Neurolipidomic insights into anxiety disorders: Uncovering lipid dynamics for potential therapeutic advances

Anxiety disorders constitute a spectrum of psychological conditions affecting millions of individuals worldwide, imposing a significant health burden. Historically, the development of anxiolytic medications has been largely focused on neurotransmitter function and modulation. However, in recent years, neurolipids emerged as a prime target for understanding psychiatric pathogenesis and developing novel medications. Neurolipids influence various neural activities such as neurotransmission and cellular functioning, as well as maintaining cell membrane integrity. Therefore, this review aims to elucidate the alterations in neurolipids associated with an anxious mental state and explore their potential as targets of novel anxiolytic medications. Existing evidence tentatively associates dysregulated neurolipid levels with the etiopathology of anxiety disorders. Notably, preclinical investigations suggest that several neurolipids, including endocannabinoids and polyunsaturated fatty acids, may hold promise as potential pharmacological targets. Overall, the current literature tentatively suggests the involvement of lipids in the pathogenesis of anxiety disorders, hinting at potential prospects for future pharmacological interventions.


Introduction
Anxiety disorders represent a diverse and prevalent mental health challenge that impacts millions of individuals around the globe (Javaid et al., 2023).These disorders encompass a range of conditions characterised by excessive fear and anxiety, where anxiety is defined as not merely a reaction to immediate threats but rather a pervasive anticipatory response to perceived future dangers (American Psychiatric Association DSMTF, 2013).Collectively, these disorders impose a significant health burden as they have the capacity to detrimentally impact an individual's quality of life (Xiong et al., 2022), and they often coexist with various psychiatric comorbidities which further complicates their treatment and management (Nel et al., 2018).The pervasive nature of anxiety disorders underscores their status as a critical public health issue, necessitating ongoing research to better understand their complex dynamics and develop more effective interventions.
Currently, anxiety disorders are predominantly diagnosed using psychometric tests, including questionnaires and interviews, rather than objective, quantitative measures (Ruscio et al., 2017).However, the symptomatology of this disorder can be variable across individuals, especially among children and adolescents (Baldwin et al., 2011); therefore, utilising a qualitative method may not be adequate when diagnosing patients with atypical presentations of anxiety-related psychiatric conditions.Moreover, it has been shown that current methods of diagnosis through a clinical and subjective assessment can have high rates of inter-reliability variability (Maes et al., 2018).This underscores the necessity for further studies to establish evidence-based biomarkers that have a broad range of clinical applications to the diagnosis of anxiety disorders.
To address the variability in the symptom presentation of anxiety disorders among individuals and the inherent limitations of qualitative diagnostic assessments, there is a pressing need to establish quantitative diagnostic measures, such as serum biomarkers, to enhance the accuracy of diagnoses in clinical practice.The use of biomarkers may be helpful in terms of understanding the specific aetiological mechanisms of anxiety in the context of the individual patient, which may then lead to the development of personalised treatments and improved treatment outcomes for patients with anxiety disorders.
Additionally, given the limitations of current treatment strategies for anxiety disorders, establishing a new pharmacological target for anxiolytic medication is essential.Currently, the treatment regime for all anxiety disorders consists of a combination of psychological and pharmacological therapy (Craske and Stein, 2016).Out of the various psychological therapies, cognitive behavioural therapy (CBT) has the strongest evidence base for treating anxiety disorders, and more recently, its accessibility has significantly improved due to the advent of online therapy (Olthuis et al., 2016).In contrast, while the currently available pharmacological interventions do offer effective management of anxiety symptoms, these medications are not without some drawbacks.Presently, the first-line medications for various anxiety disorders including Panic Disorder, Generalized Anxiety Disorder, and Social Anxiety Disorder are Selective Serotonin Reuptake Inhibitors (SSRIs) and Serotonin and Norepinephrine Reuptake Inhibitors (SNRIs).Despite their widespread use, the adverse effect profile of these medications (including nausea, headaches, sexual dysfunction, and insomnia) often discourages patients from continuing treatment (Gosmann et al., 2023).Benzodiazepines are another class of pharmacotherapy that is effective for acute anxiolysis and sedation; however, due to its high risk of dependence, its long-term use is generally not advised (Balon and Starcevic, 2020).Other pharmacological options for anxiolysis, such as buspirone (5-HT1A agonist) and mirtazapine (noradrenergic and specific serotonergic antidepressant) have been proposed to be effective treatment options for anxiety disorders; however, the evidence supporting their use is not as robust when compared to first-line medications such as SSRIs (Garakani et al., 2020).Other less commonly utilised pharmacological treatment options include medicinal cannabis, antipsychotics (such as quetiapine), and anticonvulsants (such as pregabalin and gabapentin) (Stanciu et al., 2021;Berger et al., 2022).The current evidence supporting the use of these medications is limited, and each medication class carries its own unique adverse effect profile that can negatively impact an individual's cognitive ability and daily functioning (Stanciu et al., 2021;Berger et al., 2022).Therefore, it must be acknowledged that the currently available pharmacological treatments for anxiety are not without their shortcomings, and consequently, the pursuit of a novel anxiolytic medication with a minimised adverse effect profile is imperative.
Various neuropeptides of the central nervous system have been investigated in recent years as a potential target for anxiolytic treatment.For instance, oxytocin, renowned for its impact on social cognition and behaviour, has been theorised as a potential treatment for the social impairments often observed in anxiety disorders (Kendrick et al., 2018;Neumann and Slattery, 2016).Indeed, various animal and human studies have supported a role for intranasal oxytocin in disorders such as Social Anxiety Disorder (Kendrick et al., 2018).Furthermore, neuropeptides of the limbic system, including tachykinins such as Substance P, and vasoactive peptides such as angiotensin-2 and vasopressin, have also been proposed as candidates for anxiety pharmacotherapy (Marvar et al., 2021).One experimental proof-of-concept study in humans found that SRX246 (vasopressin-1 antagonist) decreased the anxiety-potentiated startle response, with a similar anxiolytic response as that of benzodiazepines and SSRIs (Lago et al., 2021).Overall, emerging research shows promising evidence of novel pharmacological targets that may be effective at inducing anxiolysis.
In recent years, neurolipids have also become a focus of interest as a potential novel biomarker and therapeutic target for neurocognitive and psychiatric disorders (Schneider et al., 2017a) (Müller et al., 2015).Lipids, comprising over 50 % of the brain's dry weight, are a significant component within the brain structure and facilitate a wide range of neuronal activities (Miranda and Oliveira, 2015).A significant portion of the lipids within the brain tissue exist in the plasma membrane, where they function not only to maintain cell membrane integrity but also to modulate the functions of cell membrane proteins, thereby influencing synaptic throughput (Schneider et al., 2017a).Furthermore, beyond their structural role, lipids participate dynamically in cell signalling, both as agents within exo-and endocytic processes and as second messengers of signalling cascades (Müller et al., 2015).In particular, some neurolipids have been identified to traverse the cellular membrane, functioning as intracellular transmitters to relay signals within the cell or as extracellular transmitters to communicate between cells (Schneider et al., 2017a).Considering the broad range of influences lipid molecules have over neuronal functions, it has been hypothesised that neurolipids may serve a crucial role in the pathophysiology of neuropsychiatric conditions (Schneider et al., 2017a).
Emerging research has demonstrated that variations in the lipid composition within neural tissueranging from the microscopic level of intracellular compartments to the macroscopic scale of specific brain regionsare associated with impacts on perception, mood, and emotional behaviour (Müller et al., 2015;Miranda and Oliveira, 2015;Akefe et al., 2023).The current evidence suggests that neuronal lipids exert an influential force across various structural dimensions within the central nervous system, which effectively underscores the potential role neurolipids play in the pathophysiology of psychiatric conditions (Humer et al., 2020).Indeed, it has been demonstrated in human and animal-model studies that anxiety-related symptoms and behaviours are correlated with altered expressions of neurolipids, further suggesting these molecules' potential role in the pathogenesis of anxiety disorders (Demirkan et al., 2013;Oliveira et al., 2016).Consequently, this review aims to synthesise the existing literature on the relationship between neurolipidomic profiles, anxiety disorders, and anxiety-related behaviours.In addition, it seeks to identify specific lipid biomarkers and clarify the underlying biochemical pathways involved in the pathogenesis of anxiety disorders, thus guiding future research, and informing potential therapeutic interventions (Miranda and Oliveira, 2015).

