Rigorous investigation of synaptic transmission requires analysis of unitary synaptic events by simultaneous recording from presynaptic terminals and postsynaptic target neurons. However, this has been achieved at only a limited number of model synapses, including the squid giant synapse and the mammalian calyx of Held. Cortical presynaptic terminals have been largely inaccessible to direct presynaptic recording, due to their small size. Here, we describe a protocol for improved subcellular patch-clamp recording in rat and mouse brain slices, with the synapse in a largely intact environment. Slice preparation takes ~2 h, recording ~3 h and post hoc morphological analysis 2 d. Single presynaptic hippocampal mossy fiber terminals are stimulated minimally invasively in the bouton-attached configuration, in which the cytoplasmic content remains unperturbed, or in the whole-bouton configuration, in which the cytoplasmic composition can be precisely controlled. Paired pre-postsynaptic recordings can be integrated with biocytin labeling and morphological analysis, allowing correlative investigation of synapse structure and function. Paired recordings can be obtained from mossy fiber terminals in slices from both rats and mice, implying applicability to genetically modified synapses. Paired recordings can also be performed together with axon tract stimulation or optogenetic activation, allowing comparison of unitary and compound synaptic events in the same target cell. Finally, paired recordings can be combined with spontaneous event analysis, permitting collection of miniature events generated at a single identified synapse. In conclusion, the subcellular patch-clamp techniques detailed here should facilitate analysis of biophysics, plasticity and circuit function of cortical synapses in the mammalian central nervous system.

Patch clamp recording of neurons is a labor-intensive and time-consuming procedure. Here, we demonstrate a tool that fully automatically performs electrophysiological recordings in label-free tissue slices. The automation covers the detection of cells in label-free images, calibration of the micropipette movement, approach to the cell with the pipette, formation of the whole-cell configuration, and recording. The cell detection is based on deep learning. The model is trained on a new image database of neurons in unlabeled brain tissue slices. The pipette tip detection and approaching phase use image analysis techniques for precise movements. High-quality measurements are performed on hundreds of human and rodent neurons. We also demonstrate that further molecular and anatomical analysis can be performed on the recorded cells. The software has a diary module that automatically logs patch clamp events. Our tool can multiply the number of daily measurements to help brain research.

Live calcium imaging is often used as a proxy for electrophysiological measurements and has been a valuable tool that allows simultaneous analysis of neuronal activity in multiple cells at the population level. In the enteric nervous system, there are two main electrophysiological classes of neurons, after-hyperpolarizing (AH)- and synaptic (S)-neurons, which have been shown to have different calcium handling mechanisms. However, they are rarely considered separately in calcium imaging experiments. A handful of studies have shown that in guinea pig, a calcium transient will accompany a single action potential in AH-neurons, but multiple action potentials are required to generate a calcium transient in S-neurons. How this translates to different modes of cellular depolarization and whether this is consistent across species is unknown. In this study, we used simultaneous whole-cell patch-clamp electrophysiology together with calcium imaging to investigate how enteric neurons respond to different modes of depolarization. Using both traditional (4 Hz) and also high-speed (1,000 Hz) imaging techniques, we found that single action potentials elicit calcium transients in both AH-neurons and S-neurons. Subthreshold membrane depolarizations were also able to elicit calcium transients, although calcium responses were generally amplified if an action potential was present. Furthermore, we identified that responses to nicotinic acetylcholine receptor stimulation can be used to distinguish between AH- and S-neurons in calcium imaging.NEW & NOTEWORTHY Live calcium imaging is an important tool for investigating enteric nervous system (ENS) function. Previous studies have shown that multiple action potentials are needed to generate a calcium response in S-neurons, which has important implications for the interpretation of calcium imaging data. Here, we show that in mouse myenteric neurons, calcium transients are elicited by single action potentials in both AH- and S-neurons. In addition, nicotinic acetylcholine receptor stimulation can be used to distinguish between these two classes.

The chloride (Cl-) channel cystic fibrosis transmembrane conductance regulator (CFTR) is defective in cystic fibrosis (CF), and mutation of its encoding gene leads to various defects such as retention of the misfolded protein in the endoplasmic reticulum, reduced stability at the plasma membrane, abnormal channel gating with low open probability, and thermal instability, which leads to inactivation of the channel at physiological temperature. Pharmacotherapy is one major therapeutic approach in the CF field and needs sensible and fast tools to identify promising compounds. The high throughput screening assays available are often fast and sensible techniques but with lack of specificity. Few works used automated patch clamp (APC) for CFTR recording, and none have compared conventional and planar techniques and demonstrated their capabilities for different types of experiments. In this study, we evaluated the use of planar parallel APC technique for pharmacological search of CFTR-trafficking correctors and CFTR function modulators. Using optimized conditions, we recorded both wt- and corrected F508del-CFTR Cl- currents with automated whole-cell patch clamp and compared the data to results obtained with conventional manual whole-cell patch clamp. We found no significant difference in patch clamp parameters such as cell capacitance and series resistance between automated and manual patch clamp. Also, the results showed good similarities of CFTR currents recording between the two methods. We showed that similar stimulation protocols could be used in both manual and automatic techniques allowing precise control of temperature, classic I/V relationship, and monitoring of current stability in time. In conclusion, parallel patch-clamp recording allows rapid and efficient investigation of CFTR currents with a variety of tests available and could be considered as new tool for medium throughput screening in CF pharmacotherapy.

