Current Issues in Physiology II

CURRENT ISSUES IN PHYSIOLOGY II Editor Hatice YORULMAZ Lyon 2025 CURRENT ISSUES IN PHYSIOLOGY II Editor Hatice YORULMAZ Lyon 2025 Current Issues in Physiology II Editor • Prof. Dr. Hatice YORULMAZ • Orcid: 0000-0002-0550-9899 Cover Design • Motion Graphics Book Layout • Motion Graphics First Published • December 2025, Lyon e-ISBN: 978-2-38236-969-2 DOI: 10.5281/zenodo.18006901 copyright © 2025 by Livre de Lyon All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without prior written permission from the Publisher. The author or authors of the relevant section are responsible for any copyright infringement that may occur due to the images and graphics used in the book. The editor or publisher does not assume responsibility in this regard. Publisher • Livre de Lyon Address • 37 rue marietton, 69009, Lyon France website • http://www.livredelyon.com e-mail • [email protected] I PREFACE Dear Readers, Physiology is a dynamic and evolving field that deepens our understanding of life, from the cellular processes to the complexities of whole systems. This book, Current Issues in Physiology II, is a continuation of the journey initiated in the first volume. It brings together the latest advancements and pressing challenges in physiological science, providing essential insights for both academic researchers and healthcare professionals. The aim of this work is to explore cutting-edge topics that reflect the current frontiers of physiological research, presenting diverse viewpoints and serving as a reliable reference in the field. Each chapter has been meticulously crafted by experts who are leaders in their respective areas, ensuring that the content is scientifically rigorous and relevant to contemporary discussions. It is my hope that this book will be a valuable resource for graduate students, researchers, and anyone with an interest in the ever-evolving world of physiology. I extend my deepest thanks to all the authors who contributed to this book, my colleagues for their unwavering academic support, and the institutions whose resources made this work possible. May this book inspire further inquiry and contribute to the growth of knowledge in the field of physiology. Prof. Dr. Hatice YORULMAZ Haliç University Faculty of Medicine, Department of Physiology Istanbul, Türkiye III CONTENTS PREFACE I CHAPTER I. DISRUPTION OF CRITICAL PHYSIOLOGICAL PROCESSES: A CURRENT PERSPECTIVE ON SEPSIS PATHOPHYSIOLOGY 1 Hatice YORULMAZ CHAPTER II. PATHOPHYSIOLOGICAL RESPONSES OF ORGAN SYSTEMS DURING SEPSIS 19 Hatice YORULMAZ CHAPTER III. ENVIRONMENTAL POLLUTANTS AND THEIR EFFECTS ON PHYSIOLOGICAL SYSTEMS: EVIDENCE FROM EXPERIMENTAL ANIMAL MODELS 31 Ferda PERÇİN PAÇAL CHAPTER IV. ER DRIVEN MOLECULAR SIGNATURES OF 53 EPILEPTOGENESIS: A UNIFIED MODEL OF PERK, IRE1 AND ATF6 PATHWAYS Sabriye KARADENİZLİ TAŞKIN CHAPTER V. THE PHYSOLOGY OF OLFACTORY MEMORY 69 FORMATION AND RETRIEVAL IN MAMMALIAN SYSTEM Mustafa BARAN CHAPTER VI. ELECTROPHYSIOLOGICAL MEASUREMENT OFOLFACTORY FUNCTION 81 Mustafa BARAN CHAPTER VII. PHYSIOLOGY OF AUTISM SPECTRUM DISORDER: CURRENT ISSUES AND EMERGING MECHANISM 93 Burcu ÇEVRELİ CHAPTER VIII. THE DUAL DEFENSE MECHANISM OF EXERCISEINDUCED MYOKINES AGAINST NEURODEGENERATION 113 Atakan ÖZTÜRK CHAPTER IX. STZ INDUCED DIABETES MELLITUS MODELS IN EXPERIMENTAL ANIMALS 127 Deniz ÖNAL DISRUPTION OF CRITICAL PHYSIOLOGICAL PROCESSES . . .   7 Bacterial components stimulate both immune and endothelial cells, prompting cytokine production that facilitates immune cell migration. This process is accompanied by a marked upregulation of adhesion molecules, including selectins, integrins, and members of the immunoglobulin superfamily such as intercellular adhesion molecules (ICAMs) and vascular cell adhesion molecule-1 (VCAM-1). Additionally, chemokines secreted by immune and endothelial cells such as CXCL8, CXCL1, CXCL2, and CXCL5 create a gradient that recruits neutrophils from bone marrow reserves and promotes their adhesion. Endothelial P-selectin not only captures leukocytes and aids their rolling along the vessel wall but also activates integrins through P-selectin glycoprotein ligand-1 (PSGL-1), further enhancing leukocyte activation (17,18). In the context of sepsis, the inflammatory response of the endothelium contributes to oxidative damage by elevating the production of reactive oxygen species (ROS) and reactive nitrogen species like nitric oxide (NO). ROS generated by immune and endothelial cells, especially within endothelial mitochondria, impair mitochondrial function, diminish antioxidant defenses, and intensify inflammation. These combined effects cause shedding of the endothelial glycocalyx, upregulation of adhesion molecules, increased endothelial permeability, and apoptosis of endothelial cells. Chemokines secreted at the site of inflammation recruit immune cells from the bone marrow. Imbalances in endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) result in excessive NO production, causing vasodilation. Endothelial injury contributes to hemodynamic instability and may ultimately lead to organ failure (19,20). The endothelial glycocalyx, located at the boundary between blood flow and the vessel wall, is essential for preserving the integrity of the endothelial barrier. This structure is made up of a negatively charged network comprising membranebound proteoglycans, glycoproteins, glycolipids, glycosaminoglycans like heparan sulfate, and various plasma proteins (21). Alterations in the glycocalyx caused by sepsis play a significant role in promoting endothelial damage and impairing microvascular function. In particular, increased levels of matrix metalloproteinases and reactive oxygen species lead to the degradation of the glycocalyx structure. Molecules such as heparan sulfate, hyaluronan, and sialic acid, which are integral components of the glycocalyx, are released into the plasma. This process is commonly referred to as “shedding” (22). During this progression, excessive NO production, heightened ROS activity, and loss of the endothelial glycocalyx contribute significantly to endothelial dysfunction. 8   CURRENT ISSUES IN PHYSIOLOGY II 4.2 Coagulopathy and disseminated intravascular coagulation development Sepsis triggers a complex interplay between inflammation and coagulation pathways, frequently leading to coagulopathy characterized by dysregulated clot formation and breakdown. This imbalance can escalate into disseminated intravascular coagulation (DIC), a severe and often fatal complication marked by widespread microvascular thrombosis and subsequent bleeding due to consumption of clotting factors. PAMPs and DAMPs activate both monocytes/ macrophages and endothelial cells. Endothelial cells function as regulators of coagulation-anticoagulation balance through molecules such as thrombomodulin and endothelial protein C receptor (EPCR), which become disrupted during sepsis. The inflammatory response and altered release of adhesion molecules during sepsis create a prothrombotic and antifibrinolytic environment, leading to microvascular thrombosis, tissue ischemia, and multiple organ dysfunction syndrome (MODS). This activation notably increases the expression of tissue factor (TF) via proinflammatory cytokines such as IL-6, IL-1, and TNF-α. TF triggers the extrinsic coagulation pathway, resulting in thrombin generation. This process is associated with upregulated expression of endothelial adhesion molecules like VCAM-1 and ICAM-1, as well as coagulation factors including von Willebrand factor (VWF), TF and plasminogen activator inhibitor-1 (PAI-1) Moreover, coagulation factors such as thrombin and factor Xa can independently stimulate cytokine production through protease-activated receptors (PARs), thereby further amplifying the inflammatory response (23, 24). Activation of platelets and coagulation factors contributes to sepsis-induced coagulopathy and the formation of microthrombi. As these elements become depleted, the balance between coagulation and fibrinolysis is disrupted, leading to DIC. Additionally, mitochondrial damage, neuroendocrine dysregulation, and endoplasmic reticulum stress may also contribute to coagulation dysfunction. (24). Platelet activation begins when platelets bind to exposed collagen and von Willebrand factor (vWF) in the subendothelial matrix through the glycoprotein Ib-IX-V complex (GPIb-IX-V) on their surface. This interaction triggers the release of substances such as adenosine diphosphate (ADP), thromboxane A2 (TXA2), and platelet factor 4 (PF4), which enhance platelet activation and aggregation, eventually resulting in thrombus formation. Damage to the endothelium and trauma activate both intrinsic and extrinsic coagulation pathways (25). Tissue factor expression is continuously increased under proinflammatory conditions, further promoting coagulation and inflammation. DISRUPTION OF CRITICAL PHYSIOLOGICAL PROCESSES . . .   9 During sepsis, levels and activity of coagulation inhibitors like Tissue Factor Pathway Inhibitor (TFPI) decline. Additionally, proinflammatory cytokines including TNF-α and IL-6 inhibit antithrombin production and accelerate its breakdown, while also reducing its ability to bind coagulation factors effectively (26). During sepsis, the overactivation of coagulation factors results in decreased levels of Activated Protein C (APC). Like antithrombin, APC’s structure and function are altered by inflammatory cytokines, which reduce its binding affinity for coagulation factors and impair its capacity to deactivate factors Va and VIIIa, thereby worsening coagulation. Moreover, elevated levels of proinflammatory cytokines disrupt APC’s interaction with protease-activated receptor-1 (PAR-1), weakening its anti-inflammatory and anticoagulant functions (27). Additionally, while fibrinolysis can be activated, levels of plasminogen activator inhibitor-1 (PAI1) are typically elevated in sepsis, which inhibits fibrin degradation. This results in a state known as “fibrinolytic shutdown.” Such suppression leads to the accumulation of fibrin within the microvasculature and impairs organ perfusion. Moreover, the disruption of the endothelial glycocalyx compromises its antithrombotic barrier function, promoting platelet adhesion and enhancing surface-mediated coagulation factor reactions (24). Neutrophil extracellular traps (NETs), formed upon neutrophil activation, consist of DNA, histones, and granule-derived components. These structures exhibit strong procoagulant properties. Upon activation, neutrophils release NETs containing DNA and histones, which act as damage-associated molecular patterns (DAMPs), directly trigger coagulation, and amplify inflammation through mechanisms such as tissue factor (TF) induction and platelet adhesion. Notably, extracellular histones contribute significantly to endothelial damage in this context (28). Understanding the mechanisms underlying coagulopathy and DIC in sepsis is essential for early diagnosis and targeted therapeutic interventions, as these conditions significantly contribute to organ dysfunction and mortality in septic patients. 5. Mitochondrial Dysfunction and Cellular Energy Deficiency in Sepsis In sepsis, inflammation and immune responses increase the production of reactive oxygen and nitrogen species (ROS/RNS). These reactive species damage mitochondrial proteins, lipids, and mitochondrial DNA (mtDNA). The balance between the clearance of damaged mitochondria through selective 10   CURRENT ISSUES IN PHYSIOLOGY II autophagy (mitophagy) and the biogenesis of new mitochondria is crucial. Mitophagy pathways, such as the PINK1/Parkin pathway, may be suppressed in sepsis, leading to the accumulation of dysfunctional mitochondria. Confirmed decreases in the activities of mitochondrial respiratory complexes I, III, and IV, as well as reductions in oxygen consumption, have been observed. Additionally, a decline in mitochondrial inner membrane potential (ΔΨm), increased proton leak, heightened mitochondrial membrane permeability, and the opening of the mitochondrial permeability transition pore (mPTP) contribute to impaired ATP production and cellular energy imbalance. Mitochondrial dysfunction results in insufficient ATP synthesis, adversely affecting cellular processes such as ion pumping, cell repair, apoptosis, and necrosis. This energy deficit is especially detrimental in high-energy demanding tissues, such as skeletal muscle, leading to functional impairment (29,30). Under normal physiology, cells convert glucose to pyruvate in the presence of oxygen and generate ATP through oxidative phosphorylation within the mitochondria. However, in sepsis, hypoperfusion and microcirculatory disturbances impair adequate oxygen delivery to tissues. In conditions of oxygen deficiency, the conversion of pyruvate to lactate increases. During this process, ATP production decreases while lactate and hydrogen ion (H⁺) accumulation leads to metabolic acidosis (31). In sepsis, not only oxygen deficiency but also mitochondrial dysfunction plays a critical role. Even when oxygen reaches the cell, it may not be effectively utilized within the mitochondria—a condition known as cytopathic hypoxia. Consequently, oxygen consumption decreases, the cell fails to meet its energy demands, and becomes reliant on anaerobic glycolysis. Endogenous and/or therapeutically administered catecholamines (particularly adrenaline) in sepsis increase the rate of glycolysis via β₂-adrenergic receptors, leading to elevated pyruvate production. If pyruvate cannot enter the mitochondria, lactate production rises. This can result in increased lactate levels despite adequate oxygen availability. Hepatic perfusion is reduced in sepsis, impairing mitochondrial function in the liver and decreasing lactate clearance, thereby elevating plasma lactate concentrations. Recent studies have demonstrated that activated immune cells in sepsis (such as macrophages and T cells) metabolize glucose through glycolysis even in aerobic conditions a phenomenon known as the Warburg effect producing high amounts of lactate. Moreover, lactate may act as a signaling molecule in this context. (1,32,33) In sepsis, lactic acidosis is not solely a consequence of the shift to anaerobic metabolism; it also results from multiple mechanisms, including mitochondrial DISRUPTION OF CRITICAL PHYSIOLOGICAL PROCESSES . . .   11 dysfunction, catecholamine stimulation, immune cell activation, and reduced lactate clearance. Therefore, lactate levels reflect not only tissue hypoxia but also the overall status of cellular metabolism. Lactate monitoring holds critical diagnostic and prognostic value in the management of sepsis (34). In summary, sepsis is characterized by mitochondrial damage and impaired biogenesis, leading to oxidative stress, reduced ATP production, a metabolic shift toward anaerobic glycolysis, and the subsequent development of lactic acidosis. 