Search strategy
A thorough search of various databases was conducted to identify primary research that investigated the role of neurolipids in anxiety disorders and anxiety-related behaviours.The databases searched include PubMed, Embase, and PsycINFO.These databases were chosen due to their extensive coverage of biomedical and psychological literature.The following search terms were used: ("Generalized anxiety disorder" OR "anxiety disorder" OR "anxiety disorders" OR "anxiety symptoms" OR "anxiety behaviours" OR "social anxiety disorder" OR agoraphobia OR anxiety[tiab]) AND ("sphingolipids" OR "polyunsaturated fatty acid" OR "polyunsaturated fatty acids" OR PUFA* OR endocannabinoid* OR ceramide* OR ApoE OR "omega-3" OR "fish oil" OR "membrane lipid" OR "omega-6" OR "membrane lipids" OR "membrane composition" OR cholesterol* OR phosphatidyl* OR ("oxidative stress" AND "lipid metabolism") OR "lipid oxidation" OR "lipid peroxidation").
The search was limited to articles published in English from January 2010 to June 2023.
This review included peer-reviewed primary research articles that focused on the biochemical mechanisms and implications of lipidomics in the brain related to anxiety disorders and anxious behaviour.Specifically, articles were selected if they provided empirical data relevant to the study topic.Excluded from this review were non-peer-reviewed articles, conference abstracts, editorials, studies focusing solely on peripheral lipidomic profiles without relation to brain function, and articles not available in full text or not written in English.
Data were extracted methodically by three independent reviewers.Extracted information included authors, year of publication, study design, sample size, key findings, and implications for understanding anxiety disorders.Discrepancies between reviewers were resolved through discussion and consensus.The relationships between lipidomic profiles and anxiety symptoms across different studies were examined to identify patterns and discrepancies.
The quality of each selected study was assessed using the ARRIVE checklist for animal studies and the CASP checklist for human studies respectively, ensuring a thorough evaluation of research quality across the various experiment types.Selected papers that contained inherent limitations or potential publication bias were noted, and these concerns were addressed within the manuscript itself.

Neurolipids and their functions
Within the human brain, there are a wide array of neurolipids which exert a diverse range of functions.Firstly, there are the three major classes of lipids that comprise the neuronal cell membrane including sterols (e.g.cholesterol), glycerophospholipids, and sphingolipids.In addition to playing a major role as the building blocks of synaptic vesicles and neuronal membranes, these brain lipids are involved across a diverse range of neuronal functions including scaffolding (Lee et al., 2020), neurodevelopment (Zhu et al., 2016), signalling (Falomir-Lockhart et al., 2019), neuroinflammatory (Giacobbe et al., 2020), neurometabolic (Bruce et al., 2017), and cognitive functioning (de Mendoza and Pilon, 2019;Tracey et al., 2018a;Snowden et al., 2017;Egawa et al., 2016a;Derbyshire, 2018).In addition, fatty acids and endocannabinoids actively participate in cellular signalling and inflammatory pathways within the brain (Kruk-Slomka et al., 2017).These lipid compounds are involved in a range of neurophysiological processes involved in brain development (Joffre et al., 2016), neuroprotection (Zoppi et al., 2011), pain modulation (Akopian et al., 2009), and cognitive function (Kruk--Slomka et al., 2017).
In the following sections, we investigate the multifaceted functions of neurolipids with the view of further understanding their diverse roles in the context of the development of the symptoms associated with anxiety disorders.

Cholesterol
Cholesterol is an integral component of the neural cell membrane and the myelin sheath (Hussain et al., 2019;Jin et al., 2019).Due to the limited permeability of plasma lipoproteins across the blood-brain barrier, the de novo synthesis of cholesterol by neurons and glial cells is the primary source of cholesterol in the CNS (Hussain et al., 2019).Cholesterol serves a crucial role in terms of maintaining cellular membrane stability and contributes significantly to synaptic neurotransmitter exocytosis and endocytosis, which in turn enhances synaptic transmission efficiency (Li et al., 2022).Consequently, dysregulation in cholesterol levels has been associated with signs of neurodegeneration and impaired neural plasticity (Koudinov and Koudinova, 2005).For example, mice that were fed with excess cholesterol in their diets demonstrated impaired long-term potentiation (Koudinov and Koudinova, 2005).Moreover, in another study, rodents that were administered a cholesterol-rich diet exhibited changes in the brain similar to those seen in neurodegenerative disorders with decreased synaptic plasticity (Koudinov and Koudinova, 2003;Akefe et al., 2021).
Cholesterol, together with sphingolipids, is known to have an important role in terms of maintaining the stability and functioning of membrane lipid rafts (MLRs), which are small lipid-protein assemblies that float within the lipid bilayer of the cellular membrane (Sezgin et al., 2017).In this arrangement, cholesterol interacts with various lipids (e.g.sphingomyelin) using its alpha interface and simultaneously engages with transmembrane proteins including neurotransmitters through its beta interface.Through this mechanism, MLRs have been implicated across a range of cellular processes including protein membrane trafficking, receptor functioning, and neurotransmission (Mirzaei et al., 2021).Therefore, it has been hypothesised that lowering serum cholesterol concentrations may influence the function of MLRs, and thus have a positive neuromodulatory effect (Egawa et al., 2016b).

Phospholipids
Phospholipids form the fundamental structure of physiological membranes, which also include various other lipid molecules along with embedded membrane proteins (Angeline and John, 2013).Contrary to merely serving as passive barriers and structural platforms for protein-mediated interactions, phospholipids within cellular membranes undergo continual dynamic changes, influencing various properties of the membrane surface including the chemistry, fluidity, and functioning of cellular membranes (Berkecz et al., 2018).These alterations are in part, due to the activity of phospholipases, which are enzymes that catalyse the hydrolysis of phospholipid ester bonds.Phospholipases, regulated by stimuli and other cellular factors including Ca2+ levels, can be either intracellularly or extracellularly secreted, which accounts for their widespread distribution (Angeline and John, 2013).
The phospholipases cleave phospholipids into a free fatty acid and a lysophospholipid, which is a phospholipid with a singular fatty acyl chain (D'Arrigo and Servi, 2010).The Phospholipase 1 (PLA1) group typically acts on phospholipids containing monounsaturated fatty acids (MUFA) or saturated fatty acids at the sn-1 position (Choi et al., 2018;Akefe, 2022), while Phospholipase 2 (PLA2) has been characterised to preferentially act on phospholipids with unsaturated fatty acid groups on the sn-2 position (Narayana et al., 2015a).
The current literature suggests that phospholipase A1 (PLA1) group influences neuronal lipid metabolism, and thereby aids in neuronal remodelling and maintenance (Richmond and Smith, 2011).A 2015 study by Narayana et al. found that the stimulation of neuroexocytosis results in a major increase in saturated free fatty acids, which is highly suggestive of PLA1 involvement (Narayana et al., 2015a).Furthermore, a recent study found that DDHD2, an isoform of the PLA1 family, has been found to interact with STXBP1, which is a key protein in neuronal SNARE complexes that mediate neuronal endocytosis and exocytosis (Akefe et al., 2024).DDHD2 knockout mice were found to exhibit significantly reduced memory formation and fear conditioning, highlighting the role of phospholipases in synaptic and memory formation (Akefe et al., 2024).
On the other hand, Phospholipase A2 (PLA2) activity appears to influence neuronal functioning by promoting the fusion of secretory vesicles (Pellett et al., 2019).Morgan and Burgoyne (1990) found that during exocytosis, there was a concomitant increase in the levels of endogenous arachidonic acid (C20:4n-6, AA) production and catecholamine secretion (Morgan and Burgoyne, 1990).The increase in AA and stress hormones in turn may have implications in the pathogenesis of anxiety disorders, leading to novel pharmacological therapeutic approaches in the management of such conditions (Morgan and Burgoyne, 1990).
Overall, phospholipids are a diverse lipid class and are typically derived from the two common precursors which include diacylglycerol and phosphatidic acid (Tracey et al., 2018b).They can be broadly categorized into two main groups: glycerophospholipids and phosphosphingolipids.

