In Vitro Patch-Clamp.

The patch-clamp technique is one of the most useful tools to analyze the function of electrically active cells such as neurons. This technique allows for the analysis of proteins (ion channels and receptors), cells (neurons), and synapses that are the building blocks of neuronal networks. Cortical development involves coordinated changes in functional measures at each of these levels of analysis that reflect both cellular and circuit maturation. This chapter explains the technical and theoretical basis of patch-clamp methodology and introduces several examples of how this technique can be applied in the context of cortical development.

In Vivo Whole-Cell Recording from the Mouse Brain.

Measuring the membrane potential dynamics of neurons offers a comprehensive understanding of the molecular and cellular mechanisms that form their spiking activity, thus playing a crucial role in unraveling the mechanistic processes governing brain function. Techniques for intracellular recordings of membrane potentials pioneered in the 1940s have witnessed significant advancements since their inception. Among these, whole-cell patch-clamp recording has emerged as a leading method for measuring neuronal membrane potentials due to its high stability and broad applicability ranging from cultured cells to brain slices and even behaving animals. This chapter provides a detailed protocol to acquire stable whole-cell recordings from neurons in the cerebral cortex of awake, head-restrained mice. Significant enhancements to our protocol include implanting a metal head-post using adhesive resin cement and preparing a recording pipette with a long shank for targeting deeper brain regions. This protocol, once implemented, enables whole-cell recordings up to 2.5 mM beneath the cortical surface.

Electrophysiological and morphological properties of prefrontal pyramidal neurons innervated by mediodorsal thalamus.

The high-order cognitive and executive functions are necessary for an individual to survive. The densely bidirectional innervations between the medial prefrontal cortex (mPFC) and the mediodorsal thalamus (MD) play a vital role in regulating high-order functions. Pyramidal neurons in mPFC have been classified into several subclasses according to their morphological and electrophysiological properties, but the properties of the input-specific pyramidal neurons in mPFC remain poorly understood. The present study aimed to profile the morphological and electrophysiological properties of mPFC pyramidal neurons innervated by MD. In the past, the studies for characterizing the morphological and electrophysiological properties of neurons mainly relied on the electrophysiological recording of a large number of neurons and their morphologic reconstructions. But, it is a low efficient method for characterizing the circuit-specific neurons. The present study combined the advantages of traditional morphological and electrophysiological methods with machine learning to address the shortcomings of the past method, to establish a classification model for the morphological and electrophysiological properties of mPFC pyramidal neurons, and to achieve more accurate and efficient identification of the properties from a small size sample of neurons. We labeled MD-innervated pyramidal neurons of mPFC using the trans-synaptic neural circuitry tracing method and obtained their morphological properties using whole-cell patch-clamp recording and morphologic reconstructions. The results showed that the classification model established in the present study could predict the electrophysiological properties of MD-innervated pyramidal neurons based on their morphology. MD-innervated pyramidal neurons exhibit larger basal dendritic length but lower apical dendrite complexity compared to non-MD-innervated neurons in the mPFC. The morphological characteristics of the two subtypes (ET-1 and ET-2) of mPFC pyramidal neurons innervated by MD are different, with the apical dendrites of ET-1 neurons being longer and more complex than those of ET-2 neurons. These results suggest that the electrophysiological properties of MD- innervated pyramidal neurons within mPFC correlate with their morphological properties, indicating that the different roles of these two subclasses in local circuits within PFC, as well as in PFC-cortical/subcortical brain region circuits.

LRMP inhibits cAMP potentiation of HCN4 channels by disrupting intramolecular signal transduction.

