Illuminating the brain-genetically encoded single wavelength fluorescent biosensors to unravel neurotransmitter dynamics.

Understanding how neuronal networks generate complex behavior is one of the major goals of Neuroscience. Neurotransmitter and Neuromodulators are crucial for information flow between neurons and understanding their dynamics is the key to unravel their role in behavior. To understand how the brain transmits information and how brain states arise, it is essential to visualize the dynamics of neurotransmitters, neuromodulators and neurochemicals. In the last five years, an increasing number of single-wavelength biosensors either based on periplasmic binding proteins (PBPs) or on G-protein-coupled receptors (GPCR) have been published that are able to detect neurotransmitter release in vitro and in vivo with high spatial and temporal resolution. Here we review and discuss recent progress in the development of these sensors, their limitations and future directions.

Neurochemical correlates of synapse density in a Huntington's disease mouse model.

Striatal medium spiny neurons are highly susceptible in Huntington's disease (HD), resulting in progressive synaptic perturbations that lead to neuronal dysfunction and death. Non-invasive imaging techniques, such as proton magnetic resonance spectroscopy (1 H-MRS), are used in HD mouse models and patients with HD to monitor neurochemical changes associated with neuronal health. However, the association between brain neurochemical alterations and synaptic dysregulation remains unknown, limiting our ability to monitor potential treatments that may affect synapse function. We conducted in vivo longitudinal 1 H-MRS in the striatum followed by ex vivo analyses of excitatory synapse density of two synaptic circuits disrupted in HD, thalamo-striatal (T-S), and cortico-striatal (C-S) pathways, to assess the relationship between neurochemical alterations and changes in synapse density. We used the zQ175(Tg/0) HD mouse model as well as zQ175 mice lacking one allele of CK2α'(zQ175(Tg/0) :CK2α'(+/-) ), a kinase previously shown to regulate synapse function in HD. Longitudinal analyses of excitatory synapse density showed early and sustained reduction in T-S synapses in zQ175 mice, preceding C-S synapse depletion, which was rescued in zQ175:CK2α'(+/-) . Changes in T-S and C-S synapses were accompanied by progressive alterations in numerous neurochemicals between WT and HD mice. Linear regression analyses showed C-S synapse number positively correlated with 1 H-MRS-measured levels of GABA, while T-S synapse number positively correlated with levels of phosphoethanolamine and negatively correlated with total creatine levels. These associations suggest that these neurochemical concentrations measured by 1 H-MRS may facilitate monitoring circuit-specific synaptic dysfunction in the zQ175 mouse model and in other HD pre-clinical studies.

Time and Brain Region-Dependent Excitatory Neurochemical Alterations in Bilateral Common Carotid Artery Occlusion Global Ischemia Model.

Strict metabolic regulation in discrete brain regions leads to neurochemical changes in cerebral ischemia. Accumulation of extracellular glutamate is one of the early neurochemical changes that take place during cerebral ischemia. Understanding the sequential neurochemical processes involved in cerebral ischemia-mediated excitotoxicity before the clinical intervention of revascularization and reperfusion may greatly influence future therapeutic strategies for clinical stroke recovery. This study investigated the influence of time and brain regions on excitatory neurochemical indices in the bilateral common carotid artery occlusion (BCCAO) model of global ischemia. Male Wistar rats were subjected to BCCAO for 15 and 60 min to evaluate the effect of ischemia duration on excitatory neurochemical indices (dopamine level, glutamine synthetase, glutaminase, glutamate dehydrogenase, aspartate aminotransferase, monoamine oxidase, acetylcholinesterase, and Na+ K+ ATPase activities) in the discrete brain regions (cortex, striatum, cerebellum, and hippocampus). BCCAO without reperfusion caused marked time and brain region-dependent alterations in glutamatergic, glutaminergic, dopaminergic, monoaminergic, cholinergic, and electrogenic homeostasis. Prolonged BCCAO decreased cortical, striatal, and cerebellar glutamatergic, glutaminergic, dopaminergic, cholinergic, and electrogenic activities; increased hippocampal glutamatergic, glutaminergic, dopaminergic, and cholinergic activities, increased cortical and striatal monoaminergic activity; decreased cerebellar and hippocampal monoaminergic activity; and decreased hippocampal electrogenic activity. This suggests that excitatory neurotransmitters play a major role in the tissue-specific metabolic plasticity and reprogramming that takes place between the onset of cardiac arrest-mediated global ischemia and clinical intervention of recanalization. These tissue-specific neurochemical indices may serve as diagnostic and therapeutic strategies for mitigating the progression of ischemic damage before revascularization.

