\\u0026nbsp;\\u003c/strong\\u003eKadriye Yagmur Oruc, Aykut Oruc, Ruhat Arslan, Furkan Pasa Diriarin, Murat Mengi, Gamze Tanriverdi, Karolin Yanar, Mediha Ozeren Eser, Gokhan Agturk, Ali Ihsan Sonkurt, Berkay Guler\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eWriting - original draft preparation:\\u0026nbsp;\\u003c/strong\\u003eKadriye Yagmur Oruc\\u003cstrong\\u003e,\\u0026nbsp;\\u003c/strong\\u003eHakki Oktay Seymen\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eWriting - review and editing:\\u0026nbsp;\\u003c/strong\\u003eKadriye Yagmur Oruc\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSupervision:\\u0026nbsp;\\u003c/strong\\u003eHakki Oktay Seymen\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eMartami F, Holton KF (2023) Targeting Glutamate Neurotoxicity through Dietary Manipulation: Potential Treatment for Migraine. Nutrients Sep 12;15 (18):3952. https://doi.org/10.3390/nu15183952.\\u003c/li\\u003e\\n\\u003cli\\u003eArmada-Moreira A, Gomes JI, Pina CC, et al. (2020) Going the Extra (Synaptic) Mile: Excitotoxicity as the Road Toward Neurodegenerative Diseases. Front Cell Neurosci 14:90. https://doi.org/10.3389/fncel.2020.00090.\\u003c/li\\u003e\\n\\u003cli\\u003eHerman MA, Jahr CE (2007) Extracellular glutamate concentration in hippocampal slice. J Neurosci 27(36):9736-41. https://doi.org/10.1523/JNEUROSCI.3009-07.2007.\\u003c/li\\u003e\\n\\u003cli\\u003eMark LP, Prost RW, Ulmer JL, Smith MM, Daniels DL, Strottmann JM, Brown WD, Hacein-Bey L (2001) Pictorial review of glutamate excitotoxicity: fundamental concepts for neuroimaging. AJNR Am J Neuroradiol 22(10):1813-24. \\u003c/li\\u003e\\n\\u003cli\\u003eClements JD, Lester RA, Tong G, Jahr CE, Westbrook GL (1992) The time course of glutamate in the synaptic cleft. Science 258(5087):1498-501. https://doi.org/10.1126/science.1359647. \\u003c/li\\u003e\\n\\u003cli\\u003eDong XX, Wang Y, Qin ZH (2009) Molecular mechanisms of excitotoxicity and their relevance to pathogenesis of neurodegenerative diseases. Acta Pharmacol Sin 30(4):379-87. https://doi.org/10.1038/aps.2009.24. \\u003c/li\\u003e\\n\\u003cli\\u003eFontana IC, Souza DG, Souza DO, Gee A, Zimmer ER, Bongarzone S (2023) A Medicinal Chemistry Perspective on Excitatory Amino Acid Transporter 2 Dysfunction in Neurodegenerative Diseases. J Med Chem 66(4):2330-2346. https://doi.org/10.1021/acs.jmedchem.2c01572. \\u003c/li\\u003e\\n\\u003cli\\u003eO\\u0026apos;Dowd BF, Heiber M, Chan A, Heng HH, Tsui LC, Kennedy JL, Shi X, Petronis A, George SR, Nguyen T (1993) A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11. Gene. 136(1-2):355-60. https://doi.org/10.1016/0378-1119(93)90495-o. \\u003c/li\\u003e\\n\\u003cli\\u003eTatemoto K, Hosoya M, Habata Y, Fujii R, Kakegawa T, Zou MX, Kawamata Y, Fukusumi S, Hinuma S, Kitada C, Kurokawa T, Onda H, Fujino M (1998) Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor. Biochem Biophys Res Commun 251(2):471-6. https://doi.org/10.1006/bbrc.1998.9489. \\u003c/li\\u003e\\n\\u003cli\\u003eIvanov MN, Stoyanov DS, Pavlov SP, Tonchev AB (2022) Distribution, Function, and Expression of the Apelinergic System in the Healthy and Diseased Mammalian Brain. Genes (Basel) 13(11):2172. https://doi.org/10.3390/genes13112172. \\u003c/li\\u003e\\n\\u003cli\\u003eCirillo P, Ziviello F, Pellegrino G, Conte S, Cimmino G, Giaquinto A, Pacifico F, Leonardi A, Golino P, Trimarco B (2015) The adipokine apelin-13 induces expression of prothrombotic tissue factor. Thromb Haemost Feb;113(2):363-72. https://doi.org/10.1160/TH14-05-0451. \\u003c/li\\u003e\\n\\u003cli\\u003eLi A, Zhao Q, Chen L, Li Z (2023) Apelin/APJ system: an emerging therapeutic target for neurological diseases. Mol Biol Rep 50(2):1639-1653. https://doi.org/10.1007/s11033-022-08075-9. \\u003c/li\\u003e\\n\\u003cli\\u003eWan T, Fu M, Jiang Y, Jiang W, Li P, Zhou S (2022) Research Progress on Mechanism of Neuroprotective Roles of Apelin-13 in Prevention and Treatment of Alzheimer\\u0026apos;s Disease. Neurochem Res 47(2):205-217. https://doi.org/10.1007/s11064-021-03448-1. \\u003c/li\\u003e\\n\\u003cli\\u003eChen P, Wang Y, Chen L, Song N, Xie J (2020) Apelin-13 Protects Dopaminergic Neurons against Rotenone-Induced Neurotoxicity through the AMPK/mTOR/ULK-1 Mediated Autophagy Activation. Int J Mol Sci 21(21):8376. https://doi.org/10.3390/ijms21218376. \\u003c/li\\u003e\\n\\u003cli\\u003eLv SY, Chen WD, Wang YD (2020) The Apelin/APJ System in Psychosis and Neuropathy. Front Pharmacol 11:320. https://doi.org/10.3389/fphar.2020.00320. \\u003c/li\\u003e\\n\\u003cli\\u003eLi E, Deng H, Wang B, Fu W, You Y, Tian S (2016) Apelin-13 exerts antidepressant-like and recognition memory improving activities in stressed rats. Eur Neuropsychopharmacol 26(3):420-30. https://doi.org/10.1016/j.euroneuro.2016.01.007. \\u003c/li\\u003e\\n\\u003cli\\u003eLi C, Wu X, Liu S, Zhao Y, Zhu J, Liu K (2019) Roles of Neuropeptide Y in Neurodegenerative and Neuroimmune Diseases. Front Neurosci 13:869. https://doi.org/10.3389/fnins.2019.00869. \\u003c/li\\u003e\\n\\u003cli\\u003eTanaka M, Yamada S, Watanabe Y (2021) The Role of Neuropeptide Y in the Nucleus Accumbens. Int J Mol Sci 22(14):7287. https://doi.org/10.3390/ijms22147287. \\u003c/li\\u003e\\n\\u003cli\\u003eM\\u0026eacute;ndez-Couz M, Manahan-Vaughan D, Silva AP, Gonz\\u0026aacute;lez-Pardo