Glycerophospholipids
Glycerophospholipids consist of a phosphate head and two hydrophobic fatty acid tails bound to a glycerol molecule (Choi et al., 2018).The phosphate head is further bound to another organic hydrophilic molecule, which determines the class of phospholipid (e.g.phosphatidylcholine when a choline molecule is bound (Tracey et al., 2018b).The most common phospholipid class within the rat neurolipidome is phosphatidylethanolamine (PE), which accounts for 54 % of the brain's phospholipids, followed by phosphatidylcholine (PC) which accounts for 31 % (Choi et al., 2018).Studies in the human brain have found the opposite, with the human motor cortex comprising 42 % PC and 35-37 % PE (Hancock et al., 2022).It also must be noted, however, that these proportions vary in different regions of the brain (Hancock et al., 2022).The neuronal phospholipids contain variable fatty acid tails, with long-chain PUFAs being one of the major fatty acids expressed in phospholipids (Schneider et al., 2017b).These PUFA-bound phospholipids are known to preferentially localise around membrane proteins (Schneider et al., 2017b), and as a result, these phospholipids may have an important role in terms of mediating neuronal excitation, inhibitory neurotransmission and synaptic plasticity (Thalman et al., 2018).

Sphingolipids
Sphingolipids are a family of lipids that contain a sphingoid base backbone.In humans, the most common sphingoid base is sphingosine (Tracey et al., 2018b), which is found as a constituent of sphingomyelin (Leipelt and Merrill, 2004).Sphingomyelin is a major component of neuronal membranes and myelin and is known to have a critical role in early neuronal development as well as being an important modulator of synaptogenesis (Hussain et al., 2019).
Sphingolipids, along with cholesterol, have been proposed to concentrate and form membrane-protein-rich regions in the membrane known as MLRs (Sezgin et al., 2017).These structures are known to trap and oligomerise signalling proteins, thereby promoting cell signalling and leading to amplification (Sezgin et al., 2017).Furthermore, MLRs are the site of activation for acid sphingomyelinase, leading to the subsequent formation of ceramides in response to intracellular stressors.The ceramides then form macrodomains within the cell surface.These microdomains bind to membrane-related proteins such as c-Raf-1 and protein kinase-C, which consequently promotes the oligomerisation of G-protein coupled receptors (Müller et al., 2015).Through this mechanism, sphingolipids and ceramides appear to be involved in a wide range of cellular processes including inflammation, cell proliferation, cellular differentiation, and apoptosis (Futerman, 2016).
Furthermore, a range of sphingolipids are known to be bioactive and function as messengers across a range of cellular transduction pathways.An example of such a sphingolipid is sphingosine-1-phosphate, which is known to be involved in cell migration and lymphocytic trafficking (Grassi et al., 2019).

Fatty acids
Fatty acids encompass a diverse group of organic molecules which are crucial for a range of biological functions.Fatty acids consist of hydrocarbon chains with a carboxylic acid group and can vary in chain length, saturation, and structure (Joffre, 2019).
Among the varied fatty acid types, polyunsaturated fatty acids (PUFAs) have emerged as a crucial subgroup and have been demonstrated to be involved in a range of neurocircuits in the brain (Pancheva et al., 2023).PUFAs have a biochemical structure consisting of multiple double bonds in their hydrocarbon chains and form an integral part of the brain's lipidome, comprising approximately 25-30 % of the brain's fatty acid content (Joffre, 2019).Within the central nervous system, PUFAs exist in their esterified form and are bound to the phospholipids in the neural cell membrane (Bazinet and Layé, 2014).PUFAs can be broadly categorised into two groups: the n-3 PUFAs and the n-6 PUFAs.The n-3 PUFAs include eicosapentaenoic acid (C20:5n-3, EPA), docosahexaenoic acid (C22:6n-3, DHA) and alpha-linoleic acid (C18:3n-3, ALA), and the n-6 PUFAs include arachidonic acid and linoleic acid (C18:2n-6, LA).
The biosynthesis of PUFA involves a series of enzymatic steps primarily occurring in the liver and adipose tissue (Valenzuela et al., 2024).Key enzymes in this process include elongases and desaturases (Valenzuela et al., 2024).Elongases, such as the very long-chain fatty acids protein (ELOVL), add two-carbon units to the fatty acid chain (Valenzuela et al., 2024).Desaturases, including delta-5 desaturase and delta-6 desaturase, introduce double bonds at specific positions within the fatty acid chain (Videla et al., 2022).Factors influencing desaturation and elongation include dietary intake of fatty acids, environmental factors, health status, hormonal regulation (such as insulin and glucagon levels), interaction with other metabolic pathways, and genetic variations in the genes encoding for these enzymes (Wong and Sul, 2010;Bézard et al., 1994).
n-3 PUFAs have been shown to play a crucial role in early brain development and neuronal functioning (Pancheva et al., 2023).According to a study conducted by Joffre et al. (2016) (Joffre et al., 2016), DHA was strongly localised to the frontal cortex of mice brains, and when the mice were fed with n-3 deficient diet, the mice were shown to have poorer spatial memory performance, higher levels of depressive symptoms, and reduced levels of neuronal plasticity (Joffre et al., 2016).Furthermore, n-3 PUFAs may also have a role as a secondary messenger; they have been shown to inhibit inflammatory pathways (Heras-Sandoval et al., 2016) through the release of resolvins and neuroprotectins, which in turn promote neuronal survival and mitigate cell death (Kim et al., 2000).Therefore, n-3 PUFAs may have a role in the pathogenesis of anxiety disorders through their actions across different domains of brain function and metabolism (Joffre, 2019).However, there is a clinical need for further studies to ascertain the causal association between FFAs and the pathogenesis of anxiety disorders.
The n-6 PUFAs, including linoleic acid and arachidonic acid, have a greater antagonistic role to the n-3 PUFAs and act as precursors to proinflammatory messengers which are known as eicosanoids (Calder, 2020).Eicosanoids are derived from the oxidation of AA, through enzymes such as the cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 family of proteins (Calder, 2020).Mastinu et al. (2020) proposed that mast cells and astrocytes, which release AA during silent inflammation, could contribute to the symptoms that occur in depression and anxiety by altering the brain's fatty acid profile (Traina and Cocchi, 2020).These findings suggest that AA, as well as its derivatives, may be involved in the pathophysiology of the neurocircuitry involved in anxiety disorders.Moreover, recent studies indicate an intricate interplay between n-6 PUFAs, notably AA, and their interaction with modulating saturated free fatty acids (FFAs) (Narayana et al., 2015b).The complex relationship between these neurolipids may be a site for further investigation as abnormal interactions between these neurolipids may be associated with the development of anxiety disorders.

Endocannabinoids
The endocannabinoid system has been shown to mediate a range of neurological functions including neuroprotective mechanisms (Zoppi et al., 2011), learning (Marvar et al., 2021), cognition (Kruk-Slomka et al., 2017), and pain modulation (Akopian et al., 2009).The endocannabinoid system has also been implicated across various stress-related psychiatric conditions (Hill and Patel, 2013), and hence, it may be a potent future therapeutic target for anxiety symptoms.CB1 and CB2 are the two endocannabinoid receptors that are expressed within the human nervous system but are distributed across different brain networks (Kruk-Slomka et al., 2017).CB1 is mostly expressed in the axons and presynaptic terminals of the central nervous system, while CB2 has been implicated across a range of immunological and inflammatory pathways (Shahbazi et al., 2020).Uniquely, the endocannabinoid signalling system acts in a retrograde fashion, with activation of CB1 on the presynaptic terminal resulting in the inhibition of neurotransmitter release (Kruk-Slomka et al., 2017).
The endocannabinoid receptor ligands include anandamide (AEA) and 2-arachidonoylglycerol (2-AG).Broadly speaking, both AEA and 2-AG are derived from AA, although the two have distinctly different pathways of synthesis (Lu and Mackie, 2016).AEA is synthesised from N-arachidonoyl phosphatidyl ethanol (NAPE) as a consequence of the various phospholipase enzymes, and 2-AG is synthesised from the cleavage of 2-arachidonoyl containing phospholipids in the cell membranes (Lu and Mackie, 2016).Notably, 2-AG has a 1000 times greater level of concentration than AEA in the nervous system, thus making it S. Maehashi et al. the primary mediator for endocannabinoid activity (Sugiura et al., 1995).
The endocannabinoid degradation pathways are mediated through the actions of fatty acid amide hydroxylase (FAAH) and monoacylglycerol lipase (MAGL).As well as controlling synaptic endocannabinoid concentrations, these molecules also influence neuroinflammatory processes by producing arachidonic acid within the nervous system (Nomura et al., 2011).Several early studies have highlighted the possible roles of AA in neuroexocytosis, which suggests a potential association between AA and the pathogenesis of psychiatric conditions (Piomelli et al., 1987;Williams et al., 1989).Piomelli et al. (1987) found that AA and its metabolites promote synaptic plasticity (Piomelli et al., 1987), while Williams et al. (1989) have shown that it has actions in terms of mediating long-term synaptic plasticity within the hippocampus (Williams et al., 1989).In Williams et al.'s study (1989), the activity of AA was attributed to its actions across presynaptic sites although the molecular mechanism has not been defined.

