Mutations in more than half of human connexin genes encoding gap junction (GJ) subunits have been linked to inherited human diseases. Functional studies of human GJ channels are essential for revealing mechanistic insights into the etiology of disease-linked connexin mutants. However, the commonly used Xenopus oocytes, N2A, HeLa, and other model cells for recombinant expression of human connexins have different and significant limitations. Here we developed a human cell line (HEK293) with each of the endogenous connexins (Cx43 and Cx45) knocked out using the CRISPR-Cas9 system. Double knockout HEK293 cells showed no background GJ coupling, were easily transfected with several human connexin genes (such as those encoding Cx46, Cx50, Cx37, Cx45, Cx26, and Cx36) which successfully formed functional GJs and were readily accessible for dual patch clamp analysis. Single knockout Cx43 or Cx45 HEK cell lines could also be used to characterize human GJ channels formed by Cx45 or Cx43, respectively, with an expression level suitable for studying macroscopic and single channel GJ channel properties. A cardiac arrhythmia linked Cx45 mutant R184G failed to form functional GJs in DKO HEK293 cells with impaired localizations. These genetically engineered HEK293 cells are well suited for patch clamp study of human GJ channels.

OBJECTIVE: Investigation of chronic homocysteine action on the excitability and N-methyl-D-aspartate (NMDA) sensitivity of the peripheral trigeminovascular system of rats.
BACKGROUND: Migraine is a neurological disease that affects 15%-20% of the general population. Epidemiological observations show that an increase of the sulfur-containing amino acid homocysteine in plasma-called hyperhomocysteinemia-is associated with a high risk of migraine, especially migraine with aura. In animal studies, rats with hyperhomocysteinemia demonstrated mechanical allodynia, photophobia, and anxiety, and higher sensitivity to cortical spreading depression. In addition, rats with hyperhomocysteinemia were more sensitive in a model of chronic migraine induced by nitroglycerin which indicated the involvement of peripheral nociceptive mechanisms. The present work aimed to analyze the excitability of meningeal afferents and neurons isolated from the trigeminal ganglion of rats with prenatal hyperhomocysteinemia.
METHODS: Experiments were performed on male rats born from females fed with a methionine-rich diet before and during pregnancy. The activity of meningeal afferents was recorded extracellularly in hemiskull preparations ex vivo and action potentials were characterized using cluster analysis. The excitability of trigeminal ganglion neurons was assessed using whole-cell patch clamp recording techniques and calcium imaging studies. Meningeal mast cells were stained using toluidine blue.
RESULTS: The baseline extracellular recorded electrical activity of the trigeminal nerve was higher in the hyperhomocysteinemia group with larger amplitude action potentials. Lower concentrations of KCl caused an increase in the frequency of action potentials of trigeminal afferents recorded in rat hemiskull ex vivo preparations. In trigeminal ganglion neurons of rats with hyperhomocysteinemia, the current required to elicit at least one action potential (rheobase) was lower, and more action potentials were induced in response to stimulus of 2 × rheobase. In controls, short-term application of homocysteine and its derivatives increased the frequency of action potentials of the trigeminal nerve and induced Ca2+ transients in neurons, which are associated with the activation of NMDA receptors. At the same time, in rats with hyperhomocysteinemia, we did not observe an increased response of the trigeminal nerve to NMDA. Similarly, the parameters of Ca2+ transients induced by NMDA, homocysteine, and its derivatives were not changed in rats with hyperhomocysteinemia. Acute incubation of the meninges in homocysteine and homocysteinic acid did not change the state of the mast cells, whereas in the model of hyperhomocysteinemia, an increased degranulation of mast cells in the meninges was observed.
CONCLUSIONS: Our results demonstrated higher excitability of the trigeminal system of rats with hyperhomocysteinemia. Together with our previous finding about the lower threshold of generation of cortical spreading depression in rats with hyperhomocysteinemia, the present data provide evidence of homocysteine as a factor that increases the sensitivity of the peripheral migraine mechanisms, and the control of homocysteine level may be an important strategy for reducing the risk and/or severity of migraine headache attacks.

Multiple patch-clamp recordings and morphological reconstruction are powerful approaches for neuronal microcircuitry dissection and cell type classification but are challenging due to the sophisticated expertise needed. Here, we present a protocol for applying these techniques to neurons in the medial entorhinal cortex (MEC) of mice. We detail steps to prepare brain slices containing MEC and perform simultaneous multiple whole-cell recordings, followed by procedures of histological staining and neuronal reconstruction. We then describe how we analyze morphological and electrophysiological features. For complete details on the use and execution of this protocol, please refer to Shi et al.1.

Analysis of synaptic strength and plasticity provides functional insights of complicated neural circuits. Here, we describe steps for cell- and projection-specific optogenetic manipulation of divergent basal ganglia circuits using anterograde and retrograde viral vectors. We quantitatively analyze synaptic function of these circuits utilizing a patch-clamp technique. This protocol is applicable to probe potential circuit targets for treatment of brain diseases. For complete details on the use and execution of this protocol, please refer to Ji et al.1.

