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The patch clamp technique has become the gold standard for neuron electrophysiology research in brain science. Brain slices have been widely utilized as the targets of the patch clamp technique due to their higher optical transparency compared to a live brain and their intercellular connectivity in comparison to cultured single neurons. However, the narrow working space, small scope, and depth of the field of view make the positioning of the operation’s micropipette to the target neuron a time-consuming task reliant on a high level of experience, significantly slowing down operation of the patch clamp technique in brain slices. Further, the current poor controllability in gigaseal formation, which is the key to electrophysiology signal recording, significantly lowers the patch clamp success rate. In this paper, a stepwise navigation of the micropipette is conducted to accelerate the positioning process of the micropipette tip to the target neuron in the brain slice. Then, a fuzzy proportional–integral–derivative controller is designed to control the gigaseal formation process along a designed resistance curve. The experimental results demonstrate an almost doubled patch clamp technique speed, with a 25% improvement in the success rate compared to the conventional manual method. The above advantages may promote the application of our method in brain science research based on brain slice platforms.

• We combined the patch-clamp technique with ratiometric fluorescence imaging using the proton-responsive dye BCECF as a luminal probe. • Upon application of a steep cytosol-directed potassium ion (K⁺) gradient in Arabidopsis mesophyll vacuoles, a strong and reversible acidification of the vacuolar lumen was detected, whereas no associated electrical currents were observed, in agreement with electroneutral cation/H⁺ exchange. • Our data show that this acidification was generated by NHX antiport activity, because: it did not distinguish between K⁺ and sodium (Na⁺) ions; it was sensitive to the NHX inhibitor benzamil; and it was completely absent in vacuoles from nhx1 nhx2 double knockout plants. Our data further show that NHX activity could be reversed, was voltage-independent and specifically impaired by the low-abundance signaling lipid PI(3,5)P₂, which may regulate salt accumulation in plants by acting as a common messenger to coordinately shut down secondary active carriers responsible for cation and anion uptake inside the vacuole. • Finally, we developed a theory based on thermodynamics, which supports the data obtained by our novel experimental approach. • This work, therefore, represents a proof-of-principle that can be applied to the study of proton- dependent exchangers from plants and animals, which are barely detectable using conventional techniques.

This Technical Report describes new methods of transcranial magnetic stimulation (TMS) in non-human primates. By combining single neuron recording with a modified TMS coil with focused stimulation in alert macaques, the authors show that this method can reduce stimulation artifact and allow investigation into the neuronal mechanisms of TMS. Transcranial magnetic stimulation (TMS) is a widely used, noninvasive method for stimulating nervous tissue, yet its mechanisms of effect are poorly understood. Here we report new methods for studying the influence of TMS on single neurons in the brain of alert non-human primates. We designed a TMS coil that focuses its effect near the tip of a recording electrode and recording electronics that enable direct acquisition of neuronal signals at the site of peak stimulus strength minimally perturbed by stimulation artifact in awake monkeys ( Macaca mulatta ). We recorded action potentials within ∼1 ms after 0.4-ms TMS pulses and observed changes in activity that differed significantly for active stimulation as compared with sham stimulation. This methodology is compatible with standard equipment in primate laboratories, allowing easy implementation. Application of these tools will facilitate the refinement of next generation TMS devices, experiments and treatment protocols.

A distinct set of channels and transporters regulates the ion fluxes across the lysosomal membrane. Malfunctioning of these transport proteins and the resulting ionic imbalance is involved in various human diseases, such as lysosomal storage disorders, cancer, as well as metabolic and neurodegenerative diseases. As a consequence, these proteins have stimulated strong interest for their suitability as possible drug targets. A detailed functional characterization of many lysosomal channels and transporters is lacking, mainly due to technical difficulties in applying the standard patch-clamp technique to these small intracellular compartments. In this review, we focus on current methods used to unravel the functional properties of lysosomal ion channels and transporters, stressing their advantages and disadvantages and evaluating their fields of applicability.

The gold-standard clamp measurements for insulin sensitivity (cSI), β-cell function (cBCF), and disposition index (cDI = cSI × cBCF) are not practical in large-scale studies. We sought to 1) validate a mathematical model-derived DI from oral glucose tolerance tests (OGTT) with insulin (mDI) and without (mDI-woI) against cDI and oral disposition index (oDI) and 2) evaluate the ability of the novel indices to detect prediabetes and type 2 diabetes (T2D). We carried out a secondary analysis of previously reported cross-sectional observational studies. The Insulin Sensitivity and Secretion mathematical model for glucose-insulin dynamics was applied to 5-point and 3-point OGTTs synchronized with hyperinsulinemic-euglycemic and hyperglycemic clamps from 130 youth with obesity (68 normal glucose tolerance [NGT], 33 impaired glucose tolerance [IGT], 29 T2D). Model-derived DI correlated well with clamp DI (R = 0.76 [logged]). Between NGT and IGT, mDI and mDI-woI decreased more than oDI and cDI, (60% and 59% vs 29% and 27%), and by receiver operating characteristic analysis were superior at detecting IGT compared with oDI and cDI (area under the curve [AUC] 0.88-0.87 vs 0.68-0.65), as was mean glucose (AUC 0.87). mDI-woI is better than oDI or the labor-intensive cDI for detecting dysglycemia in obese youth. Bypassing insulin measurements with mDI-woI from the OGTT provides a cost-effective approach for large-scale epidemiological studies of dysglycemia in youth.

