Cannabinoid type-1 receptor signaling in dopaminergic Engrailed-1 expressing neurons modulates motivation and depressive-like behavior.

The endocannabinoid system comprises highly versatile signaling functions within the nervous system. It is reported to modulate the release of several neurotransmitters, consequently affecting the activity of neuronal circuits. Investigations have highlighted its roles in numerous processes, including appetite-stimulating characteristics, particularly for palatable food. Moreover, endocannabinoids are shown to fine-tune dopamine-signaled processes governing motivated behavior. Specifically, it has been demonstrated that excitatory and inhibitory inputs controlled by the cannabinoid type 1 receptor (CB1) regulate dopaminergic neurons in the mesocorticolimbic pathway. In the present study, we show that mesencephalic dopaminergic (mesDA) neurons in the ventral tegmental area (VTA) express CB1, and we investigated the consequences of specific deletion of CB1 in cells expressing the transcription factor Engrailed-1 (En1). To this end, we validated a new genetic mouse line EN1-CB1-KO, which displays a CB1 knockout in mesDA neurons beginning from their differentiation, as a tool to elucidate the functional contribution of CB1 in mesDA neurons. We revealed that EN1-CB1-KO mice display a significantly increased immobility time and shortened latency to the first immobility in the forced swim test of adult mice. Moreover, the maximal effort exerted to obtain access to chocolate-flavored pellets was significantly reduced under a progressive ratio schedule. In contrast, these mice do not differ in motor skills, anhedonia- or anxiety-like behavior compared to wild-type littermates. Taken together, these findings suggest a depressive-like or despair behavior in an inevitable situation and a lack of motivation to seek palatable food in EN1-CB1-KO mice, leading us to propose that CB1 plays an important role in the physiological functions of mesDA neurons. In particular, our data suggest that CB1 directly modifies the mesocorticolimbic pathway implicated in depressive-like/despair behavior and motivation. In contrast, the nigrostriatal pathway controlling voluntary movement seems to be unaffected.

Chronic Administration of Δ9-Tetrahydrocannabinol Alters Brain Glucose Uptake and Improves Waiting Impulsivity in the Rat.

Introduction: Δ9-Tetrahydrocannabinol (THC) acts as an agonist at cannabinoid receptors. Its chronic intake affects many behaviors, including cognitive processes. The aims of this study in rats are to assess the chronic effects of THC on impulsivity and on regional brain glucose uptake. Materials and Methods: For the determination of "waiting impulsivity," a total of 20 male Lister Hooded rats were trained to perform a reaction time task, followed by a baseline test of impulsivity and baseline glucose uptake measurements with [18F]-fluoro-2-deoxy-D-glucose and positron emission tomography (PET). Then, 10 rats each received 3 mg/kg THC or vehicle injected intraperitoneally daily for 21 days. Subsequently, a second behavioral test and PET measurements were performed, and blood THC concentrations were determined. Analyses of variance of brain regions of the impulsivity network with the parameter "standardized uptake value" regarding glucose uptake and correlation analyses of the collected parameters were carried out. Discussion: After chronic THC treatment, decreased glucose uptake (p-values <0.05) was found in cingulate cortex, hippocampus, amygdala, thalamus, and cerebellar cortex, as compared with vehicle-treated rats. The number of correct no-go responses (increased waiting time) significantly increased (p<0.05) in THC-treated rats. Furthermore, correct no-go responses correlated positively and strongly with the THC blood concentrations (Spearman's ρ=0.79, p<0.01). Conclusion: These findings reflect a specific reduction in impulsive behavior after chronic THC treatment, showing a functionally relevant influence of THC on "waiting impulsivity" with reduced selective glucose uptake at the same time.

Microglial cannabinoid receptor type 1 mediates social memory deficits in mice produced by adolescent THC exposure and 16p11.2 duplication.

Adolescent cannabis use increases the risk for cognitive impairments and psychiatric disorders. Cannabinoid receptor type 1 (Cnr1) is expressed not only in neurons and astrocytes, but also in microglia, which shape synaptic connections during adolescence. However, the role of microglia in mediating the adverse cognitive effects of delta-9-tetrahydrocannabinol (THC), the principal psychoactive constituent of cannabis, is not fully understood. Here, we report that in mice, adolescent THC exposure produces microglial apoptosis in the medial prefrontal cortex (mPFC), which was exacerbated in a model of 16p11.2 duplication, a representative copy number variation (CNV) risk factor for psychiatric disorders. These effects are mediated by microglial Cnr1, leading to reduction in the excitability of mPFC pyramidal-tract neurons and deficits in social memory in adulthood. Our findings suggest the microglial Cnr1 may contribute to adverse effect of cannabis exposure in genetically vulnerable individuals.

