Identification of Psychoplastogenic Tropanes Lacking Muscarinic Activity.

Tropane-containing small molecules like scopolamine are a promising class of psychoplastogens. However, their potent antagonism of all muscarinic receptor subtypes presents the potential for undesirable anticholinergic side effects. In an effort to decouple their neuroplasticity-promoting effects from their muscarinic activity, we performed phenotypic structure-activity relationship studies across a variety of structurally distinct subclasses of tropanes. We discovered several novel tropanes capable of significantly increasing cortical neuronal growth while exhibiting drastically reduced activity at all muscarinic receptor subtypes compared to scopolamine.

EFFECT OF CHRONIC ADMINISTRATION OF LOW DOSES OF POLYPEPTIDES OF CATTLE CEREBRAL CORTEX AND METHIONYL-GLUTAMYL-HISTIDYL-PHENYLALANYL-PROLYL-GLYCYL-PROLINE ON BEHAVIORAL RESPONSES OF RAT OFFSPRING.

In the modern world, anyone is susceptible to the effects of stress, regardless of age, gender, culture, and social status. Stress at an early age accelerates long-term changes in the functional properties underlying emotional perception and therefore may alter the stress response later in life. Unfortunately, the interdisciplinary approach in stress research emphasised the study of stress phenomenon in the development of this or that pathology or manifestation of appropriate reactions under the influence of this or that factor, i.e. the study of a particular case, which did not significantly affect the conceptual level of interpretation of the stress phenomenon as such. Moreover, we did not come across any publications interpreting the pathogenesis of the development of the classical triad of stress, confirming, or refuting its validity. In this study, we evaluated the effect of nootropic drugs - polypeptides of cattle cerebral cortex and methionyl-glutamyl-histidyl-phenylalanyl-prolyl-glycyl-proline on the behaviour of rat offspring under conditions of maternal deprivation. The drug affects processes related to memory formation and learning, enhances attention during learning and analysis of information, improves adaptation of the organism to hypoxia, cerebral ischaemia, anaesthesia and other damaging effects. As a result of the conducted study against the background of early postnatal maternal deprivation and the use of such drugs as methionyl-glutamyl-histidyl-phenylalanyl-prolyl-glycyl-proline and polypeptides of cattle cerebral it was noted that the latter drug showed the greatest effectiveness as a means of compensating the reaction to chronic stress under conditions of maternal deprivation.

High-throughput sensitive screening of small molecule modulators of microexon alternative splicing using dual Nano and Firefly luciferase reporters.

Disruption of alternative splicing frequently causes or contributes to human diseases and disorders. Consequently, there is a need for efficient and sensitive reporter assays capable of screening chemical libraries for compounds with efficacy in modulating important splicing events. Here, we describe a screening workflow employing dual Nano and Firefly luciferase alternative splicing reporters that affords efficient, sensitive, and linear detection of small molecule responses. Applying this system to a screen of ~95,000 small molecules identified compounds that stimulate or repress the splicing of neuronal microexons, a class of alternative exons often disrupted in autism and activated in neuroendocrine cancers. One of these compounds rescues the splicing of several analyzed microexons in the cerebral cortex of an autism mouse model haploinsufficient for Srrm4, a major activator of brain microexons. We thus describe a broadly applicable high-throughput screening system for identifying candidate splicing therapeutics, and a resource of small molecule modulators of microexons with potential for further development in correcting aberrant splicing patterns linked to human disorders and disease.

Investigations on the Ability of the Insular Cortex to Process Peripheral Immunosuppression.

