Regulation of lipid synthesis in myelin modulates neural activity and is required for motor learning.

Brain function relies on both rapid electrical communication in neural circuitry and appropriate patterns or synchrony of neural activity. Rapid communication between neurons is facilitated by wrapping nerve axons with insulation by a myelin sheath composed largely of different lipids. Recent evidence has indicated that the extent of myelination of nerve axons can adapt based on neural activity levels and this adaptive myelination is associated with improved learning of motor tasks, suggesting such plasticity may enhance effective learning. In this study, we examined whether another aspect of myelin plasticity-changes in myelin lipid synthesis and composition-may also be associated with motor learning. We combined a motor learning task in mice with in vivo two-photon imaging of neural activity in the primary motor cortex (M1) to distinguish early and late stages of learning and then probed levels of some key myelin lipids using mass spectrometry analysis. Sphingomyelin levels were elevated in the early stage of motor learning while galactosylceramide levels were elevated in the middle and late stages of motor learning, and these changes were correlated across individual mice with both learning performance and neural activity changes. Targeted inhibition of oligodendrocyte-specific galactosyltransferase expression, the enzyme that synthesizes myelin galactosylceramide, impaired motor learning. Our results suggest regulation of myelin lipid composition could be a novel facet of myelin adaptations associated with learning.

Lipidomics-based tissue heterogeneity in specimens of luminal breast cancer revealed by clustering analysis of mass spectrometry imaging: A preliminary study.

Cancer tissues reflect a greater number of pathological characteristics of cancer compared to cancer cells, so the evaluation of cancer tissues can be effective in determining cancer treatment strategies. Mass spectrometry imaging (MSI) can evaluate cancer tissues and even identify molecules while preserving spatial information. Cluster analysis of cancer tissues' MSI data is currently used to evaluate the phenotype heterogeneity of the tissues. Interestingly, it has been reported that phenotype heterogeneity does not always coincide with genotype heterogeneity in HER2-positive breast cancer. We thus investigated the phenotype heterogeneity of luminal breast cancer, which is generally known to have few gene mutations. As a result, we identified phenotype heterogeneity based on lipidomics in luminal breast cancer tissues. Clusters were composed of phosphatidylcholine (PC), triglycerides (TG), phosphatidylethanolamine, sphingomyelin, and ceramide. It was found that mainly the proportion of PC and TG correlated with the proportion of cancer and stroma on HE images. Furthermore, the number of carbons in these lipid class varied from cluster to cluster. This was consistent with the fact that enzymes that synthesize long-chain fatty acids are increased through cancer metabolism. It was then thought that clusters containing PCs with high carbon counts might reflect high malignancy. These results indicate that lipidomics-based phenotype heterogeneity could potentially be used to classify cancer for which genetic analysis alone is insufficient for classification.

Tubulin Polyglutamylation by TTLL1 and TTLL7 Regulate Glutamate Concentration in the Mice Brain.

As an important neurotransmitter, glutamate acts in over 90% of excitatory synapses in the human brain. Its metabolic pathway is complicated, and the glutamate pool in neurons has not been fully elucidated. Tubulin polyglutamylation in the brain is mainly mediated by two tubulin tyrosine ligase-like (TTLL) proteins, TTLL1 and TTLL7, which have been indicated to be important for neuronal polarity. In this study, we constructed pure lines of Ttll1 and Ttll7 knockout mice. Ttll knockout mice showed several abnormal behaviors. Matrix-assisted laser desorption/ionization (MALDI) Imaging mass spectrometry (IMS) analyses of these brains showed increases in glutamate, suggesting that tubulin polyglutamylation by these TTLLs acts as a pool of glutamate in neurons and modulates some other amino acids related to glutamate.

Spatial distribution of the Shannon entropy for mass spectrometry imaging.