Mitochondria are double-membrane organelles crucial for oxidative phosphorylation, enabling efficient ATP synthesis by eukaryotic cells. Both of the membranes, the highly selective inner mitochondrial membrane (IMM) and a relatively porous outer membrane (OMM), harbor a number of integral membrane proteins that help in the transport of biological molecules. These transporters are especially enriched in the IMM, where they help maintain transmembrane gradients for H+, K+, Ca2+, PO43-, and metabolites like ADP/ATP, citrate, etc. Impaired activity of these transporters can affect the efficiency of energy-transducing processes and can alter cellular redox state, leading to activation of cell-death pathways or metabolic syndromes in vivo. Although several methodologies are available to study ion flux through membrane proteins, the patch-clamp technique remains the gold standard for quantitatively analyzing electrogenic ion exchange across membranes. Direct patch-clamp recordings of mitoplasts (mitochondria devoid of outer membrane) in different modes, such as whole-mitoplast or excised-patch mode, allow researchers the opportunity to study the biophysics of mitochondrial transporters in the native membrane, in real time, in isolation from other fluxes or confounding factors due to changes in ion gradients, pH, or mitochondrial potential (ΔΨ). Here, we summarize the use of patch clamp to investigate several membrane proteins of mitochondria. We demonstrate how this technique can be reliably applied to record whole-mitoplast Ca2+ currents mediated via mitochondrial calcium uniporter or H+ currents mediated by uncoupling protein 1 and discuss critical considerations while recording currents from these small vesicles of the IMM (mitoplast diameter = 2-5 µm).

In vitro spinal cord preparations have been extensively used to study microcircuits involved in the control of movement. By allowing precise control of experimental conditions coupled with state-of-the-art genetics, imaging, and electrophysiological techniques, isolated spinal cords from mice have been an essential tool in detailing the identity, connectivity, and function of spinal networks. The majority of the research has arisen from in vitro spinal cords of neonatal mice, which are still undergoing important postnatal maturation. Studies from adults have been attempted in transverse slices, however, these have been quite challenging due to the poor motoneuron accessibility and viability, as well as the extensive damage to the motoneuron dendritic trees. In this work, we describe two types of coronal spinal cord preparations with either the ventral or the dorsal horn ablated, obtained from mice of different postnatal ages, spanning from preweaned to 1 mo old. These semi-intact preparations allow recordings of sensory-afferent and motor-efferent responses from lumbar motoneurons using whole cell patch-clamp electrophysiology. We provide details of the slicing procedure and discuss the feasibility of whole cell recordings. The in vitro dorsal and ventral horn-ablated spinal cord preparations described here are a useful tool to study spinal motor circuits in young mice that have reached the adult stages of locomotor development.NEW & NOTEWORTHY In the past 20 years, most of the research into the mammalian spinal circuitry has been limited to in vitro preparations from embryonic and neonatal mice. We describe two in vitro longitudinal lumbar spinal cord preparations from juvenile mice that allow the study of motoneuron properties and respective afferent or efferent spinal circuits through whole cell patch clamp. These preparations will be useful to those interested in the study of microcircuits at mature stages of motor development.

Electrophysiology studies the electrical properties of cells and tissues including bioelectrical signals and membrane ion channel activities. As an important means to reveal ion channel related physiological functions and the underlying mechanisms, electrophysiological techniques have been widely used in studies of animals, higher plants and algae that are closely related to higher plants. However, few electrophysiological studies have been carried out in red tide organisms, especially in dinoflagellates, which is mainly due to the complex surface structure of dinoflagellate amphiesma. In this study, the surface amphiesma of Alexandrium pacificum, a typical red tide species, was removed by centrifugation, low-temperature treatment and enzymatic treatment. In all three treatments, low-temperature treatment with 4 °C for 2 h had high ecdysis rate and high fixation rate, and the treated cells were easy to puncture, so low-temperature treatment was used as a preprocessing treatment for subsequent current recording. Acquired protoplasts of A. pacificum were identified by calcofluor fluorescence and immobilized by poly-lysine. A modified \"puncture\" single-electrode voltage-clamp recording was first applied to dinoflagellates, and voltage-gated currents, which had the characteristics of outward K+ current and inward Cl- current, were recorded and confirmed by ion replacement, indicating the voltage-gated currents were mixed. This method can be used as a technical basis for the electrophysiological study of dinoflagellates and provides a new perspective for the study of stress tolerance, red tide succession, and the regulation of physiological function of dinoflagellates.