Lymphoid restricted membrane protein (LRMP) is a specific regulator of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation of HCN4, but the interaction domains, mechanisms of action, and basis for isoform-specificity remain unknown. Here, we identify the domains of LRMP essential for this regulation, show that LRMP acts by disrupting the intramolecular signal transduction between cyclic nucleotide binding and gating, and demonstrate that multiple unique regions in HCN4 are required for LRMP isoform-specificity. Using patch clamp electrophysiology and Förster resonance energy transfer (FRET), we identified the initial 227 residues of LRMP and the N-terminus of HCN4 as necessary for LRMP to associate with HCN4. We found that the HCN4 N-terminus and HCN4-specific residues in the C-linker are necessary for regulation of HCN4 by LRMP. Finally, we demonstrated that LRMP-regulation can be conferred to HCN2 by addition of the HCN4 N-terminus along with mutation of five residues in the S5 region and C-linker to the cognate HCN4 residues. Taken together, these results suggest that LRMP inhibits HCN4 through an isoform-specific interaction involving the N-terminals of both proteins that prevents the transduction of cAMP binding into a change in channel gating, most likely via an HCN4-specific orientation of the N-terminus, C-linker, and S4-S5 linker.

Acid-Sensitive Outwardly Rectifying Cl- Current in OV2944 Mouse Ovarian Cancer Cells.

BACKGROUND/AIMS: Extracellular acidic conditions impair cellular activities; however, some cancer cells drive cellular signaling to adapt to the acidic environment. It remains unclear how ovarian cancer cells sense changes in extracellular pH. This study was aimed at characterizing acid-inducible currents in an ovarian cancer cell line and evaluating the involvement of these currents in cell viability. METHODS: The biophysical and pharmacological properties of membrane currents in OV2944, a mouse ovarian cancer cell line, were studied using the whole-cell configuration of the patch-clamp technique. Viability of this cell type in acidic medium was evaluated using the MTT assay. RESULTS: OV2944 had significant acid-sensitive outwardly rectifying (ASOR) Cl- currents at a pH50 of 5.3. The ASOR current was blocked by pregnenolone sulfate (PS), a steroid ion channel modulator that blocks the ASOR channel as one of its targets. The viability of the cells was reduced after exposure to an acidic medium (pH 5.3) but was slightly restored upon PS administration. CONCLUSION: These results offer first evidence for the presence of ASOR Cl- channel in ovarian cancer cells and indicate its involvement in cell viability under acidic environment.

Electrophysiology of fluoride channels in the yeasts Saccharomyces cerevisiae and Candida albicans.

Tight regulation of molecules moving through the cell membrane is particularly important for free-living microorganisms because of their small cell volumes and frequent changes in the chemical composition of the extracellular environment. This is true for nutrients, but even more so for toxic molecules. Traditionally, the transport of these diverse molecules in microorganisms has been studied on cell populations rather than on single cells, mainly because of technical difficulties. The goal of this chapter is to make available a detailed method to prepare yeast spheroplasts to study the movement of fluoride ions across the plasma membrane of single cells by the patch-clamp technique. In this procedure, three steps are critical to achieve high resistance (GΩ) seals between the membrane and the glass electrode: (1) appropriate removal of the cell wall by enzymatic treatment; (2) balance between the osmotic strength of sealing solutions and cell membrane turgor; and (3) meticulous morphological inspection of spheroplasts suitable for gigaseal formation. We show now that this method, originally developed for Saccharomyces cerevisiae, can also be applied to Candida albicans, an opportunistic human pathogen.

Structural change of the cytoplasmic N-terminus and S1 segment of voltage-sensing phosphatase reported by Anap.

