A Pathologically Friendly Strategy for Determining the Organ-specific Spatial Tumor Microenvironment Topology in Lung Adenocarcinoma Through the Integration of snRandom-seq and Imaging Mass Cytometry.

Heterogeneous organ-specific responses to immunotherapy exist in lung cancer. Dissecting tumor microenvironment (TME) can provide new insights into the mechanisms of divergent responses, the process of which remains poor, partly due to the challenges associated with single-cell profiling using formalin-fixed paraffin-embedded (FFPE) materials. In this study, single-cell nuclei RNA sequencing and imaging mass cytometry (IMC) are used to dissect organ-specific cellular and spatial TME based on FFPE samples from paired primary lung adenocarcinoma (LUAD) and metastases. Single-cell analyses of 84 294 cells from sequencing and 250 600 cells from IMC reveal divergent organ-specific immune niches. For sites of LUAD responding well to immunotherapy, including primary LUAD and adrenal gland metastases, a significant enrichment of B, plasma, and T cells is detected. Spatially resolved maps reveal cellular neighborhoods recapitulating functional units of the tumor ecosystem and the spatial proximity of B and CD4+ T cells at immunogenic sites. Various organ-specific densities of tertiary lymphoid structures are observed. Immunosuppressive sites, including brain and liver metastases, are deposited with collagen I, and T cells at these sites highly express TIM-3. This study originally deciphers the single-cell landscape of the organ-specific TME at both cellular and spatial levels for LUAD, indicating the necessity for organ-specific treatment approaches.

Development and validation of a GC-MS/MS method for the determination of iodoacetic acid in biological samples.

Iodoacetic acid (IAA) is a halogenated disinfection by-product of growing concern due to its high cytotoxicity, genotoxicity, endocrine disruptor effects, and potential carcinogenicity. However, the data on distribution and excretion of IAA after ingestion by mammals are still scarce. Here, we developed a reliable and validated method for detecting IAA in biological specimens (plasma, urine, feces, liver, kidney, and tissues) based on modified QuEChERS sample preparation combined with gas chromatography-tandem triple quadrupole mass spectrometry (GC-MS/MS). The detection method for IAA exhibited satisfactory recovery rates (62.6-108.0%) with low relative standard deviations (RSD < 12.3%) and a low detection limit for all biological matrices ranging from 0.007 to 0.032 ng/g. The study showed that the proposed method was reliable and reproducible for analyzing IAA in biological specimens. It was successfully used to detect IAA levels in biological samples from rats given gavage administration. The results indicated that IAA was found in various tissues and organs, including plasma, thyroid, the liver, the kidney, the spleen, gastrointestinal tract, and others, 6 h after exposure. This study provides the first data on the in vivo distribution in and excretion of IAA by mammals following oral exposure.

Decoction regulating phytochemicals' micromorphology changes and anti-inflammation activity enhancements originated from herb medicine supermolecules.