Lipid peroxidation
Lipid peroxidation refers to the oxidative modification and subsequent degradation of the unsaturated fatty acids present within the cell membrane (Farooqui and Horrocks, 1998;Dix and Aikens, 1993;Akefe et al., 2022).Lipid peroxidation can occur as a physiological or pathological process.In a physiological context, the oxidation of lipids is a necessary biological process involved in the synthesis of signalling molecules including eicosanoids from PUFA (Farooqui and Horrocks, 1998;Dix and Aikens, 1993).Conversely, the pathological generation of reactive oxygen species (ROS) can occur either as an unintended byproduct of various cellular processes, including the electron transport chain within the mitochondria, or as a result of enzymatic processes such as NADPH oxidase (NOX) (Angelova et al., 2021).
Extensive and uncontrolled lipid peroxidation can lead to the compromise of the integrity and functioning of healthy neuronal membranes.The products of lipid peroxidation are also highly reactive, potentially generating more ROS and can cause alteration and degradation of DNA and protein structure (Gaschler and Stockwell, 2017).Hence, it has been suggested that the process of destabilization and damage induced by lipid peroxidation may be associated with the onset of psychiatric conditions including the classically described symptoms associated with anxiety disorders.Alternatively, the products of lipid peroxidation could potentially serve as a marker of neuronal inflammation stemming from such conditions (Joshi and Praticò, 2014) and may have clinical applications as a novel biomarker of anxiety symptoms.

Methodology involved in the quantification of lipid biomarkers
Advancements in techniques for measuring and quantifying lipid metabolism have significantly enhanced our understanding of the neurolipidome.Particularly, mass spectrometry (MS) has undergone substantial refinement over the past decade, closely paralleling the burgeoning research in lipidomics (Züllig and Köfeler, 2021).Among these techniques, matrix-assisted laser desorption/ionisation imaging mass spectrometry (MALDI-MS) stands out, leveraging a matrix to preserve lipid sample structures, thereby vastly improving sensitivity and expanding the range of analysable lipids (Harvey et al., 2023).Notably, MALDI-MS offers a unique advantage in analysing histological tissue samples, providing spatial context to lipid distribution (Aichler and Walch, 2015).
Moreover, mass spectrometry-based serum lipid profiling enables a more comprehensive analysis of lipid biomarkers in clinical settings and thus facilitates the characterisation of inflammatory diseases (Matthiesen et al., 2021), cancer markers (Matthiesen et al., 2021), and psychiatric disorders (Matthiesen et al., 2021).
In recent years, improvements to spectrometry techniques, such as high-resolution time-of-flight analysis, have facilitated multiplex systems capable of analysing multiple lipids concurrently, yielding more intricate datasets (Ni et al., 2023).Furthermore, advancements have enabled the study of lipidome heterogeneity at the cellular level by utilising employing dual-polarity ionisation MS for high-throughput processing (Zhang et al., 2023).
Enhancements to MALDI matrix materials, including the use of graphite or metal nanoparticle mediums, have enabled the characterisation of free fatty acids that were previously too small for analysis (Wu et al., 2016).Consequently, there has been a need for subsequent improvements in bioinformatics tools, such as LipidFinder (O'Connor et al., 2017), which facilitates the analysis of these datasets by characterizing them based on online lipid databases like LIPID MAPS (Fahy et al., 2007).Another notable tool, BioPAN, allows for the contextualisation of quantitative lipidomics data by connecting them with known genes and metabolic pathways (Gaud et al., 2021).
Overall, the advancements in lipidomics and mass spectrometry techniques represent a rapidly expanding field with immense potential  for identifying future therapeutic targets and advancing clinical research.Table . 1

Pattern of lipid dysregulation
Rodent model studies have demonstrated that prolonged exposure to stress may lead to an altered composition and function of these neurolipids within the central nervous system (Demirkan et al., 2013;Hursitoglu and Kurutas, 2023;Bulut et al., 2013).Dysregulation of the brain lipidome may alter the homeostatic balance and may consequently result in the development of anxiety-related behaviour (Demirkan et al., 2013).However, little is known about the actual mechanism underlying the aetiology of anxiety disorders.This has prompted for exploration of the brain's neurolipidome in both healthy and pathological states to uncover its role in various neurological and psychiatric disorders.The proposed mechanisms include changes to transcriptional regulation as well as epigenetic modifications, with ongoing efforts to identify the types of epigenetic mechanisms involved in the pathogenesis of anxiety disorders (Oliveira et al., 2016).
In rodents that displayed anxiety-related behaviour, neurolipid alterations were mainly observed within the prefrontal cortex and the hippocampus (Oliveira et al., 2016).A study conducted by Oliveira et al.Reduced anxiety-type behaviour observed in mice that were administered Simvastatin and exposed to classical music.
(Milbratz de Camargo et al., 2013;Camargo et al., 2013) Male Wistar rats Simvastatin (1 or 10 mg/kg) for 4 weeks ± fluoxetine (2 or 10 mg/kg), 3 doses in 24 h prior to test, or control (saline).Rats were exposed to the forced swimming test and elevated plus maze Decreased anxious behaviour observed when simvastatin was co-administered with fluoxetine compared to either drug alone.(Santos et al., 2012) Male C57BL/6 J mice Intervention group mice were either fed with a highcholesterol diet or exposed to chronic restraint stress for 4 weeks.
Intervention group developed anxious behaviour with elevated markers of neuroinflammation and peripheral inflammation.
The effect due to high-cholesterol diet was similar to the effect due to chronic restraint stress (positive control).
The diet group also had abnormal serotonin 5-HT receptor expression.(Zou et al., 2023) Male Sprague-Dawley rats Young (three weeks old) and mature (20 weeks old) rats were fed with either a high cholesterol diet or regular diet for 8 weeks.
Then subjected to the elevated plus maze test, serum lipid profile tests and CNS neurochemical analysis.
In young rats cholesterol had anxiolytic-like behaviour with decreased corticosterone; adult rats presented with anxiety-like behaviour with increased corticosterone (different response in different ages).(Hu et al., 2014) Human; diagnosed with GAD Simvastatin (20 mg/day) for eight weeks.Participants were previously treated with SSRI for at least 8 weeks before.Anxiety levels measured using HAM-A score.
No statistically significant change was observed between the intervention and control groups.
( Acetyl-L-Carnitine (ALC) Zebrafish ALC (0.1, 1 and 10 mg/L) ALC (at 0.1 and 10 mg/L) significantly increased the time spent in the light side (anxiogenic) of the tank; ALC at all doses showed significant reversal of the effect of chronic stress exposure (Pancotto et al., 2018) (2016) aimed to assess the impact of chronic unpredictable stress or administration of exogenous corticosteroids on the neurolipidomic function of the rodent brain.It is important to recognize that these experimental paradigms are designed to simulate the psychological effects of chronic stress, such as those observed in conditions like depression and PTSD, rather than being specifically tailored to anxiety disorders (Monteiro et al., 2015;Sequeira-Cordero et al., 2019).However, the level of distress induced was quantified using the Elevated Plus Maze, which is a commonly used measure of anxiety-related behaviour in rodents (Walf and Frye, 2007).Furthermore, the overall structure of the experiment was determined to be robust with appropriate internal validity (Walf and Frye, 2007).Therefore, while the experimental model may not directly mirror anxiety disorders, the findings concerning neurolipidomic changes associated with anxiety-related behaviours remain relevant to the broader discussion of anxiety disorders.
In the rodents that were exposed to chronic unpredictable stress or exogenous corticosteroids, there was a decrease in the content of the sphingomyelin and sphingolipid level content and a concurrent increase in the ceramide levels (C16:0, C16:1, 18:1, C22:1, C26:1) within the prefrontal cortex and hippocampus (Oliveira et al., 2016).In terms of putative mechanisms, it has been hypothesised that in the setting of chronically stressed brains, there is an increase in the levels of sphingomyelin hydrolysis, which results in an increase in its breakdown product (ceramide) (Oliveira et al., 2016).Another observable trend based on the findings of this study is that within the prefrontal cortex of chronically stressed rodents, there was an overall decrease in the concentration of glycerophospholipids (such as phosphatidylcholine) and an increase in its metabolite products (lysophospholipids) (Oliveira et al., 2016).It remains unclear as to whether glycerophospholipid metabolism in the prefrontal cortex contributes to the pathophysiology underpinning anxiety disorders.