6. Impairment of Neuroendocrine and Metabolic Responses in Sepsis During the early phase of sepsis, the body undergoes metabolic and hormonal adaptations to cope with the infection. Signals reaching the brain during this process originate from various sources, including stimulation of the vagus nerve and the release of PAMPs, DAMPs, cytokines, and other inflammatory or neurotoxic molecules. Among these, the proinflammatory cytokine IL-6 plays a crucial role by activating the hypothalamic-pituitary-adrenal (HPA) axis. IL-6 also acts as a potent pyrogen by interacting with hypothalamic centers to induce fever (35). These afferent signals can then provoke efferent responses from the central nervous system, activating multiple neurohormonal pathways such as the autonomic nervous system, HPA axis, hypothalamic-pituitary-thyroid (HPT) axis, somatotropic axis, and gonadal axis. In the HPA axis, cortisol levels are markedly elevated due to both increased production and decreased cortisol degradation. Cortisol levels remain high during the chronic phase of sepsis. While elevated cortisol confers benefits such as enhanced energy production, maintenance of vascular tone, and regulation of inflammation, it may also lead to adverse effects including hyperglycemia, muscle catabolism, and increased risk of infection (36). In the hypothalamic-pituitary-thyroid (HPT) axis during the acute phase, T3 levels decrease while T4 and TSH levels initially show a slight increase before returning to normal. The reduction in T3 persists even when TSH remains within normal ranges. In the chronic phase of sepsis, with disease progression, both T3 and T4 levels further decline, accompanied by suppression of TSH and TRH. This reflects central (hypothalamic/pituitary) suppression and indicates a resetting of the HPT axis set point (37). Regarding neuroendocrine alterations, studies have shown an initial increase followed by a decrease in plasma vasopressin (AVP) concentrations, which may be associated with septic shock. Inadequate vasopressin levels during septic shock can contribute to hypotension and impaired circulation (38). In the somatotropic axis, growth hormone 12   CURRENT ISSUES IN PHYSIOLOGY II secretion is altered during the acute phase of sepsis. Lipolysis increases and glucose levels rise. In patients with sepsis and septic shock, levels of insulin-like growth factor-I (IGF-I) and its binding proteins decrease further. In the chronic phase, growth hormone secretion is significantly reduced. This contributes to hypercatabolism, impaired tissue regeneration, and the development of “wasting syndrome” characterized by muscle and energy loss (39). Hyperglycemia is a common metabolic response during sepsis. Intracellular stress, oxidative damage, and mitochondrial dysfunction can impair insulin signaling pathways. Normally, insulin promotes the translocation of GLUT4 transporters to the cell membrane in muscle and fat tissues, but this process is disrupted during sepsis. Additionally, phosphorylation of insulin receptor substrates (IRS), particularly on tyrosine residues, is reduced, weakening the insulin signaling cascade (40,41). In sepsis, levels of adipokines such as adiponectin, resistin, leptin, and MCP-1 are altered, generally contributing to reduced insulin sensitivity. Elevated leptin levels have also been reported, which can influence appetite, energy expenditure, and immune responses. Increased insulin resistance in sepsis has been associated with higher mortality and complications. When hyperglycemia and insulin resistance coexist in critically ill patients, it may lead to adverse outcomes such as kidney failure, prolonged hospital stays, and the need for mechanical ventilation (42-44). In summary, sepsis triggers a neuroendocrine response characterized by two distinct phases. Initially, acute neuroendocrine alterations appear to be adaptive, helping to reestablish balance and conserve energy. However, in the prolonged stage of critical illness, central suppression of neuroendocrine pathways may lead to maladaptive effects (41). More studies are required to assess whether administering hypothalamic releasing hormones could improve patient outcomes. Disruptions or resistance in hormonal signaling during sepsis impair immune function, thereby raising the risk of secondary infections. 7. Recent Advances in the Pathophysiology of Sepsis 7.1. Reprogramming in the Immune Response (Trained Immunity and Tolerance) In sepsis, the well-known process involves an initial hyperactivation of the immune system followed by immune paralysis. Recent studies have demonstrated that monocytes and macrophages develop “trained immunity” through epigenetic reprogramming. These findings reveal that sepsis is not DISRUPTION OF CRITICAL PHYSIOLOGICAL PROCESSES . . .   13 merely a state of hyperinflammation but also a response that is reprogrammed at the cellular level (45). 7.2. Mitochondrial Dysfunction and Metabolic Reprogramming Cellular energy imbalance in sepsis is directly linked to mitochondrial dysfunction. Recent evidence indicates that the balance between glycolysis and oxidative phosphorylation shifts, contributing to cellular dysfunction and organ failure. Mitochondria-targeted therapies, such as peptides like SS-31, have begun to emerge in clinical research (46). 7.3. Endothelial Dysfunction and Glycocalyx Damage In sepsis, disruption of the vascular endothelial barrier is exacerbated by degradation of the glycocalyx layer. This leads to increased vascular permeability and subsequent tissue hypoperfusion. New biomarkers, such as Syndecan-1, have begun to be utilized to assess this process (47). 7.4. The Role of the Microbiome The gut microbiome is essential for modulating the immune system. Alterations in the microbiota during sepsis can impact systemic inflammation and immune responses, prompting investigation into treatments like probiotics and microbiota-targeted therapies (48). 7.5. Artificial Intelligence-Assisted Diagnosis and Risk Scoring AI-based systems have enhanced the accuracy of early diagnosis and mortality prediction in sepsis. In particular, models that extract meaningful patterns from electronic health record data form the foundation of clinical decision support systems for early sepsis detection (49). 8. Conclusion: Sepsis is a complex and multifaceted pathophysiological process. It can be conceptualized as having two phases: an early phase characterized by excessive activation of the inflammatory response, and a late phase marked by lymphocyte apoptosis and immune paralysis. The early acute phase is dominated by the stress response, with active hormone-metabolic mechanisms that facilitate adaptation. This phase emphasizes energy mobilization and strategies to manage and sustain inflammation. In the critical or prolonged illness phase, immune 14   CURRENT ISSUES IN PHYSIOLOGY II suppression, hormonal deficiencies or resistance, depletion of energy reserves, chronic catabolic state, and organ dysfunction occur, during which adaptation becomes detrimental. Today, sepsis is understood not merely as an exaggerated immune response to infection but as a systemic syndrome involving profound disturbances in cellular, metabolic, and microbial homeostasis. Recent research has expanded our understanding of sepsis pathogenesis, highlighting the central roles of epigenetic modifications, mitochondrial dysfunction, microbiota interactions, and endothelial barrier damage in this process. These findings pave the way for better comprehension of sepsis and the development of personalized therapeutic strategies. However, despite these advances, sepsis remains a global health challenge with high mortality. Therefore, the effective translation of basic scientific discoveries into clinical practice and the adoption of multidisciplinary approaches are vital in the fight against sepsis. References 1. Singer M, Deutschman CS, Seymour CW, et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis3). JAMA. 2016;315(8):801810. doi:10.1001/jama.2016.0287 2. Rudd KE, Johnson SC, Agesa KM, et al. Global, regional, and national sepsis incidence and mortality, 1990–2017: analysis for the Global Burden of Disease Study. Lancet. 2020;395(10219):200-211. doi:10.1016/ S0140-6736(19)32989-7 3. Baykara N, Akalın H, Arslantaş MK, et al. Epidemiology of sepsis in intensive care units in Turkey: a multicenter, pointprevalence study. Crit Care. 2018;22(1):93. doi:10.1186/s13054-018-2013-1 4. Cicchinelli S, Pignataro G, Gemma S, et al. 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During Sepsis and COVID19, the ProInflammatory and AntiInflammatory Responses Are Concomitant. Clin Rev Allergy Immunol. 2023;65(2):183187. doi:10.1007/s12016-023-08965-1 16. Ahuja K, Nguyen AP, Lee EP, et al. TIGIT+ Regulatory T cells contribute to immune dysfunction in latestage sepsis. J Immunol. 2023;210:99110. doi:10.3390/biomedicines12112420 17. Aziz M, Jacob A, Yang WL, Matsuda A, Wang P. Current trends in inflammatory and immunomodulatory mediators in sepsis. J Leukoc Biol. 2013;93(3):329342. doi:10.1189/jlb.0912437 18. Kolaczkowska E, Kubes P. Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol. 2013;13(3):159175. doi:10.1038/ nri3399 19. Tang F, Liu D, Zhang L, et al. Targeting endothelial cells with golden spice curcumin: A promising therapy for cardiometabolic multimorbidity. Pharmacol Res. 2023;197:106953. doi:10.1016/j.phrs.2023.106953 16   CURRENT ISSUES IN PHYSIOLOGY II 20. Dolmatova EV, Wang K, Mandavilli R, Griendling KK. The effects of sepsis on endothelium and clinical implications. Cardiovasc Res. 2021;117(1):6073. doi:10.1093/cvr/cvaa070 21. Chelazzi C, Villa G, Mancinelli P, De Gaudio AR, Adembri C. Glycocalyx and sepsisinduced alterations in vascular permeability. Crit Care. 2015;19(1):26. doi:10.1186/s13054-015-0741-z 22. Sullivan RC, Rockstrom MD, Schmidt EP, Hippensteel JA. Endothelial glycocalyx degradation during sepsis: Causes and consequences. Matrix Biol Plus. 2021;12:100094. doi:10.1016/j.mbplus.2021.100094 23. Vassiliou AG, Mastora Z, Orfanos SE, et al. Elevated biomarkers of endothelial dysfunction/activation at ICU admission are associated with sepsis development. Cytokine. 2014;69(2):240247. doi:10.1016/j.cyto.2014.06.010 24. Tsantes AG, Parastatidou S, Tsantes EA, et al. SepsisInduced Coagulopathy: An Update on Pathophysiology, Biomarkers, and Current Guidelines. Life. 2023;13(2):350. doi:10.3390/life13020350 25. Bloom SI, Islam MT, Lesniewski LA, Donato AJ. Mechanisms and consequences of endothelial cell senescence. Nat Rev Cardiol. 2023;20(1):3851. doi:10.1038/s41569-022-00739-0 26. Wiedermann CJ. Clinical review: molecular mechanisms underlying the role of antithrombin in sepsis. Crit Care. 2006;10(1):209. doi:10.1186/ cc4822 27. Wilhelm AR, Parsons NA, SamelsonJones BJ, et al. Activated protein C has a regulatory role in factor VIII function. Blood. 2021;137(18):25322543. doi:10.1182/blood.2020007562 28. Zhu L, Dong H, Li L, Liu X. The Mechanisms of Sepsis Induced Coagulation Dysfunction and Its Treatment. J Inflamm Res. 2025;18:14791495. doi:10.2147/jir.s504184 29. Garrabou G, Morén C, López S, et al. The effects of sepsis on mitochondria. J Infect Dis. 2012;205(3):392400. doi:10.1093/infdis/jir764 30. Hu D, Sheeja Prabhakaran H, Zhang YY, Luo G, He W, Liou YC. Mitochondrial dysfunction in sepsis: mechanisms and therapeutic perspectives. Crit Care. 2024;28:292. doi:10.1186/s13054-024-05069-w 31. Liu S, Yang T, Jiang Q, et al. Lactate and Lactylation in Sepsis: A Comprehensive Review. J Inflamm Res. 2024;17:44054417. doi:10.2147/jir. s459185 32. GarciaAlvarez M, Marik P, Bellomo R. Sepsisassociated hyperlactatemia. Crit Care. 2014;18(5):503. doi:10.1186/s13054-014-0503-3 PATHOPHYSIOLOGICAL RESPONSES OF ORGAN SYSTEMS DURING SEPSIS   23 pathogen-associated molecules reach the liver through two circulatory routes and are recognized by pattern recognition receptors on Kupffer cells or stellate cells. This recognition triggers inappropriate immune responses or excessive inflammation. One characteristic of this immune response is the increase in acute-phase proteins, which contribute to immunosuppression and endotoxin tolerance. Sepsis induces a shift of Kupffer cells towards the pro-inflammatory M1 phenotype, amplifying the inflammatory response and acute liver injury. Studies have shown that pharmacological inhibition of M1 polarization can mitigate liver damage. Sepsis-associated cholestasis disrupts glycolipid metabolism in the liver and promotes cell death pathways. Additionally, a reduction in bile acids within the enterohepatic circulation facilitates translocation of gut bacteria to the liver, thereby increasing mortality (15,16). Cell death and impaired immune functions in hepatocytes during sepsis also result from disrupted autophagy and oxidative stress. Autophagy is responsible for clearing damaged organelles within cells; however, this process is impaired in sepsis, leading to accumulation of dysfunctional mitochondria and the generation of free radicals. These free radicals activate the NF-κB pathway, which further amplifies inflammation and exacerbates liver injury. Treatments aimed at enhancing autophagy, such as rapamycin, have been shown in animal studies to reduce hepatocyte death and improve liver function, though their efficacy in humans remains under investigation. Additionally, elevated levels of IL-6 during sepsis are particularly associated with severe acute respiratory distress syndrome (ARDS), highlighting the need for early therapies targeting IL-6. High TNF-α levels indicate damage to the heart and kidneys, suggesting that anti-cytokine treatments may help preserve myocardial and renal function. Increased IL-18 in the kidneys points to pyroptosis, a form of programmed cell death, as a potential therapeutic target. Biomarkers such as procalcitonin and C-reactive protein (CRP) assist in estimating the severity of infections and organ damage. Clinically, monitoring cytokine levels plays a crucial role in risk assessment and the development of personalized treatment plans for patients (17). During sepsis, the detrimental effects of pathogens and the inflammatory response on the liver are exacerbated by mechanisms such as disruption of the gutliver axis, M1 polarization of Kupffer cells, impaired autophagy, and increased oxidative stress. These processes contribute both to acute liver dysfunction and the worsening of systemic inflammation. Therefore, it is critically important to develop multifaceted therapeutic strategies that target inflammatory signaling 24   CURRENT ISSUES IN PHYSIOLOGY II pathways, regulate autophagy, and maintain intestinal barrier integrity in order to preserve liver function and prevent organ damage during sepsis. 5. Renal Dysfunction in Sepsis Sepsis-induced acute kidney injury is characterized by endothelial dysfunction leading to impaired renal microvascular function. This dysfunction manifests through increased expression of adhesion molecules, resulting in leukocyte adhesion to the vessel walls, excessive production of tissue factor and microthrombus formation, as well as glycocalyx degradation. Additionally, decreased endothelial nitric oxide synthase (eNOS) activity in small arterioles and glomerular endothelial cells reduces glomerular blood flow. While total renal blood flow may not always decrease in sepsis, blood flow distribution between the cortex and medulla becomes imbalanced. Furthermore, insufficient perfusion of peritubular capillaries in cortical glomeruli is associated with endothelial oxidative damage. Endothelial ion channels, especially in small arteries and glomeruli, play a critical role in regulating renal microvascular permeability. The TRPM2 channel activates the TLR4/NOX-2/ROS/NF-κB pathway during sepsis, increasing glomerular endothelial permeability. Similarly, the TRPV4 channel facilitates Ca²⁺ influx, triggering the NF-κB/IRF-3 pathway and promoting glomerular endothelial inflammation (6,18). Mitochondrial dysfunction occurs during sepsis, leading to decreased ATP production and resulting in cellular energy deficiency. This energy shortage causes cessation of cellular functions and initiates cell death pathways. In response to injury, renal tubular cells halt their cell cycle in either the G1 or G2 phase as a protective mechanism. Damage is especially pronounced in the tubulointerstitial region. While G1 phase arrest is considered an early protective response following injury, prolonged or severe damage involving G2 phase arrest is associated with maladaptive repair processes (19,20). In a multicenter study conducted in Turkey involving 776 patients, sepsis was found to play a significant role in acute kidney injury. Among the patients, 33.6% required renal replacement therapy, and during a six-month follow-up period, the mortality rate was 24.1%. Additionally, 9.5% of the patients progressed to end-stage renal disease (21). Early diagnosis and treatment focus on rapid infection control, appropriate antimicrobial therapy, fluid resuscitation to stabilize hemodynamics, and vasopressor use when necessary. Potential future therapeutic approaches under investigation include drugs targeting cell cycle arrest, agents that inhibit apoptotic PATHOPHYSIOLOGICAL RESPONSES OF ORGAN SYSTEMS DURING SEPSIS   25 pathways, removal of toxic mediators through methods such as hemofiltration and adsorption, and strategies aimed at endothelial protection (22). 