Microglial cannabinoid receptor type 1 mediates social memory deficits produced by adolescent THC exposure and 16p11.2 duplication.

Adolescent cannabis use increases the risk for cognitive impairments and psychiatric disorders. Cannabinoid receptor type 1 (Cnr1) is expressed not only in neurons and astrocytes, but also in microglia, which shape synaptic connections during adolescence. Nonetheless, until now, the role of microglia in mediating the adverse cognitive effects of delta-9-tetrahydrocannabinol (THC), the principal psychoactive constituent of cannabis, has been unexplored. Here, we report that adolescent THC exposure produces microglial apoptosis in the medial prefrontal cortex (mPFC), which was exacerbated in the mouse model of 16p11.2 duplication, a representative copy number variation (CNV) risk factor for psychiatric disorders. These effects are mediated by microglial Cnr1, leading to reduction in the excitability of mPFC pyramidal-tract neurons and deficits in social memory in adulthood. Our findings highlight the importance of microglial Cnr1 to produce the adverse effect of cannabis exposure in genetically vulnerable individuals.

NAPE-PLD deletion in stress-TRAPed neurons results in an anxiogenic phenotype.

Anandamide (AEA) is an endogenous ligand of the cannabinoid CB1 and CB2 receptors, being a component of the endocannabinoid signaling system, which supports the maintenance or regaining of neural homeostasis upon internal and external challenges. AEA is thought to play a protective role against the development of pathological states after prolonged stress exposure, including depression and generalized anxiety disorder. Here, we used the chronic social defeat (CSD) stress as an ethologically valid model of chronic stress in male mice. We characterized a genetically modified mouse line where AEA signaling was reduced by deletion of the gene encoding the AEA synthesizing enzyme N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) specifically in neurons activated at the time of CSD stress. One week after the stress, the phenotype was assessed in behavioral tests and by molecular analyses. We found that NAPE-PLD deficiency in neurons activated during the last three days of CSD stress led to an increased anxiety-like behavior. Investigating the molecular mechanisms underlying this phenotype may suggest three main altered pathways to be affected: (i) desensitization of the negative feedback loop of the hypothalamic-pituitary-adrenal axis, (ii) disinhibition of the amygdala by the prefrontal cortex, and (iii) altered neuroplasticity in the hippocampus and prefrontal cortex.

Nuclei on the Rise: When Nuclei-Based Methods Meet Next-Generation Sequencing.

In the last decade, we have witnessed an upsurge in nuclei-based studies, particularly coupled with next-generation sequencing. Such studies aim at understanding the molecular states that exist in heterogeneous cell populations by applying increasingly more affordable sequencing approaches, in addition to optimized methodologies developed to isolate and select nuclei. Although these powerful new methods promise unprecedented insights, it is important to understand and critically consider the associated challenges. Here, we provide a comprehensive overview of the rise of nuclei-based studies and elaborate on their advantages and disadvantages, with a specific focus on their utility for transcriptomic sequencing analyses. Improved designs and appropriate use of the various experimental strategies will result in acquiring biologically accurate and meaningful information.

Mitochondrial cannabinoid receptors gate corticosterone impact on novel object recognition.

Corticosteroid-mediated stress responses require the activation of complex brain circuits involving mitochondrial activity, but the underlying cellular and molecular mechanisms are scantly known. The endocannabinoid system is implicated in stress coping, and it can directly regulate brain mitochondrial functions via type 1 cannabinoid (CB1) receptors associated with mitochondrial membranes (mtCB1). In this study, we show that the impairing effect of corticosterone in the novel object recognition (NOR) task in mice requires mtCB1 receptors and the regulation of mitochondrial calcium levels in neurons. Different brain circuits are modulated by this mechanism to mediate the impact of corticosterone during specific phases of the task. Thus, whereas corticosterone recruits mtCB1 receptors in noradrenergic neurons to impair NOR consolidation, mtCB1 receptors in local hippocampal GABAergic interneurons are required to inhibit NOR retrieval. These data reveal unforeseen mechanisms mediating the effects of corticosteroids during different phases of NOR, involving mitochondrial calcium alterations in different brain circuits.

Peripheral CB1 receptor blockade acts as a memory enhancer through a noradrenergic mechanism.