H, Arias JL, Conejo NM (2021) Metaplastic contribution of neuropeptide Y receptors to spatial memory acquisition. Behav Brain Res 396:112864. https://doi.org/10.1016/j.bbr.2020.112864. \\u003c/li\\u003e\\n\\u003cli\\u003eCurdt N, Schmitt FW, Bouter C, Iseni T, Weile HC, Altunok B, Beindorff N, Bayer TA, Cooke MB, Bouter Y (2022) Search strategy analysis of Tg4-42 Alzheimer Mice in the Morris Water Maze reveals early spatial navigation deficits. Sci Rep 12(1):5451. https://doi.org/10.1038/s41598-022-09270-1. \\u003c/li\\u003e\\n\\u003cli\\u003eKeimasi M, Salehifard K, Keimasi M, Amirsadri M, Esfahani NMJ, Moradmand M, Esmaeili F, Mofid MR (2023) Alleviation of cognitive deficits in a rat model of glutamate-induced excitotoxicity, using an N-type voltage-gated calcium channel ligand, extracted from Agelena labyrinthica crude venom. Front Mol Neurosci 16:1123343. https://doi.org/10.3389/fnmol.2023.1123343. \\u003c/li\\u003e\\n\\u003cli\\u003eBehl T, Madaan P, Sehgal A, Singh S, Makeen HA, Albratty M, Alhazmi HA, Meraya AM, Bungau S (2022) Demystifying the Neuroprotective Role of Neuropeptides in Parkinson\\u0026apos;s Disease: A Newfangled and Eloquent Therapeutic Perspective. Int J Mol Sci 23(9):4565. https://doi.org/10.3390/ijms23094565. \\u003c/li\\u003e\\n\\u003cli\\u003eZheng Y, Zhang L, Xie J, Shi L (2021) The Emerging Role of Neuropeptides in Parkinson\\u0026apos;s Disease. Front Aging Neurosci 13:646726. https://doi.org/10.3389/fnagi.2021.646726. \\u003c/li\\u003e\\n\\u003cli\\u003eOruc A, Oruc KY, Yanar K, Mengi M, Caglar A, Kurt BO, Altan M, Sonmez OF, Cakatay U, Uzun H, Simsek G (2024) The Role of Glycogen Synthase Kinase-3\\u0026beta; in the Zinc-Mediated Neuroprotective Effect of Metformin in Rats with Glutamate Neurotoxicity. Biol Trace Elem Res 202(1):233-245. https://doi.org/10.1007/s12011-023-03667-3. \\u003c/li\\u003e\\n\\u003cli\\u003eArauz-Contreras J, Feria-Velasco A (1984) Monosodium-L-glutamate-induced convulsions--I. Differences in seizure pattern and duration of effect as a function of age in rats. Gen Pharmacol 15(5):391-5. https://doi.org/10.1016/0306-3623(84)90036-3. \\u003c/li\\u003e\\n\\u003cli\\u003eKatane M, Homma H (2010) D-aspartate oxidase: the sole catabolic enzyme acting on free D-aspartate in mammals. Chem Biodivers 7(6):1435-49. https://doi.org/10.1002/cbdv.200900250. \\u003c/li\\u003e\\n\\u003cli\\u003eSacchi S, Cappelletti P, Murtas G (2018) Biochemical Properties of Human D-amino Acid Oxidase Variants and Their Potential Significance in Pathologies. Front Mol Biosci 5:55. https://doi.org/10.3389/fmolb.2018.00055. \\u003c/li\\u003e\\n\\u003cli\\u003eYap SH, Lee CS, Furusho A, Ishii C, Shaharudin S, Zulhaimi NS, Kamarulzaman A, Kamaruzzaman SB, Mita M, Leong KH, Hamase K, Rajasuriar R (2022) Plasma d-amino acids are associated with markers of immune activation and organ dysfunction in people with HIV. AIDS 36(7):911-921. https://doi.org/10.1097/QAD.0000000000003207. \\u003c/li\\u003e\\n\\u003cli\\u003eBhagavan HN, Coursin DB, Stewart CN (1971) Monosodium glutamate induces convulsive disorders in rats. Nature 232(5308):275-6. https://doi.org/10.1038/232275a0. \\u003c/li\\u003e\\n\\u003cli\\u003eOru\\u0026ccedil; KY, Ağt\\u0026uuml;rk G, Oru\\u0026ccedil; A, Yanar K, Seymen HO (2025) Protective effect of Apelin-13 on D-glutamic acid-induced excitotoxicity in SH-SY5Y cell line: An in-vitro study. Neuropeptides 109:102483. https://doi.org/10.1016/j.npep.2024.102483. \\u003c/li\\u003e\\n\\u003cli\\u003eVorhees CV, Williams MT (2006) Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc 1(2):848-58. https://doi.org/10.1038/nprot.2006.116. \\u003c/li\\u003e\\n\\u003cli\\u003eSadowska-Bartosz I, Galiniak S, Bartosz G, Rachel M (2014) Oxidative modification of proteins in pediatric cystic fibrosis with bacterial infections. Oxid Med Cell Longev 2014:389629. https://doi.org/10.1155/2014/389629. \\u003c/li\\u003e\\n\\u003cli\\u003eHanasand M, Omdal R, Norheim KB, G\\u0026oslash;ransson LG, Brede C, Jonsson G (2012) Improved detection of advanced oxidation protein products in plasma. Clin Chim Acta 413(9-10):901-6. https://doi.org/10.1016/j.cca.2012.01.038. \\u003c/li\\u003e\\n\\u003cli\\u003eBuege JA, Aust SD (1978) Microsomal lipid peroxidation. Methods Enzymol 52:302-10. https://doi.org/10.1016/s0076-6879(78)52032-6. \\u003c/li\\u003e\\n\\u003cli\\u003eSedlak J, Lindsay RH (1968) Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman\\u0026apos;s reagent. Anal Biochem 25(1):192-205. https://doi.org/10.1016/0003-2697(68)90092-4. \\u003c/li\\u003e\\n\\u003cli\\u003eSingh N, Vijayanti S, Saha L, Bhatia A, Banerjee D, Chakrabarti A (2018) Neuroprotective effect of Nrf2 activator dimethyl fumarate, on the hippocampal neurons in chemical kindling model in rat. Epilepsy Res 143:98-104. https://doi.org/10.1016/j.eplepsyres.2018.02.011. \\u003c/li\\u003e\\n\\u003cli\\u003eEskandari E, Eaves CJ (2022) Paradoxical roles of caspase-3 in regulating cell survival, proliferation, and tumorigenesis. J Cell Biol 221(6):e202201159. https://doi.org/10.1083/jcb.202201159. \\u003c/li\\u003e\\n\\u003cli\\u003eBrecht S, Gelderblom M, Srinivasan A, Mielke K, Dityateva G, Herdegen T (2001) Caspase-3 activation and DNA fragmentation in primary hippocampal neurons following glutamate excitotoxicity. Brain Res