The brain and immune system communicate through complex bidirectional pathways, but the specificity by which the brain perceives or even remembers alterations in immune homeostasis is still poorly understood. Recent data revealed that immune-related information under peripheral inflammatory conditions, termed as "immunengram", were represented in specific neuronal ensembles in the insular cortex (IC). Chemogenetic reactivation of these neuronal ensembles was sufficient to retrieve the inflammatory stages, indicating that the brain can store and retrieve specific immune responses. Against this background, the current approach was designed to investigate the ability of the IC to process states of immunosuppression pharmacologically induced by the mechanistic target of rapamycin (mTOR) inhibitor rapamycin. We here show that the IC perceives the initial state of immunosuppression, reflected by increased deep-brain electroencephalography (EEG) activity during acute immunosuppressive drug treatment. Following an experienced period of immunosuppression, though, diminished splenic cytokine production as formerly induced by rapamycin could not be reinstated by nonspecific chemogenetic activation or inhibition of the IC. These findings suggest that the information of a past, or experienced status of pharmacologically induced immunosuppression is not represented in the IC. Together, the present work extends the view of immune-to-brain communication during the states of peripheral immunosuppression and foster the prominent role of the IC for interoception.

Air pollution from biomass burning disrupts early adolescent cortical microarchitecture development.

Exposure to outdoor particulate matter (PM2.5) represents a ubiquitous threat to human health, and particularly the neurotoxic effects of PM2.5 from multiple sources may disrupt neurodevelopment. Studies addressing neurodevelopmental implications of PM exposure have been limited by small, geographically limited samples and largely focus either on macroscale cortical morphology or postmortem histological staining and total PM mass. Here, we leverage residentially assigned exposure to six, data-driven sources of PM2.5 and neuroimaging data from the longitudinal Adolescent Brain Cognitive Development Study (ABCD Study®), collected from 21 different recruitment sites across the United States. To contribute an interpretable and actionable assessment of the role of air pollution in the developing brain, we identified alterations in cortical microstructure development associated with exposure to specific sources of PM2.5 using multivariate, partial least squares analyses. Specifically, average annual exposure (i.e., at ages 8-10 years) to PM2.5 from biomass burning was related to differences in neurite development across the cortex between 9 and 13 years of age.

Inhibition of cortical synaptic transmission, behavioral nociceptive, and anxiodepressive-like responses by arecoline in adult mice.

Areca nut, the seed of Areca catechu L., is one of the most widely consumed addictive substances in the world after nicotine, ethanol, and caffeine. The major effective constituent of A. catechu, arecoline, has been reported to affect the central nervous system. Less is known if it may affect pain and its related emotional responses. In this study, we found that oral application of arecoline alleviated the inflammatory pain and its induced anxiolytic and anti-depressive-like behavior. Arecoline also increased the mechanical nociceptive threshold and alleviated depression-like behavior in naïve mice. In the anterior cingulate cortex (ACC), which acts as a hinge of nociception and its related anxiety and depression, by using the multi-electrode field potential recording and whole-cell patch-clamp recording, we found that the evoked postsynaptic transmission in the ACC of adult mice has been inhibited by the application of arecoline. The muscarinic receptor is the major receptor of the arecoline in the ACC. Our results suggest that arecoline alleviates pain, anxiety, and depression-like behavior in both physiological and pathological conditions, and this new mechanism may help to treat patients with chronic pain and its related anxiety and disorder in the future.

Calycosin promotes axon growth by inhibiting PTPRS and alleviates spinal cord injury.

Our former studies have identified the alleviating effect of Calycosin (CA) on spinal cord injury (SCI). In this study, our purpose is to explore the influence of CA on SCI from the perspective of promoting axon growth. The SCI animal model was constructed by spinal cord compression, wherein rat primary cortex neuronal isolation was performed, and the axonal growth restriction cell model was established via chondroitin sulfate proteoglycan (CSPG) treatment. The expressions of axon regeneration markers were measured via immunofluorescent staining and western blot, and the direct target of CA was examined using silver staining. Finally, the expression of the protein tyrosine phosphatase receptor type S (PTPRS) was assessed using western blot. CA treatment increased neuronal process outgrowth and the expressions of axon regeneration markers, such as neurofilament H (NF-H), vesicular glutamate transporter 1 (vGlut1), and synaptophysin (Syn) in both SCI model rats and CSPG-treated primary cortical neurons, and PTPRS levels were elevated after SCI induction. In addition, PTPRS was the direct target of CA, and according to in vivo findings, exposure to CA reduced the PTPRS content. Furthermore, PTPRS overexpression inhibited CA's enhancement of axon regeneration marker content and neuronal axon lengths. CA improves SCI by increasing axon development through regulating PTPRS expression.