Peripheral inputs continuously shape brain function and can influence memory acquisition, but the underlying mechanisms have not been fully understood. Cannabinoid type-1 receptor (CB1R) is a well-recognized player in memory performance, and its systemic modulation significantly influences memory function. By assessing low arousal/non-emotional recognition memory in mice, we found a relevant role of peripheral CB1R in memory persistence. Indeed, the peripherally-restricted CB1R specific antagonist AM6545 showed significant mnemonic effects that were occluded in adrenalectomized mice, and after peripheral adrenergic blockade. AM6545 also transiently impaired contextual fear memory extinction. Vagus nerve chemogenetic inhibition reduced AM6545-induced mnemonic effect. Genetic CB1R deletion in dopamine ß-hydroxylase-expressing cells enhanced recognition memory persistence. These observations support a role of peripheral CB1R modulating adrenergic tone relevant for cognition. Furthermore, AM6545 acutely improved brain connectivity and enhanced extracellular hippocampal norepinephrine. In agreement, intra-hippocampal ß-adrenergic blockade prevented AM6545 mnemonic effects. Altogether, we disclose a novel CB1R-dependent peripheral mechanism with implications relevant for lengthening the duration of non-emotional memory.

Myeloid But Not Endothelial Expression of the CB2 Receptor Promotes Atherogenesis in the Context of Elevated Levels of the Endocannabinoid 2-Arachidonoylglycerol.

The endocannabinoid 2-arachidonoylglycerol (2-AG) is an inflammatory mediator and ligand for the cannabinoid receptors CB1 and CB2. We investigated the atherogenic mechanisms set in motion by 2-AG. Therefore, we created two atherosclerotic mouse models with distinct cell-specific knockouts of the CB2 receptor on either myeloid or endothelial cells. These mice were treated with JZL184, resulting in elevated plasma levels of 2-AG. After a high-fat high-cholesterol diet, atherosclerotic plaques were analyzed. The atherogenic effect of 2-AG was abrogated in mice lacking myeloid expression of the CB2 receptor but not in mice lacking endothelial expression of the CB2 receptor. In vitro, treatment of human monocytes with 2-AG led to the increased production of reactive oxygen species (ROS) and IL-1ß. In conclusion, 2-AG shows an atherogenic effect in vivo, dependent on the presence of the CB2 receptor on myeloid cells. In addition, our in vitro data revealed 2-AG to promote inflammatory signalling in monocytes. 2-Arachidonoylglycerol shows an atherogenic effect that is abrogated in mice lacking myeloid expression of the CB2 receptor.

Extraction and Simultaneous Quantification of Endocannabinoids and Endocannabinoid-Like Lipids in Biological Tissues.

Extraction and quantification of endocannabinoids from biological tissues is essential to unravel their changes under physiological and pathophysiological conditions. We describe here an analytical protocol for the extraction of endocannabinoids, anandamide (archidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG), and endocannabinoid-like lipids such as palmitoyl ethanolamide (PEA) and oleoyl ethanolamide (OEA), as well as arachidonic acid (AA) from biological tissues using liquid-liquid extraction method and simultaneous quantification by liquid chromatography multiple reaction monitoring (LC/MRM).

A Resilience Related Glial-Neurovascular Network Is Transcriptionally Activated after Chronic Social Defeat in Male Mice.

Upon chronic stress, a fraction of individuals shows stress resilience, which can prevent long-term mental dysfunction. The underlying molecular mechanisms are complex and have not yet been fully understood. In this study, we performed a data-driven behavioural stratification together with single-cell transcriptomics of the hippocampus in a mouse model of chronic social defeat stress. Our work revealed that in a sub-group exhibiting molecular responses upon chronic stress, the dorsal hippocampus is particularly involved in neuroimmune responses, angiogenesis, myelination, and neurogenesis, thereby enabling brain restoration and homeostasis after chronic stress. Based on these molecular insights, we applied rapamycin after the stress as a proof-of-concept pharmacological intervention and were able to substantially increase stress resilience. Our findings serve as a data resource and can open new avenues for further understanding of molecular processes underlying stress response and for targeted interventions supporting resilience.

Cannabinoid CB1 Receptor Deletion from Catecholaminergic Neurons Protects from Diet-Induced Obesity.