Mass spectrometry imaging (MSI) allows us to visualize the spatial distribution of molecular components in a sample. A large amount of mass spectrometry data comprehensively provides molecular distributions. In this study, we focus on the information in the obtained data and use the Shannon entropy as a quantity to analyze MSI data. By calculating the Shannon entropy at each pixel on a sample, the spatial distribution of the Shannon entropy is obtained from MSI data. We found that low-entropy pixels in entropy heat maps for kidneys of mice had different structures between two ages (3 months and 31 months). Such changes cannot be visualized by conventional imaging techniques. We further propose a method to find informative molecules. As a demonstration of the proposed scheme, we identified two molecules by setting a region of interest which contained low-entropy pixels and by exploring changes of peaks in the region.

Mechanical transmission at spine synapses: Short-term potentiation and working memory.

Do dendritic spines, which comprise the postsynaptic component of most excitatory synapses, exist only for their structural dynamics, receptor trafficking, and chemical and electrical compartmentation? The answer is no. Simultaneous investigation of both spine and presynaptic terminals has recently revealed a novel feature of spine synapses. Spine enlargement pushes the presynaptic terminals with muscle-like force and augments the evoked glutamate release for up to 20 min. We now summarize the evidence that such mechanical transmission shares critical features in common with short-term potentiation (STP) and may represent the cellular basis of short-term and working memory. Thus, spine synapses produce the force of learning to leave structural traces for both short and long-term memories.

Biosynthetic Gene Expression and Tissue Distribution of Diosgenin in Dioscorea japonica.

Diosgenin is an aglycone of dioscin, a major bioactive steroidal saponin found in plants, including Himalayan Paris (Paris polyphylla), fenugreek (Trigonella foenum-graecum), and yam (Dioscorea spp.). We have previously demonstrated that a species of natural yam, Dioscorea japonica, contains a promising bioactive compound diosgenin, which induces anti-carcinogenic and anti-hypertriacylglycerolemic activities. Here, we found for the first time that Japanese yam (D. japonica) bulbils are richer in diosgenin than the edible tubers (rhizomes) and leaves. LC-MS and imaging-MS analyses revealed that diosgenin accumulated in the peripheral region of D. japonica bulbils. Additionally, we performed RNA-seq analysis of D. japonica, and multiple sequence alignment identified D. japonica CYP90 (DjCYP90), the orthologous gene of CYP90G4 in P. polyphylla, CYP90B50 in T. foenum-graecum, CYP90G6 in Dioscorea zingiberensis, and CYP90G in Dioscorea villosa, which encodes a diosgenin biosynthetic rate-limiting enzyme. The expression levels of DjCYP90 were significantly upregulated in D. japonica bulbils than in its rhizomes and leaves. Since diosgenin is one of the most promising functional food factors executing several favorable bioactivities, D. japonica bulbils rich in diosgenin would be a beneficial natural resource.

Proteomic profile of nuclei containing p62-positive inclusions in a patient with neuronal intranuclear inclusion disease.

Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disease characterized by eosinophilic hyaline intranuclear inclusions in the neurons, glial cells, and other somatic cells. Although CGG repeat expansions in NOTCH2NLC have been identified in most East Asian patients with NIID, the pathophysiology of NIID remains unclear. Ubiquitin- and p62-positive intranuclear inclusions are the pathological hallmark of NIID. Targeted immunostaining studies have identified several other proteins present in these inclusions. However, the global molecular changes within nuclei with these inclusions remained unclear. Herein, we analyzed the proteomic profile of nuclei with p62-positive inclusions in a NIID patient with CGG repeat expansion in NOTCH2NLC to discover candidate proteins involved in the NIID pathophysiology. We used fluorescence-activated cell sorting and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify each protein identified in the nuclei with p62-positive inclusions. The distribution of increased proteins was confirmed via immunofluorescence in autopsy brain samples from three patients with genetically confirmed NIID. Overall, 526 proteins were identified, of which 243 were consistently quantified using MS. A 1.4-fold increase was consistently observed for 20 proteins in nuclei with p62-positive inclusions compared to those without. Fifteen proteins identified with medium or high confidence in the LC-MS/MS analysis were further evaluated. Gene ontology enrichment analysis showed enrichment of several terms, including poly(A) RNA binding, nucleosomal DNA binding, and protein binding. Immunofluorescence studies confirmed that the fluorescent intensities of increased RNA-binding proteins identified by proteomic analysis, namely hnRNP A2/B1, hnRNP A3, and hnRNP C1/C2, were higher in the nuclei with p62-positive inclusions than in those without, which were not confined to the intranuclear inclusions. We identified several increased proteins in nuclei with p62-positive inclusions. Although larger studies are needed to validate our results, these proteomic data may form the basis for understanding the pathophysiology of NIID.