Mol Brain Res 94(1-2):25-34. https://doi.org/10.1016/s0006-8993(01)02767-6. \\u003c/li\\u003e\\n\\u003cli\\u003eHazzaa SM, Abdelaziz SAM, Abd Eldaim MA, Abdel-Daim MM, Elgarawany GE (2020) Neuroprotective Potential of Allium sativum against Monosodium Glutamate-Induced Excitotoxicity: Impact on Short-Term Memory, Gliosis, and Oxidative Stress. Nutrients 12(4):1028. https://doi.org/10.3390/nu12041028. \\u003c/li\\u003e\\n\\u003cli\\u003eMohseni F, Garmabi B, Khaksari M (2021) Apelin-13 attenuates spatial memory impairment by anti-oxidative, anti-apoptosis, and anti-inflammatory mechanism against ethanol neurotoxicity in the neonatal rat hippocampus. Neuropeptides 87:102130. https://doi.org/10.1016/j.npep.2021.102130. \\u003c/li\\u003e\\n\\u003cli\\u003eShen X, Yuan G, Li B, Cao C, Cao D, Wu J, Li X, Li H, Shen H, Wang Z, Chen G (2022) Apelin-13 attenuates early brain injury through inhibiting inflammation and apoptosis in rats after experimental subarachnoid hemorrhage. Mol Biol Rep 49(3):2107-2118. https://doi.org/10.1007/s11033-021-07028-y. \\u003c/li\\u003e\\n\\u003cli\\u003eSpencer B, Potkar R, Metcalf J, Thrin I, Adame A, Rockenstein E, Masliah E (2016) Systemic Central Nervous System (CNS)-targeted Delivery of Neuropeptide Y (NPY) Reduces Neurodegeneration and Increases Neural Precursor Cell Proliferation in a Mouse Model of Alzheimer Disease. J Biol Chem 291(4):1905-1920. https://doi.org/10.1074/jbc.M115.678185. \\u003c/li\\u003e\\n\\u003cli\\u003eSmiałowska M, Domin H, Zieba B, Koźniewska E, Michalik R, Piotrowski P, Kajta M (2009) Neuroprotective effects of neuropeptide Y-Y2 and Y5 receptor agonists in vitro and in vivo. Neuropeptides 43(3):235-49. https://doi.org/10.1016/j.npep.2009.02.002. \\u003c/li\\u003e\\n\\u003cli\\u003eDomin H, Przykaza Ł, Jantas D, Kozniewska E, Boguszewski PM, Śmiałowska M (2017) Neuropeptide Y Y2 and Y5 receptors as promising targets for neuroprotection in primary neurons exposed to oxygen-glucose deprivation and in transient focal cerebral ischemia in rats. Neuroscience 344:305-325. https://doi.org/10.1016/j.neuroscience.2016.12.040. \\u003c/li\\u003e\\n\\u003cli\\u003eKhezri MR, Ghasemnejad-Berenji M, Moloodsouri D (2023) The PI3K/AKT Signaling Pathway and Caspase-3 in Alzheimer\\u0026apos;s Disease: Which One Is the Beginner? J Alzheimers Dis 92(2):391-393. https://doi.org/10.3233/JAD-221157. \\u003c/li\\u003e\\n\\u003cli\\u003eRoskoski R Jr (2012) ERK1/2 MAP kinases: structure, function, and regulation. Pharmacol Res 66(2):105-43. https://doi.org/10.1016/j.phrs.2012.04.005. \\u003c/li\\u003e\\n\\u003cli\\u003eSoriano FX, Hardingham GE (2007) Compartmentalized NMDA receptor signalling to survival and death. J Physiol 584(Pt 2):381-7. https://doi.org/10.1113/jphysiol.2007.138875. \\u003c/li\\u003e\\n\\u003cli\\u003eCook DR, Gleichman AJ, Cross SA, Doshi S, Ho W, Jordan-Sciutto KL, Lynch DR, Kolson DL (2011) NMDA receptor modulation by the neuropeptide apelin: implications for excitotoxic injury. J Neurochem 118(6):1113-23. https://doi.org/10.1111/j.1471-4159.2011.07383.x. \\u003c/li\\u003e\\n\\u003cli\\u003eIshimaru Y, Sumino A, Kajioka D, Shibagaki F, Yamamuro A, Yoshioka Y, Maeda S (2017) Apelin protects against NMDA-induced retinal neuronal death via an APJ receptor by activating Akt and ERK1/2, and suppressing TNF-\\u0026alpha; expression in mice. J Pharmacol Sci 133(1):34-41. https://doi.org/10.1016/j.jphs.2016.12.002. \\u003c/li\\u003e\\n\\u003cli\\u003eZeng Z, Li H, You M, Rong R, Xia X (2022) Dephosphorylation of ERK1/2 and DRP1 S585 regulates mitochondrial dynamics in glutamate toxicity of retinal neurons in vitro. Exp Eye Res 225:109271. https://doi.org/10.1016/j.exer.2022.109271. \\u003c/li\\u003e\\n\\u003cli\\u003eGutkind JS (2000) Regulation of mitogen-activated protein kinase signaling networks by G protein-coupled receptors. Sci STKE 2000(40):re1. https://doi.org/10.1126/stke.2000.40.re1. \\u003c/li\\u003e\\n\\u003cli\\u003ePalanivel V, Gupta V, Mirshahvaladi SSO, Sharma S, Gupta V, Chitranshi N, Mirzaei M, Graham SL, Basavarajappa D (2022) Neuroprotective Effects of Neuropeptide Y on Human Neuroblastoma SH-SY5Y Cells in Glutamate Excitotoxicity and ER Stress Conditions. Cells 11(22):3665. https://doi.org/10.3390/cells11223665. \\u003c/li\\u003e\\n\\u003cli\\u003eManning BD, Toker A (2017) AKT/PKB Signaling: Navigating the Network. Cell 169(3):381-405. https://doi.org/10.1016/j.cell.2017.04.001. \\u003c/li\\u003e\\n\\u003cli\\u003eZhou XM, Liu CY, Liu YY, Ma QY, Zhao X, Jiang YM, Li XJ, Chen JX (2021) Xiaoyaosan Alleviates Hippocampal Glutamate-Induced Toxicity in the CUMS Rats via NR2B and PI3K/Akt Signaling Pathway. Front Pharmacol 12:586788. https://doi.org/10.3389/fphar.2021.586788. \\u003c/li\\u003e\\n\\u003cli\\u003eZeng XJ, Yu SP, Zhang L, Wei L (2010) Neuroprotective effect of the endogenous neural peptide apelin in cultured mouse cortical neurons. Exp Cell Res 316(11):1773-83. https://doi.org/10.1016/j.yexcr.2010.02.005. \\u003c/li\\u003e\\n\\u003cli\\u003eJacques D, Dumont Y, Fournier A, Quirion R (1997) Characterization of neuropeptide Y receptor subtypes in the normal human brain, including the hypothalamus. Neuroscience 79(1):129-48. https://doi.org/10.1016/s0306-4522(96)00639-2. \\u003c/li\\u003e\\n\\u003cli\\u003eStanić D, Brumovsky P, Fetissov S, Shuster