BACKGROUND: Voltage-sensing phosphatase contains a structurally conserved S1-S4-based voltage-sensor domain, which undergoes a conformational transition in response to membrane potential change. Unlike that of channels, it is functional even in isolation and is therefore advantageous for studying the transition mechanism, but its nature has not yet been fully elucidated. This study aimed to address whether the cytoplasmic N-terminus and S1 exhibit structural change. METHODS: Anap, an environment-sensitive unnatural fluorescent amino acid, was site-specifically introduced to the voltage sensor domain to probe local structural changes by using oocyte voltage clamp and photometry. Tetramethylrhodamine was also used to probe some extracellularly accessible positions. In total, 51 positions were investigated. RESULTS: We detected robust voltage-dependent signals from widely distributed positions including N-terminus and S1. In addition, response to hyperpolarization was observed at the extracellular end of S1, reflecting the local structure flexibility of the voltage-sensor domain in the down-state. We also found that the mechanical coupling between the voltage-sensor and phosphatase domains affects the depolarization-induced optical signals but not the hyperpolarization-induced signals. CONCLUSIONS: These results fill a gap between the previous interpretations from the structural and biophysical approaches and should provide important insights into the mechanisms of the voltage-sensor domain transition as well as its coupling with the effector.

Using automated patch clamp electrophysiology platforms in ion channel drug discovery: an industry perspective.

INTRODUCTION: Automated patch clamp (APC) is now well established as a mature technology for ion channel drug discovery in academia, biotech and pharma companies, and in contract research organizations (CRO), for a variety of applications including channelopathy research, compound screening, target validation and cardiac safety testing. AREAS COVERED: Ion channels are an important class of drugged and approved drug targets. The authors present a review of the current state of ion channel drug discovery along with new and exciting developments in ion channel research involving APC. This includes topics such as native and iPSC-derived cells in ion channel drug discovery, channelopathy research, organellar and biologics in ion channel drug discovery. EXPERT OPINION: It is our belief that APC will continue to play a critical role in ion channel drug discovery, not only in 'classical' hit screening, target validation and cardiac safety testing, but extending these applications to include high throughput organellar recordings and optogenetics. In this way, with advancements in APC capabilities and applications, together with high resolution cryo-EM structures, ion channel drug discovery will be re-invigorated, leading to a growing list of ion channel ligands in clinical development.

Protocol to study microcircuits in the medial entorhinal cortex in mice using multiple patch-clamp recordings and morphological reconstruction.

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.

Whole-Cell Configuration of the Patch-Clamp Technique in the hERG Channel Assay.

In vitro electrophysiological safety studies have become an integral part of the drug development process because, in many instances, compound-induced QT prolongation has been associated with a direct block of human ether-a-go-go-related gene (hERG) potassium channels or their native current, the rapidly activating delayed rectifier potassium current (IKr ). Therefore, according to the ICH S7B guideline, the in vitro hERG channel patch-clamp assay is commonly used as an early screen to predict the ability of a compound to prolong the QT interval prior to first-in-human testing. The protocols described in this article are designed to assess the effects of acute or long-term exposure to new chemical entities on the amplitude of IKr in HEK293 cells stably transfected with the hERG channel (whole-cell configuration of the patch-clamp technique). Examples of results obtained with moxifloxacin, terfenadine, arsenic, pentamidine, erythromycin, and sotalol are provided for illustrative purposes. © 2024 Wiley Periodicals LLC. Basic Protocol: Measurement of the acute effects of test items in the hERG channel test Alternate Protocol: Measurement of the long-term effects of test items in the hERG channel test.

Patch-seq: Multimodal Profiling of Single-Cell Morphology, Electrophysiology, and Gene Expression.

Cells exhibit diverse morphologic phenotypes, biophysical and functional properties, and gene expression patterns. Understanding how these features are interrelated at the level of single cells has been challenging due to the lack of techniques for multimodal profiling of individual cells. We recently developed Patch-seq, a technique that combines whole-cell patch clamp recording, immunohistochemistry, and single-cell RNA-sequencing (scRNA-seq) to comprehensively profile single cells. Here we present a detailed step-by-step protocol for obtaining high-quality morphological, electrophysiological, and transcriptomic data from single cells. Patch-seq enables researchers to explore the rich, multidimensional phenotypic variability among cells and to directly correlate gene expression with phenotype at the level of single cells.

Electrophysiological Methods to Measure Ca2+ Current.