6. Acute Respiratory Failure and ARDS in Sepsis: Effects on Pulmonary Physiology In sepsis, the lungs are among the earliest and most severely affected organs. The clinical presentation of lung injury typically manifests as acute lung injury (ALI) or its more severe form, acute respiratory distress syndrome (ARDS). Mortality rates are high. The injury leads to disruption of the alveolarcapillary barrier integrity, resulting in increased vascular permeability and leakage of fluid and proteins into the alveoli. Interleukin-6 (IL-6) plays a critical role in endothelial barrier breakdown and the development of pulmonary edema. Neutrophil extracellular traps (NETs) contribute to excessive vascular inflammation, capillary obstruction, and impaired oxygen exchange in sepsisassociated lung injury (23) Microvascular occlusions, fibrin deposition, and the development of antifibrinolytic states cause damage within the pulmonary microvascular bed. Surfactant protein production in alveolar epithelial cells is impaired, leading to a reduced capacity for alveolar fluid clearance. Disruption of cell-to-cell junctions, epithelial injury, and apoptosis/necroptosis severely affect alveolar functions. Notably, necroptosis is associated with mitochondrial damage and mitochondrial DNA leakage, activating the STING and RIPK1-RIPK3-MLKL signaling pathways. This exacerbates injury in both alveolar and endothelial cells. Recent studies around 2025 indicate that modifications such as H3K14 histone lactylation in respiratory endothelial cells contribute to the regulation of genes related to ferroptosis. Inhibition of these pathways has been observed to reduce endothelial activation and mitigate lung injury (23-25). Recent molecular studies have demonstrated that the Spns2/S1P (Sphingosine-1-Phosphate) signaling pathway regulates immune responses in sepsis-induced lung injury. S1P is a bioactive lipid mediator that controls the migration and function of immune cells. During sepsis, the Spns2 transporter modulates the release of S1P from alveolar macrophages, thereby enhancing their bacterial clearance capacity. Interestingly, inhibition of this pathway (Spns2 suppression) has been shown to increase macrophage phagocytic activity without triggering excessive inflammation. This suggests that targeting the Spns2/S1P axis could offer a novel therapeutic approach for regulating organspecific immune responses in ARDS treatment (26,27). 26   CURRENT ISSUES IN PHYSIOLOGY II In summary, ARDS developing during sepsis results from complex immune responses, cytokine release, neutrophil activation, increased vascular permeability, and epithelial damage. Efforts are ongoing to discover agents that protect endothelial barrier function by targeting pathways such as histone modification regulation, ferroptosis inhibition, and NET modulation. 7. Conclusion Sepsis is more than a systemic inflammatory response to infection; it is a multifaceted process involving integrated dysfunction of multiple organs and systems, including the immune, coagulation, endocrine, circulatory, and nervous systems. Dysfunction in one organ system (such as acute kidney injury) adversely affects the function of other organs (such as the lungs or heart) through both direct metabolic imbalances (electrolyte, acid-base disturbances) and inflammatory signaling. These interactions are referred to as “organ-organ cross-talk.” For example, acute kidney injury leads to increased inflammatory cytokines, resulting in pulmonary edema and ARDS; sepsisassociated cardiac dysfunction causes hypoperfusion, which in turn may lead to kidney and liver failure. MODS (Multiple Organ Dysfunction Syndrome) in sepsis is a progressive and often irreversible clinical condition. Therefore, early physiological monitoring is crucial to detect “silent organ dysfunctions” before they fully develop. Instead of relying solely on biomarkers that directly reflect organ damage, regular monitoring of real-time physiological parameters such as hemodynamics, perfusion, respiratory mechanics, oxygen consumption (VO₂), lactate levels, and urine output is essential (28,29) In particular, physiologically targeted treatment strategies include preserving endothelial stability (targeting molecules such as adrenomedullin, S1P, angiopoietins), immunomodulation (cytokine blockers, treatments for immune paralysis), managing coagulopathy (preventing microangiopathy related to DIC), and correcting mitochondrial dysfunction (e.g., mitochondriatargeted antioxidants). Personalized therapeutic approaches should be shaped by the patient’s physiological status and pathophysiological responses, rather than relying solely on classical protocols (30-32). Sepsis is not merely a singular infection response but a systemic process involving a complex physiological chain reaction among interacting organ systems. This dynamic network of interactions sets the stage for progressive organ dysfunction, ultimately leading to MODS. Therefore, clinical monitoring requires not only the evaluation of organ outputs but also a systems-level approach PATHOPHYSIOLOGICAL RESPONSES OF ORGAN SYSTEMS DURING SEPSIS   27 grounded in understanding the underlying pathophysiological mechanisms. In the prevention and management of MODS, early interventions aimed at preserving physiological integrity and integrated with pathophysiological insights have become one of the most critical factors determining patient prognosis. In this context, sepsis management must be reconsidered from a multidisciplinary perspective, where physiological monitoring data form the foundation for personalized and targeted interventions. References 1. Borges A, Bento L. Organ crosstalk and dysfunction in sepsis. Ann Intensive Care. 2024;14(1):147. doi:10.1186/s13613-024-01377-0 2. Singer M, Deutschman CS, Seymour CW, et al. 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Therapeutic effects of orexin-A in sepsisassociated encephalopathy in mice. J Neuroinflammation. 2024;21(1):131. doi:10.1186/s12974-024-03111-w 15. Zhang X, Liu H, Hashimoto K, Yuan S, Zhang J. The gut-liver axis in sepsis: interaction mechanisms and therapeutic potential. Crit Care. 2022;26:213. doi:10.1186/s13054-022-04090-1 16. Shi L, Jin L, Huang W. Bile acids, intestinal barrier dysfunction, and related diseases. Cells. 2023;12:1888. doi:10.3390/cells12141888 17. Beyer D, Hoff J, Sommerfeld O, Zipprich A, Gaßler N, Press AT. The liver in sepsis: molecular mechanism of liver failure and their potential for clinical translation. Mol Med. 2022;28:84. 18. Molema G, Zijlstra JG, van Meurs M, Kamps JAAM. Renal microvascular endothelial cell responses in sepsis-induced acute kidney injury. Nat Rev Nephrol. 2022;18(2):95–112. 19. Moonen L, D’Haese PC, Vervaet BA. Epithelial cell cycle behaviour in the injured kidney. Int J Mol Sci. 2018;19(7):2038. doi:10.3390/ijms19072038 20. Wang WG, Sun WX, Gao BS, Lian X, Zhou HL. Cell cycle arrest as a therapeutic target of acute kidney injury. Curr Protein Pept Sci. 2017;18(12):12241231. doi:10.2174/1389203717666160915162238 PATHOPHYSIOLOGICAL RESPONSES OF ORGAN SYSTEMS DURING SEPSIS   29 21. Gursu M, Yegenaga I, Tuglular S, et al. Acute kidney injury in Turkey: epidemiological characteristics, etiology, clinical course, and prognosis. BMC Nephrol. 2022;23(1):326. doi:10.1186/s12882-022-02933-1 22. Kounatidis D, Tzivaki I, Daskalopoulou S, et al. Sepsis-associated acute kidney injury: what’s new regarding its diagnostics and therapeutics? Diagnostics. 2024;14(24):2845. doi:10.3390/diagnostics14242845 23. Xu X, Zhang Q, Lv Z, et al. Unraveling the deadly dance: endothelial cells and neutrophils in sepsis-induced acute lung injury/acute respiratory distress syndrome. Front Cell Dev Biol. 2025;13:1551138. doi:10.3389/ fcell.2025.1551138 24. Sun B, Lei M, Zhang J, Kang H, Liu H, Zhou F. 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Respir Res. 2025;26(1):54. doi:10.1186/s12931-025-03137-5 30. HoeyerNielsen AK, Holmberg MJ, Grossestreuer AV, et al. Association between the oxygen consumption: lactate ratio and survival in critically ill patients with sepsis. Shock. 2021;55(6):775-781. doi:10.1097/ shk.0000000000001661 31. Li B, Lin W, Hu R, et al. Crosstalk between lung and extrapulmonary organs in sepsisrelated acute lung injury/acute respiratory distress syndrome. Ann Intensive Care. 2025;15:97. doi:10.1186/s13613-025-01513-4 32. Chen K, Wang D, Qian M, et al. Endothelial cell dysfunction and targeted therapeutic drugs in sepsis. Heliyon. 2024;10(13):e33340. doi:10.1016/j. heliyon.2024.e33340 31 CHAPTER III ENVIRONMENTAL POLLUTANTS AND THEIR EFFECTS ON PHYSIOLOGICAL SYSTEMS: EVIDENCE FROM EXPERIMENTAL ANIMAL MODELS Ferda PERÇİN PAÇAL (Assoc. Prof. Dr.), Department of Laboratory Animal Science, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul, Turkiye e-mail:fer[email protected] ORCID:0000-0001-8583-2217 1. Introduction Rapidly intensifying anthropogenic activities, in combination with climate change and various natural processes, place increasing pressure on ecosystems and the organisms that depend on them. A wide spectrum of environmental stressors including rising global temperatures that disrupt terrestrial and aquatic habitats, and the accumulation of pollutants such as heavy metals, pesticides, plastics, pharmaceuticals, and persistent organic compounds pose substantial risks to biological systems (1). Airborne contaminants introduce toxic gases and particulates that impair respiratory and cardiovascular function, while water pollution destabilizes aquatic homeostasis and food-web Dynamics (2-4). Soil contamination further alters nutrient availability and biochemical conditions, affecting both plant communities and terrestrial fauna. At the cellular and systemic levels, these pollutants disrupt physiological homeostasis through mechanisms involving oxidative imbalance, endocrine dysregulation, metabolic pathway disturbances, and inflammatory responses. Such disruptions compromise essential biological functions including respiration, circulation, reproduction, and detoxification ultimately undermining an organism’s capacity to maintain internal stability under environmental stress (1). When these perturbations overwhelm adaptive physiological reserves, they may precipitate behavioral abnormalities, metabolic dysfunction, and organ-level 32   CURRENT ISSUES IN PHYSIOLOGY II toxicity, with cascading consequences for populations and entire ecosystems (5). The growing number of studies addressing the biological consequences of air, water, and soil pollution reflects increasing global awareness of these threats and underscores the urgency of understanding the mechanistic links between environmental degradation and organismal health. 1.1. The Role of Experimental Animal Models in Environmental Physiology Research Environmental physiology seeks to explain how organisms regulate and adjust their physiological functions in response to external variables such as temperature, humidity, oxygen levels, and atmospheric pressure. This framework is essential for interpreting how environmental stressors including pollutants shape health, disease susceptibility, and adaptive capacity. Experimental animal models have long been central to unraveling the physiological and molecular pathways through which environmental contaminants exert adverse effects on organ systems (6). By providing controlled conditions in which exposure parameters can be precisely manipulated, these models enable researchers to dissect pollutant-induced alterations in cellular signaling, organ function, metabolism, and whole-body homeostasis. A range of species including mice, rats, guinea pigs, rabbits, and zebrafish has been extensively utilized due to their well-characterized physiology, genetic tractability, and translational relevance to human health (7-10). Beyond traditional mammalian systems, fish embryos, particularly those of zebrafish, have emerged as powerful tools for environmental risk assessment. Their rapid development, optical transparency, and compatibility with small-scale, high-throughput assays allow efficient evaluation of chemical toxicity across multiple biological endpoints (11). Each animal model offers unique strengths and limitations, and appropriate selection of species, exposure routes, and study designs is critical for ensuring physiological relevance and translational value (Table 1) . Physiological approaches using experimental models allow detailed assessment of how pollutants influence cardiovascular, respiratory, endocrine, and neural systems, thereby connecting mechanistic toxicology with broader ecological and biomedical implications (12,13). As research on environmental contamination expands, animal models are increasingly applied to investigate the toxic effects of airborne, waterborne, and soil-derived pollutants including heavy metals, pesticides, microplastics, and industrial chemicals. Depending on the contaminant and exposure scenario, administration methods may include ENVIRONMENTAL POLLUTANTS AND THEIR EFFECTS ON PHYSIOLOGICAL . . .   39 that 45-day exposure to lead acetate reduced testicular mass, sperm counts, and antioxidant defenses while histological analyses revealed germ cell depletion and maturation arrest (58). Collectively, these findings consistently demonstrate that mercury, cadmium, and lead readily accumulate in reproductive tissues and trigger multi-level toxicity. The convergence of outcomes across different species and exposure routes underscores the exceptional sensitivity of reproductive organs to heavy metals and highlights the translational relevance of animal models for understanding mechanisms of human reproductive and developmental injury. 4.2. Phthalates and Endocrine Disruption Phthalates particularly DEHP represent another major class of reproductive toxicants due to their widespread use and well-established endocrine-disrupting properties. They are detectable in various environmental and dietary sources, contributing to near-universal exposure (59,60). Animal studies have provided robust mechanistic evidence demonstrating how DEHP can compromise reproductive and developmental processes (61-64). In a controlled study by XuaXia et al. (2023), male mice treated with DEHP (200 mg/kg/day, intragastric, 35 days) exhibited increased oxidative stress in testicular and sperm cells, impaired germ cell proliferation, and substantial declines in sperm quality hallmarks of subfertility (63). Extending these findings, Zhang et al. (2025) employed a paternal transgenerational exposure model in which male C57BL/6J mice were treated with DEHP (5–500 mg/kg/day) for four weeks (64). Offspring from these males, across both F1 and F2 generations, displayed altered sperm parameters, disrupted hormone profiles, and increased testicular apoptosis, demonstrating that DEHP-induced reproductive toxicity can persist beyond the directly exposed generation. Female reproductive systems also exhibit marked sensitivity. Wu et al. (2023) documented that combined exposure to polystyrene microplastics and DEHP for 35 days compromised ovarian granulosa cells, enhanced follicular atresia, and disrupted ovarian integrity (62). These findings highlight the potential for pollutant mixtures to amplify reproductive hazards through synergistic mechanisms. Overall, experimental animal models provide essential mechanistic insight into how environmental contaminants including heavy metals, phthalates, and microplastics disturb reproductive and developmental physiology. These studies not only reveal cellular and molecular pathways underlying toxicity but also 40   CURRENT ISSUES IN PHYSIOLOGY II inform risk assessment frameworks and public health strategies by identifying early biomarkers of reproductive dysfunction relevant to both animal and human health. 