S, Herzog H, H\\u0026ouml;kfelt T (2006) Characterization of neuropeptide Y2 receptor protein expression in the mouse brain. I. Distribution in cell bodies and nerve terminals. J Comp Neurol 499(3):357-90. https://doi.org/10.1002/cne.21046. \\u003c/li\\u003e\\n\\u003cli\\u003eSchlicker E, Kathmann M (2008) Presynaptic neuropeptide receptors. Handb Exp Pharmacol (184):409-34. https://doi.org/10.1007/978-3-540-74805-2_13. \\u003c/li\\u003e\\n\\u003cli\\u003eSilva AP, Carvalho AP, Carvalho CM, Malva JO (2001) Modulation of intracellular calcium changes and glutamate release by neuropeptide Y1 and Y2 receptors in the rat hippocampus: differential effects in CA1, CA3 and dentate gyrus. J Neurochem 79(2):286-96. https://doi.org/10.1046/j.1471-4159.2001.00560.x. \\u003c/li\\u003e\\n\\u003cli\\u003eSilva AP, Xapelli S, Grouzmann E, Cavadas C (2005) The putative neuroprotective role of neuropeptide Y in the central nervous system. Curr Drug Targets CNS Neurol Disord 4(4):331-47. https://doi.org/10.2174/1568007054546153. \\u003c/li\\u003e\\n\\u003cli\\u003eFatoba O, Kloster E, Reick C, Saft C, Gold R, Epplen JT, Arning L, Ellrichmann G (2018) Activation of NPY-Y2 receptors ameliorates disease pathology in the R6/2 mouse and PC12 cell models of Huntington\\u0026apos;s disease. Exp Neurol 302:112-128. https://doi.org/10.1016/j.expneurol.2018.01.001. \\u003c/li\\u003e\\n\\u003cli\\u003eDecressac M, Pain S, Chabeauti PY, Frangeul L, Thiriet N, Herzog H, Vergote J, Chalon S, Jaber M, Gaillard A (2012) Neuroprotection by neuropeptide Y in cell and animal models of Parkinson\\u0026apos;s disease. Neurobiol Aging 33(9):2125-37. https://doi.org/10.1016/j.neurobiolaging.2011.06.018. \\u003c/li\\u003e\\n\\u003cli\\u003eKloster E, Saft C, Akkad DA, Epplen JT, Arning L (2014) Association of age at onset in Huntington disease with functional promoter variations in NPY and NPY2R. J Mol Med (Berl) 92(2):177-84. https://doi.org/10.1007/s00109-013-1092-3. \\u003c/li\\u003e\\n\\u003cli\\u003eSilva AP, Pinheiro PS, Carvalho AP, Carvalho CM, Jakobsen B, Zimmer J, Malva JO (2003) Activation of neuropeptide Y receptors is neuroprotective against excitotoxicity in organotypic hippocampal slice cultures. FASEB J 17(9):1118-20. https://doi.org/10.1096/fj.02-0885fje. \\u003c/li\\u003e\\n\\u003cli\\u003eXapelli S, Bernardino L, Ferreira R, Grade S, Silva AP, Salgado JR, Cavadas C, Grouzmann E, Poulsen FR, Jakobsen B, Oliveira CR, Zimmer J, Malva JO (2008) Interaction between neuropeptide Y (NPY) and brain-derived neurotrophic factor in NPY-mediated neuroprotection against excitotoxicity: a role for microglia. Eur J Neurosci 27(8):2089-102. https://doi.org/10.1111/j.1460-9568.2008.06172.x. \\u003c/li\\u003e\\n\\u003cli\\u003eDomin H (2021) Neuropeptide Y Y2 and Y5 receptors as potential targets for neuroprotective and antidepressant therapies: Evidence from preclinical studies. Prog Neuropsychopharmacol Biol Psychiatry 111:110349. https://doi.org/10.1016/j.pnpbp.2021.110349. \\u003c/li\\u003e\\n\\u003cli\\u003eXapelli S, Silva AP, Ferreira R, Malva JO (2007) Neuropeptide Y can rescue neurons from cell death following the application of an excitotoxic insult with kainate in rat organotypic hippocampal slice cultures. Peptides 28(2):288-94. https://doi.org/10.1016/j.peptides.2006.09.031. \\u003c/li\\u003e\\n\\u003cli\\u003eKraeuter AK, Guest PC, Sarnyai Z (2019) The Open Field Test for Measuring Locomotor Activity and Anxiety-Like Behavior. Methods Mol Biol 1916:99-103. https://doi.org/10.1007/978-1-4939-8994-2_9. \\u003c/li\\u003e\\n\\u003cli\\u003eTian SW, Xu F, Gui SJ (2018) Apelin-13 reverses memory impairment and depression-like behavior in chronic social defeat stressed rats. Peptides 108:1-6. https://doi.org/10.1016/j.peptides.2018.08.009. \\u003c/li\\u003e\\n\\u003cli\\u003eWang W, Xu T, Chen X, Dong K, Du C, Sun J, Shi C, Li X, Yang Y, Li H, Xu ZD (2019) NPY Receptor 2 Mediates NPY Antidepressant Effect in the mPFC of LPS Rat by Suppressing NLRP3 Signaling Pathway. Mediators Inflamm 2019:7898095. https://doi.org/10.1155/2019/7898095. \\u003c/li\\u003e\\n\\u003cli\\u003eVillarreal-Silva EE, Gonz\\u0026aacute;lez-Navarro AR, Salazar-Ybarra RA, Quiroga-Garc\\u0026iacute;a O, Cruz-Elizondo MAJ, Garc\\u0026iacute;a-Garc\\u0026iacute;a A, Rodr\\u0026iacute;guez-Rocha H, Morales-G\\u0026oacute;mez JA, Quiroga-Garza A, Elizondo-Oma\\u0026ntilde;a RE, de Le\\u0026oacute;n \\u0026Aacute;RM, Guzm\\u0026aacute;n-L\\u0026oacute;pez S (2022) Aged rats learn Morris Water maze using non-spatial search strategies evidenced by a parameter-based algorithm. Transl Neurosci 13(1):134-144. https://doi.org/10.1515/tnsci-2022-0221. \\u003c/li\\u003e\\n\\u003cli\\u003eNegr\\u0026oacute;n-Oyarzo I, Espinosa N, Aguilar-Rivera M, Fuenzalida M, Aboitiz F, Fuentealba P (2018) Coordinated prefrontal-hippocampal activity and navigation strategy-related prefrontal firing during spatial memory formation. Proc Natl Acad Sci U S A 115(27):7123-7128. https://doi.org/10.1073/pnas.1720117115. \\u003c/li\\u003e\\n\\u003cli\\u003eJin H, Yang C, Jiang C, Li L, Pan M, Li D, Han X, Ding J (2022) Evaluation of Neurotoxicity in BALB/c Mice following Chronic Exposure to Polystyrene Microplastics. Environ Health Perspect 130(10):107002. https://doi.org/10.1289/EHP10255. \\u003c/li\\u003e\\n\\u003cli\\u003eKutlu