The ability to sense changes in oxygen availability is fundamentally important for the survival of all aerobic organisms. However, cellular oxygen sensing mechanisms and pathologies remain incompletely understood and studies of acute oxygen sensing, in particular, have produced inconsistent results. Current methods cannot simultaneously measure the key cellular events in acute hypoxia (i.e., changes in redox state, electrophysiological properties, and mechanical responses) at controlled partial pressures of oxygen (pO2 ). The lack of such a comprehensive method essentially contributes to the discrepancies in the field. A sealed microfluidic system that combines i) Raman spectroscopy, ii) patch-clamp electrophysiology, and iii) live-cell imaging under precisely controlled pO2 have therefore been developed. Merging these modalities allows label-free and simultaneous observation of oxygen-dependent alterations in multiple cellular redox couples, membrane potential, and cellular contraction. This technique is adaptable to any cell type and allows in-depth insight into acute oxygen sensing processes underlying various physiologic and pathologic conditions.

Recently, there have been great advances in cardiovascular channelopathy modeling and drug safety pharmacology using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The automated patch-clamp (APC) technique overcomes the disadvantages of the manual patch-clamp (MPC) technique, which is labor intensive and gives low output. However, the application of the APC platform is still limited in iPSC-CM based research, due to the difficulty in maintaining the high quality of single iPSC-CMs during dissociation and recording. In this study, we improved the method for single iPSC-CM preparation by applying 2.5 µM blebbistatin (BB, an excitation-contraction coupling uncoupler) throughout APC procedures (dissociation, filtration, storage, and recording). Under non-BB buffered condition, iPSC-CMs in suspension showed a severe bleb-like morphology. However, BB-supplement led to significant improvements in morphology and INa recording, and we even obtained several CMs that showed spontaneous action potentials with typical morphology. Furthermore, APC faithfully recapitulated the single-cell electrophysiological phenotypes of iPSC-CMs derived from Brugada syndrome patients, as detected with MPC. Our study indicates that APC is capable of replacing MPC in the modeling of cardiac channelopathies using human iPSC-CMs by providing high-quality data with higher throughput.

Patch clamp is a technique that can measure weak current in the level of picoampere (pA). It has been widely used for cellular electrophysiological recording in fundamental medical researches, such as membrane potential and ion channel currents recording, etc. In order to obtain accurate measurement results, both the resistance and capacitance of the pipette are required to be compensated. Capacitance compensations are composed of slow and fast capacitance compensation. The slow compensation is determined by the lipid bilayer of cell membrane, and its magnitude usually ranges from a few picofarads (pF) to a few microfarads (μF), depending on the cell size. The fast capacitance is formed by the distributed capacitance of the glass pipette, wires and solution, mostly ranging in a few picofarads. After the pipette sucks the cells in the solution, the positions of the glass pipette and wire have been determined, and only taking once compensation for slow and fast capacitance will meet the recording requirements. However, when the study needs to deal with the temperature characteristics, it is still necessary to make a recognition on the temperature characteristic of the capacitance. We found that the time constant of fast capacitance discharge changed with increasing temperature of bath solution when we studied the photothermal effect on cell membrane by patch clamp. Based on this phenomenon, we proposed an equivalent circuit to calculate the temperature-dependent parameters. Experimental results showed that the fast capacitance increased in a positive rate of 0.04 pF/℃, while the pipette resistance decreased. The fine data analysis demonstrated that the temperature rises of bath solution determined the kinetics of the fast capacitance mainly by changing the inner solution resistance of the glass pipette. This result will provide a good reference for the fine temperature characteristic study related to cellular electrophysiology based on patch clamp technique.
膜片钳（Patch Clamp）是一种皮安培（pA）级弱电流的测量技术，在基础医学研究中广泛用于细胞电生理记录，如细胞膜电位和离子通道电流的研究等。为了得到准确的离子通道测量结果，需要同时进行微电极的串联阻抗和相关电容补偿。电容补偿包括慢电容补偿和快电容补偿：慢电容源于细胞脂双层，其幅度依细胞大小从几皮法拉（pF）到几微法拉（μF）不等。快电容由玻璃微电极、导线和溶液及接地线等因素决定，幅值在几个皮法拉。一般当微电极完成与细胞的吸附，导线和电极位置确定后，只需在实验前进行一次快、慢电容补偿即可记录到可靠的离子通道电流。但对于伴随溶液温度改变的相关研究来说，细胞浴液的变化是如何影响快电容变化的尚没有报道。本论文在使用膜片钳研究细胞光热效应的实验中，观察到溶液的温升可以改变系统快电容的放电特性。基于此提出了一个考虑温度因素的快电容等效电路，并计算了相关参量变化情况及温度特性。实验数据表明快电容以约 0.04 pF/℃ 的速率随温度增加而增加，而微电极的串联阻抗却随温度增加而减小。进一步分析表明：溶液温度升高对微电极快电容放电时间特性的改变，主要是通过改变微电极内溶液阻抗实现的。这一结果对基于膜片钳技术开展细胞层面的精细温度特性研究有参考意义。.