Changes of Mass Spectra Patterns on a Brain Tissue Section Revealed by Deep Learning with Imaging Mass Spectrometry Data.

The characteristic patterns of mass spectra in imaging mass spectrometry (IMS) strongly reflect the tissue environment. However, the boundaries formed where different tissue environments collide have not been visually assessed. In this study, IMS and convolutional neural network (CNN), one of the deep learning methods, were applied to the extraction of characteristic mass spectra patterns from training brain regions on rodents' brain sections. CNN produced classification models with high accuracy and low loss rate in any test data sets of mouse coronal sections measured by desorption electrospray ionization (DESI)-IMS and of mouse and rat sagittal sections by matrix-assisted laser desorption (MALDI)-IMS. On the basis of the extracted mass spectra pattern features, the histologically plausible segmentation and classification score imaging of the brain sections were obtained. The boundary imaging generated from classification scores showed the extreme changes of mass spectra patterns between the tissue environments, with no significant buffer zones for the intermediate state. The CNN-based analysis of IMS data is a useful tool for visually assessing the changes of mass spectra patterns on a tissue section, and it will contribute to a comprehensive view of the tissue environment.

Persistent elevation of lysophosphatidylcholine promotes radiation brain necrosis with microglial recruitment by P2RX4 activation.

Brain radiation necrosis (RN) or neurocognitive disorder is a severe adverse effect that may occur after radiation therapy for malignant brain tumors or head and neck cancers. RN accompanies inflammation which causes edema or micro-bleeding, and no fundamental treatment has been developed. In inflammation, lysophospholipids (LPLs) are produced by phospholipase A2 and function as bioactive lipids involved in sterile inflammation in atherosclerosis or brain disorders. To elucidate its underlying mechanisms, we investigated the possible associations between lysophospholipids (LPLs) and RN development in terms of microglial activation with the purinergic receptor P2X purinoceptor 4 (P2RX4). We previously developed a mouse model of RN and in this study, measured phospholipids and LPLs in the brains of RN model by liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses. We immune-stained microglia and the P2RX4 in the brains of RN model with time-course. We treated RN model mice with ivermectin, an allosteric modulator of P2RX4 and investigate the effect on microglial activation with P2RX4 and LPLs' production, and resulting effects on overall survival and working memory. We revealed that LPLs (lysophosphatidylcholine (LPC), lysophosphatidyl acid, lysophosphatidylserine, lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylglycerol) remained at high levels during the progression of RN with microglial accumulation, though phospholipids elevations were limited. Both microglial accumulation and activation of the P2RX4 were attenuated by ivermectin. Moreover, the elevation of all LPLs except LPC was also attenuated by ivermectin. However, there was limited prolongation of survival time and improvement of working memory disorders. Our findings suggest that uncontrollable increased LPC, even with ivermectin treatment, promoted the development of RN and working memory disorders. Therefore, LPC suppression will be essential for controlling RN and neurocognitive disorder after radiation therapy.

Decreased sphingomyelin (t34:1) is a candidate predictor for lung squamous cell carcinoma recurrence after radical surgery: a case-control study.