To achieve the most accurate assessment of functional Ca2+ channel or modulator properties and their regulation, a patch-clamp technique to record membrane currents is required. This technique has wide applications ranging from recording the activity of native channels in their natural environment to that of recombinant channels expressed in heterologous cells. This chapter introduces the methods that have been used for the detection of calcium release-activated calcium (CRAC) currents, one of the store-operated calcium entry pathways, in human primary T cells. This standard protocol is for laboratories already equipped with a full patch-clamp set-up or for investigators collaborating with laboratories experienced in patch clamp.

Immediate reuse of patch-clamp pipettes after ultrasonic cleaning.

The patch-clamp technique has revolutionized neurophysiology by allowing to study single neuronal excitability, synaptic connectivity, morphology, and the transcriptomic profile. However, the throughput in recordings is limited because of the manual replacement of patch-pipettes after each attempt which are often also unsuccessful. This has been overcome by automated cleaning the tips in detergent solutions, allowing to reuse the pipette for further recordings. Here, we developed a novel method of automated cleaning by sonicating the tips within the bath solution wherein the cells are placed, reducing the risk of contaminating the bath solution or internal solution of the recording pipette by any detergent and avoiding the necessity of a separate chamber for cleaning. We showed that the patch-pipettes can be used consecutively at least ten times and that the cleaning process does not negatively impact neither the brain slices nor other patched neurons. This method, combined with automated patch-clamp, highly improves the throughput for single and especially multiple recordings.

Layer 1 neocortex: Gating and integrating multidimensional signals.

Layer 1 (L1) of the neocortex acts as a nexus for the collection and processing of widespread information. By integrating ascending inputs with extensive top-down activity, this layer likely provides critical information regulating how the perception of sensory inputs is reconciled with expectation. This is accomplished by sorting, directing, and integrating the complex network of excitatory inputs that converge onto L1. These signals are combined with neuromodulatory afferents and gated by the wealth of inhibitory interneurons that either are embedded within L1 or send axons from other cortical layers. Together, these interactions dynamically calibrate information flow throughout the neocortex. This review will primarily focus on L1 within the primary sensory cortex and will use these insights to understand L1 in other cortical areas.

Voltage-clamp fluorometry to record flow-activated PIEZO1 currents and fluorometric signals.

PIEZO channels sense mechanical forces through conformational rearrangements of a mechanosensory domain called blade. To probe these rearrangements in real time, we have inserted conformational-sensitive cyclic-permuted GFP into several positions of PIEZO1's blade. Here, we describe the step-by-step experimental procedure we developed to simultaneously measure flow-activated ionic currents and fluorometric signals in cells expressing these engineered constructs. We describe steps for performing transfection, seeding cells on coverslips, setting up a perfusion-based fluid shear application system, and performing voltage-clamp fluorometry. For complete details on the use and execution of this protocol, please refer to Ozkan et al. (2023).1.

Advances in ion channel high throughput screening: where are we in 2023?

INTRODUCTION: Automated Patch Clamp (APC) technology has become an integral element in ion channel research, drug discovery and development pipelines to overcome the use of the highly time-consuming manual patch clamp (MPC) procedures. This automated technology offers increased throughput and promises a new model in obtaining ion channel recordings, which has significant relevance to the development of novel therapies and safety profiling of candidate therapeutic compounds. AREAS COVERED: This article reviews the recent innovations in APC technology, including platforms, and highlights how they have facilitated usage in both industry and academia. The review also provides an overview of the ion channel research endeavors and how APC platforms have contributed to the understanding of ion channel research, pharmacological tools and therapeutics. Furthermore, the authors provide their opinion on the challenges and goals for APC technology going forward to accelerate academic research and drug discovery across a host of therapeutic areas. EXPERT OPINION: It is clear that APC technology has progressed drug discovery programs, specifically in the field of neuroscience and cardiovascular research. The challenge for the future is to keep pace with fundamental research and improve translation of the large datasets obtained.