5. Experimental Animal Models in Investigating Pollutant-Induced Alterations in Neurophysiology Environmental pollutants are increasingly recognized as major drivers of neurophysiological disruption, giving rise to a broad spectrum of neurotoxic outcomes. Neurotoxicity defined as structural or functional damage to the central nervous system induced by chemical, biological, or physical agents is closely linked to conditions such as Alzheimer’s and Parkinson’s diseases, cognitive decline, neurodevelopmental disorders including attention-deficit/hyperactivity disorder and autism spectrum disorder, and various neurovascular impairments (65-67). Industrial emissions, mining operations, intensive agricultural practices and insufficient environmental controls particularly in developing regions release hazardous substances into the air, water, and soil (68-70). Consequently, humans and animals are chronically exposed to complex pollutant mixtures, including heavy metals, pesticides, herbicides, and airborne particulate matter, many of which bioaccumulate and elicit neurotoxic effects even at low doses (71-73). Although thousands of chemicals with potential neurotoxicity have been identified, mechanistic understanding of most remains limited (74,75). Within this context, experimental animal models represent indispensable tools for clarifying how pollutants disrupt neurophysiology across exposure routes, developmental stages, and biological systems. 5.1. Air Pollution and Pollution-Induced Neuroinflammation A clear example of pollution-induced neurophysiological disruption comes from controlled inhalation studies using APP/PS1 transgenic mice and wild-type counterparts. Animals exposed to fine particulate matter (PM2.5) for six hours daily over three months exhibited pronounced Alzheimer-like changes, including microglial and astrocytic activation and upregulation of pro-inflammatory cytokines. These findings indicate that long-term particulate exposure accelerates hallmark Alzheimer’s pathology by exacerbating Aβ plaque deposition, promoting gliosis, and enhancing neuroinflammation (76). Supporting evidence comes from another study demonstrating that traffic-related nanoscale particulate matter can induce early neurobiological disturbances relevant to Alzheimer’s disease. In female C57BL/6J mice, 150 ENVIRONMENTAL POLLUTANTS AND THEIR EFFECTS ON PHYSIOLOGICAL . . .   41 hours of nanoscale PM exposure over 10 weeks produced neuronal atrophy, altered myelin basic protein levels, and increased Iba1 immunoreactivity specifically within the CA1 region of the hippocampus. These structural and inflammatory alterations were accompanied by impaired spatial memory, highlighting the exceptional vulnerability of hippocampal circuits to pollutioninduced injury (77). 5.2.Pesticide-Induced Neurotoxicity and Parkinsonian Mechanisms Beyond Alzheimer-related pathways, environmental toxicants are increasingly implicated in molecular cascades characteristic of Parkinson’s disease (PD), including oxidative stress, mitochondrial dysfunction, apoptosis, neuroinflammation, dysregulated neurotransmission, and compromised antioxidant defenses (78). Rotenone exposure in rodents provides a wellestablished model reproducing these PD-like features: complex I inhibition, reduced ATP synthesis, elevated reactive oxygen species, proteasomal impairment, and cytoplasmic α-synuclein accumulation closely mirroring human PD pathology (79,80). Similarly, perinatal exposure to low-dose dieldrin (0.3–3 mg/kg every three days) disrupts dopaminergic neurochemistry in offspring and heightens susceptibility to MPTP-induced neurotoxicity, demonstrating that early-life pesticide exposure can potentiate later Parkinsonian pathology (81). Comparable dopaminergic toxicity has also been demonstrated following exposure to trichloroethylene (TCE). Fischer 344 rats treated with 500–1000 mg/ kg TCE for six weeks showed substantial mitochondrial impairment, elevated oxidative stress, and increased α-synuclein accumulation (79). Together, these animal studies provide compelling mechanistic evidence that diverse pollutants converge on mitochondrial and oxidative pathways leading to neurodegeneration, reinforcing their relevance to human PD. 5.3.Heavy Metals and Neurodevelopmental and Neurodegenerative Outcomes Heavy metals also constitute a significant class of neurotoxic environmental contaminants. Early-life exposure to mixtures of arsenic, cadmium, and lead disrupts long-term potentiation a process essential for memory formation and compromises blood–brain barrier integrity, leading to cognitive impairments in rodent models (82). Lead exposure, in particular, has been extensively studied: developmental exposure to low Pb levels during gestation and lactation results in enduring deficits in learning, memory, hyperactivity, and increased anxiety. 42   CURRENT ISSUES IN PHYSIOLOGY II Additional studies across species show that early Pb exposure accelerates β-amyloid accumulation, a hallmark of Alzheimer-like neuropathology (83). Aluminum is similarly recognized as a neurotoxic metal with strong links to neurodegenerative disease. Chronic oral exposure to aluminum (2 mM) in a tauopathy mouse model accelerates tau accumulation, enhances neuronal apoptosis, and induces severe neurological dysfunction effects associated with glutamate dysregulation, increased free radical generation, and impaired antioxidant defense systems (84,85). 5.4. Zebrafish as a High-Throughput Model for Neurotoxicology Zebrafish have emerged as a powerful vertebrate system for highthroughput screening and mechanistic studies of pollutant-induced neurotoxicity. Recent research demonstrates that graphene-family nanomaterials (GFNs) induce distinct neurodevelopmental abnormalities exposure of zebrafish embryos to carboxylated graphene oxide (GO-COOH; 10–100 mg/L) resulted in pronounced neural defects and altered locomotor behavior, emphasizing the sensitivity of the developing nervous system to nanomaterial-driven toxicity (86). Zebrafish studies have also illuminated pesticide-induced dopaminergic vulnerability; exposure to the dithiocarbamate pesticide ziram selectively damages dopaminergic neurons in a synuclein-dependent manner, suggesting a mechanistic pathway through which pesticides may contribute to PD (87). Additional evidence highlights how heavy metal toxicity manifests in zebrafish models. Daily AlCl₃ exposure (200 mg/kg) induces oxidative stress, reduces antioxidant capacity, alters neurotransmitter balance, and impairs learning and memory (88). Similarly, developmental lead exposure disrupts the expression of sorl1 an established Alzheimer’s risk gene in zebrafish embryos, suggesting that Pb may initiate early molecular events contributing to neurodegeneration (85,89). Collectively, these findings underscore the versatility of zebrafish in detecting pollutant-induced behavioral, biochemical, and genetic alterations relevant to human neurodegenerative disease. 6. Conclusion Experimental animal models remain indispensable for elucidating how environmental pollutants disrupt physiological, developmental, and neurobiological systems across the lifespan. Evidence across diverse species from rodents to zebrafish demonstrates that environmental contaminants such ENVIRONMENTAL POLLUTANTS AND THEIR EFFECTS ON PHYSIOLOGICAL . . .   43 as heavy metals, pesticides, phthalates, microplastics, industrial chemicals, and airborne particulate matter can accumulate in target tissues and induce doseand time-dependent toxicity. These exposures impair key biological processes, including reproductive function, embryonic development, immune and endocrine regulation, and central nervous system homeostasis, ultimately contributing to pathologies resembling human disorders such as infertility, metabolic disease, neurodevelopmental deficits, and neurodegeneration. 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Evaluating the protective effects of melatonin on di(2-ethylhexyl) phthalate-induced testicular injury in adult mice. Biomed Pharmacother. 2018;108:515-523. doi:10.1016/j.biopha.2018.09.044 60. Aviles A, Boulogne I, Durand N, et al. Effects of DEHP on postembryonic development, nuclear receptor expression, metabolite and ecdysteroid concentrations of the moth Spodoptera littoralis. Chemosphere. 2019;215:725738. doi:10.1016/j.chemosphere.2018.10.102 61. Ge X, Weis K, Flaws J, Raetzman L. Prenatal exposure to the phthalate DEHP impacts reproduction-related gene expression in the pituitary. Reprod Toxicol. 2022;108:18-27. doi:10.1016/j.reprotox.2021.12.008 62. Wu H, Liu Q, Yang N, Xu S. Polystyrene-microplastics and DEHP co-exposure induced DNA damage, cell cycle arrest and necroptosis of ovarian ER DRIVEN MOLECULAR SIGNATURES OF EPİLEPTOGENESIS . . .   55 The primary objective of the UPR is to alleviate the burden of misfolded proteins or unfolded proteins within the ER. To accomplish this, the UPR: · Temporarily attenuates global protein synthesis to reduce the influx of newly synthesized proteins, · Enhances the degradation of aberrant proteins through ER-associated degradation (ERAD), · and upregulates ER chaperones that facilitate proper protein folding. Through these coordinated actions, the UPR attempts to restore ER homeostasis. However, if ER stress becomes chronic or severe, maladaptive responses ensue, ultimately leading to apoptosis, mitochondrial dysfunction, and inflammation (15-18). Figure 1. An overview of the molecular foundations of ER stress (Illustrated using ChatGPT5.0). 3. The role of PERK Pathway in Epileptogenesis PERK is a transmembrane protein kinase located in the ER membrane. It represents one of three major signaling branches of the unfolded protein response (UPR) activated during ER stress. Upon accumulation of misfolded 56   CURRENT ISSUES IN PHYSIOLOGY II proteins, GRP78 dissociates from PERK, permitting its dimerization and autophosphorylation (p-PERK). Activated PERK subsequently phosphorylates eukaryotic initiation factor 2α (eIF2α), leading to a global reduction in protein synthesis, except for specific mRNAs (such as ATF4) whose translation is enhanced under these conditions. In this manner, the PERK pathway functions as a protective “molecular switch” that limits ER protein load and allows time for proper protein folding (19-21). Activation of the PERK-eIF2α-ATF4 axis regulates diverse cellular outcomes, including antioxidant responses, cell cycle arrest, apoptosis, and ERAD. Through ATF4-driven transcriptional programs, cells attempt to adapt to stress; however, if ER stress persists, ATF4 induces CHOP (C/EBP homologous protein) expression, shifting the balance toward apoptosis (17). From a neurophysiological perspective, PERK activation and subsequent eIF2α phosphorylation at Ser51 reduce global translation, conserving cellular energy and enhancing protein quality control mechanisms. When ER stress is prolonged, alterations in the repertoire of synaptic proteins and glial signaling influence neuronal excitability, seizure threshold, and cognitive processes such as learning and memory. Epileptogenetic insults and recurrent seizures impose significant cellular stress, thereby activating UPR signalingincluding the PERK pathway-as part of neuronal adaptive response (17, 22). Findings from the genetic absence epilepsy model, WAG/Rij rats, demonstrated that mild ER stress induced by Thapsigargin (Tg) significantly increased. eif2ak3 (PERK) mRNA levels in the thalamus, accompanied by an increase in spike-wave discharges (SWD) activity. These results suggest that under mild ER stress, activation of GRP78 and PERK pathways may contribute to SWD modulation and neuronal excitability (23). The PERK pathway also plays a pivotal role in neuronal injury associated with status epilepticus (SE). Studies have shown that PERK activation and subsequent CHOP induction contribute to neuronal apoptosis and structural brain damage following SE (24). PERK dysregulation, including increased p-PERK, p-eIF2α, and CHOP expression, has been observed in both human temporal lobe epilepsy (TLE) tissue and experimental seizure models such as SE and pentylenetetrazolium (PTZ) induced epilepsies. Chronic ER stress-driven CHOP expression appears to be a major contributor to neuronal loss during epileptogenesis (24-26). A second key contributor to the PERK pathway to epileptogenesis involves its interaction with inflammation and neuroimmune signaling. In TLE patients, ER DRIVEN MOLECULAR SIGNATURES OF EPİLEPTOGENESIS . . .   57 increased PERK activation in the temporal neocortex correlates positively with NLRP3 inflammasome components localized to neurons, astrocytes, and microglia. Pharmacological inhibition of NLRP3 with MCC950 reduces PERK pathway markers, while ER stress inhibitor TUDCA suppresses NLRP3 expression in experimental SE models (27). These findings imply bidirectional crosstalk between ER stress and neuroinflammation during epileptogenesis. The PERK pathway also influences blood–brain barrier (BBB) integrity, which is critical to seizure susceptibility. ER stress contributes to BBB breakdown, increasing vascular permeability, neuroinflammation, and heightened neuronal excitability. In the pilocarpine-induced SE model, endothelial cells exhibited increased expression of GRP78 and p-PERK, suggesting that PERK is involved in vasogenic edema and barrier dysfunction. Thus, targeting ER stress may help restore endothelial function and mitigate SE-induced BBB disruption (28). In summary, the PERK pathway exerts a dose-dependent and contextdependent dual effect in epileptogenesis. Mild activation may promote adaptation and influence seizure activity. Severe or chronic activation leads to apoptosis, BBB dysfunction, inflammation, and neurodegeneration. These multifaceted roles of PERK are both mediators of neuronal adaptation and drivers of pathological progression during epileptogenesis. 4. The role of the IRE1 pathway in Epileptogenesis During ER stress, activation of the IRE1 pathway is a critical cellular mechanism that restores homeostasis. Under physiological conditions, the ER chaperone GRP78 binds to the luminal domain of IRE1, thereby stabilizing IRE1. When misfolded proteins accumulate in the ER lumen, GRP78 dissociates, allowing IRE1 to dimerize/oligomerize and autophosphorylate. This phosphorylation event allosterically activates the endoribonuclease (RNAase) domain located in the C-terminal cytosolic region of IRE1. Activated IRE1 initiates a unique splicing reaction in the mRNA of X-box binding protein 1 (XBP1) by excising a 26-nucleotide intron. This intron splicing event alters the translational reading frame, generating the transcriptionally active form XBP1s, which translocates into the nucleus to regulate UPR target genes involved in ER biogenesis, lipid synthesis, ERAD, and chaperone production (22, 29). This adaptive response promotes cellular survival under moderate ER stress. Under severe or prolonged stress, IRE1 RNase activity also targets specific ER-localized mRNAs for degradation via regulated IRE1-dependent 58   CURRENT ISSUES IN PHYSIOLOGY II decay (RIDD), thereby reducing the protein-folding load. However, persistent hyperactivation of IRE1 shifts signaling toward apoptosis. Phosphorylated IRE1 directly interacts with TRAF2 (TNF receptor–associated factor 2), initiating downstream activation of ASK1 (apoptosis signal-regulating kinase 1) and its effector kinase JNK (c-Jun N-terminal kinase). JNK promotes apoptosis by activating the pro-apoptotic protein BIM and inhibiting anti-apoptotic Bcl-2, thereby amplifying cell death pathways (30). Thus, the IRE1–XBP1 axis functions as a dual-acting signaling branch that supports adaptation under mild stress but transitions to destructive pro-apoptotic and pro-inflammatory signaling when stress becomes excessive or chronic. The involvement of the IRE1 pathway in epileptogenesis stems from its bidirectional influence on adaptive and apoptotic responses and its interactions with genes associated with neurotransmission. In genetically absence epileptic WAG/Rij rats, ER stress induced by thapsigargin demonstrated dose-dependent effects on seizure activity, wherein mild ER stress increased GRP78, PERK, and XBP1 mRNA levels, accompanied by increased spike-wave discharge (SWD) activity. The same study also reported elevated expression of the T-type Ca²⁺ channel gene CACNA1H, correlating with XBP1 expression, suggesting that the adaptive IRE1–XBP1 response may indirectly modulate neuronal excitability through ion channel regulation. Under high ER stress, XBP1 mRNA levels also increased, accompanied by elevated NF-κB and TNF-α, consistent with enhanced pro-inflammatory and pro-apoptotic signaling and the onset of neurodegeneration (23). The IRE1 pathway has also been shown to regulate neurotransmission. Studies indicate that IRE1 signaling contributes to the surface trafficking and functional expression of the α1(A322D) subunit of GABA_A receptors, the primary inhibitory ion channels in the CNS (31). Such findings highlight the potential role of IRE1 signaling in the fine-tuning of excitatory–inhibitory balance, a crucial determinant of epileptogenesis. In human temporal lobe epilepsy (TLE), IRE1α-mediated signaling is markedly upregulated in the hippocampus. This increased activation appears to exert both pro-apoptotic and anti-apoptotic effects. Activation of the TRAF2– ASK1–JNK cascade may promote neuronal loss, particularly in the CA1–CA3 hippocampal subfields, contributing to structural remodeling associated with epileptogenesis (32). Conversely, increased XBP1 expression enhances protein folding, stabilizes Ca²⁺ homeostasis, and supports synaptic protein integrity, thereby promoting neuronal survival and resilience under stress. ER DRIVEN MOLECULAR SIGNATURES OF EPİLEPTOGENESIS . . .   