Brain Chimeroids reveal individual susceptibility to neurotoxic triggers.

Interindividual genetic variation affects the susceptibility to and progression of many diseases1,2. However, efforts to study how individual human brains differ in normal development and disease phenotypes are limited by the paucity of faithful cellular human models, and the difficulty of scaling current systems to represent multiple people. Here we present human brain Chimeroids, a highly reproducible, multidonor human brain cortical organoid model generated by the co-development of cells from a panel of individual donors in a single organoid. By reaggregating cells from multiple single-donor organoids at the neural stem cell or neural progenitor cell stage, we generate Chimeroids in which each donor produces all cell lineages of the cerebral cortex, even when using pluripotent stem cell lines with notable growth biases. We used Chimeroids to investigate interindividual variation in the susceptibility to neurotoxic triggers that exhibit high clinical phenotypic variability: ethanol and the antiepileptic drug valproic acid. Individual donors varied in both the penetrance of the effect on target cell types, and the molecular phenotype within each affected cell type. Our results suggest that human genetic background may be an important mediator of neurotoxin susceptibility and introduce Chimeroids as a scalable system for high-throughput investigation of interindividual variation in processes of brain development and disease.

Chemical effects on neural network activity: Comparison of acute versus network formation exposure in microelectrode array assays.

New approach methodologies (NAMs) can address information gaps on potential neurotoxicity or developmental neurotoxicity hazard for data-poor chemicals. Two assays have been previously developed using microelectrode arrays (MEA), a technology which measures neural activity. The MEA acute network function assay (AcN) uses dissociated rat cortical cells cultured at postnatal day 0 and evaluates network activity during a 40-minute chemical exposure on day in vitro (DIV)13 or 15. In contrast, the MEA network formation assay (NFA) uses a developmental exposure paradigm spanning DIV0 through DIV12. Measures of network activity over time at DIV5, 7, 9, and 12 in the NFA are reduced to an estimated area under the curve to facilitate concentration-response evaluation. Here, we evaluated the hypothesis that chemicals with effects in the AcN also perturb the NFA by examining quantitative and qualitative concordance between assays. Out of 243 chemicals screened in both assays, we observed 70.3% concordance between the AcN and NFA after eliminating activity inferred to be cytotoxic (selective activity), with the majority of discordance explained by chemicals that altered selective activity in the AcN but not NFA. The NFA detected more active chemicals when evaluating activity associated with cytotoxicity. Median potency values were lower in the NFA compared to the AcN, but within-chemical potency values were not uniformly lower in the NFA than the AcN. Lastly, the AcN and NFA captured unique bioactivity fingerprints; the AcN was more informative for identifying chemicals with a shared mode of action, while the NFA provided information relevant to developmental exposure. Taken together, this analysis provides a rationale for using both approaches for chemical evaluation with consideration of the context of use, such as screening/ prioritization, hazard identification, or to address questions regarding biological mechanism or function.

Sodium para-aminosalicylic acid attenuates combined manganese/iron-induced cortical synaptic damage in rats.

We established experimental models of manganese (Mn) and iron (Fe) exposure in vitro and in vivo, and addressed the effects of manganese and iron combined exposure on the synaptic function of pheochromocytoma derived cell line 12 (PC12) cells and rat cortex, respectively. We investigated the protective effect of sodium para-aminosalicylate (PAS-Na) on manganese and iron combined neurotoxicity, providing a scientific basis for the prevention and treatment of ferromanganese combined neurotoxicity. Western blot and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were performed to detect the expression levels of protein and mRNA related to synaptic damage. Y-maze novelty test and balance beam test were used to evaluate the motor and cognitive function of rats. Haematoxylin and eosin (H&E) and Nissl staining were performed to observe the cortical damage of rats. The results showed that the combined exposure of Mn and Fe in rats led to a synergistic effect, attenuating growth and development, and altering learning and memory as well as motor function. The combination of Mn and Fe also caused damage to the synaptic structure of PC12 cells, which is manifested as swelling of dendrites and axon terminals, and even lead to cell death. PAS-Na displayed some antagonistic effects against the Mn- and Fe-induced synaptic structural damage, growth, learning and memory impairment.