In vivo patch-clamp recording techniques provide access to the sub- and suprathreshold membrane potential dynamics of individual neurons during behavior. However, maintaining recording stability throughout behavior is a significant challenge, and while methods for head restraint are commonly used to enhance stability, behaviorally related brain movement relative to the skull can severely impact the success rate and duration of whole-cell patch-clamp recordings.

Patch-clamp and multi-electrode array electrophysiology techniques are used to measure dynamic functional properties of neurons. Whole-cell and cell-attached patch-clamp recordings in brain slices can be performed in voltage-clamp and current-clamp configuration to reveal cell-type-specific synaptic and cellular parameters governing neurotransmission. Multi-electrode array electrophysiology can provide spike activity recordings from multiple neurons, enabling larger sample sizes, and long-term recordings. We provide our guide to preparing acute rodent brain slices with example experiments and analyses intended for novice and expert electrophysiologists. For complete details on the use and execution of this protocol, please refer to Manz et al. (2020b).

Standard high throughput screening projects using automated patch-clamp instruments often fail to grasp essential details of the mechanism of action, such as binding/unbinding dynamics and modulation of gating. In this study, we aim to demonstrate that depth of analysis can be combined with acceptable throughput on such instruments. Using the microfluidics-based automated patch clamp, IonFlux Mercury, we developed a method for a rapid assessment of the mechanism of action of sodium channel inhibitors, including their state-dependent association and dissociation kinetics. The method is based on a complex voltage protocol, which is repeated at 1 Hz. Using this time resolution we could monitor the onset and offset of both channel block and modulation of gating upon drug perfusion and washout. Our results show that the onset and the offset of drug effects are complex processes, involving several steps, which may occur on different time scales. We could identify distinct sub-processes on the millisecond time scale, as well as on the second time scale. Automated analysis of the results allows collection of detailed information regarding the mechanism of action of individual compounds, which may help the assessment of therapeutic potential for hyperexcitability-related disorders, such as epilepsies, pain syndromes, neuromuscular disorders, or neurodegenerative diseases.

Pancreatic ATP-sensitive K+ channels (KATP) comprise four inward rectifier subunits (Kir6.2), each associated with a sulphonylurea receptor (SUR1). ATP/ADP binding to Kir6.2 shuts KATP. Mg-nucleotide binding to SUR1 stimulates KATP. In the absence of Mg2+, SUR1 increases the apparent affinity for nucleotide inhibition at Kir6.2 by an unknown mechanism. We simultaneously measured channel currents and nucleotide binding to Kir6.2. Fits to combined data sets suggest that KATP closes with only one nucleotide molecule bound. A Kir6.2 mutation (C166S) that increases channel activity did not affect nucleotide binding, but greatly perturbed the ability of bound nucleotide to inhibit KATP. Mutations at position K205 in SUR1 affected both nucleotide affinity and the ability of bound nucleotide to inhibit KATP. This suggests a dual role for SUR1 in KATP inhibition, both in directly contributing to nucleotide binding and in stabilising the nucleotide-bound closed state.

Patch-clamp recording techniques have revolutionized understanding of the function and sub-cellular location of ion channels in excitable cells. The cell-attached patch-clamp configuration represents the method of choice to describe the endogenous properties of voltage-activated ion channels in the axonal, somatic and dendritic membrane of neurons, without disturbance of the intracellular milieu. Here, we directly examine the errors associated with cell-attached patch-clamp measurement of ensemble ion channel activity. We find for a number of classes of voltage-activated channels, recorded from the soma and dendrites of neurons in acute brain-slices and isolated cells, that the amplitude and kinetics of ensemble ion channel activity recorded in cell-attached patches is significantly distorted by transmembrane voltage changes generated by the flow of current through the activated ion channels. We outline simple error-correction procedures that allow a more accurate description of the density and properties of voltage-activated channels to be incorporated into computational models of neurons.

Single-molecule research techniques such as patch-clamp electrophysiology deliver unique biological insight by capturing the movement of individual proteins in real time, unobscured by whole-cell ensemble averaging. The critical first step in analysis is event detection, so called "idealisation", where noisy raw data are turned into discrete records of protein movement. To date there have been practical limitations in patch-clamp data idealisation; high quality idealisation is typically laborious and becomes infeasible and subjective with complex biological data containing many distinct native single-ion channel proteins gating simultaneously. Here, we show a deep learning model based on convolutional neural networks and long short-term memory architecture can automatically idealise complex single molecule activity more accurately and faster than traditional methods. There are no parameters to set; baseline, channel amplitude or numbers of channels for example. We believe this approach could revolutionise the unsupervised automatic detection of single-molecule transition events in the future.