59 Experimental seizure models further support this duality: kainic acid– induced status epilepticus significantly elevates spliced XBP1 levels, which have been shown to protect neurons against excitotoxic injury (33). The IRE1 pathway also interfaces with neuroinflammatory mechanisms. In the temporal cortex of TLE patients, a positive correlation has been observed between NLRP3 inflammasome components and ER stress–related proteins, including IRE1. By promoting reactive oxygen species (ROS) production, IRE1 signaling may facilitate NLRP3 localization to mitochondria, thereby activating caspase-2 and amplifying inflammation (27). In summary; taken together, the IRE1 pathway acts as a finely tuned molecular regulator during epileptogenesis. Under moderate ER stress, it promotes neuronal survival by inducing XBP1s and supporting ER proteostasis. Under severe or chronic stress, it activates TRAF2–JNK and NLRP3-mediated pathways that drive apoptosis, inflammation, and neurodegeneration. Thus, IRE1 signaling occupies a central position at the intersection of adaptive proteostasis and pathological stress responses, making it a pivotal determinant of neuronal outcome during the development and progression of epilepsy. 5. The Role of the ATF6 Pathway in Epileptogenesis The activating transcription factor 6 (ATF6) pathway is one of the three major sensors of the unfolded protein response (UPR) activated under endoplasmic reticulum (ER) stress. ATF6 is a type II transmembrane protein embedded in the ER and includes two principal isoforms, ATF6α and ATF6β, of which ATF6α is the more extensively characterized and functionally dominant variant (34). Under physiological conditions, the ER chaperone GRP78 (BiP) binds to ATF6, preventing its activation. Accumulation of misfolded proteins in the ER lumen dissociates GRP78, exposing Golgi localization sequences. ATF6 then translocates from the ER to the Golgi apparatus, where it undergoes sequential cleavage by Site-1 protease (S1P) and Site-2 protease (S2P). This processing liberates the cytosolic fragment ATF6f, a basic leucine zipper (bZIP) transcription factor that enters the nucleus and binds to ER stress response elements (ERSEs) to regulate target genes involved in ER chaperones, ER-associated degradation (ERAD), and other UPR mediators such as XBP1 and CHOP (17). The cleavage mechanism of ATF6 resembles that of sterol regulatory element-binding proteins (SREBPs), linking ATF6 activation to lipid metabolism. ATF6 also interacts with peroxisome proliferator–activated receptor α (PPARα), 60   CURRENT ISSUES IN PHYSIOLOGY II participating in fatty acid oxidation and modulating SREBP2-mediated lipogenesis. Additionally, ATF6 functions downstream of the PERK pathway by enhancing ATF4 translation in response to eIF2α phosphorylation (35). Mild to moderate ER stress promotes ATF4-dependent transcription of ER chaperones, antioxidant proteins, and autophagy-related factors that preserve cellular homeostasis (36). Notably, ATF4 is required for ER-phagy (reticulophagy), acting by inducing genes such as p62, Nbr1, Atg7, and Atg5, and by interacting with ER surface proteins like CCPG1 (17). However, under severe or prolonged ER stress, ATF4 shifts toward pro-apoptotic signaling by inducing CHOP (C/EBP homologous protein), a key regulator of ER stress–mediated apoptosis (37). CHOP upregulates proapoptotic proteins such as BIM and suppresses anti-apoptotic BCL-2, activating the mitochondrial apoptosis cascade via caspases-2, -9, and -3 (38). CHOP also enhances GADD34 expression, restoring protein synthesis but increasing reactive oxygen species (ROS) and ERO1α, which promote oxidative protein folding but can lead to apoptotic cell death (39). Epileptogenic insults and recurrent seizures lead to the accumulation of misfolded proteins within the ER lumen, activating the ATF6 pathway as part of the neuronal stress response. During ER stress, GRP78 dissociation triggers ATF6 trafficking to the Golgi apparatus and subsequent cleavage into its active transcription factor form. The downstream effects of ATF6 signaling are complex, encompassing both adaptive and deleterious outcomes depending on the severity and duration of ER stress (17). In kainic acid–induced status epilepticus (SE) models, elevated levels of cleaved ATF6 (ATF6f) have been detected in the hippocampus. Genetic deletion of ATF6α exacerbates neuronal degeneration following kainate-induced seizures, suggesting a neuroprotective role for ATF6α in excitotoxic conditions, potentially through the regulation of Ca²⁺ homeostasis (40-41). In contrast, severe ER stress can activate ATF6-dependent apoptotic signaling. In WAG/Rij rats, high-dose thapsigargin increased ATF6 mRNA expression in the thalamus, accompanied by elevations in NF-κB and TNF-α levels, despite no significant change in GRP78 expression. A positive correlation between ATF6 mRNA and NF-κB levels suggests that ATF6 may promote proinflammatory and translational suppression pathways via NF-κB induction, contributing to neuronal vulnerability. Interestingly, these alterations were associated with decreased SWD activity, reflecting stress-dependent modulation of thalamocortical network dynamics (23, 42-43). ER DRIVEN MOLECULAR SIGNATURES OF EPİLEPTOGENESIS . . .   61 The ATF6 pathway also influences neurotransmission. ATF6 signaling has been shown to support surface trafficking of the GABA_A receptor α1(A322D) subunit, suggesting that ATF6 may regulate inhibitory neurotransmission and excitatory–inhibitory balance (31). Human studies further support a role for ATF6 in epileptogenesis. In the temporal neocortex of TLE patients, ER stress–associated markers—including GRP78, PERK, p-PERK, eIF2α, p-eIF2α, ATF4, and CHOP—are upregulated. Positive correlations between ER stress markers (including downstream ATF6 targets) and NLRP3 inflammasome components suggest that ATF6 activation may contribute indirectly to neuroinflammation (27). In summary, ATF6 signaling serves as a crucial regulator in epileptogenesis, mediating adaptive responses, including regulation of Ca²⁺ homeostasis, promotion of ER proteostasis, and attenuation of excitotoxic injury. Pathological responses, including NF-κB-mediated inflammation and CHOP-driven apoptosis, occur during chronic or severe ER stress. Given its dual functionality, ATF6α is a promising therapeutic target in epilepsy, with the potential to enhance neuroprotection while limiting maladaptive stress responses. 6. Conclusion Epileptogenesis is increasingly recognized as a multifactorial process in which traditional concepts of excitatory–inhibitory imbalance and synaptic reorganization intersect with intracellular stress responses, particularly those originating from the endoplasmic reticulum (ER). The unfolded protein response (UPR), activated by ER stress, emerges as a central molecular network that can either preserve neuronal integrity or exacerbate pathology, depending on the severity and duration of stress. The three canonical UPR pathways—PERK, IRE1, and ATF6—operate in parallel yet are intricately interconnected, shaping neuronal fate throughout the epileptogenic cascade. The PERK pathway plays a dual role, attenuating global protein translation to alleviate ER stress while selectively enhancing stress-response gene expression via ATF4. Although mild PERK activation may support neuronal adaptation, chronic or excessive activation induces CHOP-mediated apoptosis, neuroinflammation, and blood–brain barrier dysfunction, contributing to neuronal vulnerability and seizure propagation. The IRE1 pathway is the most evolutionarily conserved arm of the UPR, with a potent adaptive capacity through XBP1s-mediated enhancement of proteostasis, lipid metabolism, and synaptic protein regulation. However, 62   CURRENT ISSUES IN PHYSIOLOGY II prolonged or severe stress shifts IRE1 signaling toward TRAF2–ASK1–JNK activation, promoting apoptosis, inflammation, and oxidative stress. The involvement of IRE1 in modulating ion channel expression and inhibitory neurotransmission underscores its significance in shaping neuronal excitability in epilepsy. The ATF6 pathway also demonstrates context-dependent roles in epileptogenesis. Under moderate stress, ATF6 activation promotes ER homeostasis, Ca²⁺ regulation, and resistance to excitotoxic injury. Yet under persistent ER stress, ATF6 synergizes with CHOP, NF-κB, and inflammatory mediators to intensify neuronal degeneration. Human temporal lobe epilepsy tissue further reveals coordinated upregulation of ATF6-related stress markers alongside inflammasome components, highlighting the translational relevance of this pathway. Collectively, evidence from experimental models, molecular studies, and human epilepsy demonstrates that ER stress and UPR signaling are not merely secondary consequences of seizures. Still, active drivers of epileptogenesis, influencing neuronal survival, synaptic remodeling, neuroimmune activation, and network excitability. These pathways represent promising therapeutic targets: pharmacological modulation of UPR components may restore cellular homeostasis, mitigate seizure-induced damage, and potentially alter the course of epileptogenesis. Future research integrating transcriptomics, proteomics, and neurophysiological modeling will be essential to delineate the precise temporal dynamics of ER stress responses during epilepsy development. Understanding how PERK, IRE1, and ATF6 signaling networks interact within vulnerable neuronal circuits may open the door to mechanism-based, disease-modifying therapies for epilepsy. ER DRIVEN MOLECULAR SIGNATURES OF EPİLEPTOGENESIS . . .   63 Figure 2. Dual effects of ER stress pathways on Epilepsy (Illustrated using NotebookLM). 64   CURRENT ISSUES IN PHYSIOLOGY II 8. AI disclosure The sentence progressed in academic fluency, and translation errors were checked and corrected with the Grammarly and ChatGPT 5.0 programs. 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Nat Neurosci. 2015;18(3):344-350. doi.org/10.1038/nn.3933 THE PHYSOLOGY OF OLFACTORY MEMORY FORMATION . . .   71 systems in general. This specific reaction, known as the bradycardic response and reflected in the decrease of the heart rate, can be elicited in reaction to novel olfactory stimuli, and it features the phenomenon of habituation in reaction to repeated exposure (11). Another equally valid behavioral approach in exploring the sense of olfactory habituation is monitoring the amount of observation time for scented stimuli (12, 13). In this approach, each individual is exposed to novel scented stimuli, and the amount of observation time for each stimulus is measured. By carrying out comparisons between successive observations of the very same scent, it can be observed that the amount of investigation time features significantly scaled-down observations, indicating the process of scentrelated habituation. Habituation paradigms are helpful in understanding memory formation for simple stimuli, memory durations, stimulus specificity, and neural mechanism underlying habituation memory (14). A prime application of such paradigms is in determining an individual animal’s ability to discriminate between familiar and novel stimuli following habituation, and novel stimulus responses can reveal the extent of such familiarization (12). In addition, evaluating memory performance for different retention intervals makes it possible to accurately estimate memory retention periods. In the case of experimental work on mice and rats, it has been indicated that memory for habituation and memory specificity can be influenced in different ways for different retention intervals (15) and by factors such as the role of neuromodulators like acetylcholine and norepinephrine in such memory tasks (16, 17). Habituation paradigms are very useful in understanding speciesspecific communication systems and individual and sex-specific recognition and scent marking (18,19) Olfactory sensory neurons adapt to repeated or sustained olfactory stimuli, and behavioral olfactory habituation is an important aspect of this process. Central olfactory neurons are known to exhibit a more prominent response decrement compared to peripheral neurons. In specific, piriform cortical neurons exhibit rapid and near-complete response adaptation within seconds or minutes of olfactory stimulus exposure, as reported in both experimental animals and human subjects. Experiments recording second-order mitral cells and their olfactory target cells in the piriform cortical pyramidal layer simultaneously have depicted that the pyramidal cells adapt to odors more rapidly and thoroughly compared to mitral cells. Mitral cells are known to adapt, whereas in their downstreams, the adaptation observed appears to be rapid and almost complete. In addition, intracellular recording in awake, behaving animals has shown that this cortical cell adaptation correlates well with the elimination of 72   CURRENT ISSUES IN PHYSIOLOGY II the glutamatergic synapse between mitral and pyramidal cells. This elimination of mitral and pyramidal cells’ glutamatergic synapses has a specific recovery period that parallels the adaptation of postsynaptic potentials induced by odors and has been described to occur in relation to the short-term adaptation of potentials industriously initiated due to odors (20). In specific, it is known that glutamate III-metabotropic glutamate receptors are mediators of this short-term adaptation. In addition, exposure to odors for prolonged periods or in brief pulses may invoke LTH in mice. In human participants, daily exposure to odors in environments such as the home and workplace may invoke LTH and augmented vigilance thresholds that last for several weeks following exposure (21). In addition, electrophysiological recordings in awake rat subjects, employing identical paradigms to those used in behavioral recording, have described that mitral cells in the rat olfactory bulb decrease responsiveness to repeated pulses of specific odors when delivered at frequency rates of every five minutes. In specific, NMDA receptors in the olfactory bulb are required for this decrease in responsiveness to persist in awake rat subjects (22). In spite of the known linkage between olfactory habituation and the olfactory bulb and cortex of mice and rat models, extensive work done on the role of neural correlates in higher-order areas of the brain like the enthorinal cortex, frontal cortex, and amygdala of the hamster has indicated extensive involvement in producing habituation memory. Based on work completed by researchers such as Maras and Petrulis on the role of multiple areas of the brain involved in the creation of habituation memory, it can be concluded that memory functions like those of habituation are not straightforward processes (23). 