Distinct neural activities of the cortical layer 2/3 across isoflurane anesthesia: A large-scale simultaneous observation of neurons.

Anesthesia inhibits neural activity in the brain, causing patients to lose consciousness and sensation during the surgery. Layers 2/3 of the cortex are important structures for the integration of information and consciousness, which are closely related to normal cognitive function. However, the dynamics of the large-scale population of neurons across multiple regions in layer 2/3 during anesthesia and recovery processes remains unclear. We conducted simultaneous observations and analysis of large-scale calcium signaling dynamics across multiple cortical regions within cortical layer 2/3 during isoflurane anesthesia and recovery in vivo by high-resolution wide-field microscopy. Under isoflurane-induced anesthesia, there is an overall decrease in neuronal activity across multiple regions in the cortical layer 2/3. Notably, some neurons display a paradoxical increase in activity during anesthesia. Additionally, the activity among multiple cortical regions under anesthesia was homogeneous. It is only during the recovery phase that variability emerges in the extent of increased neural activity across different cortical regions. Within the same duration of anesthesia, neural activity did not return to preanesthetic levels. To sum up, anesthesia as a dynamic alteration of brain functional networks, encompassing shifts in patterns of neural activity, homogeneousness among cortical neurons and regions, and changes in functional connectivity. Recovery from anesthesia does not entail a reversal of these effects within the same timeframe.

Elevated intrinsic cortical curvature in treatment-resistant schizophrenia: Evidence of structural deformation in functional connectivity areas and comparison with alternate indices of structure.

BACKGROUND: Research suggests structural and connectivity abnormalities in patients with treatment-resistant schizophrenia (TRS) compared to first-line responders and healthy-controls. However, measures of these abnormalities are often influenced by external factors like nicotine and antipsychotics, limiting their clinical utility. Intrinsic-cortical-curvature (ICC) presents a millimetre-scale measure of brain gyrification, highly sensitive to schizophrenia differences, and associated with TRS-like traits in early stages of the disorder. Despite this evidence, ICC in TRS remains unexplored. This study investigates ICC as a marker for treatment resistance in TRS, alongside structural indices for comparison. METHODS: We assessed ICC in anterior cingulate, dorsolateral prefrontal, temporal, and parietal cortices of 38 first-line responders, 30 clozapine-resistant TRS, 37 clozapine-responsive TRS, and 52 healthy-controls. For comparative purposes, Fold and Curvature indices were also analyzed. RESULTS: Adjusting for age, sex, nicotine-use, and chlorpromazine equivalence, principal findings indicate ICC elevations in the left hemisphere dorsolateral prefrontal (p < 0.001, η2partial = 0.142) and temporal cortices (LH p = 0.007, η2partial = 0.060; RH p = 0.011, η2partial = 0.076) of both TRS groups, and left anterior cingulate cortex of clozapine-resistant TRS (p = 0.026, η2partial = 0.065), compared to healthy-controls. Elevations that correlated with reduced cognition (p = 0.001) and negative symptomology (p < 0.034) in clozapine-resistant TRS. Fold and Curvature indices only detected group differences in the right parietal cortex, showing interactions with age, sex, and nicotine use. ICC showed interactions with age. CONCLUSION: ICC elevations were found among patients with TRS, and correlated with symptom severity. ICCs relative independence from sex, nicotine-use, and antipsychotics, may support ICC's potential as a viable marker for TRS, though age interactions should be considered.

[Effect of Huanglian Jiedu Decoction on TREM2/Akt/GSK3ß pathway in cerebral cortex of APP/PS1 transgenic mice].