MG, Gould TJ (2016) Effects of drugs of abuse on hippocampal plasticity and hippocampus-dependent learning and memory: contributions to development and maintenance of addiction. Learn Mem 23(10):515-33. https://doi.org/10.1101/lm.042192.116. \\u003c/li\\u003e\\n\\u003cli\\u003eKim EJ, Pellman B, Kim JJ (2015) Stress effects on the hippocampus: a critical review. Learn Mem 22(9):411-6. https://doi.org/10.1101/lm.037291.114. \\u003c/li\\u003e\\n\\u003cli\\u003eVorhees CV, Williams MT (2006) Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc 1(2):848-58. https://doi.org/10.1038/nprot.2006.116. \\u003c/li\\u003e\\n\\u003cli\\u003eGallagher M, Burwell R, Burchinal M (1993) Severity of spatial learning impairment in aging: development of a learning index for performance in the Morris water maze. Behav Neurosci 107(4):618-26. https://doi.org/10.1037//0735-7044.107.4.618. \\u003c/li\\u003e\\n\\u003cli\\u003eHern\\u0026aacute;ndez-Mercado K, Zepeda A (2022) Morris Water Maze and Contextual Fear Conditioning Tasks to Evaluate Cognitive Functions Associated With Adult Hippocampal Neurogenesis. Front Neurosci 15:782947. https://doi.org/10.3389/fnins.2021.782947. \\u003c/li\\u003e\\n\\u003cli\\u003eGeerts JP, Chersi F, Stachenfeld KL, Burgess N (2020) A general model of hippocampal and dorsal striatal learning and decision making. Proc Natl Acad Sci U S A 117(49):31427-31437. https://doi.org/10.1073/pnas.2007981117. \\u003c/li\\u003e\\n\\u003cli\\u003eInostroza M, Cid E, Brotons-Mas J, Gal B, Aivar P, Uzcategui YG, Sandi C, Menendez de la Prida L (2011) Hippocampal-dependent spatial memory in the water maze is preserved in an experimental model of temporal lobe epilepsy in rats. PLoS One 6(7):e22372. https://doi.org/10.1371/journal.pone.0022372. \\u003c/li\\u003e\\n\\u003cli\\u003eHaghparast E, Esmaeili-Mahani S, Abbasnejad M, Sheibani V (2018) Apelin-13 ameliorates cognitive impairments in 6-hydroxydopamine-induced substantia nigra lesion in rats. Neuropeptides 68:28-35. https://doi.org/10.1016/j.npep.2018.01.001. \\u003c/li\\u003e\\n\\u003cli\\u003eShen P, Yue Q, Fu W, Tian SW, You Y (2019) Apelin-13 ameliorates chronic water-immersion restraint stress-induced memory performance deficit through upregulation of BDNF in rats. Neurosci Lett 696:151-155. https://doi.org/10.1016/j.neulet.2018.11.051. \\u003c/li\\u003e\\n\\u003cli\\u003eAntunes M, Biala G (2012) The novel object recognition memory: neurobiology, test procedure, and its modifications. Cogn Process 13(2):93-110. https://doi.org/10.1007/s10339-011-0430-z. \\u003c/li\\u003e\\n\\u003cli\\u003eKornhuber J, Zoicas I (2017) Neuropeptide Y prolongs non-social memory and differentially affects acquisition, consolidation, and retrieval of non-social and social memory in male mice. Sci Rep 7(1):6821. https://doi.org/10.1038/s41598-017-07273-x. \\u003c/li\\u003e\\n\\u003cli\\u003eOsredkar J, Gosar D, Maček J, Kumer K, Fabjan T, Finderle P, \\u0026Scaron;terpin S, Zupan M, Jekovec Vrhov\\u0026scaron;ek M (2019) Urinary Markers of Oxidative Stress in Children with Autism Spectrum Disorder (ASD). Antioxidants (Basel) 8(6):187. https://doi.org/10.3390/antiox8060187. \\u003c/li\\u003e\\n\\u003cli\\u003eZgutka K, Tkacz M, Tomasiak P, Tarnowski M (2023) A Role for Advanced Glycation End Products in Molecular Ageing. Int J Mol Sci 24(12):9881. https://doi.org/10.3390/ijms24129881. \\u003c/li\\u003e\\n\\u003cli\\u003ePrasad C, Davis KE, Imrhan V, Juma S, Vijayagopal P (2017) Advanced Glycation End Products and Risks for Chronic Diseases: Intervening Through Lifestyle Modification. Am J Lifestyle Med 13(4):384-404. https://doi.org/10.1177/1559827617708991. \\u003c/li\\u003e\\n\\u003cli\\u003eKothandan D, Singh DS, Yerrakula G, D B, N P, Santhana Sophia B V, A R, Ramya Vg S, S K, M J (2024) Advanced Glycation End Products-Induced Alzheimer\\u0026apos;s Disease and Its Novel Therapeutic Approaches: A Comprehensive Review. Cureus 16(5):e61373. https://doi.org/10.7759/cureus.61373. \\u003c/li\\u003e\\n\\u003cli\\u003eDu H, Ma Y, Wang X, Zhang Y, Zhu L, Shi S, Pan S, Liu Z (2023) Advanced glycation end products induce skeletal muscle atrophy and insulin resistance via activating ROS-mediated ER stress PERK/FOXO1 signaling. Am J Physiol Endocrinol Metab 324(3):E279-E287. https://doi.org/10.1152/ajpendo.00218.2022. \\u003c/li\\u003e\\n\\u003cli\\u003eWan L, Bai X, Zhou Q, Chen C, Wang H, Liu T, Xue J, Wei C, Xie L (2022) The advanced glycation end-products (AGEs)/ROS/NLRP3 inflammasome axis contributes to delayed diabetic corneal wound healing and nerve regeneration. Int J Biol Sci 18(2):809-825. https://doi.org/10.7150/ijbs.63219. \\u003c/li\\u003e\\n\\u003cli\\u003eDeng S, He R, Yue Z, Li B, Li F, Xiao Q, Wang X, Li Y, Chen R, Rong S (2024) Association of Advanced Glycation End Products with Cognitive Function: HealthyDance Study. J Alzheimers Dis 100(2):551-562. https://doi.org/10.3233/JAD-240296. \\u003c/li\\u003e\\n\\u003cli\\u003ePeppa M, Uribarri J, Vlassara H (2008) Aging and glycoxidant stress. Hormones (Athens) 7(2):123-32. https://doi.org/10.1007/BF03401503. \\u003c/li\\u003e\\n\\u003cli\\u003eOleniuc M, Secara I, Onofriescu M, Hogas S, Voroneanu L, Siriopol D, Covic A (2011) Consequences of Advanced Glycation End Products Accumulation in Chronic Kidney Disease and Clinical Usefulness