BACKGROUND: Mahuang Fuzi decoction (MGF) is composed of three herb medicines that has been clinically used to treat inflammatory diseases for a long history. At present, more and more active phytochemicals' aggregations have been found during the thermodynamic process of herb medicine decoction, and revealing the clinical efficacy of herb medicine through supramolecular strategies is the focus of current research. However, it is not clear whether decoction induced supermolecules' morphological changes to modify activity. METHODS: Dynamic light scattering (DLS) and field emission scanning electron microscopy (FESEM) were used to analyze the micromorphology of MGF, MGF SA (MGF supermolecules), and MIX (physical mixture of MGF single decoction). The interaction and thermodynamic parameters of single herbs in a decoction were investigated by Isothermal titration calorimetry (ITC). The phytochemicals were systematically analyzed by ultra high performance liquid chromatography-Q Exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry (UHPLC-Q-Orbitrap HRMS). Under the safe dose on RAW264.7 cells, NO, IL-6 and TNF-α were determined by Enzyme-Linked ImmunoSorbent Assay (ELISA) method. NF-κB p65 translocation from the cytoplasm into the nucleus was examined using the immunofluorescence assay and the western blot, respectively. Furthermore, Metabolomics was used to discover potential biomarkers and the associated metabolic pathways of MGF SA treatment. RESULTS: There were nanoscale aggregations in MGF, and the micromorphology of the extracted MGF SA consisted of uniform particles; while the MIX micromorphology had no uniformity. ITC showed that the interaction MH-GC and FZ-GC were a spontaneous exothermic reaction, indicating that their phytochemicals had the property of self-assembly. Though the micromorphology between MGF, MGF SA, and MIX was obviously different, UHPLC-Q-Orbitrap HRMS results displayed that the main phytochemicals of MGF and MIX had nearly the same components. Interestingly, MGF and MGF SA could significantly inhibit the production of NO, and had better inhibition effect on the expression of nuclear protein NF-κB p65 than MIX, among which MGF SA had the best effect. Further investigation indicated that the perturbance of metabolic profiling in RAW264.7 inflammatory cells was obviously reversed by MGF SA. CONCLUSIONS: The decoction enriched the key active phytochemicals and regulated the formation of homogeneous nanoparticles in MGF SA. The supermolecules in MGF SA significantly enhanced its anti-inflammatory activity, primarily affecting the NF-κB signaling pathway and the biosynthesis and metabolism of arginine in RAW264.7 inflammatory cells. Current study displayed that co-decocting herbal medicine were beneficial to the treatment of diseases than the mixture of the single herbs' extraction.

A metal ions-mediated natural small molecules carrier-free injectable hydrogel achieving laser-mediated photo-Fenton-like anticancer therapy by synergy apoptosis/cuproptosis/anti-inflammation.

Tumor microenvironment (TME) plays an important role in the tumorigenesis, proliferation, invasion and metastasis. Thereby developing synergistic anticancer strategies with multiple mechanisms are urgent. Copper is widely used in the treatment of tumor chemodynamic therapy (CDT) due to its excellent laser-mediated photo-Fenton-like reaction. Additionally, copper can induce cell death through cuproptosis, which is a new modality different from the known death mechanisms and has great promise in tumor treatment. Herein, we report a natural small molecules carrier-free injectable hydrogel (NCTD Gel) consisted of Cu2+-mediated self-assembled glycyrrhizic acid (GA) and norcantharidin (NCTD), which are mainly governed by coordination and hydrogen bonds. Under 808 nm laser irradiation, NCTD Gel can produce reactive oxygen species (ROS), consume glutathione (GSH) and overcome hypoxia in TME, leading to synergistically regulate TME via apoptosis, cuproptosis and anti-inflammation. In addition, NCTD Gel's CDT display high selectivity and good biocompatibility as it relies on the weak acidity and H2O2 overexpression of TME. Notably, NCTD Gel's components are originated from clinical agents and its preparation process is easy, green and economical, without any excipients. This study provides a new carrier-free hydrogel synergistic antitumor strategy, which has a good prospect in industrial production and clinical transformation.

SETD4 Confers Cancer Stem Cell Chemoresistance in Nonsmall Cell Lung Cancer Patients via the Epigenetic Regulation of Cellular Quiescence.

Increasing evidence indicates that quiescent cancer stem cells (CSCs) are a root cause of chemoresistance. SET domain-containing protein 4 (SETD4) epigenetically regulates cell quiescence in breast cancer stem cells (BCSCs), and SETD4-positive BCSCs are chemoradioresistant. However, the role of SETD4 in chemoresistance, tumor progression, and prognosis in nonsmall cell lung cancer (NSCLC) patients is unclear. Here, SETD4-positive cells were identified as quiescent lung cancer stem cells (qLCSCs) since they expressed high levels of ALDH1 and CD133 and low levels of Ki67. SETD4 expression was significantly higher in advanced-stage NSCLC tissues than in early-stage NSCLC tissues and significantly higher in samples from the chemoresistant group than in those from the chemosensitive group. Patients with high SETD4 expression had shorter progression-free survival (PFS) times than those with low SETD4 expression. SETD4 facilitated heterochromatin formation via H4K20me3, thereby leading to cell quiescence. RNA-seq analysis showed upregulation of genes involved in cell proliferation, glucose metabolism, and PI3K-AKT signaling in activated qLCSCs (A-qLCSCs) compared with qLCSCs. In addition, SETD4 overexpression facilitated PTEN-mediated inhibition of the PI3K-mTOR pathway. In summary, SETD4 confers chemoresistance, tumor progression, and a poor prognosis by regulating CSCs in NSCLC patients.