The Chinese medical mechanism of Huanglian Jieduo Decoction on treating Alzheimer's disease(AD) characterized by "toxin damaging brain collateral" is still unclear. This study aims to explore the mechanism of Huanglian Jieduo Decoction on regulating triggering receptor expressed on myeloid cells 2(TREM2)/protein kinase B(Akt)/glycogen synthase kinase 3ß(GSK3ß) pathway to improve the cognitive deficit in APP/PS1 transgenic mice. APP/PS1 mice of approximately nine months old were randomly divided into the model group, the low, medium, and high(2.5, 5, and 10 g·kg~(-1)) groups of Huanglian Jiedu Decoction, and 0.75 mg·kg~(-1) donepezil hydrochloride group, and the C57BL/6J mice with the same age were taken as the normal group. After one month of continuous oral administration, a Morris water maze was performed to detect the learning and memory ability of mice. Hematoxylin-eosin(HE) staining was applied to observe the morphology of neuronal cells in the cortical area of mice. Immunofluorescence was used to detect the protein expressions of ß-amyloid(Aß_(1-42)), CD86, and arginase 1(Arg1). The mRNA levels of interleukin(IL)-1ß, IL-6, and IL-10 in the cortex of mice were detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR). The protein expressions of TREM2, phosphoinositide-3 kinase(PI3K), Akt, GSK3ß, and beta-catenin(ß-catenin) in mouse cortex were determined by Western blot. The results indicated that the escape latency of the model group was significantly prolonged, and the residence time in the target quadrant and the number of crossing the platform were significantly reduced compared with the normal group. Mice in the model group had a significantly lower number of neurons in the cortex and showed nuclear pyknosis and a significant increase in the expressions of Aß_(1-42) and CD86. The mRNA levels of IL-1ß and IL-6 in tissue were significantly increased, IL-10 were increased, while Arg1 were significantly decreased. The expression of TREM2, p-PI3K(Y607), p-Akt(T308), p-GSK3ß(Ser9), and ß-catenin in the cortex were significantly down-regulated. Compared with the model group, the escape latency of the mice in the administration group was significantly shortened, and the number of crossing the platform and the residence time in the target quadrant were significantly increased. Furthermore, the number of neurons in the cortex of mice was increased, and nuclear pyknosis was improved. Aß_(1-42) deposition was decreased significantly. The mRNA levels of IL-1ß, IL-6 and CD86 were significantly decreased, while IL-10 and Arg1 levels were significantly increased. The expression of TREM2, p-PI3K(Y607), p-Akt(T308), p-GSK3ß(Ser9), and ß-catenin protein in the cortex of each administration group was significantly up-regulated compared with the model group. In conclusion, Huanglian Jiedu Decoction reduced the expression of Aß_(1-42) and neuroinflammation to a neuro-protective effect, thereby improving the learning and memory ability in APP/PS1 mice, which may be related to the TREM2/Akt/GSK3ß signaling pathway.

Modulation of contextual fear acquisition and extinction by acute and chronic relaxin-3 receptor (RXFP3) activation in the rat retrosplenial cortex.

The retrosplenial cortex (RSC) plays a central role in processing contextual fear conditioning. In addition to corticocortical and thalamocortical projections, the RSC receives subcortical inputs, including a substantial projection from the nucleus incertus in the pontine tegmentum. This GABAergic projection contains the neuropeptide, relaxin-3 (RLN3), which inhibits target neurons via its Gi/o-protein-coupled receptor, RXFP3. To assess this peptidergic system role in contextual fear conditioning, we bilaterally injected the RSC of adult rats with an adeno-associated-virus (AAV), expressing the chimeric RXFP3 agonist R3/I5 or a control AAV, and subjected them to contextual fear conditioning. The R3/I5 injected rats did not display any major differences to control-injected and naïve rats but displayed a significantly delayed extinction. Subsequently, we employed acute bilateral injections of the specific RXFP3 agonist peptide, RXFP3-Analogue 2 (A2), into RSC. While the administration of A2 before each extinction trial had no impact on the extinction process, treatment with A2 before each acquisition trial resulted in delayed extinction. In related anatomical studies, we detected an enrichment of RLN3-immunoreactive nerve fibers in deep layers of the RSC, and a higher level of co-localization of RXFP3 mRNA with vesicular GABA transporter (vGAT) mRNA than with vesicular glutamate transporter-1 (vGLUT1) mRNA across the RSC, consistent with an effect of RLN3/RXFP3 signalling on the intrinsic, inhibitory circuits within the RSC. These findings suggest that contextual conditioning processes in the RSC involve, in part, RLN3 afferent modulation of local inhibitory neurons that provides a stronger memory acquisition which, in turn, retards the extinction process.