The use of single-cell assays has emerged as a cutting-edge technique during the past decade. Although single-cell mass spectrometry (MS) has recently achieved remarkable results, deep biological insights have not yet been obtained, probably because of various technical issues, including the unavoidable use of matrices, the inability to maintain cell viability, low throughput because of sample pretreatment, and the lack of recordings of cell physiological activities from the same cell. In this study, we describe a patch clamp/MS-based platform that enables the sensitive, rapid, and in situ chemical profiling of single living neurons. This approach integrates modified patch clamp technique and modified MS measurements to directly collect and detect nanoliter-scale samples from the cytoplasm of single neurons in mice brain slices. Abundant possible cytoplasmic constituents were detected in a single neuron at a relatively fast rate, and over 50 metabolites were identified in this study. The advantages of direct, rapid, and in situ sampling and analysis enabled us to measure the biological activities of the cytoplasmic constituents in a single neuron, including comparing neuron types by cytoplasmic chemical constituents; observing changes in constituent concentrations as the physiological conditions, such as age, vary; and identifying the metabolic pathways of small molecules.

The brain’s extracellular matrix (ECM) is a macromolecular network composed of glycosaminoglycans, proteoglycans, glycoproteins, and fibrous proteins. In vitro studies often use purified ECM proteins for cell culture coatings, however these may not represent the molecular complexity and heterogeneity of the brain’s ECM. To address this, we compared neural network activity (over 30 days in vitro ) from primary neurons co-cultured with glia grown on ECM coatings from decellularized brain tissue (bECM) or MaxGel, a non-tissue-specific ECM. Cells were grown on a multi-electrode array (MEA) to enable noninvasive long-term interrogation of neuronal networks. In general, the presence of ECM accelerated the formation of networks without affecting the inherent network properties. However, specific features of network activity were dependent on the type of ECM: bECM enhanced network activity over a greater region of the MEA whereas MaxGel increased network burst rate associated with robust synaptophysin expression. These differences in network activity were not attributable to cellular composition, glial proliferation, or astrocyte phenotypes, which remained constant across experimental conditions. Collectively, the addition of ECM to neuronal cultures represents a reliable method to accelerate the development of mature neuronal networks, providing a means to enhance throughput for routine evaluation of neurotoxins and novel therapeutics.

Mechanosensitive (MS) channels of small (MscS) and large (MscL) conductance are the major players in the protection of bacterial cells against hypoosmotic shock. Although a great deal is known about structure and function of these channels, much less is known about how membrane lipids may influence their mechanosensitivity and function. In this study, we use liposome coreconstitution to examine the effects of different types of lipids on MscS and MscL mechanosensitivity simultaneously using the patch-clamp technique and confocal microscopy. Fluorescence lifetime imaging (FLIM)-FRET microscopy demonstrated that coreconstitution of MscS and MscL led to clustering of these channels causing a significant increase in the MscS activation threshold. Furthermore, the MscL/MscS threshold ratio dramatically decreased in thinner compared with thicker bilayers and upon addition of cholesterol, known to affect the bilayer thickness, stiffness and pressure profile. In contrast, application of micromolar concentrations of lysophosphatidylcholine (LPC) led to an increase of the MscL/MscS threshold ratio. These data suggest that differences in hydrophobic mismatch and bilayer stiffness, change in transbilayer pressure profile, and close proximity of MscL and MscS affect the structural dynamics of both channels to a different extent. Our findings may have far-reaching implications for other types of ion channels and membrane proteins that, like MscL and MscS, may coexist in multiple molecular complexes and, consequently, have their activation characteristics significantly affected by changes in the lipid environment and their proximity to each other.

Aims/hypothesis While it is well known that diet-induced obesity causes insulin resistance, the precise mechanisms underpinning the initiation of insulin resistance are unclear. To determine factors that may cause insulin resistance, we have performed a detailed time-course study in mice fed a high-fat diet (HFD). Methods C57Bl/6 mice were fed chow or an HFD from 3 days to 16 weeks and glucose tolerance and tissue-specific insulin action were determined. Tissue lipid profiles were analysed by mass spectrometry and inflammatory markers were measured in adipose tissue, liver and skeletal muscle. Results Glucose intolerance developed within 3 days of the HFD and did not deteriorate further in the period to 12 weeks. Whole-body insulin resistance, measured by hyperinsulinaemic–euglycaemic clamp, was detected after 1 week of HFD and was due to hepatic insulin resistance. Adipose tissue was insulin resistant after 1 week, while skeletal muscle displayed insulin resistance at 3 weeks, coinciding with a defect in glucose disposal. Interestingly, no further deterioration in insulin sensitivity was observed in any tissue after this initial defect. Diacylglycerol content was increased in liver and muscle when insulin resistance first developed, while the onset of insulin resistance in adipose tissue was associated with increases in ceramide and sphingomyelin. Adipose tissue inflammation was only detected at 16 weeks of HFD and did not correlate with the induction of insulin resistance. Conclusions/interpretation HFD-induced whole-body insulin resistance is initiated by impaired hepatic insulin action and exacerbated by skeletal muscle insulin resistance and is associated with the accumulation of specific bioactive lipid species.

Brain functions are fundamental for the survival of organisms, and they are supported by neural circuits consisting of a variety of neurons. To investigate the function of neurons at the single-cell level, researchers often use whole-cell patch-clamp recording techniques. These techniques enable us to record membrane potentials (including action potentials) of individual neurons of not only anesthetized but also actively behaving animals. This whole-cell recording method enables us to reveal how neuronal activities support brain function at the single-cell level. In this review, we introduce previous studies using in vivo patch-clamp recording techniques and recent findings primarily regarding neuronal activities in the hippocampus for behavioral function. We further discuss how we can bridge the gap between electrophysiology and biochemistry.