Discovery, traceability, formation mechanism, metal and organic components analysis of supramolecules from Maxing Shigan decoction.

Traditional Chinese medicine (TCM) decoction is a complex polydispersed phase system containing colloid solution, emulsion and suspension, which maybe induced by the supramolecular phenomenon in decoction. However, until now there is no systematic analysis of composition and formation mechanism of supramolecules in TCM decoction contained mineral drug and herb medicines. Maxing Shigan Decoction (MXSGT), one of the classic TCM recipes, has been widely used in the treatment of fever in clinic. In this study, we obtained the supramolecular part of MXSGT (MXSGT NPs). And its traceability, formation mechanism, metal and organic components were further analyzed. The morphology was characterized by scanning electron microscopy (SEM) and dynamic light scattering (DLS); and the lipopolysaccharides (LPS) induced rats' fever model was established to evaluate the antipyretic effect of MXSGT NPs. Furthermore, interaction of the disassembled groups was studied to explore the traceability and formation mechanism of MXSGT NPs by isothermal titration calorimeter (ITC). Due to the combination of mineral gypsum and herb medicines, both ICP-OES and UHPLC-Q-Orbitrap HRMS were used to analyze metal and organic components of MXSGT and MXSGT NPs, respectively. The results showed that MXSGT NPs was regular spherical nanoparticles and had the same antipyretic effect as MXSGT. Moreover, MXSGT NPs was formed by the interaction between metal and organic components, resulted in enriching the main active compounds of MXSGT. This study would provide a new idea of studying TCM decoction, especially clarifying the connotation with the participation of mineral gypsum.

Low-dose blood BTEX are associated with pulmonary function through changes in inflammatory markers among US adults: NHANES 2007-2012.

The effects of blood benzene, toluene, ethylbenzene, and xylenes (BTEX) on lung function among general adults remain unknown. We enrolled 5519 adults with measured blood BTEX concentrations and lung function from the US National Health and Nutrition Examination Survey 2007-2012. Weighted linear models were fitted to assess the associations of BTEX with lung function and inflammation parameters (white blood cell five-part differential count and C-reactive protein). The mediating effect of inflammation between BTEX and lung function was also examined. Blood BTEX concentrations decreased yearly from 1999 and were extremely low from 2007 to 2012. Benzene and toluene exerted the greatest influence on lung function in terms of forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), calculated FEV1:FVC ratio, peak expiratory flow rate (PEFR), and forced mid expiratory flow (FEF25-75%). Both ethylbenzene and all xylene isomers had no effects on FVC but reduced FEV1, FEV1:FVC ratio, PEFR, and FEF25-75%. Weighted quantile analyses demonstrated that BTEX mixture was associated with decreases in FVC, FEV1, FEV1:FVC ratio, PEFR, and FEF25-75%, with benzene weighted most heavily for all lung function parameters. BTEX also increased the levels of inflammation indicated by white blood cell five-part differential count and C-reactive protein, and increased levels of inflammation also reduced lung function. From multiple mediation analysis, inflammation mediated the effects of benzene on FEV1 and PEFR, the effects of toluene on FEV1, and the effects of ethylbenzene on FEV1 and PEFR. Low-dose exposure to BTEX was associated with reduced pulmonary function both in large and small airways. Inflammation could be involved in this pathogenesis.

Lipid analysis of meat from Bactrian camel (Camelus bacterianus), beef, and tails of fat-tailed sheep using UPLC-Q-TOF/MS based lipidomics.