High sugar consumption for seven days in adult mice increased blood glucose variability, induced an anxiolytic effect and triggered oxidative stress in cerebral cortex.

Brain function is highly altered by glucose toxicity related to diabetes. High consumption of sugar in normal conditions is suspected to affect as well brain integrity. The present study investigates the possible effects of short-term exposure to high sugar diet on brain redox homeostasis in healthy mice. Male adult healthy mice were divided into two groups: control (CG) and sugar-exposed group (SG), that was exposed continually to 10% of glucose in drinking water for 7 days and 20% sucrose pellets food. Behavior, blood glucose variability and cerebral cortex oxidative stress biomarkers were measured at the end of exposure. Animals exposed to the high sugar diet expressed a significant increase in blood glucose levels and high glucose variability compared to control. These animals expressed as well anxiolytic behavior as revealed by the plus maze test. Exposure to the sugar diet altered redox homeostasis in the brain cortex as revealed by an increase in lipid peroxidation and the activity of antioxidant enzymes superoxide dismutase (SOD) and glutathione-s-transferase (GST). On the other hand, catalase (CAT) activity was decreased, and reduced glutathione (GSH) level was not altered compared to control. Further studies are required to understand the mechanisms trigging oxidative stress (OS) in the brain in response to short term exposure to high sugar diet and glucose fluctuations.

In vivo whole-cortex marker of excitation-inhibition ratio indexes cortical maturation and cognitive ability in youth.

A balanced excitation-inhibition ratio (E/I ratio) is critical for healthy brain function. Normative development of cortex-wide E/I ratio remains unknown. Here, we noninvasively estimate a putative marker of whole-cortex E/I ratio by fitting a large-scale biophysically plausible circuit model to resting-state functional MRI (fMRI) data. We first confirm that our model generates realistic brain dynamics in the Human Connectome Project. Next, we show that the estimated E/I ratio marker is sensitive to the gamma-aminobutyric acid (GABA) agonist benzodiazepine alprazolam during fMRI. Alprazolam-induced E/I changes are spatially consistent with positron emission tomography measurement of benzodiazepine receptor density. We then investigate the relationship between the E/I ratio marker and neurodevelopment. We find that the E/I ratio marker declines heterogeneously across the cerebral cortex during youth, with the greatest reduction occurring in sensorimotor systems relative to association systems. Importantly, among children with the same chronological age, a lower E/I ratio marker (especially in the association cortex) is linked to better cognitive performance. This result is replicated across North American (8.2 to 23.0 y old) and Asian (7.2 to 7.9 y old) cohorts, suggesting that a more mature E/I ratio indexes improved cognition during normative development. Overall, our findings open the door to studying how disrupted E/I trajectories may lead to cognitive dysfunction in psychopathology that emerges during youth.

Keep calm and make neurons: The effects of glucocorticoids on human cortical neurogenesis.

Maternal well-being is important for the development of the fetus, with a key influence on its nervous system. In this issue of Neuron, Krontira et al.1 implicate glucocorticoids, the stress hormones, in the regulation of neural stem cell identity and proliferation, with long-lasting consequences on brain architecture and educational attainment.

Sex differences in neuronal activation in the cortex and midbrain during quinine-adulterated alcohol intake.