High-calorie diets and chronic stress are major contributors to the development of obesity and metabolic disorders. These two risk factors regulate the activity of the sympathetic nervous system (SNS). The present study showed a key role of the cannabinoid type 1 receptor (CB1) in dopamine ß-hydroxylase (dbh)-expressing cells in the regulation of SNS activity. In a diet-induced obesity model, CB1 deletion from these cells protected mice from diet-induced weight gain by increasing sympathetic drive, resulting in reduced adipogenesis in white adipose tissue and enhanced thermogenesis in brown adipose tissue. The deletion of CB1 from catecholaminergic neurons increased the plasma norepinephrine levels, norepinephrine turnover, and sympathetic activity in the visceral fat, which coincided with lowered neuropeptide Y (NPY) levels in the visceral fat of the mutant mice compared with the controls. Furthermore, the mutant mice showed decreased plasma corticosterone levels. Our study provided new insight into the mechanisms underlying the roles of the endocannabinoid system in regulating energy balance, where the CB1 deletion in dbh-positive cells protected from diet-induced weight gain via multiple mechanisms, such as increased SNS activity, reduced NPY activity, and decreased basal hypothalamic-pituitary-adrenal (HPA) axis activity.

Association of polygenic risk scores and hair cortisol with mental health trajectories during COVID lockdown.

The COVID-19 pandemic is a global stressor with inter-individually differing influences on mental health trajectories. Polygenic Risk Scores (PRSs) for psychiatric phenotypes are associated with individual mental health predispositions. Elevated hair cortisol concentrations (HCC) and high PRSs are related to negative mental health outcomes. We analyzed whether PRSs and HCC are related to different mental health trajectories during the first COVID lockdown in Germany. Among 523 participants selected from the longitudinal resilience assessment study (LORA), we previously reported three subgroups (acute dysfunction, delayed dysfunction, resilient) based on weekly mental health (GHQ-28) assessment during COVID lockdown. DNA from blood was collected at the baseline of the original LORA study (n = 364) and used to calculate the PRSs of 12 different psychopathological phenotypes. An explorative bifactor model with Schmid-Leiman transformation was calculated to extract a general genetic factor for psychiatric disorders. Hair samples were collected quarterly prior to the pandemic for determining HCC (n = 192). Bivariate logistic regressions were performed to test the associations of HCC and the PRS factors with the reported trajectories. The bifactor model revealed 1 general factor and 4 sub-factors. Results indicate a significant association between increased values on the general risk factor and the allocation to the acute dysfunction class. The same was found for elevated HCC and the exploratorily tested sub-factor "childhood-onset neurodevelopmental disorders". Genetic risk and long-term cortisol secretion as a potential indicator of stress, indicated by PRSs and HCC, respectively, predicted different mental health trajectories. Results indicate a potential for future studies on risk prediction.

Dynamic Changes in the Endocannabinoid System during the Aging Process: Focus on the Middle-Age Crisis.

Endocannabinoid (eCB) signaling is markedly decreased in the hippocampus (Hip) of aged mice, and the genetic deletion of the cannabinoid receptor type 1 (CB1) leads to an early onset of cognitive decline and age-related histological changes in the brain. Thus, it is hypothesized that cognitive aging is modulated by eCB signaling through CB1. In the present study, we detailed the changes in the eCB system during the aging process using different complementary techniques in mouse brains of five different age groups, ranging from adolescence to old age. Our findings indicate that the eCB system is most strongly affected in middle-aged mice (between 9 and 12 months of age) in a brain region-specific manner. We show that 2-arachidonoylglycerol (2-AG) was prominently decreased in the Hip and moderately in caudate putamen (CPu), whereas anandamide (AEA) was decreased in both CPu and medial prefrontal cortex along with cingulate cortex (mPFC+Cg), starting from 6 months until 12 months. Consistent with the changes in 2-AG, the 2-AG synthesizing enzyme diacylglycerol lipase α (DAGLα) was also prominently decreased across the sub-regions of the Hip. Interestingly, we found a transient increase in CB1 immunoreactivity across the sub-regions of the Hip at 9 months, a plausible compensation for reduced 2-AG, which ultimately decreased strongly at 12 months. Furthermore, quantitative autoradiography of CB1 revealed that [3H]CP55940 binding markedly increased in the Hip at 9 months. However, unlike the protein levels, CB1 binding density did not drop strongly at 12 months and at old age. Furthermore, [3H]CP55940 binding was significantly increased in the lateral entorhinal cortex (LEnt), starting from the middle age until the old age. Altogether, our findings clearly indicate a middle-age crisis in the eCB system, which could be a potential time window for therapeutic interventions to abrogate the course of cognitive aging.

Brain-derived neurotrophic factor expression in serotonergic neurons improves stress resilience and promotes adult hippocampal neurogenesis.