Gut brain regulation using psychobiotics for improved neuropsychological illness.

"Psychobiotics" are a novel class of probiotics that are beneficial to the health and functional efficiency of our brain and psychology. The main hold on command in ill conditions of the brain and psychology is overtaken by these psychobiotic bacteria (a dietary supplement) via the action/determined role of bacterial neurochemicals or neuroactive substances that are released by them in the intestinal epithelium after their ingestion. Although these psychobiotics flourish in the gut of the host consuming them, the effect is widely spread to the brain due to the communication between the gut and the brain via the bidirectional gut-brain axis. The nervous system involved in this directional process includes both the enteric nervous system and the central nervous system. With time, several corroborations have proved the effectiveness of psychobiotics in terms of mental illnesses and brain disorders. In the prevailing situation of the coronavirus pandemic, psychobiotics may serve as an aid because a majority of the population worldwide is already suffering from psychological issues due to changes in lifestyle and dietary habits, and in need of an immediate solution to cope with it. Moreover, the in silico approach is also vital for the development of biological relevance to neurosubstances.

Antibiotic cocktail-induced gut microbiota depletion in different stages could cause host cognitive impairment and emotional disorders in adulthood in different manners.

Gut microbiota depletion may result in cognitive impairment and emotional disorder. This study aimed to determine the possible association between host gut microbiota, cognitive function, and emotion in various life stages and its related underlying mechanisms. Seventy-five neonatal mice were randomly divided into five groups (n = 15 per group). Mice in the vehicle group were administered distilled water from birth to death, and those in the last four groups were administered antibiotic cocktail from birth to death, from birth to postnatal day (PND) 21 (infancy), from PND 21 to 56 (adolescence), and from PND 57 to 84 (adulthood), respectively. Antibiotic exposure consistently altered the gut microbiota composition and decreased the diversity of gut microbiota. Proteobacteria were the predominant bacteria instead of Firmicutes and Bacteroidetes after antibiotic exposure in different life stages. Long-term and infant gut microbiota depletion resulted in anxiety- and depression-like behaviors, memory impairments, and increased expression of γ-aminobutyric acid type A receptor α1 of adult mice. Long-term antibiotic exposure also significantly decreased serum interleukin (IL)-1ß, IL-10, and corticosterone of adult mice. Gut microbiota depletion in adolescence resulted in anxiety-like behaviors, short-term memory decline, decreased serum interferon-γ (IFN-γ), mRNA expression of 5-hydroxytryptamine receptor 1A, and neuropeptide Y receptor Y2 in the prefrontal cortex of adult mice. Antibiotic exposure in adulthood damaged short-term memory and decreased serum IL-10, IFN-γ, and increased γ-aminobutyric acid type B receptor 1 mRNA expression of adult mice. These results suggest that antibiotic-induced gut microbiota depletion in the long term and infancy resulted in the most severe cognitive and emotional disorders followed by depletion in adolescence and adulthood. These results also suggest that gut microbes could influence host cognitive function and emotion in a life stage-dependent manner by affecting the function of the immune system, hypothalamic-pituitary-adrenal axis, and the expression of neurochemicals in the brain.