3. Long-term Olfactory Memory Long-term memory is conceptualized as the retention of past representations over extended periods and is often seen as distinct from working memory (24). By contrast, some different models of memory propose that there is only one storage system for memory, emphasizing the role of activation of long-term processes as primitive (25,26). These models propose that the experience of odors is inextricably connected to olfactory experience and knowledge and that the role of long-term processing in this sensory modality cannot be overstated (27,28). “Explicit” or “declarative” memory is described as memory that can be consciously accessed and recalled. It is an intricately complex process that involves the coordinated participation of different areas of the human brain. THE PHYSOLOGY OF OLFACTORY MEMORY FORMATION . . .   73 Evidence of activation of different areas of the human brain that are associated with explicit memory functions can be derived from human imaging and case analysis of similar lesion paradigms in animals. Some of the work that has emerged concerning the intricacies of the neural circuitry that supports explicit memory functions has been identified in some studies (29,30). In recent years, work on odor recognition memory has utilized findings in cognitive science and neuroscience, particularly in the application of signal detection analysis. There is substantial agreement that the process of recognition consists of two main steps: the first is episodic memory for those stimuli that have been seen previously, and the second is the sensation of familiarity for those stimuli that were seen just recently. In the conventional approach in human olfactory memory and in applying the framework of signal detection theory, the process of a “recognition” paradigm is followed, in which the participant rates the odors as “old” (previously seen and “new” (never seen previously). In this experimental approach, the Receiver Operating Characteristic (ROC) curves illustrate the relationship between the amount of “hits” (the amount of correctly identified “old” stimuli) and false alarms (instances in which the “new” stimuli are identified as “old”). In 2004, the ROC approach in determining rat odor recognition memory was used in a study conducted by researchers (31). Odors such as lemon and thyme were paired with sand in small plastic containers. In the study phase, the rat had 10 stimuli, and in each cup, some of their favorite sweetened cereal had been placed under some sand in each cup. They were left for 30 minutes and then given another test phase of 20 target cups, each cup containing a random combination of 10 familiar and 10 novel scents from the common pool, together with some sand. In the test phase, the rats were required to dig in the target cups containing novel scents and avoid digging in the cups containing old scents in order to receive rewards. In addition to this, the authors attempted to alter the preference of the rats towards digging in target cups by varying the height of the target cups and the amount of rewards in relation to the target cups and an additional reward cup present at the back of the cage. The findings indicated that when the rewards were small or required considerable effort, the rats avoided digging in the cup containing novel scents (old), while those that contained higher rewards or easier access demonstrated an increased inclination towards digging (novel), marking the gradual approach of human participants’ liberal threshold for “old” responses. In contrast, when confronted with higher rewards or easier access 74   CURRENT ISSUES IN PHYSIOLOGY II in target cups, the rats demonstrated an increased preference towards digging (novel), marking the approach of human participants’ conservative threshold for “old” responses. In order to overcome the possible risk that the rat participants were detecting the rewards present in the target cups, the authors performed the probe trials in which no rewards were present in the novel scent-containing cups to ensure that rewards were provided only after the rat initiated digging behavior. The experiment had a protocol in place to analyze the ROC curve of whole rats that revealed the existence of an asymmetrical part (a positive Y-intercept, in particular), and a large and nonlinear one. There had to be an associative memory test for rat recognition in order to assess the separability of the asymmetry concerning recollection from the nonlinear part correlated with familiarity in the olfactory ROC. Pairs of stimuli were shown that were comprised of different scents blended together with different digging materials such as wood chips, beads, and sand in a cup (32). Everyday during the experiment, the rats were accustomed to 10 different odor-medium combinations. Then, after a 30-minute waiting period, the rat subjects were required to distinguish between the re-presentation of the previously experienced sets of 10 and the newly prepared sets of 10 that comprised different combinations of the same scents and media. In this process of odor-medium identification, the concept of non-matching and biases was applied in much the same way that it had been in former item recognition tasks. The ROC function that emerged showed obvious markers of asymmetry, emphasizing the preponderant role of recollection in this process. In turn, the ROC had a linear form, indicating that it lacked any prominent familiarity component. Indeed, this finding fits well within the parameters of what has been described for the ROC function in human memory paradigms testing recollection in associative recognition and source memory (32). In any case, this work increases understanding of olfactory memory in the rat and its similarities to human memory in associative tasks. In order to evaluate the dissociation between the familiarity and recollection components of the ROC, this experiment proposed the use of a novel experimental paradigm that tested the hypothesis based on dual process theory. There is a distinction made within this paradigm between fast, goalconcurrently executed familiarity that benefits perceptually driven and generally functions on the pattern-matching level, and the slower process of recollection that is conceptually driven and depends on organization. THE PHYSOLOGY OF OLFACTORY MEMORY FORMATION . . .   75 In this training and testing phase, the animals were given a task of item recognition that lacked any response deadline, and this generated an asymmetric and nonlinear ROC function in conformity with past observations. In this phase, the central focal point that this experiment sought to evaluate had to do with the amount of time it took for the animals to dig into novel-smelling test cups compared to the amount of time it took for them to turn away from old-smelling containers. In this case, the experiment introduced response deadlines, which were half of the natural response latencies. Results indicated that performance was comprised mainly of familiarity, as indicated by the curvilinear ROC function, when under the influence of the deadline. In contrast, the ROC function for the no-deadline condition indicated that recollection contributed significantly, and in contrast to the no-deadline condition, the ROC function in the deadline condition indicated absolute symmetry. Thus, the findings confirmed the dual process theory in emphasizing that fast responses in terms of familiarity are possible, and recollection requires the intentional process. Studies conducted using Receiver Operating Characteristic (ROC) analysis have helped clarify the hippocampus’ crucial role in episodic recollection and not just familiarity. The theory proposed that the hippocampus played an essential role in recollection and that it is not required for basic rule-game acquisition in non-matching tasks or for sensitivity to response biases. Findings confirmed that the rat group with isolated hippocampal damage was able to perform the non-matching task, showing that response biases were unchanged. In this case, the animals failed to demonstrate any deficiency in the familiarity aspect of the ROC function, indicated by curvlinearity, and were shown to be deficient in the recollective component, indicated by asymmetry. Evidence supporting this claim has been given in the work Fortin et al. (31), showing that hippocampally damaged rats are capable of showing symmetry and curvlinarity in the ROC curve, reflecting preserved familiarity and impaired recollection abilities. Further validation of this nexus came from the work of Cabeza et al., who reinforced that it is familiarity that grew when normal hippocampal function failed (32). Normal animals were seen to have extreme asymmetry and linearity in their ROC function in associative recognition memory tasks, marking them highly for recollection yet low on familiarity. In contrast, animals that were hippocampally impaired had extreme reductions in ROC asymmetry, yet this indicated that those animals were obviously memory-impaired. In their impaired state, their ROC function became curvilinear, meaning that it indicated that those animals were employing familiarity to compensate for what was lost. 76   CURRENT ISSUES IN PHYSIOLOGY II Double dissociation of impaired recollection and increased familiarity solidly confirms the hippocampus’ role in uniquely enhancing recollection in memory tasks and points towards the compensatory role of familiarity when it fails. Another study investigated the role of the amygdala in recognizing odors and odour familiarity memory employing a signal detection approach. The amygdala has been shown to have highly interconnected pathways to the perirhinal cortex and hippocampus, which may influence their functions in memory tasks involving the recognition of things. In fact, the reorganization of memory due to damage in the hippocampus and the role of the amygdala in linking odors and rewards have led to the belief that the amygdala may have some special role in enhancing familiarity-related responses to odors, particularly in relation to processes mediated for “mere exposure” and rewarding stimuli. Evidence in favor of this theory emerged when it observed that bilateral damage in the amygdala led to less curvilinear ROC functions, reflecting decreased familiarity of odors. Significantly, this damage failed to significantly alter the symmetry of the ROC functions, suggesting that the parameters of recollection memory were unaffected. Together, the findings of this and hippocampal damage findings illustrate that these two structures share different functions: while the hippocampus is vital for remembering scents, the role of the amygdala is vital for scent recognition. 4. Conclusions Olfactory memory and learning are very complex and include multiple memory systems in the brain that are vital for memory learning strategies (33). The hippocampus is very significant in this process as it allows us to remember scents better than recognize them, which is mediated solely by the amygdala (31,34). The hippocampus is vital for associative memory and transitive inference and shows its significance in understanding relationships between scents (35,36). Other areas of the brain, such as the medio-dorsal thalamus and the orbitofrontal cortex, are also highly influential in memory formation, particularly in relation to learning associations between odors and other stimuli and changes in the values of reinforcement (37,38). Another area that is highly influential in the memory of odors is the olfactory bulb and the piriform cortex, which are responsible for encoding and remembering odors (35, 39). The scent of coffee is one such case because it is only one aspect that prompts you to remember the scent of coffee, and it also prompts you to remember the word “coffee,” as well as another memory that may or may not be connected to coffee THE PHYSOLOGY OF OLFACTORY MEMORY FORMATION . . .   77 in any manner. In turn, this raises further questions about the nature of human scent memory and whether it remains independent in relation to semantic markers. In contrast, the implicit paradigms for learning give us data that is much more like what happens in our daily episodic experiences of smell. This is another approach that may be taken towards understanding memory for smell (40). In view of the clear and vital role that memory for odors plays in our daily functioning, it is necessary that the parameters of learning and memory for odors continue to be investigated on an ongoing basis. 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The Medial Temporal Lobe and Recognition Memory. Annual Review of Neuroscience. 2007;30(1):123. doi:10.1146/annurev.neuro.30.051606.094328 37. Herz RS, Engen T. Odor memory: Review and analysis. Psychonomic Bulletin & Review. 1996;3(3):300. doi:10.3758/bf03210754 38. Rolls ET. The Orbitofrontal Cortex and Reward. Cerebral Cortex. 2000;10(3):284. doi:10.1093/cercor/10.3.284 39. Zelano C, Montag JL, Khan R, Sobel N. A Specialized Odor Memory Buffer in Primary Olfactory Cortex. PLoS ONE. 2009;4(3). doi:10.1371/journal. pone.0004965 40. Degel J, Köster EP. Implicit Memory for Odors: A Possible Method for Observation. Perceptual and Motor Skills. 1998;86(3):943. doi:10.2466/ pms.1998.86.3.943 ELECTROPHYSIOLOGICAL MEASUREMENT OFOLFACTORY FUNCTION   87 stems mainly from their ability to objectively identify olfactory responses in individuals who may be hesitant or unable to provide reliable data through behavioral assessments. Also, as a psychophysiological measure, CSERPs can reveal subtle responses that behavioral tests may miss. CSERPs have been successfully recorded in patients with olfactory disorders from various causes, such as nasal issues, upper respiratory infections, drug effects, traumatic brain injuries, neurological conditions, and unknown factors. So far, the main diagnostic use of CSERPs has focused on determining their presence or absence, usually evaluated by trained observers. The absence of CSERP responses to olfactory stimuli has been observed in anosmic individuals. However, it is important to note that these potentials can remain undetectable in patients even when behavioral evaluations suggest some level of preserved olfactory function. (33) 3. Continuous Time-Frequency EEG The existing literature shows that limited research has used continuous timefrequency measures at the onset of olfactory stimuli. Only two studies are known to have employed this method. One study, presented as a conference abstract by Lorig and Randol, reported changes in power across various frequency bands just 1.5 seconds after olfactory stimulation. They found that only low-frequency modulations showed a variable response depending on the specific odor. These initial findings hint that the timing of frequency power changes may be slower than the modulations of event-related potentials, suggesting that EEG and ERP changes may reflect different parts of the olfactory network. However, given the early stage of this report, a fuller understanding of the connection between these two components will require simultaneous calculations of EEG and ERP changes from the same EEG signal in response to stimuli. (10,34) A study by Huart et al. examined EEG changes in relation to both olfactory and trigeminal stimulation among eleven individuals with normal olfactory function. Their research outlined both the phase-locked component of the EEG and the continuous, non-phase-locked changes in the frequency spectrum. Participants were exposed to carbon dioxide for trigeminal stimulation and phenylethyl alcohol for olfactory stimulation, delivered using an olfactometer. Huart et al. applied wavelet analysis to assess the changes in EEG power during olfactory stimulation across a frequency range of 0.3 to 30 Hz. They found a sustained increase in power within the theta frequency band, followed by a brief drop in alpha power. These power changes were most evident over the central 88   CURRENT ISSUES IN PHYSIOLOGY II electrode Cz, matching the distribution seen in olfactory event-related potentials. The authors suggested that this shared distribution indicates both ERP and timefrequency changes represent a common cortical response to stimulation. (10) The study showed that trigeminal nerve stimulation first caused an increase in high-frequency power, followed by a prolonged dip in alpha power, a pattern also noted with olfactory stimulation. This initial high-frequency component was tentatively seen as a specific processing mechanism for trigeminal input, while the alpha power change likely indicated reduced neuronal idling linked to cortical processing of the trigeminal stimulus. The consistency in cortical responses, shown by similar alpha power changes during both olfactory and trigeminal nerve stimulation, may indicate a shared approach in their processing pathways. This shared processing suggests a connected relationship between how nasal somatosensory and olfactory information is handled, supporting recent research claiming that “the act of sniffing is integral to the olfactory percept.” (35) Huart et al. further examined the ability of time-frequency changes caused by olfactory and trigeminal stimulation to distinguish between background EEG noise and stimulus processing. The capacity to differentiate between the presence or absence of olfactory cortical processing is critical for clinical evaluations of olfactory function and supports the overall reliability of this approach. Notably, the time-frequency signal showed better discriminative power than olfactory event-related potentials, successfully differentiating between background noise and stimulus-induced olfactory changes with a peak sensitivity of 81.8% and a specificity of 90.9%. (10) 4. Conclusion Although there has been progress in assessing olfactory function clinically, significant uncertainties remain, especially regarding the prognosis and differential diagnosis of patients with olfactory disorders. While specific causes are clear for some patients, many have unidentified reasons for their conditions. Also, dependable prognostic tools are still necessary. Future improvements in electrophysiological techniques, like time-frequency analysis, and imaging methods, such as fMRI and cortical thickness measures, are expected to fill these important gaps. The field of olfaction has advanced significantly in recent years. Currently, olfactory dysfunction can be diagnosed or confirmed in clinical practice using reliable and validated psychophysical, electrophysiological, and imaging ELECTROPHYSIOLOGICAL MEASUREMENT OFOLFACTORY FUNCTION   89 techniques. These methods provide accurate structural representations of the parts involved in smell perception. 