The neurotrophin brain-derived neurotrophic factor (BDNF) stimulates adult neurogenesis, but also influences structural plasticity and function of serotonergic neurons. Both, BDNF/TrkB signaling and the serotonergic system modulate behavioral responses to stress and can lead to pathological states when dysregulated. The two systems have been shown to mediate the therapeutic effect of antidepressant drugs and to regulate hippocampal neurogenesis. To elucidate the interplay of both systems at cellular and behavioral levels, we generated a transgenic mouse line that overexpresses BDNF in serotonergic neurons in an inducible manner. Besides displaying enhanced hippocampus-dependent contextual learning, transgenic mice were less affected by chronic social defeat stress (CSDS) compared to wild-type animals. In parallel, we observed enhanced serotonergic axonal sprouting in the dentate gyrus and increased neural stem/progenitor cell proliferation, which was uniformly distributed along the dorsoventral axis of the hippocampus. In the forced swim test, BDNF-overexpressing mice behaved similarly as wild-type mice treated with the antidepressant fluoxetine. Our data suggest that BDNF released from serotonergic projections exerts this effect partly by enhancing adult neurogenesis. Furthermore, independently of the genotype, enhanced neurogenesis positively correlated with the social interaction time after the CSDS, a measure for stress resilience.

Cannabinoid CB1 receptor gene inactivation in oligodendrocyte precursors disrupts oligodendrogenesis and myelination in mice.

Cannabinoids are known to modulate oligodendrogenesis and developmental CNS myelination. However, the cell-autonomous action of these compounds on oligodendroglial cells in vivo, and the molecular mechanisms underlying these effects have not yet been studied. Here, by using oligodendroglial precursor cell (OPC)-targeted genetic mouse models, we show that cannabinoid CB1 receptors exert an essential role in modulating OPC differentiation at the critical periods of postnatal myelination. We found that selective genetic inactivation of CB1 receptors in OPCs in vivo perturbs oligodendrogenesis and postnatal myelination by altering the RhoA/ROCK signaling pathway, leading to hypomyelination, and motor and cognitive alterations in young adult mice. Conversely, pharmacological CB1 receptor activation, by inducing E3 ubiquitin ligase-dependent RhoA proteasomal degradation, promotes oligodendrocyte development and CNS myelination in OPCs, an effect that was not evident in OPC-specific CB1 receptor-deficient mice. Moreover, pharmacological inactivation of ROCK in vivo overcomes the defects in oligodendrogenesis and CNS myelination, and behavioral alterations found in OPC-specific CB1 receptor-deficient mice. Overall, this study supports a cell-autonomous role for CB1 receptors in modulating oligodendrogenesis in vivo, which may have a profound impact on the scientific knowledge and therapeutic manipulation of CNS myelination by cannabinoids.

Chronic Pain and the Endocannabinoid System: Smart Lipids - A Novel Therapeutic Option?

The development of a high-end cannabinoid-based therapy is the result of intense translational research, aiming to convert recent discoveries in the laboratory into better treatments for patients. Novel compounds and new regimes for drug treatment are emerging. Given that previously unreported signaling mechanisms for cannabinoids have been uncovered, clinical studies detailing their high therapeutic potential are mandatory. The advent of novel genomic, optogenetic, and viral tracing and imaging techniques will help to further detail therapeutically relevant functional and structural features. An evolutionarily highly conserved group of neuromodulatory lipids, their receptors, and anabolic and catabolic enzymes are involved in a remarkable variety of physiological and pathological processes and has been termed the endocannabinoid system (ECS). A large body of data has emerged in recent years, pointing to a crucial role of this system in the regulation of the behavioral domains of acquired fear, anxiety, and stress-coping. Besides neurons, also glia cells and components of the immune system can differentially fine-tune patterns of neuronal activity. Dysregulation of ECS signaling can lead to a lowering of stress resilience and increased incidence of psychiatric disorders. Chronic pain may be understood as a disease process evoked by fear-conditioned nociceptive input and appears as the dark side of neuronal plasticity. By taking a toll on every part of your life, this abnormal persistent memory of an aversive state can be more damaging than its initial experience. All strategies for the treatment of chronic pain conditions must consider stress-related comorbid conditions since cognitive factors such as beliefs, expectations, and prior experience (memory of pain) are key modulators of the perception of pain. The anxiolytic and anti-stress effects of medical cannabinoids can substantially modulate the efficacy and tolerability of therapeutic interventions and will help to pave the way to a successful multimodal therapy. Why some individuals are more susceptible to the effects of stress remains to be uncovered. The development of personalized prevention or treatment strategies for anxiety and depression related to chronic pain must also consider gender differences. An emotional basis of chronic pain opens a new horizon of opportunities for developing treatment strategies beyond the repeated sole use of acutely acting analgesics. A phase I trial to determine the pharmacokinetics, psychotropic effects, and safety profile of a novel nanoparticle-based cannabinoid spray for oromucosal delivery highlights a remarkable innovation in galenic technology and urges clinical studies further detailing the huge therapeutic potential of medical cannabis (Lorenzl et al.; this issue).