IPSC-Derived Human Neurons with GCaMP6s Expression Allow In Vitro Study of Neurophysiological Responses to Neurochemicals.

The study of human neurons and their interaction with neurochemicals is difficult due to the inability to collect primary biomaterial. However, recent advances in the cultivation of human stem cells, methods for their neuronal differentiation and chimeric fluorescent calcium indicators have allowed the creation of model systems in vitro. In this paper we report on the development of a method to obtain human neurons with the GCaMP6s calcium indicator, based on a human iPSC line with the TetON-NGN2 transgene complex. The protocol we developed allows us quickly, conveniently and efficiently obtain significant amounts of human neurons suitable for the study of various neurochemicals and their effects on specific neurophysiological activity, which can be easily registered using fluorescence microscopy. In the neurons we obtained, glutamate (Glu) induces rises in [Ca2+]i which are caused by ionotropic receptors for Glu, predominantly of the NMDA-type. Taken together, these facts allow us to consider the model we have created to be a useful and successful development of this technology.

Protective Role of Capsaicin in Neurological Disorders: An Overview.

Different pathological conditions that begin with slow and progressive deformations, cause irreversible affliction by producing loss of neurons and synapses. Commonly it is referred to as 'protein misfolding' diseases or proteinopathies and comprises the latest definition of neurological disorders (ND). Protein misfolding dynamics, proteasomal dysfunction, aggregation, defective degradation, oxidative stress, free radical formation, mitochondrial dysfunctions, impaired bioenergetics, DNA damage, neuronal Golgi apparatus fragmentation, axonal transport disruption, Neurotrophins (NTFs) dysfunction, neuroinflammatory or neuroimmune processes, and neurohumoral changes are the several mechanisms that embark the pathogenesis of ND. Capsaicin (8-Methyl-N-vanillyl-6-nonenamide) one of the major phenolic components in chili peppers (Capsicum) distinctively triggers the unmyelinated C-fiber and acts on Transient Receptor Potential Vanilloid-1, which is a Ca2+ permeable, non-selective cation channel. Several studies have shown the neuroprotective role of capsaicin against oxidative damage, behavioral impairment, with 6-hydroxydopamine (6-OHDA) induced Parkinson's disease, pentylenetetrazol-induced seizures, global cerebral ischemia, and streptozotocin-induced Alzheimer's disease. Based on these lines of evidence, capsaicin can be considered as a potential constituent to develop suitable neuro-pharmacotherapeutics for the management and treatment of ND. Furthermore, exploring newer horizons and carrying out proper clinical trials would help to bring out the promising effects of capsaicin to be recommended as a neuroprotectant.

Characterization of sleep-related neurochemicals at different developmental stages and insomnia models of Drosophila melanogaster.

Neurotransmitters play an important role in regulating the physiological activity of the animal, especially in emotion and sleep, whereas nucleotides are involved in almost all cellular processes. However, the characteristics of sleep-related neurochemicals under different life cycles and environments remain poorly understood. A rapid and sensitive analytical method was established with LC-MS/MS to determine eight endogenous neurochemicals in Drosophila melanogaster, and their levels in the different developmental stages of D. melanogaster were evaluated. The results indicated that there were significant discrepancies among different stages, especially from the pupal stage to the adult stage. The levels of these compounds in the caffeine-induced insomnia model of D. melanogaster were investigated. Compared with the normal group, the eight endogenous metabolites did not fluctuate significantly in insomnia D. melanogaster, which may be due to the mechanism of caffeine-induced insomnia through other pathways, such as adenosine. The results provide a reference for decoding neurochemicals involved in the development of the full cycle of mammalian life and the exploration of insomnia and even other mental diseases induced by exogenous substances in the future.

Normalization of Cerebral Blood Flow, Neurochemicals, and White Matter Integrity after Kidney Transplantation.