BACKGROUND: To reduce disease recurrence after radical surgery for lung squamous cell carcinomas (SQCCs), accurate prediction of recurrent high-risk patients is required for efficient patient selection for adjuvant chemotherapy. Because treatment modalities for recurrent lung SQCCs are scarce compared to lung adenocarcinomas (ADCs), accurately selecting lung SQCC patients for adjuvant chemotherapy after radical surgery is highly important. Predicting lung cancer recurrence with high objectivity is difficult with conventional histopathological prognostic factors; therefore, identification of a novel predictor is expected to be highly beneficial. Lipid metabolism alterations in cancers are known to contribute to cancer progression. Previously, we found that increased sphingomyelin (SM)(d35:1) in lung ADCs is a candidate for an objective recurrence predictor. However, no lipid predictors for lung SQCC recurrence have been identified to date. This study aims to identify candidate lipid predictors for lung SQCC recurrence after radical surgery. METHODS: Recurrent (n = 5) and non-recurrent (n = 6) cases of lung SQCC patients who underwent radical surgery were assigned to recurrent and non-recurrent groups, respectively. Extracted lipids from frozen tissue samples of primary lung SQCC were analyzed by liquid chromatography-tandem mass spectrometry. Candidate lipid predictors were screened by comparing the relative expression levels between the recurrent and non-recurrent groups. To compare lipidomic characteristics associated with recurrent SQCCs and ADCs, a meta-analysis combining SQCC (n = 11) and ADC (n = 20) cohorts was conducted. RESULTS: Among 1745 screened lipid species, five species were decreased (≤ 0.5 fold change; P < 0.05) and one was increased (≥ 2 fold change; P < 0.05) in the recurrent group. Among the six candidates, the top three final candidates (selected by AUC assessment) were all decreased SM(t34:1) species, showing strong performance in recurrence prediction that is equivalent to that of histopathological prognostic factors. Meta-analysis indicated that decreases in a limited number of SM species were observed in the SQCC cohort as a lipidomic characteristic associated with recurrence, in contrast, significant increases in a broad range of lipids (including SM species) were observed in the ADC cohort. CONCLUSION: We identified decreased SM(t34:1) as a novel candidate predictor for lung SQCC recurrence. Lung SQCCs and ADCs have opposite lipidomic characteristics concerning for recurrence risk. TRIAL REGISTRATION: This retrospective study was registered at the UMIN Clinical Trial Registry ( UMIN000039202 ) on January 21, 2020.

Desorption ionization using through-hole alumina membrane offers higher reproducibility than 2,5-dihydroxybenzoic acid, a widely used matrix in Fourier transform ion cyclotron resonance mass spectrometry imaging analysis.

RATIONALE: DIUTHAME (desorption ionization using through-hole alumina membrane), a recently developed matrix-free ionization-assisting substrate, was examined for reproducibility in terms of mass accuracy and intensity using standard lipid and mouse brain sections. The impregnation property of DIUTHAME significantly improved the reproducibility of mass accuracy and intensity compared with 2,5-dihydroxybenzoic acid (DHB). METHODS: Frozen tissue sections were mounted on indium tin oxide-coated glass slides. DIUTHAME and DHB were applied to individual sections. Subsequently, a solution of a phosphatidylcholine standard, PC(18:2/18:2), was poured onto the DIUTHAME and matrix. Finally, the samples were subjected to laser desorption ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry. The reproducibility was tested by calculating the mean ± standard deviation values of mass errors and intensities of individual ion species. RESULTS: Analysis of the PC(18:2/18:2) standard showed significantly (p < 0.01) lower mass error for DIUTHAME-MS than for MALDI-MS. Endogenous PC(36:4) analysis in mouse brain section also showed significantly (p < 0.05) lower mass errors for DIUTHAME-MS. Furthermore, we investigated the mass error of some abundant lipid ions in brain sections and observed similar results. DIUTHAME-MS displayed lower signal intensity in standard PC analysis. Interestingly, it offered higher signal intensities for all the endogenous lipid ions. Lower fluctuations of both mass accuracies and signal intensities were observed in DIUTHAME-MS. CONCLUSIONS: Our results demonstrated that DIUTHAME-MS offers higher reproducibility for mass accuracies and intensities than MALDI-MS in both standard lipid and mouse brain tissue analyses. It can potentially be used instead of conventional MALDI-MS and mass spectrometry imaging analyses to achieve highly reproducible data for mass accuracy and intensity.