of Their Assessment Using a Non-invasive Technique - Skin Autofluorescence. Maedica (Bucur) 6(4):298-307. \\u003c/li\\u003e\\n\\u003cli\\u003eWu B, Yu L, Hu P, Lu Y, Li J, Wei Y, He R (2018) GRP78 protects CHO cells from ribosylation. Biochim Biophys Acta Mol Cell Res 1865(4):629-637. https://doi.org/10.1016/j.bbamcr.2018.02.001. \\u003c/li\\u003e\\n\\u003cli\\u003eBayarsaikhan E, Bayarsaikhan D, Lee J, Son M, Oh S, Moon J, Park HJ, Roshini A, Kim SU, Song BJ, Jo SM, Byun K, Lee B (2016) Microglial AGE-albumin is critical for neuronal death in Parkinson\\u0026apos;s disease: a possible implication for theranostics. Int J Nanomedicine 10 Spec Iss(Spec Iss):281-92. https://doi.org/10.2147/IJN.S95077. \\u003c/li\\u003e\\n\\u003cli\\u003eMalarkey EB, Parpura V (2008) Mechanisms of glutamate release from astrocytes. Neurochem Int 52(1-2):142-54. https://doi.org/10.1016/j.neuint.2007.06.005. \\u003c/li\\u003e\\n\\u003cli\\u003eKamynina A, Esteras N, Koroev DO, Angelova PR, Volpina OM, Abramov AY (2021) Activation of RAGE leads to the release of glutamate from astrocytes and stimulates calcium signal in neurons. J Cell Physiol 236(9):6496-6506. https://doi.org/10.1002/jcp.30324. \\u003c/li\\u003e\\n\\u003cli\\u003eKoerich S, Parreira GM, de Almeida DL, Vieira RP, de Oliveira ACP (2023) Receptors for Advanced Glycation End Products (RAGE): Promising Targets Aiming at the Treatment of Neurodegenerative Conditions. Curr Neuropharmacol 21(2):219-234. https://doi.org/10.2174/1570159X20666220922153903. \\u003c/li\\u003e\\n\\u003cli\\u003eLamichhane S, Bastola T, Pariyar R, Lee ES, Lee HS, Lee DH, Seo J (2017) ROS Production and ERK Activity Are Involved in the Effects of d-\\u0026beta;-Hydroxybutyrate and Metformin in a Glucose Deficient Condition. Int J Mol Sci 18(3):674. https://doi.org/10.3390/ijms18030674. \\u003c/li\\u003e\\n\\u003cli\\u003eAlderman CJ, Shah S, Foreman JC, Chain BM, Katz DR (2002) The role of advanced oxidation protein products in regulation of dendritic cell function. Free Radic Biol Med 32(5):377-85. https://doi.org/10.1016/s0891-5849(01)00735-3. \\u003c/li\\u003e\\n\\u003cli\\u003eYilmazer UT, Pehlivan B, Guney S, Yar-Saglam AS, Balabanli B, Kaltalioglu K, Coskun-Cevher S (2024) The combined effect of morin and hesperidin on memory ability and oxidative/nitrosative stress in a streptozotocin-induced rat model of Alzheimer\\u0026apos;s disease. Behav Brain Res 471:115131. https://doi.org/10.1016/j.bbr.2024.115131. \\u003c/li\\u003e\\n\\u003cli\\u003eLi L, Renier G (2006) Activation of nicotinamide adenine dinucleotide phosphate (reduced form) oxidase by advanced glycation end products links oxidative stress to altered retinal vascular endothelial growth factor expression. Metabolism 55(11):1516-23. https://doi.org/10.1016/j.metabol.2006.06.022. \\u003c/li\\u003e\\n\\u003cli\\u003eHeijnis WH, Dekker HL, de Koning LJ, Wierenga PA, Westphal AH, de Koster CG, Gruppen H, van Berkel WJ (2011) Identification of the peroxidase-generated intermolecular dityrosine cross-link in bovine \\u0026alpha;-lactalbumin. J Agric Food Chem 59(1):444-9. https://doi.org/10.1021/jf104298y. \\u003c/li\\u003e\\n\\u003cli\\u003eAl-Hilaly YK, Williams TL, Stewart-Parker M, Ford L, Skaria E, Cole M, Bucher WG, Morris KL, Sada AA, Thorpe JR, Serpell LC (2013) A central role for dityrosine crosslinking of Amyloid-\\u0026beta; in Alzheimer\\u0026apos;s disease. Acta Neuropathol Commun 1:83. https://doi.org/10.1186/2051-5960-1-83. \\u003c/li\\u003e\\n\\u003cli\\u003eWang J, Swanson RA (2020) Superoxide and Non-ionotropic Signaling in Neuronal Excitotoxicity. Front Neurosci 4:861. https://doi.org/10.3389/fnins.2020.00861. \\u003c/li\\u003e\\n\\u003cli\\u003ePennathur S, Jackson-Lewis V, Przedborski S, Heinecke JW (1999) Mass spectrometric quantification of 3-nitrotyrosine, ortho-tyrosine, and o,o\\u0026apos;-dityrosine in brain tissue of 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine-treated mice, a model of oxidative stress in Parkinson\\u0026apos;s disease. J Biol Chem 274(49):34621-8. https://doi.org/10.1074/jbc.274.49.34621. \\u003c/li\\u003e\\n\\u003cli\\u003ePathak S, Nadar R, Kim S, Liu K, Govindarajulu M, Cook P, Watts Alexander CS, Dhanasekaran M, Moore T (2024) The Influence of Kynurenine Metabolites on Neurodegenerative Pathologies. Int J Mol Sci 25(2):853. https://doi.org/10.3390/ijms25020853. \\u003c/li\\u003e\\n\\u003cli\\u003eSorgdrager FJH, Vermeiren Y, Van Faassen M, van der Ley C, Nollen EAA, Kema IP, De Deyn PP (2019) Age- and disease-specific changes of the kynurenine pathway in Parkinson\\u0026apos;s and Alzheimer\\u0026apos;s disease. J Neurochem 151(5):656-668. https://doi.org/10.1111/jnc.14843. \\u003c/li\\u003e\\n\\u003cli\\u003eGanong AH, Cotman CW (1986) Kynurenic acid and quinolinic acid act at N-methyl-D-aspartate receptors in the rat hippocampus. J Pharmacol Exp Ther 236(1):293-9. \\u003c/li\\u003e\\n\\u003cli\\u003eDharmajaya R, Sari DK (2022) Malondialdehyde value as radical oxidative marker and endogenous antioxidant value analysis in brain tumor. Ann Med Surg (Lond) 77:103231. https://doi.org/10.1016/j.amsu.2021.103231. \\u003c/li\\u003e\\n\\u003cli\\u003ePe\\u0026ntilde;a-Bautista C, Vento M, Baquero M, Ch\\u0026aacute;fer-Peric\\u0026aacute;s C (2019) Lipid peroxidation in neurodegeneration. Clin Chim