BACKGROUND: CKD is associated with abnormalities in cerebral blood flow, cerebral neurochemical concentrations, and white matter integrity. Each of these is associated with adverse clinical consequences in the non-CKD population, which may explain the high prevalence of dementia and stroke in ESKD. Because cognition improves after kidney transplantation, comparing these brain abnormalities before and after kidney transplantation may identify potential reversibility in ESKD-associated brain abnormalities. METHODS: In this study of patients with ESKD and age-matched healthy controls, we used arterial spin labeling to assess the effects of kidney transplantation on cerebral blood flow and magnetic resonance spectroscopic imaging to measure cerebral neurochemical concentrations (N-acetylaspartate, choline, glutamate, glutamine, myo-inositol, and total creatine). We also assessed white matter integrity measured by fractional anisotropy (FA) and mean diffusivity (MD) with diffusion tensor imaging. We used a linear mixed model analysis to compare longitudinal, repeated brain magnetic resonance imaging measurements before, 3 months after, and 12 months after transplantation and compared these findings with those of healthy controls. RESULTS: Study participants included 29 patients with ESKD and 19 controls; 22 patients completed post-transplant magnetic resonance imaging. Cerebral blood flow, which was higher in patients pretransplant compared with controls (P=0.003), decreased post-transplant (P<0.001) to values in controls. Concentrations of neurochemicals choline and myo-inositol that were higher pretransplant compared with controls (P=0.001 and P<0.001, respectively) also normalized post-transplant (P<0.001 and P<0.001, respectively). FA increased (P=0.001) and MD decreased (P<0.001) post-transplant. CONCLUSIONS: Certain brain abnormalities in CKD are reversible and normalize with kidney transplantation. Further studies are needed to understand the mechanisms underlying these brain abnormalities and to explore interventions to mitigate them even in patients who cannot be transplanted. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Cognitive Impairment and Imaging Correlates in End Stage Renal Disease, NCT01883349.

Neurochemical Alterations in Social Anxiety Disorder (SAD): A Systematic Review of Proton Magnetic Resonance Spectroscopic Studies.

(1) Objective: Considering that current knowledge of mechanisms involved in the molecular pathogenesis of Social Anxiety Disorder (SAD) is limited, we conducted a systematic review to evaluate cumulative data obtained by Proton Magnetic Resonance Spectroscopic (1H MRS) studies. (2) Methods: A computer-based literature search of Medline, EMBASE, PsycInfo, and ProQuest was performed. Only cross-sectional studies using 1H MRS techniques in participants with SAD and healthy controls (HCs) were selected. (3) Results: The search generated eight studies. The results indicated regional abnormalities in the 'fear neurocircuitry' in patients with SAD. The implicated regions included the anterior cingulate cortex (ACC), dorsomedial prefrontal cortex (dmPFC), dorsolateral prefrontal cortex (dlPFC), insula, occipital cortex (OC), as well as the subcortical regions, including the thalamus, caudate, and the putamen. (4) Conclusions: The evidence derived from eight studies suggests that possible pathophysiological mechanisms of SAD include impairments in the integrity and function of neurons and glial cells, including disturbances in energy metabolism, maintenance of phospholipid membranes, dysregulations of second messenger systems, and excitatory/inhibitory neurocircuitry. Conducting more cross-sectional studies with larger sample sizes is warranted given the limited evidence in this area of research.

The cross-sectional interplay between neurochemical profile and brain connectivity.