Patch-seq: Advances and Biological Applications.

Multimodal analysis of gene-expression patterns, electrophysiological properties, and morphological phenotypes at the single-cell/single-nucleus level has been arduous because of the diversity and complexity of neurons. The emergence of Patch-sequencing (Patch-seq) directly links transcriptomics, morphology, and electrophysiology, taking neuroscience research to a multimodal era. In this review, we summarized the development of Patch-seq and recent applications in the cortex, hippocampus, and other nervous systems. Through generating multimodal cell type atlases, targeting specific cell populations, and correlating transcriptomic data with phenotypic information, Patch-seq has provided new insight into outstanding questions in neuroscience. We highlight the challenges and opportunities of Patch-seq in neuroscience and hope to shed new light on future neuroscience research.

Signature morphoelectric properties of diverse GABAergic interneurons in the human neocortex.

Human cortex transcriptomic studies have revealed a hierarchical organization of γ-aminobutyric acid-producing (GABAergic) neurons from subclasses to a high diversity of more granular types. Rapid GABAergic neuron viral genetic labeling plus Patch-seq (patch-clamp electrophysiology plus single-cell RNA sequencing) sampling in human brain slices was used to reliably target and analyze GABAergic neuron subclasses and individual transcriptomic types. This characterization elucidated transitions between PVALB and SST subclasses, revealed morphological heterogeneity within an abundant transcriptomic type, identified multiple spatially distinct types of the primate-specialized double bouquet cells (DBCs), and shed light on cellular differences between homologous mouse and human neocortical GABAergic neuron types. These results highlight the importance of multimodal phenotypic characterization for refinement of emerging transcriptomic cell type taxonomies and for understanding conserved and specialized cellular properties of human brain cell types.

Efficient sampling-based Bayesian Active Learning for synaptic characterization.

Bayesian Active Learning (BAL) is an efficient framework for learning the parameters of a model, in which input stimuli are selected to maximize the mutual information between the observations and the unknown parameters. However, the applicability of BAL to experiments is limited as it requires performing high-dimensional integrations and optimizations in real time. Current methods are either too time consuming, or only applicable to specific models. Here, we propose an Efficient Sampling-Based Bayesian Active Learning (ESB-BAL) framework, which is efficient enough to be used in real-time biological experiments. We apply our method to the problem of estimating the parameters of a chemical synapse from the postsynaptic responses to evoked presynaptic action potentials. Using synthetic data and synaptic whole-cell patch-clamp recordings, we show that our method can improve the precision of model-based inferences, thereby paving the way towards more systematic and efficient experimental designs in physiology.

Architectural organization of ∼1,500-neuron modular minicolumnar disinhibitory circuits in healthy and Alzheimer's cortices.

Acquisition of neuronal circuit architectures, central to understanding brain function and dysfunction, remains prohibitively challenging. Here I report the development of a simultaneous and sequential octuple-sexdecuple whole-cell patch-clamp recording system that enables architectural reconstruction of complex cortical circuits. The method unveils the canonical layer 1 single bouquet cell (SBC)-led disinhibitory neuronal circuits across the mouse somatosensory, motor, prefrontal, and medial entorhinal cortices. The ∼1,500-neuron modular circuits feature the translaminar, unidirectional, minicolumnar, and independent disinhibition and optimize cortical complexity, subtlety, plasticity, variation, and redundancy. Moreover, architectural reconstruction uncovers age-dependent deficits at SBC-disinhibited synapses in the senescence-accelerated mouse prone 8, an animal model of Alzheimer's disease. The deficits exhibit the characteristic Alzheimer's-like cortical spread and correlation with cognitive impairments. These findings decrypt operations of the elementary processing units in healthy and Alzheimer's mouse cortices and validate the efficacy of octuple-sexdecuple patch-clamp recordings for architectural reconstruction of complex neuronal circuits.