Mass spectrometry imaging reveals glycine distribution in the developing and adult mouse brain.

Glycine is an important amino acid in the central nervous system. The aberrant conditions of glycine concentrations cause sever neurological disorders, such as nonketotic-hyperglycinemia (NKH), also known as glycine encephalopathy. Therefore, a better understanding of its relative abundance and distribution in the developing and adult brains would provide insights into the pathogeneses of this kind of disorders. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) imaging has been used for direct molecular-specific compound detection, distribution mapping, and identifying molecular species in tissue sections. Although a few reports have already shown the imaging of glycine using MALDI-MS in the adult mouse brain, they lack detailed neuroanatomical and developmental evaluations. We, thus, investigated the detailed distribution and abundance of glycine not only in the adult mouse brain but also in the developing mouse brain using this technique. In both brains, we detected derivatized glycine throughout the mouse brain. Interestingly, in both brains, derivatized glycine was abundantly detected in the brain stem. The other areas showed relatively lower signal intensities. As many model mice are used for glycine-related diseases, MALDI-MS is a suitable technique to analyze the pathogenesis of these diseases.

Region-specific effects of Scrapper on the abundance of glutamate and gamma-aminobutyric acid in the mouse brain.

The brain consists of various areas with anatomical features. Neurons communicate with one another via excitatory or inhibitory synaptic transmission. Altered abundance of neurotransmitters, including glutamate and gamma-aminobutyric acid (GABA), in specific brain regions is closely involved in severe neurological diseases, such as schizophrenia and obsessive-compulsive disorder. SCRAPPER, a ubiquitin E3 ligase, regulates synaptic transmission. Scrapper gene deficiency results in defective neurotransmission due to excessive secretion of neurotransmitters. The present study employed matrix-assisted laser desorption/ionization imaging mass spectrometry to analyze the abundance of amino acid neurotransmitters in Scrapper knockout (SCR-KO) mice. SCR-KO mice exhibited significantly increased glutamate levels in the isocortex (CTX), corpus callosum (CC), thalamus (TH), midbrain (MB), cerebellar cortex (CBX), and caudoputamen (CP) and increased GABA levels in the CTX, CC, TH, MB, CBX and hypothalamus (HY) compared with wild-type mice. These findings indicate that Scrapper deficiency leads to upregulated glutamate and GABA levels in multiple regions. Our results show a differential, region-specific effect of Scrapper on the abundance of glutamate and GABA.

Mass spectrometry-based phospholipid imaging: methods and findings.

Introduction: Imaging is a technique used for direct visualization of the internal structure or distribution of biomolecules of a living system in a two-dimensional or three-dimensional fashion. Phospholipids are important structural components of biological membranes and have been reported to be associated with various human diseases. Therefore, the visualization of phospholipids is crucial to understand the underlying mechanism of cellular and molecular processes in normal and diseased conditions. Areas covered: Mass spectrometry imaging (MSI) has enabled the label-free imaging of individual phospholipids in biological tissues and cells. The commonly used MSI techniques include matrix-assisted laser desorption ionization-MSI (MALDI-MSI), desorption electrospray ionization-MSI (DESI-MSI), and secondary ion mass spectrometry (SIMS) imaging. This special report described those methods, summarized the findings, and discussed the future development for the imaging of phospholipids. Expert opinion: Phospholipids imaging in complex biological samples has been significantly benefited from the development of MSI methods. In MALDI-MSI, novel matrix that produces homogenous crystals exclusively with polar lipids is important for phospholipids imaging with greater efficiency and higher spatial resolution. DESI-MSI has the potential of live imaging of the biological surface while SIMS is expected to image at the subcellular level in the near future.

Muscarinic M1 receptors stimulated by intracerebroventricular administration of McN-A-343 reduces the nerve injury-induced mechanical hypersensitivity via GABAB receptors rather than GABAA receptors in mice.