Neurochemical profile and brain connectivity are both critical aspects of brain function. However, our knowledge of their interplay across development is currently poor. We combined single-voxel magnetic resonance spectroscopy and resting functional magnetic resonance imaging in a cross-sectional sample spanning from childhood to adulthood which was reassessed in ~1.5 years (N = 293). We revealed the developmental trajectories of 20 neurochemicals in two key developmental brain regions (the intraparietal sulcus, IPS, and the middle frontal gyrus, MFG). We found that certain neurochemicals exhibited similar developmental trajectories across the two regions, while other trajectories were region-specific. Crucially, we mapped the connectivity of the brain regions IPS and MFG to the rest of the brain across development as a function of regional glutamate and GABA concentration. We demonstrated that glutamate concentration within the IPS is modulated by age in explaining IPS connectivity with frontal, temporal and parietal regions. In mature participants, higher glutamate within the IPS was related to more negative connectivity while the opposite pattern was found for younger participants. Our findings offer specific developmental insights on the interplay between the brain's resting activity and the glutamatergic system both of which are crucial for regulating normal functioning and are dysregulated in several clinical conditions.

Identification of markers of depression and neurotoxicity in pesticide exposed agriculture workers.

Earlier, we reported that chronic exposure to pesticides causes a reduction in the acetylcholinesterase activity and hematological and biochemical alterations in agriculture workers. In continuation with that, the present study aimed to investigate the pesticide-induced neurochemical imbalance and its association with behavior alterations in agricultural workers. A significant increase in depressive symptoms, assessed by the Beck Depression Inventory-II was observed in pesticide exposed workers as compared to the unexposed. A decrease in the level of dopamine in plasma and levels of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acids, norepinephrine, serotonin, and hydroxyindoleacetic acid in urine was also observed. An increase in the levels of MAO-A and MAO-B has also been observed in these individuals. The decreased levels of neurotransmitters in the blood and urine have been linked with increased levels of MAO and pesticide residues in plasma and urine. Furthermore, these changes were associated with a higher incidence of depression in agricultural workers.

Broad targeted analysis of neurochemicals in rat serum using liquid chromatography tandem mass spectrometry with chemical derivatization.

In this study, an efficient and sensitive assay for the detection of 42 polar neurochemicals, including neurotransmitters, amino acids, and biogenic amines, was established by combining reversed-phase liquid chromatography tandem mass spectrometry with chemical derivatization. An optimally designed benzoyl chloride derivatization was easily conducted in a one-pot reaction and stable neurochemical derivatives were obtained under mild conditions within 5 min (except for acetylcholine and melatonin). Derivatization also enabled the introduction of heavy labeling of the analytes through the use of labeled derivatization agents. Chromatography separation was performed on an HSS T3 column within 15 min by gradient elution. Multiple reaction monitoring acquisition mode enabled quantitation of neurochemicals with limits of detection of 0.05 to 11.63 nM and lower limits of quantitation of 0.09 to 46.50 nM in rat serum. The assay was well validated in terms of linearity and extraction recovery. Furthermore, the instrumental precision, specificity, matrix effect, accuracy, precision, stability, dilution effect, and carry-over effect were also validated. Finally, the overall efficacy of the assay was experimentally tested using serum from six Sprague-Dawley rats. The results demonstrated that the developed method is effective for broad targeted analysis of 42 neurochemicals in serum.

A Generalizable and Noncovalent Strategy for Interfacing Aptamers with a Microelectrode for the Selective Sensing of Neurotransmitters In†Vivo.

The selective sensing of neurochemicals is essential for understanding the chemical basis of brain function and pathology. Interfacing the excellent recognition features of aptamers with in vivo compatible carbon fiber microelectrode (CFE)-based electroanalytical systems offers a plausible means to achieve this end. However, this is challenging in terms of coupling chemistry, stability, and versatility. Here, we present a new interfacial functionalization strategy based on the assembly of aptamer cholesterol amphiphiles (aptCAs) on the alkyl chain-functionalized CFE. The noncovalent cholesterol-alkyl chain interactions effectively immobilize aptamers onto the CFE surface, allowing the generation of a highly selective system for probing neurochemical dynamics in living systems and opening up a vast array of new opportunities for designing in vivo sensors for exploring brain chemistry.

Effect of nisin on microbiome-brain-gut axis neurochemicals by Escherichia coli-induced diarrhea in mice.