Serum lipid biomarkers for anxiety disorder
Currently, the only method that is available to analyse the impact of and severity of anxiety symptoms is through the use of qualitative interviews (Ruscio et al., 2017).However, as previously mentioned, these methods have their inherent limitations and studies have shown that qualitative diagnostic approaches can have high rates of inter-reliability variability (Maes et al., 2018).Therefore, integrating serum biomarkers related to anxiety could serve as an objective tool to monitor and track the development of anxiety disorders across clinical contexts.Several studies have suggested elevated levels of specific neurolipid biomarkers in patients with anxiety disorders and animal models (Demirkan et al., 2013;Hursitoglu and Kurutas, 2023), underscoring the importance of further laboratory-based and human studies to explore neurolipidomic dysregulation and its association with symptoms of anxiety.

Lipid peroxidation markers
There are recent studies that have demonstrated that increased levels of neuronal oxidative stress may be associated with the pathogenesis of Generalized Anxiety Disorder (GAD) (Maes et al., 2018).Maes et al. (2018) found that individuals with GAD were likely to have elevated concentrations of lipid hydroperoxide (LOOH) and lowered levels of high-density lipoprotein cholesterol (HDL) and paraoxonase 1 (PON-1) (Maes et al., 2018).Lipid hydroperoxide is a molecule that is formed in the setting of lipid peroxidation, and HDL is an anti-inflammatory cardioprotective molecule that functions to transport cholesterol within the bloodstream (Maes et al., 2018).PON-1 is also an HDL-associated enzyme (Maes et al., 2018).Following the theory that oxidative stress and GAD occur in conjunction, it has been hypothesised that GAD may be associated with a corresponding elevated level of lipid oxidation products (LOOH) as well as a lowered concentration of anti-inflammatory factors (HDL and PON-1).Other papers corroborate the findings of Maes et al. (2018), where in one study, it was found that an increased concentration of LOOH was detected among adolescent patients with GAD (Ceylan et al., 2014).A further study found that individuals with GAD had a reduced PON-1 concentration as well as increased LOOH levels (Bulut et al., 2013).Additionally, a further study found that serum concentration of Raftlin (a membrane lipid raft) and 8-iso-PGF2α (a lipid peroxidation marker) were elevated among patients with GAD (Hursitoglu and Kurutas, 2023;Ceylan et al., 2014).
However, despite the surmounting evidence for lipid peroxidation markers being a potentially effective biomarker for anxiety disorders, the evidence must be interpreted with some caution.For example, some of the aforementioned studies may have had sampling bias for control groups due to the sparing details included in the control group selection processes (Hursitoglu and Kurutas, 2023;Bulut et al., 2013), and others have also failed to account for confounding factors within the study itself (Hursitoglu and Kurutas, 2023;Ceylan et al., 2014).These inherent limitations within the study structures may compromise the external validity and applicability of the evidence in a clinical context.
Thus, lipid peroxidation is emerging as a potential cellular mechanism implicated in the pathophysiology of GAD, and consequently, lipid peroxidation markers may offer a valuable approach for assessing, monitoring, and tracking symptoms associated with anxiety disorders in the future.

PUFA
The current evidence regarding serum concentrations of PUFA as an indicator of anxiety appears to be inconclusive (Thesing et al., 2018).One cross-sectional study with a cohort of over 12,000 participants has shown an inverse correlation between the n-6:n-3 serum PUFA ratio and the severity of anxiety symptoms (Natacci et al., 2018).In another cross-sectional study, it was found that there was an inverse correlation between serum concentrations of DHA (n-3 PUFA) and anxiety symptoms among pregnant women (Verly-Miguel et al., 2015a).However, another cross-sectional study conducted by Thesing et al. (2018) found no significant difference in the levels of PUFA across individuals with anxiety and non-clinical controls (Thesing et al., 2018).
It must also be acknowledged that some studies contained unclear methodology regarding participant selection (Verly-Miguel et al., 2015a) or employed non-random sampling methods (Thesing et al., 2018), thus potentially limiting the generalisability of the evidence.Further investigations are indicated to clarify the potential relationship between serum PUFAs, and the symptomatology associated with anxiety.

Sphingolipids
In a recent study among human participants, an inverse correlation was found between anxiety symptoms and the corresponding serum concentrations of phosphatidylcholine (PC O 36:4) and sphingomyelin (SPM 23:1) (Demirkan et al., 2013).Furthermore, anxiety symptoms were shown to be positively correlated with the serum levels of ceramides (C 22:0, C20:0, C18:0) (Demirkan et al., 2013).Additionally, in a case-control study, it was found that lower plasma concentrations of N-(hexadecanoyl)-deoxysphing-4-enine-1-sulfonate (a sphingolipid) were inversely correlated with the severity of anxiety-related symptoms (Dong et al., 2021).It must be noted however that this study's participants only included individuals afflicted by Parkinson' Disease, and thus the evidence may not be generalisable for anxiety disorders (Dong et al., 2021).
Further clinical research has the potential to advance our understanding as to how neurolipids may be used as a novel type of biomarkers to track the severity of anxiety-related symptoms among clinical populations.Further work involving both rodent-based and human-based studies has the potential to lead to new advances, both in terms of our understanding of the pathophysiology of anxiety disorders and also leading to new and effective individualised pharmacotherapeutic strategies tailored to specific symptom subtypes.

