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• 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.

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.

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.

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.

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 mammalian cerebellum is a highly multimodal structure, receiving inputs from multiple sensory modalities and integrating them during complex sensorimotor coordination tasks. Previously, using cell-type-specific anatomical projection mapping, it was shown that multimodal pathways converge onto individual cerebellar granule cells (Huang et al., 2013). Here we directly measure synaptic currents using in vivo patch-clamp recordings and confirm that a subset of single granule cells receive convergent functional multimodal (somatosensory, auditory, and visual) inputs via separate mossy fibers. Furthermore, we show that the integration of multimodal signals by granule cells can enhance action potential output. These recordings directly demonstrate functional convergence of multimodal signals onto single granule cells.

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.

Calcium-dependent chloride channels are required for normal electrolyte and fluid secretion, olfactory perception, and neuronal and smooth muscle excitability. The molecular identity of these membrane proteins is still unclear. Treatment of bronchial epithelial cells with interleukin-4 (IL-4) causes increased calcium-dependent chloride channel activity, presumably by regulating expression of the corresponding genes. We performed a global gene expression analysis to identify membrane proteins that are regulated by IL-4. Transfection of epithelial cells with specific small interfering RNA against each of these proteins shows that TMEM16A, a member of a family of putative plasma membrane proteins with unknown function, is associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent proteins, short-circuit current, and patch-clamp techniques. Our results indicate that TMEM16A is an intrinsic constituent of the calcium-dependent chloride channel. Identification of a previously unknown family of membrane proteins associated with chloride channel function will improve our understanding of chloride transport physiopathology and allow for the development of pharmacological tools useful for basic research and drug development.

Simultaneous performance of two tasks often leads to deficits in the component tasks, an effect thought to depend on the lateral prefrontal cortex (LPFC). Here, the authors recorded single-neuron activities in monkey LPFC during two simultaneous tasks, providing direct neurophysiological evidence for models of dual-task interference and capacity limitation. Simultaneous performance of two tasks often leads to performance deficits in the component tasks. This effect, known as dual-task interference, is thought to be a proof of capacity limitation in cognition, and the lateral prefrontal cortex (LPFC) has been highlighted as its putative neural substrate. Here we recorded single-neuron activities in LPFC while monkeys performed dual tasks that required the simultaneous performance of a varying-load spatial attention task and a spatial memory task. We found that the performance of the monkeys exhibited dual-task interference, and prefrontal neuron activities showed a decreased ability to represent task-relevant information to a degree proportional to the increased demand of the concurrent counterpart task. The locus of the interference was shown to originate in the simultaneous, overloaded recruitment of the same LPFC neural population by the two tasks. These results provide direct neurophysiological evidence for, and constraints to, psychological models of dual-task interference and capacity limitation.