Introduction: As a source of low-cost and high-quality meat for human beings, the consumption of camel meat was increasing, and beef has similar texture and nutritional characteristics with camel meat. Camel hump and fatty-tails are important parts of fat storage for camels and fat-tailed lambs, respectively, which were to adapt and endure harsh environments. Considering their similar physiological functions, their fat composition might be similar. Lipidomics is a system-level analysis of lipids method, which play an important role in the determination and quantification of individual lipid molecular specie, food adulteration and labeling. Methods: A GC/MS was used to analyze fatty acids composition of Xinjiang Bactrian camel meat, hump, beef, and fatty-tails. UPLC-Q-TOF/MS based on lipidomics approach was used to analyze lipid composition, characterize and examine the lipid differences in Xinjiang Bactrian camel meat, hump, beef, and fatty-tails. Results and discussion: The major fatty acids of the four samples were C16:0, C18:0, and C18:1cis, and camel meat had a significant low SFA content and high MUFA content. A total of 342 lipid species were detected, 192, 64, and 79 distinguishing lipids were found in the groups camel hump compared to camel meat, camel meat compared to beef, and camel hump compared to fatty-tails, respectively. Lipid metabolisms of ether lipid, glycerophospholipid, glycerolipid, and sphingolipid were the most influential pathways revealed by KEGG analysis. The results contributed to enrich the lipid information of Bactrian camel meat, and indicated that UPLC-Q-TOF/MS based on lipidomics was an alternative method to distinguish meat samples.

Integrative multi-omics unravels the amelioration effects of Zanthoxylum bungeanum Maxim. on non-alcoholic fatty liver disease.

BACKGROUND: The effect of Zanthoxylum bungeanum Maxim. (ZBM) on anti-obesity, lipid-lowering and liver protection has been identified, but the effect on the development of NAFLD induced by high-fat diet remains unclear. PURPOSE: To evaluate the alleviation effect of ZBM on NAFLD in vivo and explore the mechanisms by analyzing the liver transcriptome, microbiota and fecal metabolites. METHODS: NAFLD model was induced in C57BL/6J mice by feeding with high-fat diet (HFD). The potential mechanism of ZBM in improving NAFLD was studied by liver transcriptome analysis, real-time PCR, immunofluorescence, 16s rRNA sequencing and non-targeted metabonomics. RESULTS: ZBM has alleviation effects on HFD-induced NAFLD. The liver transcriptome, real-time PCR and immunofluorescence analysis showed that ZBM could efficiently regulate fatty acid and cholesterol metabolism. The 16S rRNA sequencing and LC-MS based metabonomic demonstrated that ZBM could rebalance gut microbiota dysbiosis and regulate metabolic profiles in HFD-induced NAFLD mice. Spearman correlation analysis revealed a strong correlation between gut microbiota and biochemical, pathological indexes and differential metabolic biomarkers. CONCLUSION: ZBM ameliorates HFD-induced NAFLD by regulating fatty acid and cholesterol metabolism, gut microbiota and metabolic profile.

Physiologically based pharmacokinetic (PBPK) modeling of BDE-209 following oral exposure in Chinese population.

The wide usage of decabromodiphenyl ether (BDE-209) as additive brominated flame retardant has caused its widespread occurrence in the environment and high exposure risk in humans. Estimating its internal exposure dose and reconstruction of external exposure dose using physiologically based pharmacokinetic (PBPK) modelling approach is a key step in the risk assessment of BDE-209. However, the PBPK model for BDE-209 is currently unavailable. This study has established two oral permeability-limited PBPK models of BDE-209 without enterohepatic recirculation (EHR) (model 1) and with EHR (model 2) for Chinese population. Using the in vitro experiments, the average binding of BDE-209 to human plasma protein (99.64% ± 2.97%) was obtained. Moreover, blood sample analysis and systematic literature review were performed to obtain internal and external exposure data of BDE-209 used for model calibration and validation. The predictions of both models were within 2-fold of the observed, and a longer half-life of serum BDE-209 was observed in model 2 than model 1. Based on the models, a human biomonitoring guidance value (HBM-GV) of 93.61 µg/g lw was derived for BDE-209, and there is no health risk found for Chinese population currently. This study provides new quantitative assessment tools for health risk assessment of BDE-209.

TROY Modulates Cancer Stem-Like Cell Properties and Gefitinib Resistance Through EMT Signaling in Non-Small Cell Lung Cancer.