Polyunsaturated fatty acids
Emerging evidence from animal-based studies indicates that targeting lipids may be a novel treatment intervention for individuals with anxiety disorders (Müller et al., 2015).One potential therapeutic target for treating anxiety-related symptoms is polyunsaturated fatty acids (Su et al., 2018), which are known to contribute to the structure of the phospholipid bilayer of cells including the neuronal membrane.Within the lipid bilayer, PUFAs have been observed to be preferentially organised to surround protein structures including receptors and ion channels (Schneider et al., 2017a).It has been hypothesised that the variation in the subtypes and structures of PUFA may effectively modulate the function of the proteins, and may have a central role in healthy physiological neuronal function and neurotransmission (Schneider et al., 2017a).
n-3 PUFAs, including EPA and DHA, have been extensively studied as a potential therapeutic target for various mood disorders, including anxiety disorders (Müller et al., 2015).Aside from the potential effects on neuromodulation, n-3 s have also been shown to have anti-inflammatory, vasodilatory, and neuroprotective effects on the brain tissue (Mariamenatu and Abdu, 2021).Various studies have demonstrated the positive effect of dietary supplementation of n-3 PUFA on mood.Multiple animal-model experiments have shown that increased levels of n-3 PUFA intake may have anxiolytic effects (Demers et al., 2020;Ferraz et al., 2011;Rutkowska et al., 2016;Vinot et al., 2011), while lower levels of n-3 PUFA are more likely to induce anxiety-like responses (Bondi et al., 2014).
However, the effect of n-3 PUFA supplementation in the context of clinical studies remains inconclusive.For example, in a study conducted by Natacci et al. (2018), no correlation could be established between the development of anxiety disorder and n-3 PUFA intake (Natacci et al., 2018).Additionally, a meta-analysis undertaken by Su et al. (2018) found that the anxiolytic effects of n-3 PUFA administration were only observed among individuals with GAD who were administered high doses (>2000 mg daily) (Su et al., 2018).In view of the low turnover rate of neural lipids, the findings of Su et al. (2018) indicate that high concentrations of serum n-3 PUFA may be required for substantial changes to occur within the neural circuitry.However, it remains unclear whether these alterations in n-3 PUFA levels across the neural circuitry directly correlate with symptoms of anxiety disorders.There is also a clinical requirement to establish the correlation between serum and brain levels of n-3 PUFA.Further work will be needed to identify the content of n-3 PUFA across the different brain regions and networks, and whether differences in n-3 PUFA levels are associated with any distinct symptoms associated with anxiety disorders.
Interestingly, the effects of dietary PUFA intake may have an intergenerational effect.The intergenerational impact of PUFA has been examined among rodents and their progeny (Queiroz et al., 2019;Pudell et al., 2014).One study in particular examined the impact of maternal linoleic acid (n-6 PUFA) supplementation on the psychological state of their rat offspring (Queiroz et al., 2019).It was found that rodents whose mothers received supplementary conjugated linoleic acid (1 or 3 % of their dietary intake) during gestation and lactation displayed decreased levels of anxious-like behaviours in the 1 and 3 % groups (Queiroz et al., 2019).Furthermore, another study investigated the effect of fish oil (n-3 PUFA) supplementation in female rodents during mating, gestation and lactation (3 g/kg, with 12 % EPA and 18 % DHA) (Pudell et al., 2014); in this study, the rodent offspring exhibited reduced anxiety following an olfactory bulbectomy, which is an anxiogenic procedure (Pudell et al., 2014).Both studies demonstrated a robust study structure with adequate internal validity, with appropriate statistical analyses conducted (Queiroz et al., 2019;Pudell et al., 2014).These studies suggest that maternal intake of PUFA, both n-6 and n-3, may play a crucial role in the early phase of neuronal development, and may in turn, influence the psychological state of the offspring through their impact on neurophysiological signalling pathways (Pudell et al., 2014).These findings are surprisingand point to the need for further studies to investigate the impact of how lipid intake may potentially have an impact on the progeny, thus highlighting the need for further research into neurolipids, early life experience, and epigenetics.

Free fatty acids
The role of saturated free fatty acids on mood and anxiety-like behaviours has been investigated across several rodent-based studies (García-Ríos et al., 2013;Contreras et al., 2014).In a study conducted by Garcia-Rios's group (2013), mice were fed with either amniotic fluid or an artificial mixture of eight fatty acids which mimicked the concentrations of fatty acids that are found in amniotic fluid (García-Ríos et al., 2013).It was found that mice that were fed with either of these fluids had an attenuated response to stress-inducing environments (García-Ríos et al., 2013).In another study undertaken by Contreras et al. (2014), it was demonstrated that among eight fatty acids within the mixture, myristic acid (C14:0) was the primary fatty acid that was found to have an anxiolytic effect in mice (Contreras et al., 2014).The findings of this study indicate that administering myristic acid produced a behavioural response in mice that was comparable to the effects observed with diazepam administration (Contreras et al., 2014).In interpreting the results of this study, however, it is important to consider that the intraperitoneal administration of fatty acid mixtures bypasses the gastrointestinal metabolism route (Contreras et al., 2014).Consequently, the effects of fatty acids may differ from those administered orally, suggesting that the findings of this study may have limited generalisability to orally administered fatty acids.Additionally, it must be noted that the two behavioural tests were conducted one after the other on the same rodent subjects, which may have confounded the results and, therefore, limited the applicability of the findings (Contreras et al., 2014).The mechanisms that account for the anxiolytic effect of myristic acid continues to remain unclear and further work is needed to identify the physiological mechanisms of action at a molecular and synaptic level.

Cholesterol
A novel line of investigations that has been investigated across both rodent and human-based studies involves the pharmacotherapeutic use of statins to modulate the levels of the different types of neurolipids in the brain (Cruz et al., 2017).Statins reduce the rate of de novo cholesterol synthesis by inhibiting 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase.Statins are commonly used in clinical practice for their enzymatic actions in terms of lowering serum cholesterol concentrations and managing patients with metabolic conditions including hyperlipidaemia (Milbratz de Camargo et al., 2013).
Rodent model studies have suggested that modulating the serum cholesterol concentration may influence the function of membrane lipid rafts (MLRs), which in turn may have an impact on the functions of the N-methyl-D-aspartate (NMDA) ion receptors (Cruz et al., 2017).In particular, mouse model studies have demonstrated that the administration of a statin (and the subsequent lowered serum cholesterol level) can induce an anxiolytic effect (Milbratz de Camargo et al., 2013;Santos et al., 2012).A study conducted by Santos et al. (2012) demonstrated that Wistar rats that were co-administered with fluoxetine and simvastatin displayed decreased anxious behaviour compared to either drug alone (Santos et al., 2012).The rats were not exposed to prior stressors, and anxious behaviour was quantified via the forced swimming test and the elevated plus maze (Santos et al., 2012).Furthermore, a study conducted by Milbratz de Camargo et al. (2013) demonstrated that rats that were exposed to classical music and Simvastatin for four weeks had reduced anxiety-related behaviour when assessed by the elevated plus maze and the open field test (Milbratz de Camargo et al., 2013).
The authors' hypothesis to account for this effect is that the lowered cholesterol concentration leads to a reduced association of MLRs to the NMDA receptors, which in turn causes a reduction of Ca 2+ entry via the NMDA-R channel (Milbratz de Camargo et al., 2013).This reduction of Ca 2+ entry into the cell may, in turn, lead to a reduction in the excitation of the neuron possibly leading to anxiolytic effects (Egawa et al., 2016b;Cruz et al., 2017).However, it must also be noted that the two studies were conducted within the same laboratory and that these experiments have not been replicated in the decade since their publication.Furthermore, it must be noted that one of the studies lacked blinding in its methodology and had incorporated rodent exposure to classical music as a study model, (Santos et al., 2012) which are potential limiting factors for broader clinical applicability.Therefore, these findings warrant cautious interpretation and underscore the imperative for additional research and investigation into the relationship between statin use and anxiety.
The current clinical evidence suggests that the use of statins for the management of anxiety disorders is limited (Mirzaei et al., 2021).A recent randomised controlled trial conducted by Mirzaei et al. (2021), which involved the daily administration of a statin (simvastatin 20 mg daily) to patients with GAD, found that there were no statistically significant differences among reported outcomes across the treatment and control groups (Mirzaei et al., 2021).Additionally, a comprehensive cohort study conducted by Molero et al. (2020) examined the association between statin use and anxiety disorders; overall, no association between symptoms of anxiety and statin use could be established (Molero et al., 2020).Further translational research and clinical trials are needed in this area to establish an evidence base as to whether or not statins may have a therapeutic application across the spectrum of anxiety disorders (Cruz et al., 2017).