Cholinergic neurons play an important role in the higher functions of the brain, such as the memory, cognition, and nociception. However, the exact mechanism behind how the stimulation of all the muscarinic M1 receptors in the entire brain results in the alleviation of partial sciatic nerve ligation (PSNL)-induced mechanical hypersensitivity has not been investigated. Thus, we examined which subtype of GABA receptor was involved in the alleviation of PSNL-induce mechanical hypersensitivity produced by an intracerebroventricular administration of a muscarinic M1 receptor agonist, McN-A-343. Administering a GABAA receptor antagonist, bicuculline, resulted in no changes to the McN-A-343-induced anti-hypersensitivity in PSNL mice whereas a GABAB receptor antagonist, CGP35348, dose-dependently inhibited the anti-hypersensitivity. Furthermore, CGP35348 increased mechanical hypersensitivity in naïve mice, and the hypersensitivity was blocked by NMDA receptor antagonists, MK-801 and D-AP5. Additionally, muscarinic M1 receptors colocalized with GABAB1 receptors and an NMDA receptor subunit, GluN2A, in a large region of the brain. Consequently, these results suggest that the activation of muscarinic M1 receptors in the entire brain reduces nerve injury-induced mechanical hypersensitivity via the GABAB receptors, and the activation of the GABAB receptors regulates glutamatergic transmission via NMDA receptors.

Development of sheet-enhanced technique (Set) method for matrix-assisted laser desorption/ionization imaging mass spectrometry.

RATIONALE: The key to successful experiments in matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is to apply the matrix uniformly to the sample. With the development of automated equipment, uniform matrix application has made great progress while the sample preparation required to acquire a better image becomes complicated. METHODS: The approach is to apply the matrix uniformly to tape and adhere it to the tissue section. We call this the sheet-enhanced technique (Set) method. RESULTS: The Set method promotes ionization of biomolecules as well as the spray method. This procedure does not require the preparation and application of a matrix solution for each experiment, dramatically reducing the time and effort of matrix deposition. CONCLUSIONS: In the present study, we have developed the Set method as a new matrix application method. The method promotes ionization of biomolecules as well as the spray method for MALDI-IMS.

Dietary Intake of Green Nut Oil or DHA Ameliorates DHA Distribution in the Brain of a Mouse Model of Dementia Accompanied by Memory Recovery.

Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, has significant healthbenefits. Previous studies reported decreased levels of DHA and DHA-containing phosphatidylcholines inthe brain of animals suffering from Alzheimer's disease, the most common type of dementia; furthermore,DHA supplementation has been found to improve brain DHA levels and memory efficiency in dementia. Oilextracted from the seeds of Plukenetia volubilis (green nut oil; GNO) is also expected to have DHA like effectsas it contains approximately 50% α-linolenic acid, a precursor of DHA. Despite this, changes in the spatialdistribution of DHA in the brain of animals with dementia following GNO or DHA supplementation remainunexplored. In this study, desorption electrospray ionization imaging mass spectrometry (DESI-IMS) wasapplied to observe the effects of GNO or DHA supplementation upon the distribution of DHA in the brain ofmale senescence-accelerated mouse-prone 8 (SAMP8) mice, a mouse model of dementia. DESI-IMS revealedthat brain DHA distribution increased 1.85-fold and 3.67-fold in GNO-fed and DHA-fed SAMP8 mice,respectively, compared to corn oil-fed SAMP8 mice. Memory efficiency in SAMP8 mice was also improvedby GNO or DHA supplementation. In summary, this study suggests the possibility of GNO or DHAsupplementation for the prevention of dementia.

Stimulating muscarinic M1 receptors in the anterior cingulate cortex reduces mechanical hypersensitivity via GABAergic transmission in nerve injury rats.