Targeted therapy has made breakthrough progress in the treatment of advanced non-small cell lung cancer (NSCLC) in the last 20 years. Despite that, acquired resistance of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is an urgent clinical problem. Our study established an acquired gefitinib-resistant cell line, which exhibited epithelial-mesenchymal transition (EMT) and stem cell-like properties. Transcriptional sequencing and bioinformatics analysis revealed that TROY was significantly increased in gefitinib-resistant cells. Gene set enrichment analysis (GSEA) showed EMT was the core enriched hallmark in the resistant cells. TROY siRNA interference could overcome the gefitinib resistance with the downregulated expression of EMT and CSC markers. In addition, immunohistochemistry indicated that TROY was overexpressed in tumor samples from patients who acquired resistance to first-generation EGFR-TKI without T790M mutation and the expression of TROY was associated with poor prognosis in LUAD. Here, we provided the potential role of TROY in the resistance of targeted therapy and a new strategy to overcome the acquired resistance to EGFR-TKI in NSCLC.

Efficacy of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) combined with bevacizumab for advanced non-squamous non-small-cell lung cancer patients with gradual progression on EGFR-TKI treatment: A cohort study.

ABSTRACT: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) significantly improve outcomes of patients with EGFR-mutated non-small-cell lung cancer (NSCLC). However, acquired resistance inevitably emerges and remains a major challenge. The present study aimed to evaluate the efficacy of EGFR-TKIs plus bevacizumab in advanced non-squamous NSCLC patients with gradual progression on EGFR-TKIs.Advanced non-squamous EGFR-mutated NSCLC patients with gradual progression on EGFR-TKIs were administered bevacizumab while EGFR-TKIs were continued until disease progression occurred. Tumor lesions were assessed, and blood samples were collected at the start of the combination treatment and every 6 weeks until disease progression.Among the 15 included patients, there were no grade 3 or higher adverse events (AEs). Partial response (PR) and stable disease (SD) were achieved in 1 and 13 patients, respectively, with an objective response rate (ORR) of 6.7% and a disease control rate (DCR) of 93.3%. The median progression-free survival 2 (PFS2), defined as the time from the initiation of combination treatment to disease progression, was 5.0 (95% confidence interval [CI]: 4.0-6.0) months. Additionally, Spearman correlation analysis revealed that PFS2 was positively correlated with the serum vascular endothelial growth factor (VEGF) level at baseline (r = 0.7212, P = .0234). Patients with high baseline serum VEGF levels showed a better median PFS2 than those with low baseline serum VEGF levels (5.5 months vs 3.6 months, P = .0333).EGFR-TKIs plus bevacizumab led to a durable prolongation of PFS in non-squamous NSCLC patients with gradual progression on EGFR-TKIs. This therapeutic regimen was well tolerated and could be a promising strategy for these patients. Serum VEGF could be a potential biomarker to predict a subset of patients who are likely to benefit from EGFR-TKIs combined with bevacizumab.

Mechanism of acquired resistance to nivolumab in lung squamous cell carcinoma: case report and review of the literature.

Background: Immune checkpoint inhibitors targeting PD-1 and PD-L1 have noticeably improved the treatment landscape of advanced non-small-cell lung cancer, including lung squamous cell carcinoma (SCC). Although patients with immune checkpoint therapy can achieve long-term survival, acquired resistance has been recognized more frequently, while the underlying mechanisms are currently poorly understood. Materials and methods: Here, we report a patient with metastatic lung SCC treated with nivolumab as a first-line treatment for 28 months. Conclusion: The analysis of specimens prenivolumab and postnivolumab treatment suggests that genetic alterations in SOX2 and CDKN2A/CDKN2B and changes in the tumor microenvironment could be reasons for the acquired resistance to nivolumab observed in the lung SCC patient.

Polo-like kinase 1 inhibitor BI 6727 induces DNA damage and exerts strong antitumor activity in small cell lung cancer.