Acquisition of threat responses are associated with elevated plasma concentration of endocannabinoids in male humans.

Endocannabinoids (eCBs) are involved in buffering threat and stress responses. Elevation of circulating eCBs in humans was reported to strengthen inhibition (i.e., extinction) of threat responses and to reduce effects of stressors. However, it remains unclear whether the acquisition of threat responses involves a physiological change in circulating eCBs. Here, we demonstrate in male human volunteers that the plasma concentration of the eCB N-arachidonoylethanolamine (AEA) and its metabolite arachidonic acid (AA) are increased during acquisition of threat responses. Furthermore, elevated responses to a learned threat cue (e.g., rating of fear) were associated with individual increases in plasma concentration of the eCB 2-arachidonoylglycerol (2-AG). In complementing these observations, we found individual increases in AEA associated with elevated neural responses during threat learning in the amygdala. Our results thereby suggest that physiological increases in circulating eCB levels are part of a response mechanism to learned threats.

The Microbiome and Gut Endocannabinoid System in the Regulation of Stress Responses and Metabolism.

The endocannabinoid system, with its receptors and ligands, is present in the gut epithelium and enteroendocrine cells, and is able to modulate brain functions, both indirectly through circulating gut-derived factors and directly through the vagus nerve, finally acting on the brain's mechanisms regarding metabolism and behavior. The gut endocannabinoid system also regulates gut motility, permeability, and inflammatory responses. Furthermore, microbiota composition has been shown to influence the activity of the endocannabinoid system. This review examines the interaction between microbiota, intestinal endocannabinoid system, metabolism, and stress responses. We hypothesize that the crosstalk between microbiota and intestinal endocannabinoid system has a prominent role in stress-induced changes in the gut-brain axis affecting metabolic and mental health. Inter-individual differences are commonly observed in stress responses, but mechanisms underlying resilience and vulnerability to stress are far from understood. Both gut microbiota and the endocannabinoid system have been implicated in stress resilience. We also discuss interventions targeting the microbiota and the endocannabinoid system to mitigate metabolic and stress-related disorders.

Lipidomics and Transcriptomics in Neurological Diseases.

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with various signaling or ligand function that can underscore the onset and progression of diseases. Often changing at the presymptomatic level, lipids are an emerging source of drug targets and biomarkers. Many neurological diseases exhibit neuroinflammation, neurodegeneration, and neuronal excitability as common hallmarks, partly modulated by specific lipid signaling systems. The interdependence and interrelation of synthesis of various lipids prompts a multilipid, multienzyme, and multireceptor analysis in order to derive the commonalities and specificities of neurological contexts and to expedite the unravelling of mechanistic aspects of disease onset and progression. Ascribing lipid roles to distinct brain regions advances the determination of lipid molecular phenotype and morphology associated with a neurological disease. Presented here is a modular protocol suitable for the analysis of membrane lipids and downstream lipid signals along with mRNA of enzymes and mediators underlying their functionality, extracted from discrete brain regions that are relevant for a particular neurological disease and/or condition. To ensure accurate comparative lipidomic profiling, the workflows and operating criteria were optimized and standardized for: i) brain sampling and dissection of regions of interest, ii) co-extraction of multiple lipid signals and membrane lipids, iii) dual lipid/mRNA extraction, iv) quantification by liquid chromatography multiple reaction monitoring (LC/MRM), and v) standard mRNA profiling. This workflow is amenable for the low tissue amounts obtained by sampling of the functionally discrete brain subregions (i.e. by brain punching), thus preventing bias in multimolecular analysis due to tissue heterogeneity and/or animal variability. To reveal peripheral consequences of neurological diseases and establish translational molecular readouts of neurological disease states, peripheral organ sampling, processing, and their subsequent lipidomic analysis, as well as plasma lipidomics, are also pursued and described. The protocol is demonstrated on an acute epilepsy mouse model.