Endocannabinoids
The endocannabinoid system exhibits a diverse range of actions across the central and peripheral nervous systems, thereby influencing feeding behaviour, pain processing, cognition, and emotional regulation (Hill and Patel, 2013).It has been well established that endocannabinoid receptor agonists have pleiotropic effects, and it has been shown that while low doses of endocannabinoids may have anxiolytic effects, high doses may trigger a range of anxiogenic pathways within the brain (Fowler, 2015).Multiple systematic reviews have been conducted to determine the anxiolytic effects of administering exogenous cannabinoids (such as D9-tetrahydrocannabinol (D9-THC)), but the evidence in relation to their clinical effectiveness remains inconclusive (Sarris et al., 2020;Black et al., 2019;Botsford et al., 2020).
A potential therapeutic target within the endocannabinoid system is linked to factors that inhibit the breakdown of the endocannabinoid neurotransmitters.Anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are cannabinoid receptor ligands that are endogenously expressed within the central nervous system (Fowler, 2015).Anandamide and 2-AG are catabolised by FAAH and MAGL, respectively (Bedse et al., 2018).It has been demonstrated in human neuroimaging studies that lower levels of FAAH in the amygdala, medial prefrontal cortex, cingulate, and hippocampus are associated with an attenuated amygdala reaction to a perceived threat (Green et al., 2022).Therefore, it has been hypothesised that inhibiting FAAH and MAGL would enhance the endogenous endocannabinoid signalling pathways, which in turn may have an anxiolytic effect.This hypothesis has indeed been evidenced by the fact that genetic deletions of FAAH have resulted in reduced anxious behaviour in mice; these FAAH -/-mice exhibited increased time spent in the open arms of the elevated plus maze, and greater exploratory behaviour in the open field test (Bambico et al., 2010).
Numerous subtypes of FAAH and MAGL inhibitors have been shown to have an anxiolytic effect in the context of animal-based studies.
A study conducted by Marco et al. (2015) investigated how ST4070, a FAAH inhibitor, influences the behaviour of naïve mice (mice that had not been previously subjected to stressors) (Marco et al., 2015;Moreira et al., 2008).In this study, mice were administered ST4070 and placed in an elevated plus maze (Marco et al., 2015;Moreira et al., 2008).The elevated plus maze is a commonly used tool that helps to quantify rodents' anxiety-related behaviour (Walf and Frye, 2007).The maze is composed of an X-shaped elevated pathway with two enclosed and two open arms, and the experiment model relies on rodents' natural aversion to open and exposed areas, which are typically anxiogenic (Walf and Frye, 2007).Consequently, the anxiolytic effect induced by the treatment intervention is assessed by measuring how frequently the mice enter the open arms of the maze (Walf and Frye, 2007).Overall, Marco et al.'s study demonstrated that mice that were administered with ST4070 spent increased time in the open arms of the maze, thus indicating an attenuation of the mice's anxiety-related behaviour by the FAAH inhibitor (Marco et al., 2015).However, the evidence for FAAH inhibitors in anxiolysis does not appear to be consistent.For example, in Griebel et al.'s study (2018), rodents were exposed to various anxiety models, including the punished drinking test and light-dark test.The study could not establish a statistically significant difference in the behaviour of the control group and the group administered a FAAH inhibitor (SSR411298) (Griebel et al., 2018).Additionally, a study conducted by Busquets-Garcia et al. (2011) examined the anxiolytic effects of URB597 (FAAH inhibitor) and JZL184 (MAGL inhibitor) in naïve mice (Busquets-Garcia et al., 2011).It was found that a separate single-dose administration of URB597 and JZL184 had both resulted in increased time spent in the open arms of the elevated plus maze (Busquets-Garcia et al., 2011), thus indicating an anxiolytic effect induced by the treatments.
It is important to note that these aforementioned rodent model studies, despite their robust internal validity, appropriate statistical analyses, and methodology, have small sample sizes (n=8-12) (Marco et al., 2015;Griebel et al., 2018;Busquets-Garcia et al., 2011).As a result, the generalisability of these findings to the human population may be limited.
Therefore, collectively, it must be acknowledged that currently there is mixed evidence present as to the effectiveness of FAAH and MAGL inhibitors in eliciting an anxiolytic effect (Marco et al., 2015;Griebel et al., 2018;Busquets-Garcia et al., 2011), and further research is warranted in this domain.
Moreover, multiple studies have examined the effect of FAAH or MAGL inhibitors in attenuating the rodent's behavioural response to acute and chronic stress.It must be noted that the model of stress induction is not directly applicable to anxiety disorders; rather, it is more closely correlated with Post-Traumatic Stress Disorder (Verbitsky et al., 2020) and depression (although the validity of the latter has been highly debated in recent years (de Kloet and Molendijk, 2016)).However, despite this distinction, the behavioural changes measured within the stress induction model, such as those assessed by the elevated plus maze, primarily reflect anxiety-related behaviour in rodents (Walf and Frye, 2007).Consequently, although the methodology of stress induction may not directly apply to anxiety disorders, the observed effects of FAAH and MAGL inhibitors in attenuating these anxiety-related responses remain pertinent.Nonetheless, given the incongruence of the stress model with anxiety disorders, it is important to interpret the findings of the following studies with caution.
One study examined the effect of PF-3845 (FAAH inhibitor), JZL184 (MAGL inhibitor), and JZL195 (dual FAAH-MAGL inhibitor) on mice that were exposed to acute stress in the form of restraint and foot shock (Bedse et al., 2018).The extent of behaviour alteration caused by the treatments was measured by the light-dark box test, where the rodent's anxiety-like behaviour is inferred from its reluctance to enter the brightly lit area of the box (Bourin and Hascoët, 2003).The findings of this study revealed that both PF-3845 and JZL184 individually led to an increase in exploration time in the brightly lit area, suggesting an anxiolytic response (Bedse et al., 2018).Interestingly, JZL195 (dual FAAH-MAGL inhibitor) did not cause a reduction in anxiety-related behaviour (Bedse et al., 2018).An additional study also examined the effect of PF3845 in attenuating the behavioural changes elicited by inescapable swim stress, which is an acute stressor (Duan et al., 2017).When the mice's behaviour was assessed by the elevated plus maze, it was shown that PF3845 had effectively induced a rapid anxiolytic effect in the subjects (Duan et al., 2017).Similarly, Sciolino et al. (2011) examined the effect of JZL184 in the context of acute stress, where rodents were exposed to varying levels of environmental stress prior to having their behaviour assessed by the elevated plus maze (Sciolino et al., 2011).The findings of this study indicate that JZL184 only attenuated anxiety-like behaviour in the context of a highly aversive environment (Sciolino et al., 2011).
Numerous studies have been conducted to assess the effect of FAAH and MAGL inhibitors in attenuating the anxiety-like response induced by chronic stress.One study investigated the effect of URB597 (FAAH inhibitor) on mice that were chronically stressed via social defeat (Rossi et al., 2010(Rossi et al., , 2012)).The finding of this study indicated that when mice were injected with 0.3 mg/kg dosage of URB597, the mice demonstrated increased entries into the open arms of the elevated plus maze (Rossi et al., 2010(Rossi et al., , 2012)).The findings of Dargahi et al. (2023) appear to be in agreement with this study; when URB597 was administered at 0.6 mg/kg to chronically stressed Wistar rats, the rodents demonstrated an increased number of entries in the open arms of the elevated plus maze (Dargahi et al., 2023;Danandeh et al., 2018).Additionally, the results of Danandeh et al.'s experiment (2018) further corroborate the evidence yielded from these two studies.Rodents that were previously exposed to an olfactory stressor that was then administered with URB597 demonstrated increased time and instances spent in the open arms of the elevated plus maze (Danandeh et al., 2018).Overall, it appears that there is accumulating evidence from animal model studies for the use of FAAH inhibitors in the context of attenuating anxiety-like behaviour induced by chronic stress.
Furthermore, a study conducted by Viana et al. (2019) examined the effect of URB602 (MAGL inhibitor) within the context of a chemically induced panic-like state (Viana et al., 2019).In this experiment, Wistar rats were injected with a single dose of NMDA receptor agonists to induce panic-like behaviour; it was found that local injection of URB602 into the dorsomedial hypothalamus at concentrations of 300 and 1000pmol successfully reversed this anxiety-like response (Viana et al., 2019).
Over recent years, the accumulating evidence about the role of FAAH inhibitors from rodent-based models has been investigated further as part of translational human clinical studies (Paulus et al., 2021;Schmidt et al., 2021).A randomised clinical trial that was conducted in 2021 assessed the effect of FAAH inhibitor (JNJ-42165279) administration in healthy males.The study investigated the effect of this medication on the modulation of emotional reactions in response to stressful stimuli (Paulus et al., 2021).The results of this study demonstrated that those administered with JNJ-42165279 led to an attenuated activation of the limbic system when individuals were presented with an emotionally stressful task, but JNJ-42165279 did not result in a reduction in the fear responses in the setting of conditioned stimuli (Paulus et al., 2021).It should be noted that despite the small sample size (n=21 and 22 for placebo and drug), this study adopted rigorous screening criteria to eliminate any potential confounding factors, including single nucleotide polymorphisms of the FAAH gene (Paulus et al., 2021).Another study evaluated the effectivity of JNJ-42165279 in the management of social anxiety disorder (SAD), where they found that JNJ-42165279 did result in a decrease in anxiety scoring and was well-tolerated by the participants, although this finding was not statistically significant (Schmidt et al., 2021).The findings of this particular study indicate that JNJ-42165279 does not appear to elicit an anxiolytic effect in subjects with SAD; however, the dose the participants received (25 mg) appeared to have been insufficient in inhibiting FAAH activity completely (Schmidt et al., 2021).Therefore, future research should include additional clinical trials that utilise various doses of FAAH and MAGL inhibitors across different anxiety disorders.These trials are essential for assessing the effectiveness of these medications in alleviating anxiety symptoms.
In summary, although there are some preliminary findings from rodent and human studies about the role of FAAH and MAGL inhibitors in attenuating anxious responses (Bedse et al., 2018;Griebel et al., 2018;Paulus et al., 2021;Schmidt et al., 2021), there is an ongoing need for further clinical studies in order to definitively establish the efficacy of FAAH and MAGL inhibitors as novel pharmacological interventions for individuals with anxiety disorders.