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Chemical Senses. 2005;31(2):181. doi:10.1093/chemse/bjj012 93 CHAPTER VII PHYSIOLOGY OF AUTISM SPECTRUM DISORDER: CURRENT ISSUES AND EMERGING MECHANISM Burcu ÇEVRELİ (Asst. Prof. Dr.), Üsküdar University Faculty of Medicine, Department of Physiology, E-mail: [email protected] ORCID: 0000-0001-6337-4999 1. Introduction Autism Spectrum Disorder (ASD) is a complex neurodevelopmental condition characterized by persistent difficulties in social communication and restricted, repetitive behaviors. Although historically conceptualized as a primarily behavioral or cognitive disorder, increasing evidence situates ASD within a broader physiological framework shaped by genetic, neuroendocrine, immune, and environmental interactions (1,2). Current prevalence estimates indicate that ASD affects approximately 1 in 31 children, highlighting the urgency of moving beyond descriptive symptomatology toward underlying biological mechanisms (1). Genetic variants involved in synaptic scaffolding and neurotransmitter regulation (e.g., SHANK3, NRXN1, GAD1) influence neuronal differentiation and network organization; however, their expression is dynamically modulated by prenatal physiological factors such as maternal hormones, inflammatory cytokines, oxidative stress, and metabolic status (2). Dysregulation of the hypothalamic–pituitary–adrenal axis, maternal immune activation—particularly IL-6 and IL-17A—and prenatal sleep disturbance converge with excitatory–inhibitory imbalance, mitochondrial dysfunction, and epigenetic modifications to shape atypical brain development. Collectively, these findings support conceptualizing ASD as a systems-level physiological disorder and provide a rationale for biomarker-based early detection and precision-oriented interventions. 94   CURRENT ISSUES IN PHYSIOLOGY II 1.1 From Genetics to Physiology Although twin and family studies suggest heritability rates exceeding 70%, purely genetic models fail to account for the heterogeneity and dynamic nature of ASD (3) Physiological research bridges this gap by identifying mechanisms through which genes shape neural circuits under environmental influence. For example, neuroimaging and postmortem data demonstrate that genes associated with synaptic scaffolding (e.g., SHANK3, NRXN1), neurotransmitter signaling (e.g., GAD1, SLC6A4), and neuroinflammatory control (IL6, TNF) affect early neuronal differentiation and connectivity. However, their expression patterns are highly sensitive to maternal hormones, cytokines, and stress mediators during prenatal life (4). These findings highlight that genetic susceptibility operates within a physiologically active developmental landscape, where environmental signals modulate gene expression and ultimately shape ASDrelated neural circuitry. Over the past decade, the physiological model of autism has expanded to encompass endocrine, immune, and metabolic dimensions. The HPA axis—a central regulator of stress and homeostasis—has emerged as a key mediator of brain-environment interactions. Dysregulation of cortisol and corticosterone secretion disrupts fetal neurogenesis, glial differentiation, and synaptic pruning, thereby predisposing offspring to behavioral abnormalities (5, 6). Elevated interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and reactive oxygen species (ROS) levels in maternal circulation induce oxidative stress, mitochondrial dysfunction, and microglial overactivation in the fetal brain (7, 8). Collectively, these endocrine and immune disruptions converge on shared developmental pathways, highlighting how diverse maternal physiological stressors ultimately shape neural circuit formation and increase vulnerability to ASD. 1.2 Physiological Impact of Maternal Sleep and Stress Maternal sleep quality and stress levels during pregnancy exert critical physiological influences that directly shape fetal development and longterm offspring health. Disrupted maternal sleep—characterized by insomnia, fragmented sleep, or circadian misalignment—activates the hypothalamic– pituitary–adrenal (HPA) axis, resulting in elevated cortisol levels, inflammatory cytokine release, and altered autonomic balance. (9, 10). These biological alterations modify placental function, reduce uteroplacental blood flow, and dysregulate melatonin secretion, a key synchronizer of fetal circadian and neurodevelopmental processes (11, 12). Maternal stress further intensifies these PHYSIOLOGY OF AUTISM SPECTRUM DISORDER: CURRENT ISSUES . . .   95 physiological disruptions by amplifying neuroendocrine activation and oxidative stress, which can compromise fetal neuronal proliferation, synaptogenesis, and epigenetic programming (13, 14). Evidence from both human cohorts and animal models demonstrates that chronic prenatal stress and sleep deprivation are associated with altered brain connectivity, impaired emotional regulation, and increased vulnerability to neurodevelopmental disorders in offspring, including anxiety, attention deficits, and autism-related behavioral phenotypes (15). Overall, sleep disturbances and psychological stress in pregnancy function as potent environmental modulators that influence fetal brain maturation and metabolic programming. Their combined impact underscores the importance of maternal sleep hygiene, stress reduction interventions, and prenatal monitoring strategies to optimize offspring neurobehavioral and physiological outcomes (16, 17). Together, these findings illustrate that maternal sleep disruption and psychological stress act through intertwined endocrine, inflammatory, and circadian pathways, creating a physiological milieu that can recalibrate fetal brain development and heighten susceptibility to ASD-related outcomes. 2. Neurodevelopmental and Structural Physiology 2.1 Early Brain Development and Neurogenesis Early brain development represents one of the most vulnerable and tightly regulated phases of human neurobiology, and disruptions during this period can have profound lifelong consequences. ASD is increasingly understood to originate from deviations in these foundational neurodevelopmental processes, particularly during the prenatal and perinatal windows when neural proliferation, migration, and circuit formation occur at their peak. During gestation, key neurodevelopmental events—neurogenesis, neuronal migration, and cortical layering—are orchestrated by dynamic molecular pathways such as Wnt/βcatenin, Notch, and Sonic Hedgehog (Shh) (18). Wnt/β-catenin signaling is especially critical for progenitor proliferation and differentiation. Animal studies have shown that aberrant activation or suppression of Wnt signaling during mid-gestation leads to altered hippocampal morphology and impaired synaptic density—changes closely resembling those found in ASD brains (19, 20). 2.2 Hippocampal and Cortical Contribution The hippocampus, a region essential for memory and emotional regulation, displays consistent structural anomalies in ASD. Reduced neuronal density in 96   CURRENT ISSUES IN PHYSIOLOGY II the CA1–CA3 regions, altered dendritic arborization, and smaller pyramidal neuron soma sizes have been reported in both clinical and preclinical studies (21). In addition to the hippocampus, aberrant cortical folding (gyrification) and disrupted axonal guidance in the prefrontal and temporal regions contribute to impaired social and executive functioning. Recent diffusion tensor imaging (DTI) studies reveal decreased fractional anisotropy in white matter tracts connecting the amygdala and prefrontal cortex, suggesting that abnormal axonal myelination and tract integrity are physiological hallmarks of ASD (22). Importantly, these connectivity disruptions align with contemporary network models of ASD, which posit that altered long-range communication—rather than focal lesions— underlies many of the behavioral and cognitive manifestations of the condition. Taken together, hippocampal microstructural deficits and cortical connectivity disruptions form an interrelated physiological architecture that shapes ASD phenotypes. These findings support the view that ASD arises not from isolated regional abnormalities, but from distributed network-level alterations emerging early in neurodevelopment. 2.3 Cerebellar and Amygdalar Contribution Among subcortical structures, the cerebellum and amygdala are consistently implicated in ASD physiology. Cerebellar Purkinje-cell loss and reduced GABAergic input have been identified in postmortem tissue (23). Functional MRI studies indicate hypoactivation in cerebellar lobules VI and Crus I— regions associated with sensorimotor integration and social cognition (18). The amygdala, central to emotional learning, shows volumetric enlargement in early childhood but hypoactivity in adolescence, reflecting abnormal synaptic pruning trajectories (24). These observations converge on the notion that ASD represents a distributed network disorder involving disrupted interregional communication rather than focal deficit. Such patterns may contribute to heightened reactivity in early development and blunted socioemotional responsiveness later in life, reflecting a maladaptive developmental course. Current Issues and Emerging Mechanisms. Physiological heterogeneity complicates translation. Integrative omics highlight convergence on neuroimmune and synaptic pathways (19). Electroencephalographic (EEG) studies consistently reveal abnormal gammaband synchronization, indicative of disrupted excitatory/inhibitory (E/I) balance during information processing (26). These oscillatory abnormalities reflect the physiological outcome of early structural perturbations, bridging the micro (cellular) and macro (network) levels of ASD pathology. In summary, PHYSIOLOGY OF AUTISM SPECTRUM DISORDER: CURRENT ISSUES . . .   103 Although each experimental model captures a different facet of ASD pathophysiology, together they offer a multidimensional understanding of how environmental, immunological, endocrine, and metabolic disturbances can converge on common neurodevelopmental pathways. A comparative synthesis of these models provides insight into how ASD-like traits emerge from distinct yet intersecting biological mechanisms. 5.2.1 Epigenetic vs. Immune Pathways The VPA model and MIA model represent two mechanistically different but complementary routes toward ASD-like phenotypes. Experimental rodent models remain indispensable in uncovering physiological pathways in ASD. Among these, valproic acid (VPA) exposure, maternal immune activation (MIA) paradigms provide complementary insights. VPA exposure alters chromatin structure and epigenetic programming during early neural tube development, leading to long-term deficits in synaptic architecture. Replicates altered histone acetylation and oxidative stress. Offspring display reduced sociability and repetitive grooming, correlating with downregulated BDNF (42). (Maternal immune activation, by contrast, exerts its effects predominantly through IL-6 and IL-17A–driven inflammatory cascades, altering neuronal migration and microglial function. Mimics inflammatory cytokine elevation (IL-6, IL-17a) and microglial overactivation, producing sensory hypersensitivity (7). Notably, both models ultimately demonstrate disruptions in synaptic connectivity, cortical organization, and excitatory/inhibitory (E/I) balance, which are central features observed in ASD. 5.2.2 Neuroendocrine Stress Pathways Maternal sleep deprivation presents a distinct mechanism through which chronic stress and HPA axis hyperactivity shape fetal brain development. (34, 37) Excessive glucocorticoid exposure during gestation impairs hippocampal plasticity, reduces neurogenesis, and alters BDNF–TrkB signaling—mechanisms strongly implicated in ASD and anxiety phenotypes (35). Compared to immune or epigenetic models, MSD uniquely captures stress-induced fetal programming, reflecting real-world risk factors such as prenatal insomnia, shift work, or maternal psychosocial stress (16, 17) 104   CURRENT ISSUES IN PHYSIOLOGY II 5.2.3 Hypoxia, Placental Dysfunction, and Oxidative Stress Pre-eclampsia models highlight the importance of placental insufficiency, disrupted oxygenation, and redox imbalance in shaping neurodevelopment (43). Fetal hypoxia interferes with neuronal migration, cortical lamination, and synaptogenesis—processes that are critically timed during late gestation (50). These models provide a strong translational link to human clinical conditions known to elevate ASD risk (42). 6. Environmental and Neurodevelopmental Modulators Environmental physiology exerts profound effects on neurodevelopment. Maternal infection, stress, malnutrition, and circadian disruption all modulate fetal brain physiology via the placenta. Cytokines and glucocorticoids act as biochemical messengers, reshaping gene expression in the developing nervous system (42). Vitamin D deficiency during pregnancy reduces synaptic density and enhances neuroinflammation (45). Pre-eclampsia, through hypoxia-induced oxidative stress, interferes with cortical angiogenesis and leads to neuronal apoptosis (43). Moreover, the gut–brain axis has emerged as an important physiological interface: alterations in the maternal microbiome modify immune tone and metabolite availability, influencing fetal neurophysiology (44). Vitamin-D deficiency impairs synaptogenesis. Pre-eclampsia triggers oxidative stress and epigenetic dysregulation (45). Established that maternal sleep apnea similarly alters placental physiology, linking endocrine stress to ASD-like behaviors (17, 18). As summarized in Table 4, a wide range of maternal physiological conditions exert significant influence on fetal neurodevelopment through distinct yet converging biological pathways. The placenta functions as the central physiological interface between mother and fetus. It regulates the transfer of nutrients, hormones, and cytokines. Chronic maternal stress reduces placental 11β-HSD2 activity, thereby decreasing its capacity to inactivate glucocorticoids (5). Consequently, excess maternal cortisol crosses into the fetal circulation, where it alters neuronal proliferation and myelination.Emerging transcriptomic studies demonstrate altered placental expression of HSD11B2, CRH, and VEGF genes in pregnancies associated with ASD risk (43). PHYSIOLOGY OF AUTISM SPECTRUM DISORDER: CURRENT ISSUES . . .   