The effects of nisin on the neurochemicals, Aquaporin-3 (AQP-3) and intestinal microorganisms in the brain-gut axis of mice were analyzed by using enzyme linked immune sorbent assay (ELISA) and high throughput sequencing in this investigation, to further revealed the relationship between intestinal flora abundance in mice and neurochemicals in the brain-gut axis. Using HE staining found damage of structure of small intestine villi in the model group (Escherichia coli O1, E. coli O1). Compared with normal control and ciprofloxacin groups, using ELISA showed that nisin increased the highest norepinephrine (NE) expression in the brain, expression of 5-hydroxytryptamine (5-HT) and dopamine (DA) in the duodenum, and increased the expression of AQP-3 in jejunum. Using high-throughput sequencing showed the highest diversity of cecal microflora in nisin group (ACE-index = 1417.25, Chao1-index = 1378.45), but the cecal microflora in the negative control group (ACE-index = 969.54, Chao1-index = 340.29) exhibited the lowest species diversity. Our data indicated that nisin regulates neurochemicals, AQP-3 and cecal microflora imbalance in mice.

Microbial Endocrinology: An Ongoing Personal Journey.

The development of microbial endocrinology is covered from a decidedly personal perspective. Specific focus is given to the role of microbial endocrinology in the evolutionary symbiosis between man and microbe as it relates to both health and disease. Since the first edition of this book series 5 years ago, the role of microbial endocrinology in the microbiota-gut-brain axis is additionally discussed. Future avenues of research are suggested.

Advancements in Brain Research: The In Vivo/In Vitro Electrochemical Detection of Neurochemicals.

Neurochemicals, crucial for nervous system function, influence vital bodily processes and their fluctuations are linked to neurodegenerative diseases and mental health conditions. Monitoring these compounds is pivotal, yet the intricate nature of the central nervous system poses challenges. Researchers have devised methods, notably electrochemical sensing with micro-nanoscale electrodes, offering high-resolution monitoring despite low concentrations and rapid changes. Implantable sensors enable precise detection in brain tissues with minimal damage, while microdialysis-coupled platforms allow in vivo sampling and subsequent in vitro analysis, addressing the selectivity issues seen in other methods. While lacking temporal resolution, techniques like HPLC and CE complement electrochemical sensing's selectivity, particularly for structurally similar neurochemicals. This review covers essential neurochemicals and explores miniaturized electrochemical sensors for brain analysis, emphasizing microdialysis integration. It discusses the pros and cons of these techniques, forecasting electrochemical sensing's future in neuroscience research. Overall, this comprehensive review outlines the evolution, strengths, and potential applications of electrochemical sensing in the study of neurochemicals, offering insights into future advancements in the field.

Vanillin attenuates oxidative stress and neurochemical balance in MPTP-induced Parkinson's disease mice by regulating the TLR-4 inflammatory pathway.

This study investigated the protective effect of vanillin against Parkinson's disease (PD). 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 30 mg/kg) was administered s.c. for 6 consecutive days to induce PD and mice were treated with vanillin (100 and 200 mg/kg, p.o.) for 15 days. Cognitive, motor and non-motor functions were assessed to evaluate the effect of vanillin in PD mice. Levels of dopamine and glutamate and activity of monoamine oxidaseB (MAO-B) were estimated in vanillin-treated PD mice. The effect of vanillin on the level of lipid peroxidation and superoxide dismutase in brain tissue of PD mice was estimated. Data of the study revealed that vanillin reversed the altered cognitive, motor and non-motor function in PD mice. Activity of MAO-B and neurochemical level were attenuated with vanillin in PD mice. Inflammatory cytokines, nuclear factor kappa B (NF-kB) and Toll-like receptor 4 (TLR-4) levels were lower in the vanillin-treated group compared to the PD group of mice. Data of the study suggest that vanillin protects against neuronal injury and recovers the altered behaviour in PD mice by regulating neurochemical balance and the TLR-4/NF-kB pathway.