The prognosis of small cell lung cancer (SCLC) is poor despite its good initial response to chemotherapy. Polo-like kinase 1 (PLK1) is a crucial mitotic regulator that is overexpressed in many tumors, and its overexpression is associated with tumor aggressiveness and a poor prognosis. However, its role in SCLC is still poorly characterized. Based on immunohistochemistry findings, the PLK1 protein is expressed at higher levels in SCLC tumor samples than in normal lung tissue samples. The selective PLK1 inhibitor BI 6727 significantly induced the inhibition of proliferation and apoptosis in a dose-dependent manner in SCLC cell lines. FACS analysis showed an increase in the population of cells in the G2/M phase, followed by DNA damage and the consequent activation of the ataxia telangiectasia and Rad3-related (ATR)/ataxia telangiectasia mutated (ATM)-Chk1/Chk2 checkpoint pathway. In addition, BI 6727 treatment resulted in clearly attenuated growth and apoptosis in NCI-H446 xenografts. The level of histone H2AX phosphorylation at serine-139 (γH2AX) was markedly increased both in vitro and in vivo. Our findings indicate that BI 6727 has therapeutic potential for SCLC patients.

Properties of a distinct subpopulation of GABAergic commissural interneurons that are part of the locomotor circuitry in the neonatal spinal cord.

Commissural inhibitory interneurons (INs) are integral components of the locomotor circuitry that coordinate left-right motor activity during movements. We have shown that GABA-mediated synaptic transmission plays a key role in generating alternating locomotor-like activity in the mouse spinal cord (Hinckley et al., 2005a). The primary objective of our study was to determine whether properties of lamina VIII (LVIII) GABAergic INs in the spinal cord of GAD67::GFP transgenic mice fit the classification of rhythm-coordinating neurons in the locomotor circuitry. The relatively large green fluorescent protein-expressing (GFP(+)) INs had comparable morphological and electrophysiological properties, suggesting that they comprised a homogenous neuronal population. They displayed multipolar and complex dendritic arbors in ipsilateral LVII-LVIII, and their axonal projections crossed the ventral commissure and branched into contralateral ventral, medial, and dorsal laminae. Putative synaptic contacts evident as bouton-like varicosities were detected in close apposition to lateral motoneurons, Renshaw cells, other GFP(+) INs, and unidentified neurons. Exposure to a rhythmogenic mixture triggered locomotor-like rhythmic firing in the majority of LVIII GFP(+) INs. Their induced oscillatory activity was out-of-phase with bursts of contralateral motoneurons and in-phase with bouts of ipsilateral motor activity. Membrane voltage oscillations were elicited by rhythmic increases in excitatory synaptic drive and might have been augmented by three types of voltage-activated cationic currents known to increase neuronal excitability. Based on their axonal projections and activity pattern, we propose that this population of GABAergic INs forms a class of local commissural inhibitory interneurons that are integral component of the locomotor circuitry.

[Treatment and prognosis of extrapulmonary small cell carcinoma of 243 cases].

OBJECTIVE: The extrapulmonary small cell carcinoma (EPSCC), a uncommon malignant tumor, has seldom been reported. The aim of this study was to analyze the clinical characteristics, treatment and prognosis of EPSCC. METHODS: The clinical data of 243 patients admitted in our hospital from 1977 to 2007 were reviewed. The survival rate was calculated by the Kaplan-Meier method and log-rank test. RESULTS: The median age of the patients was 58 years and the male-to-female ratio was 2.47:1. According to VALSG criteria, 209 patients had limited disease (LD) and 34 had extensive disease (ED). 170 patients received chemotherapy-based multimodal therapy, 73 received surgery, and/or radiotherapy. The 6, 12, 24, 36 and 60-month survival rates of these patients were 88.9%, 67.2%, 36.8%, 27.3% and 18.3%, respectively. The clinical stage, vessel involvement and regional lymph node metastases were independent prognostic factors of EPSCC. Patients with LD had a median overall survival of 18.6 months compared with 14.0 months in patients with ED (P = 0.030). The median survival was 19.2 months for the patients without vessel involvement and 14.4 months with vessel involvement (P = 0.026). The median survival of the patients with regional lymph node metastases was 13.9 months, while 39.5 months without regional lymph node metastases (P = 0.000). Among different primary sites, patients with gynecologic small cell cancer had a median survival of 28.0 months, head and neck 20.1 months and gastrointestinal tract 14.3 months. Brain metastasis was observed in a lower number of patients with EPSCC compared with that in patients with SCLC. There were no statistically significant differences in overall survival between patients with pure and mixed EPSCC (P = 0.396). CONCLUSION: EPSCC is an uncommon malignant tumor with early metastasis and poor prognosis. The clinical characteristics of EPSCC and SCLC were similar in some aspects, however, there are some differences in etiology, clinic course, survival and frequency of brain metastases. These differences may influence the choice of therapeutic strategy. Multimodal therapy, combination of chemo- and radio-therapy after surgical resection may improve the outcome of EPSCC.