Sphingolipids
Sphingosine-1-phosphate (S1P) is a sphingomyelin metabolite that is involved across a range of various neurological processes, including neuronal survival, axon guidance, and myelination (Mendelson et al., 2014).One particular study investigated the impact of S1P on varying levels of anxiety by locally infusing S1P into the cerebral ventricle of rodents (Jang et al., 2011).Findings from this study indicated that mice who were administered with S1P demonstrated increased levels of anxious-like behaviour and had elevated levels of tyrosine hydroxylase in the amygdala, indicating a stress-like response (Jang et al., 2011).
Another study examined the effects of FTY720 (also known as Fingolimod, a modulator of S1P receptors) on anxiety-like behaviour in rodents (Corbett et al., 2021).Typically, Fingolimod is used as a disease-modifying therapy for patients with relapsing-remitting multiple sclerosis (RRMS) (Chun and Hartung, 2010).It was shown that single and repeated intraperitoneal administration of FTY720 was found to lead to a reduction of social anxiety-like and despair-like behaviours in mice, as evaluated through the social interaction test and the open field test (Corbett et al., 2021).However, it must be noted that within this study, the sample sizes were relatively small (n=5-10, depending on the experiment group) and data that were considered outliers (greater than 3 standard deviations from the mean) were discarded, both of which are potential confounders for bias (Corbett et al., 2021).Further experimental investigation into the modulation of the different sphingolipids' functions could offer valuable insights into their therapeutic potential and their ability to induce anxiolytic effects in the future (Bernal-Vega et al., 2023).
As an initial step, further research is required to investigate the mechanisms and timing of naturally occurring sphingomyelin modifications in the healthy brain.Moreover, as part of future interdisciplinary inquiries, it is essential to elucidate the molecular and cellular mechanisms underlying these interactions.Additionally, there is a necessity for further exploration into how early life experiences might influence the development and function of sphingomyelins in the brain.

Acetyl-L-carnitine
Acetyl-L-carnitine (ALC) is another potentially important therapeutic target for novel anxiolytic medications (Pancotto et al., 2018).ALC is a molecule that mediates a range of cellular functions, including fatty acid oxidation and membrane phospholipid synthesis (Chapela et al., 2009).
In one study, ALC was administered to Zebrafish to investigate its potential anxiolytic effects (Pancotto et al., 2018).The authors evaluated these effects by observing the behaviour of the zebrafish, specifically focusing on their inclination to enter the brighter (anxiogenic) environment of the tank.The results revealed that ALC administration led to an increased number of Zebrafish entering the light area, indicating the anxiolytic properties of ALC (Pancotto et al., 2018).It was also found that administering ALC prevented neural lipid peroxidation induced by acute stress (Pancotto et al., 2018).
Hence, these initial findings indicate that ALC may act as a neuroprotective factor against neurolipid peroxidation, thereby reducing the impact of acute stress and anxiety in the zebrafish brain.It must be commented that despite the strong internal validity and appropriate methodology and statistical analyses conducted in this study, the applicability of this study's findings to mammalian and human contexts is low.Therefore, ALC may need to be investigated further as part of future translational studies involving both animal-based studies and human-based studies in order to investigate its therapeutic efficacy in the treatment of anxiety disorders.

Conclusion
Based on the current literature review, neurolipids are involved in a range of diverse range of neurophysiological pathways (Schneider et al., 2017a), and hence, may be a viable candidate as a future clinical biomarker and therapeutic target in anxiety disorders (Miranda and Oliveira, 2015).However, it must be acknowledged that the research is still within its preliminary stages and the clinical applicability of neurolipids as biomarkers or therapeutic targets for anxiety disorders remains limited.Further translational studies that extend beyond animal-based studies are needed as part of future work.
One of the major limitations in terms of the clinical use of neurolipids as potential clinical biomarkers for anxiety disorders is that there is currently no known normal reference range for the amount and concentration of the various neurolipids in the brain of healthy individuals (Natacci et al., 2018).We currently do not have any reference norms in relation to what would constitute healthy amounts and concentrations of the various neurolipids within the serum and across the different regions of the brain.Further work is needed to identify the normal patterns and concentrations of neurolipids (including the various subtypes of neurolipids) across the different areas of the healthy brain.When establishing a reference point, external influencers and variables including lifestyle, age, gender, diet, and medication use must be considered.Further research is needed in terms of lipidomic profiling among healthy individuals in order to establish the practicality of introducing lipids as a novel clinical biomarker among individuals with anxiety disorders.Additionally, research should be conducted on lipid biomarkers among individuals with anxiety disorders across the treatment and remission period in order to establish how neurolipids may change and be modulated across the different patient groups.
In terms of further studies, integrating lipidomics along with metabolomics, proteomics, transcriptomics, and genomics may hold immense potential in terms of further identifying the different processes that lipids take part in contributing to the symptomology of anxiety.The integration of lipidomics with genome-wide association studies (GWASs) holds promise in identifying the connections between alterations in the brain lipidome and common single-nucleotide polymorphisms (SNPs) in psychiatric disorders.This approach may elucidate the genomic loci associated with complex traits in the population and their relationship to lipid changes in common psychiatric disorders.This will also be beneficial in opening novel opportunities for the development of new lipid biomarkers, and novel diagnostic and therapeutic approaches in the management of various anxiety disorders (Correia et al., 2021).
Additionally, the scope of this review did not cover the role of neurolipids in the peripheral nervous system (PNS); however, there are likely bidirectional relationships between the PNS and central nervous system (CNS) which may have an important contribution to the pathogenesis of anxiety disorders.Hence, there is a need for further studies to investigate the interactions between the PNS and CNS including the llipid systems at the level of PNS.This could involve additional research endeavors focused on exploring the interactions between the peripheral nervous system (PNS) and central nervous system (CNS) neurotransmitter systems, including any alterations in their interplay.Such investigations may enhance our comprehension of the factors underlying the symptomatology observed in various anxiety disorders.
Overall, further interdisciplinary research is required to characterise the aetiopathogenesis of neurolipids within anxiety disorders.It will be important to investigate the efficacy of neurolipids as biomarkers and therapeutic targets for these psychiatric conditions.Basic biochemical studies and neurophysiological studies offer new avenues for further therapeutic approaches among individuals with anxiety.Such an approach will require ongoing experimental work using rodent-based models alongside translational clinical studies as well as a greater understanding of the contributions of the specific subtypes of neurolipids to the different biochemical and cellular pathways in the healthy developing and adult brain.Such studies have the potential to lead to a greater understanding of how these pathways may be disrupted among individuals across the spectrum of anxiety disorders.

Fig. 1 Summary•
Emerging research into neurolipids suggests their implication in the pathogenesis of anxiety disorders.•Specific serum lipids, such as sphingolipids and lipid peroxidation markers, show promise as potential biomarkers for anxiety disorders.• Various potential pharmacological neurolipid targets have been identified; in particular, MAGL and FAAH inhibitors appear to have the most convincing evidence as therapeutic targets.• Interdisciplinary research combining biochemical, neurophysiological, and translational clinical studies is crucial to further explore the role of neurolipids in anxiety disorders.

Table 1 -
A summary of the neurolipid dysregulation observed in mice and human models.

Table 2 -
A summary of the various potential therapeutic targets within the scope of neurolipids.
(Schmidt et al., 2021)al anxiety measure) score, however not statistically significant.Significantly increased proportion of patients with >30 % improvement from baseline in both LSAS and CGI-I.(Schmidtetal., 2021)Cholesterol (continued on next page) S.Maehashi et al.