Concentrations of glutamate and N-acetylaspartate detected by magnetic resonance spectroscopy in the rat hippocampus correlate with hippocampal-dependent spatial memory performance.

Magnetic resonance spectroscopy (MRS) has been employed to investigate brain metabolite concentrations in vivo, and they vary during neuronal activation, across brain activity states, or upon disease with neurological impact. Whether resting brain metabolites correlate with functioning in behavioral tasks remains to be demonstrated in any of the widely used rodent models. This study tested the hypothesis that, in the absence of neurological disease or injury, the performance in a hippocampal-dependent memory task is correlated with the hippocampal levels of metabolites that are mainly synthesized in neurons, namely N-acetylaspartate (NAA), glutamate and GABA. Experimentally naïve rats were tested for hippocampal-dependent spatial memory performance by measuring spontaneous alternation in the Y-maze, followed by anatomical magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) in the hippocampus and cortex. Memory performance correlated with hippocampal concentrations of NAA (p = 0.024) and glutamate (p = 0.014) but not GABA. Concentrations of glutamate in the cortex also correlated with spatial memory (p = 0.035). In addition, memory performance was also correlated with the relative volume of the hippocampus (p = 0.041). Altogether, this exploratory study suggests that levels of the neuronal maker NAA and the main excitatory neurotransmitter glutamate are associated with physiological functional capacity.

Oral Cannabis consumption and intraperitoneal THC:CBD dosing results in changes in brain and plasma neurochemicals and endocannabinoids in mice.

BACKGROUND: While the use of orally consumed Cannabis, cannabidiol (CBD) and tetrahydrocannabinol (THC) containing products, i.e. "edibles", has expanded, the health consequences are still largely unknown. This study examines the effects of oral consumption of whole Cannabis and a complex Cannabis extract on neurochemicals, endocannabinoids (eCB), and physiological parameters (body temperature, heart rate) in mice. METHODS: In this pilot study, C57BL/6 J mice were treated with one of the following every other day for 2 weeks: a complex Cannabis extract by gavage, whole Cannabis mixed with nutritional gel through free feeding, or purified THC/CBD by intraperitoneal (i.p.) injection. Treatments were conducted at 4 doses ranging from 0-100 mg/kg/day of CBD with THC levels of ≤ 1.2 mg/kg/day for free feeding and gavage and 10 mg/kg/day for i.p. Body temperature and heart rate were monitored using surgically implanted telemetry devices. Levels of neurochemicals, eCB, THC, CBD, and 11-OH-THC were measured using mass spectrometry 48 h after the final treatment. Statistical comparisons were conducted using ANOVA and t-tests. RESULTS: Differences were found between neurochemicals in the brains and plasma of mice treated by i.p. (e.g. dopamine, p < 0.01), gavage (e.g., phenylalanine, p < 0.05) and in mice receiving whole Cannabis (e.g., 3,4-dihydroxyphenylacetic DOPAC p < 0.05). Tryptophan trended downward or was significantly decreased in the brain and/or plasma of all mice receiving Cannabis or purified CBD/THC, regardless of dose, compared to controls. Levels of the eCB, arachidonoyl glycerol (2-AG) were decreased in mice receiving lowest doses of a complex Cannabis extract by gavage, but were higher in mice receiving highest doses compared to controls (p < 0.05). Plasma and brain levels of THC and 11-OH-THC were higher in mice receiving 1:1 THC:CBD by i.p. compared to those receiving 1:5 or 1:10 THC:CBD. Nominal changes in body temperature and heart rate following acute and repeated exposures were seen to some degree in all treatments. CONCLUSIONS: Changes to neurochemicals and eCBs were apparent at all doses regardless of treatment type. Levels of neurochemicals seemed to vary based on the presence of a complex Cannabis extract, suggesting a non-linear response between THC and neurochemicals following repeated oral dosing.