Persistent sodium current contributes to induced voltage oscillations in locomotor-related hb9 interneurons in the mouse spinal cord.

Neurochemically induced membrane voltage oscillations and firing episodes in spinal excitatory interneurons expressing the HB9 protein (Hb9 INs) are synchronous with locomotor-like rhythmic motor outputs, suggesting that they contribute to the excitatory drive of motoneurons during locomotion. Similar to central pattern generator neurons in other systems, Hb9 INs are interconnected via electrical coupling, and their rhythmic activity does not depend on fast glutamatergic synaptic transmission. The primary objective of this study was to determine the contribution of fast excitatory and inhibitory synaptic transmission and subthreshold voltage-dependent currents to the induced membrane oscillations in Hb9 INs in the postnatal mouse spinal cord. The non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) reduced the amplitude of voltage oscillations but did not alter their frequency. CNQX suppressed rhythmic motor activity. Blocking glycine and GABAA receptor-mediated inhibitory synapses as well as cholinergic transmission did not change the properties of CNQX-resistant membrane oscillations. However, disinhibition triggered new episodes of slow motor bursting that were not correlated with induced locomotor-like rhythms in Hb9 INs. Our observations indicated that fast excitatory and inhibitory synaptic inputs did not control the frequency of induced rhythmic activity in Hb9 INs. We next examined the contribution of persistent sodium current (INaP) to subthreshold membrane oscillations in the absence of primary glutamatergic, GABAergic and glycinergic synaptic drive to Hb9 INs. Low concentrations of riluzole that blocked the slow-inactivating component of sodium current gradually suppressed the amplitude and reduced the frequency of voltage oscillations. Our finding that INaP regulates locomotor-related rhythmic activity in Hb9 INs independently of primary synaptic transmission supports the concept that these neurons constitute an integral component of the rhythmogenic locomotor network in the mouse spinal cord.

Locomotor-like rhythms in a genetically distinct cluster of interneurons in the mammalian spinal cord.

Electrophysiological and morphological properties of genetically identified spinal interneurons were examined to elucidate their possible contribution to locomotor-like rhythmic activity in 1- to 4-day-old mice. In the transgenic mice used in our study, the HB9 promotor controlled the expression of the reporter gene enhanced green fluorescent protein (eGFP), giving rise to GFP+ motoneurons and ventral interneurons. However, only motoneurons and a small group of bipolar, GFP+ interneurons expressed the HB9 protein. The HB9(+)/GFP+ interneurons were clustered close to the medial surface in lamina VIII along segments L1-L3. The correlation between activity pattern in these visually identified interneurons and motoneuron output was examined using simultaneous whole cell and ventral root recordings. Neurochemically induced rhythmic membrane depolarizations in HB9/GFP interneurons were synchronous with ventral root rhythms, indicating that the interneurons received synaptic inputs from rhythm-generating networks. The frequency of excitatory postsynaptic currents significantly increased during ventral root bursts, but there was no change in the frequency of inhibitory postsynaptic currents during the cycle period. These data implied that HB9/GFP interneurons received primarily excitatory inputs from rhythmogenic interneurons. Neurobiotin-filled axon terminals were in close apposition to other neurons in the cluster and to motoneuron dendrites, raising the possibility that HB9/GFP interneurons formed synaptic connections with each other and with motoneurons. The expression of the vesicular glutamate transporter 2 in axon terminals of HB9/GFP interneurons indicated that these were glutamatergic interneurons. Our findings suggest that the visually identified HB9/GFP interneurons are premotor excitatory interneurons and putative constituents of networks generating locomotor rhythms in the mammalian spinal cord.