105 Table 4. Maternal Factors and Neurodevelopmental Outcomes Maternal Condition Physiological Pathway Offspring Effect Stress HPA activation Anxiety-like behavior Sleep Apnea Corticosterone ↑ → Neuroinflammation Social deficit Vitamin D Deficiency Synaptic dysgenesis Cortical anomalies Pre-eclampsia Hypoxia Impaired cognition The developmental timing of physiological insult is crucial. Disruptions during neurogenesis (gestational days 10–20 in rodents; first–second trimesters in humans) are more detrimental than those occurring postnatally (47). The brain’s physiological sensitivity varies across regions—hippocampal neurons and prefrontal interneurons exhibit the highest vulnerability to glucocorticoid and cytokine exposure. Biomarker-driven subtyping—distinguishing ASD individuals by cortisol profiles, inflammatory markers, or neuroimaging phenotypes—may enable precision therapies (see table 2) (48, 51). 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PHYSIOLOGY OF AUTISM SPECTRUM DISORDER: CURRENT ISSUES . . .   111 51. Abi-Dargham A, Moeller SJ, Ali F, DeLorenzo C, Domschke K, Horga G, Jutla A, Kotov R, Paulus MP, Rubio JM, Sanacora G, Veenstra-VanderWeele J, Krystal JH. Candidate biomarkers in psychiatric disorders: state of the field. World Psychiatry. 2023 Jun;22(2):236-262. doi: 10.1002/wps.21078. THE DUAL DEFENSE MECHANISM OF EXERCISE-INDUCED MYOKINES . . .   119 mechanism of action is viewed as a fundamental element in reversing cognitive decline and compensating for neuronal damage and synaptic loss (23,24,28). CTSB facilitates neurogenesis and synaptic plasticity in brain regions that support performance in specific cognitive domains, including memory, learning, and executive functions (41). 4. Pharmacological and Clinical Strategies The fact that skeletal muscles are the most highly perfused and energyconsuming organs during physical activity places this tissue in a central position in the fight against the aging process (7). At this point, it can be said that skeletal muscles are not only responsible for movement but are also a resource for maintaining general health and combating diseases. The triggering of myokine release with exercise can play a key role in the prevention and treatment of metabolic and neurological diseases. Myokines are signaling molecules secreted by skeletal muscles that facilitate inter-organ communication, especially during exercise. Due to these characteristics, they can be therapeutic targets for many diseases, including age-related conditions (42). Given the age-related decline in physical activity, the necessity of researching novel strategies that can provide effects similar to exercise has emerged. These agents, called exercise mimetics, are pharmacological or nutritional molecules aiming to imitate the beneficial signaling pathways provided by exercise, such as neuroplasticity, metabolic improvement, and muscle hypertrophy (42). Emphasizing the importance of exercise and exercise mimetics’ effects on organs like the liver, brain, bone, and the cardiovascular and gastrointestinal systems, the recent development of myokine mimetics is likely to lead to an increase in the use of beneficial signals secreted by muscle as potential drugs for systemic diseases (42). Irisin, a cleaved form of the FNDC5 protein, is an important myokine due to its ability to mimic the metabolic benefits of physical activity. Ranging from its role in fat and energy metabolism to its protective effects on the musculoskeletal, cardiac, renal, and nervous systems, Irisin has been evaluated as a potential “exercise mimetic” for future clinical applications (23,43). However, despite the pleiotropic effects of Irisin on many tissues, the lack of a precise identification of its receptor is the most significant structural barrier restricting this molecule’s transition to clinical application (44). BDNF, in addition to supporting neuroplasticity within the central nervous system, is secreted by skeletal muscle and affects both neurological and metabolic functions (28,45). A study using 7,8-dihydroxyflavone, a highly 120   CURRENT ISSUES IN PHYSIOLOGY II bioavailable BDNF mimetic, has proven that this compound exhibits effects that imitate exercise-induced adaptations (45). The aging process and sarcopenia disrupt the balance between positive regulators that support muscle growth and regeneration and negative regulators that induce muscle atrophy (46,47). Protective myokines like IGF-1 decrease due to sarcopenia, while pathological myokines such as Myostatin increase (47). To restore this balance, a better understanding of the roles of myokines like Irisin, Myostatin, and BDNF in the development of sarcopenia offers a promising opportunity for early diagnosis and intervention (47). The potential use of myokines as positive and negative biomarkers is expected to turn personalized treatment strategies based on these molecules into an important tool for tackling the challenges of aging and improving the quality of life in the elderly population (46). Myostatin is the key negative regulator whose levels are generally elevated in sarcopenic individuals, inhibiting muscle hypertrophy and promoting muscle fiber atrophy. Myostatin inhibition has emerged as a therapeutic strategy for disorders causing muscle loss, such as Duchenne muscular dystrophy (DMD), sarcopenia, and cachexia (48). In contrast, Irisin, which is critical for normal muscle metabolism and regeneration via the Akt/mTOR signaling pathway, is a myokine considered a positive biomarker for the early diagnosis of sarcopenia, as its levels are typically low in sarcopenia patients (49). Therefore, organizing myokine signaling can be a target for managing age-related muscle loss. This involves increasing the activation of positively characterized myokines (such as Irisin and BDNF) while inhibiting—or in other words, balancing—the effects of negative myokines (Myostatin) (47). For this reason, pharmacological interventions like myostatin inhibitors, alongside non-pharmacological approaches such as nutritional and lifestyle changes and personalized exercise programs, can demonstrate beneficial effects in achieving this balance (47). Exercise establishes an integrated, two-pronged defense mechanism via myokines to preserve systemic health. This mechanism is an optimized physiological response against neurodegenerative processes and metabolic disorders. Increases in exercise-induced levels of Irisin and BDNF are linked to improved neuroplasticity, reduced anxiety, and enhanced mood, while also supporting metabolic recovery (50). The second defense mechanism of exercise is the suppression of Myostatin expression, which triggers muscle loss and is associated with adverse neuroinflammatory or metabolic effects (51). THE DUAL DEFENSE MECHANISM OF EXERCISE-INDUCED MYOKINES . . .   121 5. Conclusion Skeletal muscles are not merely organs that facilitate movement, as traditionally understood, but are also a vital resource for maintaining overall health and combating disease. In fact, specialized signaling molecules termed myokines are secreted from skeletal muscle during exercise. These myokines establish a complex communication network with other organs throughout the body. Through this “cross-talk,” they play a key role in the prevention and even treatment of metabolic and neurological disorders. This positioning makes myokines a potential therapeutic target for a wide range of conditions, including those associated with aging. Specifically, their protective effects against neurodegenerative (ND) diseases place both regular exercise and these molecules at the center of novel treatment approaches. However, a critical challenge exists. Different types of exercise may trigger the release of distinct myokine profiles. In other words, the level of each myokine can vary depending on the type of exercise performed. Consequently, the observed benefits of exercise on ND diseases may also vary accordingly. If we aim to develop myokines as direct therapeutic agents, understanding this diversity and specificity will be one of the greatest obstacles to overcome. 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Introduction Public health concerns of the 21st century are concentrated around noncommunicable diseases. Obesity, diabetes mellitus (DM), and a broad spectrum of metabolic diseases are leading causes of morbidity and mortality worldwide(1). These diseases are not isolated, but rather reinforcing diseases that exacerbate each other’s effects. Metabolic diseases are a group of chronic diseases resulting from disruptions in the biochemical pathways involved in converting nutrients into energy(2). These disruptions lead to a range of complex and interrelated complications. Diabetes Mellitus (DM) and Metabolic Syndrome occupy a central position within this group, which has become one of the most common and significant health problems in the modern world. DM, which is characterized by chronic hyperglycemia, is a global epidemic with increasing proportions(3). DM is a complex metabolic disease with an absolute or relative deficiency of insulin. Insulin is the key hormone that allows glucose to enter cells from the bloodstream to be used as an energy source. Disruption of this system leads to glucose accumulation in the bloodstream(4). 2. Classification of Diabetes Mellitus While type 1 diabetes mellitus (T1DM) is characterized by a lack of insulin production by pancreatic beta cells due to autoimmune processes or toxicity, type 2 diabetes mellitus (T2DM) is known as non-insulin-dependent diabetes, which is caused by reduced sensitivity (resistance) to insulin(5). 128   CURRENT ISSUES IN PHYSIOLOGY II There are also less common forms of DM, such as gestational diabetes, monogenic diabetes, and secondary diabetes caused by pancreatitis or medications(6). 2.1 Type 1 Diabetes Mellitus (T1DM) Pathogenesis of T1DM is concentrated around absolute insulin deficiency due to autoimmune disruption of pancreatic beta cells. Genetically, a strong association with HLA-DR3/DR4 genotypes is found(7). Immunologically, autoantibodies such as glutamic acid decarboxylase (GAD) and IA-2 are found to be positive(8,9). T1DM has a sudden onset, generally in childhood or adolescence. Polyuria, polydipsia, weight loss, and weakness are prominent (10). Hyperglycemia, ketonemia, and metabolic acidosis cause the formation of ketoacidosis (11). Neurological symptoms of diabetic ketoacidosis in T1DM include, but are not limited to, lethargy, confusion, and coma. Hyperglycemia and ketosis disrupt cerebral high-energy phosphates, blood flow, and cortical water distribution, causing cerebral edema, headache, papilledema, and bilateral abducens palsy(12). There are also reports suggesting that ketoacidosis can cause ischemic optic neuropathy(13). Individuals with T1DM are dependent on lifelong insulin therapy. T2DM is the most common form of diabetes (90-95%). 2.2 Type 2 Diabetes Mellitus (T2DM) Resistance to the effects of insulin is prominent in T2DM. Beta-cell dysfunction and relative insulin deficiency develop over time in T2DM. Genetic predisposition and environmental factors such as obesity and sedentary lifestyle trigger a series of pathophysiological mechanisms that result in T2DM(14). T2DM has an insidious onset. It may be asymptomatic at diagnosis or manifest with nonspecific symptoms such as fatigue and blurred vision(15). T2DM diagnosis is usually made during routine screening or when complications like neuropathy or cardiovascular problems arise. Extreme hyperglycemia (>600 mg/dL), hyperosmolarity (>320 mOsm/L), dehydration, and the absence of ketosis and significant acidosis can cause hyperosmolar nonketotic coma in T2DM patients(16). Neurological symptoms of T2DM include, but are not limited to, decreased level of consciousness, focal neurological deficits such as hemiparesis or aphasia, focal, tonic, or epilepsia partialis continua type seizures, and various movement dysfunctions(17-20). Some work reports hemiballismus/hemichorea in patients with hyperglycemia(21). In addition, hallucinations, tonic eye deviation, nystagmus, STZ INDUCED DIABETES MELLITUS MODELS IN EXPERIMENTAL ANIMALS   135 Table 1: The summary of the most frequently used application doses in STZ-induced diabetes mellitus models, the pathological processes that develop as a result of application and the similarity of the developed model to its human counterpart Model Type Protocol Pathology Similarity to human counterpart Single HighDose (SHD) (T1DM) A single intraperitoneal (i.p.) or intravenous (i.v.) injection. (Rat: 50-65 mg/kg; Mice: 100-200 mg/kg). Rapid, prominent betacell disruption, severe insulin deficiency. Classical Type 1 diabetes. Multiple LowDose (MLD) (T1DM) Daily low-dose injections for 5 consecutive days(e.g., 20-40 mg/kg) Progressive loss of beta cells,autoimmunelike insulitis. Autoimmune, progressive type 1 diabetes. High Fat Diet (HFD) + Low-Dose STZ (T2DM) 3-8 weeks of HFD feeding, followed by a single or consequent low-dose of STZ injection (e.g., 35-45 mg/kg). Insulin resistance + partial beta-cell disruption Type 2 diabetes with insulin resistance and obesity. Nicotinamide (NA)+STZ (T2DM) NA (e.g., 230 mg/ kg) is followed by a low-dose STZ (e.g., 65 mg/kg). NA partially blocks STZ toxicity, resulting in partial beta-cell disruption. Type 2 diabetes with impaired insulin secretion without obesity. The success and reproducibility of the STZ-induced diabetes mellitus model depend on some important factors. First of all, the sex of the animals to be used directly affects the success of the implemented model. Males show a higher incidence of diabetes and more prominent hyperglycemia than females due to the protective effects of estrogen(66). Secondly, young adults (e.g., 6-8 weeks old) and C57BL/6J mice, Sprague-Dawley or Wistar rats are commonly used(63). However, using younger animals can cause a handicap due to the higher regenerative capabilities of pancreatic cells in younger animals, which can prevent the negative effects of acute high-dose injections(67). Thirdly, proper preparation and administration of STZ is utmost critical for the model to 136   CURRENT ISSUES IN PHYSIOLOGY II achieve success. STZ is unstable under light and at neutral pH. So, STZ should always be dissolved in a cold (0-4°C) and freshly prepared citrate buffer with pH around 4.2 to 4.5. STZ injections are generally given via the i.p. route. So, the prepared STZ solution should be kept on ice to minimize the degradation and also should be used within 30 minutes(42,50). On the other hand, some work suggests that the α and β anomers of freshly dissolved STZ are unstable. It has been suggested that allowing the freshly prepared STZ solution to stand on ice for 45-60 minutes allows the anomers to reach equilibrium and provides a more reproducible diabetogenic effect(68). Traditionally, fasting of animals before any injection to experimental animals is recommended. However, current evidence from literature suggests that injecting fed animals reduces the risk of severe hypoglycemia and mortality due to the sudden insulin release present in the first 24 hours following STZ injection. Therefore, for the STZ model to achieve success and decrease the mortality rates, administration without fasting is recommended(62). After the induction of diabetes model with STZ, validation with blood glucose measurements are upmost importance for the validity and reproducibility of the experiment. Fasting or random blood glucose measurements should be taken 48–72 hours after STZ injection. 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