Cholinergic systems modulate synaptic transmission across the neuraxis and play an important role in higher brain function including cognition, arousal and nociception. The anterior cingulate cortex (ACC) is a fundamental brain region for nociception and chronic pain, and receives cholinergic projections mainly from basal forebrain. Recently, we found that the activation of muscarinic M1 receptors in the ACC produced antinociceptive behavior in response to mechanical stimulation. However, it has not been tested whether stimulating muscarinic receptors in the ACC can reduce mechanical hypersensitivity in animal models of chronic pain. Here, we tested whether the activation of muscarinic M1 receptors in the ACC can alleviate mechanical hypersensitivity in a nerve injury model. The activation of muscarinic M1/M4 receptors by McN-A-343 injected into the contralateral side of the ACC, but not into the ventral posterolateral nucleus, was found to dose-dependently reduce mechanical hypersensitivity 7 days following partial sciatic nerve ligation in rats. The reduction of mechanical hypersensitivity by McN-A-343, was blocked by a selective muscarinic M1 antagonist, but not a M4 receptor antagonist. Importantly, the nerve injury model did not change the protein expression of muscarinic M1 receptors in the ACC. Additionally, a type A γ-aminobutyric acid (GABAA) receptor agonist injected into the ACC reduced the mechanical hypersensitivity in this injury model. Finally, a GABAA receptor antagonist blocked the reduction of mechanical hypersensitivity by McN-A-343 in the injury model. Collectively, these results suggest that activations of muscarinic M1 receptors in the ACC reduce nerve injury-induced mechanical hypersensitivity through GABAergic transmission via GABAA receptors.

Lysophosphatidic acid precursor levels decrease and an arachidonic acid-containing phosphatidylcholine level increases in the dorsal root ganglion of mice after peripheral nerve injury.

In the current study, we aimed to analyze the lipid changes in the dorsal root ganglion (DRG) after sciatic nerve transection (SNT) using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). We found that the arachidonic acid-containing phosphatidylcholine (AA-PC), PC(16:0/20:4) largely increased, while PC(16:0/18:1), PC(18:0/18:1) and phosphatidic acid (PA)(36:2) levels largely decreased in the DRG following nerve injury. Previous studies show that the increase in PC(16:0/20:4) was associated with neuropathic pain and that decrease in PC(16:0/18:1), PC(18:0/18:1), and PA(36:2) were due to producing lysophosphatidic acid (LPA), an initiator for neuropathic pain. These results suggest that the lipid changes in DRG after SNT could be the result of changes for the cause of neuropathic pain. Thus, blocking of LPA could be potential for treatment of neuropathic pain.

Activations of muscarinic M1 receptors in the anterior cingulate cortex contribute to the antinociceptive effect via GABAergic transmission.

Background Cholinergic systems regulate the synaptic transmission resulting in the contribution of the nociceptive behaviors. Anterior cingulate cortex is a key cortical area to play roles in nociception and chronic pain. However, the effect of the activation of cholinergic system for nociception is still unknown in the cortical area. Here, we tested whether the activation of cholinergic receptors can regulate nociceptive behaviors in adult rat anterior cingulate cortex by integrative methods including behavior, immunohistochemical, and electrophysiological methods. Results We found that muscarinic M1 receptors were clearly expressed in the anterior cingulate cortex. Using behavioral tests, we identified that microinjection of a selective muscarinic M1 receptors agonist McN-A-343 into the anterior cingulate cortex dose dependently increased the mechanical threshold. In contrast, the local injection of McN-A-343 into the anterior cingulate cortex showed normal motor function. The microinjection of a selective M1 receptors antagonist pirenzepine blocked the McN-A-343-induced antinociceptive effect. Pirenzepine alone into the anterior cingulate cortex decreased the mechanical thresholds. The local injection of the GABAA receptors antagonist bicuculline into the anterior cingulate cortex also inhibited the McN-A-343-induced antinociceptive effect and decreased the mechanical threshold. Finally, we further tested whether the activation of M1 receptors could regulate GABAergic transmission using whole-cell patch-clamp recordings. The activation of M1 receptors enhanced the frequency of spontaneous and miniature inhibitory postsynaptic currents as well as the amplitude of spontaneous inhibitory postsynaptic currents in the anterior cingulate cortex. Conclusions These results suggest that the activation of muscarinic M1 receptors in part increased the mechanical threshold by increasing GABAergic transmitter release and facilitating GABAergic transmission in the anterior cingulate cortex.