Acta 497:178-188. https://doi.org/10.1016/j.cca.2019.07.037. \\u003c/li\\u003e\\n\\u003cli\\u003eHaro Gir\\u0026oacute;n S, Monserrat Sanz J, Ortega MA, Garcia-Montero C, Fraile-Mart\\u0026iacute;nez O, G\\u0026oacute;mez-Lahoz AM, Boaru DL, de Leon-Oliva D, Guijarro LG, Atienza-Perez M, Diaz D, Lopez-Dolado E, \\u0026Aacute;lvarez-Mon M (2023) Prognostic Value of Malondialdehyde (MDA) in the Temporal Progression of Chronic Spinal Cord Injury. J Pers Med 13(4):626. https://doi.org/10.3390/jpm13040626. \\u003c/li\\u003e\\n\\u003cli\\u003eLi J, Zhou Q, Yang T, Li Y, Zhang Y, Wang J, Jiao Z (2018) SGK1 inhibits PM2.5-induced apoptosis and oxidative stress in human lung alveolar epithelial A549 cells. Biochem Biophys Res Commun 496(4):1291-1295. https://doi.org/10.1016/j.bbrc.2018.02.002. \\u003c/li\\u003e\\n\\u003cli\\u003eAjsuvakova OP, Tinkov AA, Aschner M, Rocha JBT, Michalke B, Skalnaya MG, Skalny AV, Butnariu M, Dadar M, Sarac I, Aaseth J, Bj\\u0026oslash;rklund G (2020) Sulfhydryl groups as targets of mercury toxicity. Coord Chem Rev 417:213343. https://doi.org/10.1016/j.ccr.2020.213343. \\u003c/li\\u003e\\n\\u003cli\\u003eEroglu N, Sahin G, Yesil S, Fettah A, Yildiz YT, Erel O (2023) Thiol disulfide homeostasis in ionizing radiation and chemotherapeutic drug exposure. North Clin Istanb 10(1):53-58. https://doi.org/10.14744/nci.2021.59913. \\u003c/li\\u003e\\n\\u003cli\\u003ePham TK, Buczek WA, Mead RJ, Shaw PJ, Collins MO (2021) Proteomic Approaches to Study Cysteine Oxidation: Applications in Neurodegenerative Diseases. Front Mol Neurosci 14:678837. https://doi.org/10.3389/fnmol.2021.678837. \\u003c/li\\u003e\\n\\u003cli\\u003eKazama M, Kato Y, Kakita A, Noguchi N, Urano Y, Masui K, Niida-Kawaguchi M, Yamamoto T, Watabe K, Kitagawa K, Shibata N (2020) Astrocytes release glutamate via cystine/glutamate antiporter upregulated in response to increased oxidative stress related to sporadic amyotrophic lateral sclerosis. Neuropathology 40(6):587-598. https://doi.org/10.1111/neup.12716. \\u003c/li\\u003e\\n\\u003cli\\u003eShivasharan BD, Nagakannan P, Thippeswamy BS, Veerapur VP (2013) Protective Effect of Calendula officinalis L. Flowers Against Monosodium Glutamate Induced Oxidative Stress and Excitotoxic Brain Damage in Rats. Indian J Clin Biochem 28(3):292-8. https://doi.org/10.1007/s12291-012-0256-1. \\u003c/li\\u003e\\n\\u003cli\\u003eTopcu A, Saral S, Mercantepe T, Akyildiz K, Tumkaya L, Yilmaz A (2023) The effects of apelin-13 against cisplatin-induced nephrotoxicity in rats. Drug Chem Toxicol 46(1):77-87. https://doi.org/10.1080/01480545.2021.2011309. \\u003c/li\\u003e\\n\\u003cli\\u003eSingh P, Barman B, Thakur MK (2022) Oxidative stress-mediated memory impairment during aging and its therapeutic intervention by natural bioactive compounds. Front Aging Neurosci 14:944697. https://doi.org/10.3389/fnagi.2022.944697. \\u003c/li\\u003e\\n\\u003cli\\u003eFukui K, Omoi NO, Hayasaka T, Shinnkai T, Suzuki S, Abe K, Urano S (2002) Cognitive impairment of rats caused by oxidative stress and aging, and its prevention by vitamin E. Ann N Y Acad Sci 959:275-84. https://doi.org/10.1111/j.1749-6632.2002.tb02099.x. \\u003c/li\\u003e\\n\\u003cli\\u003eWang B, Yang Q, Sun YY, Xing YF, Wang YB, Lu XT, Bai WW, Liu XQ, Zhao YX (2014) Resveratrol-enhanced autophagic flux ameliorates myocardial oxidative stress injury in diabetic mice. J Cell Mol Med 18(8):1599-611. https://doi.org/10.1111/jcmm.12312. \\u003c/li\\u003e\\n\\u003cli\\u003eNegahdar H, Hosseini SR, Parsian H, Kheirkhah F, Mosapour A, Khafri S, Haghighi AH (2015) Homocysteine, trace elements and oxidant/antioxidant status in mild cognitively impaired elderly persons: a cross-sectional study. Rom J Intern Med 53(4):336-42. https://doi.org/10.1515/rjim-2015-0043. \\u003c/li\\u003e\\n\\u003cli\\u003eSolvang SH, Nordrehaug JE, Tell GS, Nyg\\u0026aring;rd O, McCann A, Ueland PM, Midttun \\u0026Oslash;, Meyer K, Vedeler CA, Aarsland D, Refsum H, Smith AD, Giil LM (2019) The kynurenine pathway and cognitive performance in community-dwelling older adults. The Hordaland Health Study. Brain Behav Immun 75:155-162. https://doi.org/10.1016/j.bbi.2018.10.003. \\u003c/li\\u003e\\n\\u003cli\\u003eLogan S, Royce GH, Owen D, Farley J, Ranjo-Bishop M, Sonntag WE, Deepa SS (2019) Accelerated decline in cognition in a mouse model of increased oxidative stress. Geroscience 41(5):591-607. https://doi.org/10.1007/s11357-019-00105-y. \\u003c/li\\u003e\\n\\u003cli\\u003eMandour DA, Bendary MA, Alsemeh AE (2021) Histological and imunohistochemical alterations of hippocampus and prefrontal cortex in a rat model of Alzheimer like-disease with a preferential role of the flavonoid \\u0026quot;hesperidin\\u0026quot;. J Mol Histol 52(5):1043-1065. https://doi.org/10.1007/s10735-021-09998-6. \\u003c/li\\u003e\\n\\u003cli\\u003eFricker M, Tolkovsky AM, Borutaite V, Coleman M, Brown GC (2018) Neuronal Cell Death. Physiol Rev 98(2):813-880. https://doi.org/10.1152/physrev.00011.2017. \\u003c/li\\u003e\\n\\u003cli\\u003eEsmaeili-Mahani S, Haghparast E, Nezhadi A, Abbasnejad M, Sheibani V (2020) Apelin-13 prevents hippocampal synaptic plasticity impairment in Parkinsonism rats. J Chem Neuroanat 