AIMS: Continued alcohol consumption despite negative consequences is a core symptom of alcohol use disorder. This is modeled in mice by pairing negative stimuli with alcohol, such as adulterating alcohol solution with quinine. Mice consuming alcohol under these conditions are considered to be engaging in aversion-resistant intake. Previously, we have observed sex differences in this behavior, with females more readily expressing aversion-resistant consumption. We also identified three brain regions that exhibited sex differences in neuronal activation during quinine-alcohol drinking: ventromedial prefrontal cortex (vmPFC), posterior insular cortex (PIC), and ventral tegmental area (VTA). Specifically, male mice showed increased activation in vmPFC and PIC, while females exhibited increased activation in VTA. In this study, we aimed to identify what specific type of neurons are activated in these regions during quinine-alcohol drinking. METHOD: We assessed quinine-adulterated alcohol intake using the two-bottle choice procedure. We also utilized RNAscope in situ hybridization in the three brain regions that previously exhibited a sex difference to examine colocalization of Fos, glutamate, GABA, and dopamine. RESULT: Females showed increased aversion-resistant alcohol consumption compared to males. We also found that males had higher colocalization of glutamate and Fos in vmPFC and PIC, while females had greater dopamine and Fos colocalization in the VTA. CONCLUSIONS: Collectively, these experiments suggest that glutamatergic output from the vmPFC and PIC may have a role in suppressing, and dopaminergic activity in the VTA may promote, aversion-resistant alcohol consumption. Future experiments will examine neuronal circuits that contribute to sex differences in aversion resistant consumption.

Interplay between Energy Supply and Glutamate Toxicity in the Primary Cortical Culture.

Limited substrate availability because of the blood-brain barrier (BBB) has made the brain develop specific molecular mechanisms to survive, using lactate synthesized by astrocytes as a source of energy in neurons. To understand if lactate improves cellular viability and susceptibility to glutamate toxicity, primary cortical cells were incubated in glucose- or lactate-containing media and toxic concentrations of glutamate for 24 h. Cell death was determined by immunostaining and lactate dehydrogenase (LDH) release. Mitochondrial membrane potential and nitric oxide (NO) levels were measured using Tetramethylrhodamine, methyl ester (TMRM) and 4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate (DAF-FM) live staining, respectively. LDH activity was quantified in single cells in the presence of lactate (LDH substrate) and oxamate (LDH inhibitor). Nuclei of cells were stained with DAPI and neurons with MAP2. Based on the distance between neurons and glial cells, they were classified as linked (<10 µm) and non-linked (>10 µm) neurons. Lactate increased cell death rate and the mean value of endogenous NO levels compared to glucose incubations. Mitochondrial membrane potential was lower in the cells cultured with lactate, but this effect was reversed when glutamate was added to the lactate medium. LDH activity was higher in linked neurons compared to non-linked neurons, supporting the hypothesis of the existence of the lactate shuttle between astrocytes and at least a portion of neurons. In conclusion, glucose or lactate can equally preserve primary cortical neurons, but those neurons having a low level of LDH activity and incubated with lactate cannot cover high energetic demand solely with lactate and become more susceptible to glutamate toxicity.

Alleviation of migraine related pain and anxiety by inhibiting calcium-stimulating AC1-dependent CGRP in the insula of adult rats.

BACKGROUND: Recent animal and clinical findings consistently highlight the critical role of calcitonin gene-related peptide (CGRP) in chronic migraine (CM) and related emotional responses. CGRP antibodies and receptor antagonists have been approved for CM treatment. However, the underlying CGRP-related signaling pathways in the pain-related cortex remain poorly understood. METHODS: The SD rats were used to establish the CM model by dural infusions of inflammatory soup. Periorbital mechanical thresholds were assessed using von-Frey filaments, and anxiety-like behaviors were observed via open field and elevated plus maze tests. Expression of c-Fos, CGRP and NMDA GluN2B receptors was detected using immunofluorescence and western blotting analyses. The excitatory synaptic transmission was detected by whole-cell patch-clamp recording. A human-used adenylate cyclase 1 (AC1) inhibitor, hNB001, was applied via insula stereotaxic and intraperitoneal injections in CM rats. RESULTS: The insular cortex (IC) was activated in the migraine model rats. Glutamate-mediated excitatory transmission and NMDA GluN2B receptors in the IC were potentiated. CGRP levels in the IC significantly increased during nociceptive and anxiety-like activities. Locally applied hNB001 in the IC or intraperitoneally alleviated periorbital mechanical thresholds and anxiety behaviors in migraine rats. Furthermore, CGRP expression in the IC decreased after the hNB001 application. CONCLUSIONS: Our study indicated that AC1-dependent IC plasticity contributes to migraine and AC1 may be a promising target for treating migraine in the future.