111:101884. https://doi.org/10.1016/j.jchemneu.2020.101884. \\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\":\"info@researchsquare.com\",\"identity\":\"molecular-neurobiology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"moln\",\"sideBox\":\"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)\",\"snPcode\":\"12035\",\"submissionUrl\":\"https://submission.nature.com/new-submission/12035/3\",\"title\":\"Molecular Neurobiology\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"Excitotoxicity, Apelin-13, Neuropeptide Y, Allocentric Memory, Spatial Memory\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6635799/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6635799/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eGlutamate-mediated excitotoxicity causes mitochondrial dysfunction, apoptosis, neuronal death. Aim of this study is to investigate whether Apelin-13, together with NPY2 and NPY5 receptors, plays a cooperative role in neuroprotection and in preventing learning and memory impairments under excitotoxic conditions.\\u003c/p\\u003e\\n\\u003cp\\u003eD-Glutamic acid-induced excitotoxicity was established in 42-male Sprague-Dawley rats (6-8 weeks, 200-250g). Animals were randomly divided into six-groups(n=7); Control (C; 0.9% NaCl, i.p), D-Glutamic Acid (G; 4 mg/kg, i.p), Apelin-13 (A; 300 µg/kg, i.p), D-Glutamic Acid+Apelin-13 (GA), GA+NPY2 receptor(NPY2R) antagonist (GAN2; 1,5 mg/kg, i.p) and GA+NPY5 receptor(NPY5R) antagonist (GAN5; 1,5 mg/kg, i.p). Short-long term memory, learning performance, allocentric-egocentric orientation, locomotor activity were evaluated with Open field (OFT), novel object recognition (NORT), Morris water maze (MWM) tests.\\u003c/p\\u003e\\n\\u003cp\\u003eIn group G, there was an increase in Caspase-3 level(p\\u0026lt;0.001), while significant decrease (p\\u0026lt;0.001) was observed in Extracellular Signal Regulatory Kinase (ERK1/2) and Protein Kinase B-1(AKT-1) levels. Increased mitochondrial dysfunction indicated neurodegeneration due to excitotoxicity. In MWM an increase latency to the target quadrant (p\\u0026lt;0.001), a decrease in the NORT discrimination index (p\\u0026lt;0.001) were found. Apelin-13 was observed to have a neuroprotective role by alleviating the damage in GA group. While the protective effect of Apelin-13 was not observed in the presence of NPY2R antagonist; when NPY5R antagonist was applied, more pronounced neuroprotection was detected in GAN5 group compared to GAN2, since NPY2R activity continued. Histochemical staining-scorings showed that protection of Apelin-13 was mediated by NPY2R.\\u003c/p\\u003e\\n\\u003cp\\u003eApelin-13 exerts its neuroprotective effects primarily through NPY2R, its modulatory influence via NPY5R appears to be comparatively limited.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Apelin-13 confers Neuropeptide Y–mediated neuroprotection and preserves learning and allocentric memory in D-glutamic acid-induced excitotoxicity in rats\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-06-02 15:17:49\",\"doi\":\"10.21203/rs.3.rs-6635799/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2025-06-20T08:21:55+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-06-19T21:22:56+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-06-18T10:53:40+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-06-16T20:24:32+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"224454659573252277109415674122657713138\",\"date\":\"2025-06-11T17:56:29+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"323773878736201485751697255417560601209\",\"date\":\"2025-06-10T13:56:07+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"221180614710899122735898056422297943386\",\"date\":\"2025-06-10T06:20:15+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"14281903907587485870560549004621039087\",\"date\":\"2025-06-02T09:41:01+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-05-30T13:36:27+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-05-24T01:08:12+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2025-05-24T01:07:13+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Molecular Neurobiology\",\"date\":\"2025-05-10T15:41:57+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"molecular-neurobiology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"moln\",\"sideBox\":\"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)\",\"snPcode\":\"12035\",\"submissionUrl\":\"https://submission.nature.com/new-submission/12035/3\",\"title\":\"Molecular Neurobiology\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"3831d7b7-0657-472d-b4f9-76b35ddb706d\",\"owner\":[],\"postedDate\":\"June 2nd, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-01-26T16:02:00+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-6635799\",\"link\":\"https://doi.org/10.1007/s12035-026-05685-3\",\"journal\":{\"identity\":\"molecular-neurobiology\",\"isVorOnly\":false,\"title\":\"Molecular Neurobiology\"},\"publishedOn\":\"2026-01-22 15:57:26\",\"publishedOnDateReadable\":\"January 22nd, 2026\"},\"versionCreatedAt\":\"2025-06-02 15:17:49\",\"video\":\"\",\"vorDoi\":\"10.1007/s12035-026-05685-3\",\"vorDoiUrl\":\"https://doi.org/10.1007/s12035-026-05685-3\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6635799\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6635799\",\"identity\":\"rs-6635799\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}