Thoracic paravertebral sympathetic postganglionic neurons (tSPNs) comprise the final integrative output of the distributed sympathetic nervous system controlling vascular and thermoregulatory systems. Considered a non-integrating relay, what little is known of tSPN intrinsic excitability has been determined by sharp microelectrodes with presumed impalement injury. We thus undertook the first electrophysiological characterization of tSPN cellular properties using whole-cell recordings and coupled results with a conductance-based model to explore the principles governing their excitability in adult mice of both sexes. Recorded membrane resistance and time constant values were an order of magnitude greater than values previously obtained, leading to a demonstrable capacity for synaptic integration in driving recruitment. Variation in membrane resistivity was the primary determinant controlling cell excitability with vastly lower currents required for tSPN recruitment. Unlike previous microelectrode recordings in mouse which observed inability to sustain firing, all tSPNs were capable of repetitive firing. Computational modeling demonstrated that observed differences are explained by introduction of a microelectrode impalement injury conductance. Overall, tSPNs largely linearly encoded injected current magnitudes over a broad frequency range with distinct subpopulations differentiable based on repetitive firing signatures. Thus, whole-cell recordings reveal tSPNs have more dramatically amplified excitability than previously thought, with greater intrinsic capacity for synaptic integration and with the ability for maintained firing to support sustained actions on vasomotor tone and thermoregulatory function. Rather than acting as a relay, these studies support a more responsive role and possible intrinsic capacity for tSPNs to drive sympathetic autonomic function.

1. Simultaneous dendritic and somatic patch-clamp recordings were made from Purkinje cells in cerebellar slices from 12- to 21-day-old rats. Voltage responses to current impulses injected via either the dendritic or the somatic pipette were obtained in the presence of the selective I(h) blocker ZD 7288 and blockers of spontaneous synaptic input. Neurons were filled with biocytin for subsequent morphological reconstruction. 2. Four neurons were reconstructed and converted into detailed compartmental models. The specific membrane capacitance (C(m)), specific membrane resistance (R(m)) and intracellular resistivity (R(i)) were optimized by direct fitting of the model responses to the electrophysiological data from the same cell. Mean values were: C(m), 0.77 +/- 0.17 microF cm(-2) (mean +/- S.D.; range, 0.64-1.00 microF cm(-2)), R(m), 122 +/- 18 kOmega cm(2) (98-141 kOmega cm(2)) and R(i), 115 +/- 20 Omega cm (93-142 Omega cm). 3. The steady-state electrotonic architecture of these cells was compact under the experimental conditions used. However, somatic voltage-clamp recordings of parallel fibre and climbing fibre synaptic currents were substantially filtered and attenuated. 4. The detailed models were compared with a two-compartment model of Purkinje cells. The range of synaptic current kinetics that can be faithfully recorded using somatic voltage clamp is predicted fairly well by the two-compartment model, even though some of its underlying assumptions are violated. 5. A model of I(h) was constructed based on voltage-clamp data, and inserted into the passive compartmental models. Somatic EPSP amplitude was substantially attenuated compared to the amplitude of dendritic EPSPs at their site of generation. However, synaptic efficacy of the same quantal synaptic conductance, as measured by the somatic EPSP amplitude, was only weakly dependent on synaptic location on spiny branchlets. 6. The passive electrotonic structure of Purkinje cells is unusual in that the steady-state architecture is very compact, while voltage transients such as synaptic potentials and action potentials are heavily filtered.

In the present study we have characterized the biophysical properties of wild-type (WT) α1β2 and α3β2 GABAA receptors and probed the molecular basis for the observed differences. The activation and desensitization behavior and the residual currents of the receptors expressed in HEK293 cells were determined in whole-cell patch clamp recordings. Kinetic parameters of α1β2 and α3β2 activation differed significantly, with α1β2 and α3β2 exhibiting rise times (10-90%) of 24 ± 2 ms and 51 ± 7 ms, respectively. In contrast, the two receptors exhibited largely comparable desensitization behavior with decay currents that could be fitted to exponential functions with two or three components. Most notably, the two receptor compositions displayed different degrees of desentization, with the residual currents of α1β2 and α3β2 constituting 34 ± 2% and 21 ± 2% of the peak current, respectively. The respective contributions of the extracellular domains and the transmembrane/intracellular domains of the α-subunit to these physiological profiles were next assessed in recordings from cells expressing αβ2 receptors comprising chimeric α-subunits. The rise times displayed by α1ECD/α3TMDβ2 and α3ECD/α1TMDβ2 receptors were intermediate to those of WT α1β2 and WT α3β2, and the distribution of the different components of the current decays exhibited by the two chimeric receptors followed the same pattern as the two WT receptors. The residual current exhibited by α1ECD/α3TMDβ2 (23 ± 3%) was similar to that of α3β2 but significantly different from that of α1β2, whereas the residual current displayed by α3ECD/α1TMDβ2 (27 ± 2%) was intermediate to and did not differ significantly from either of the WT receptors. This points to molecular differences in the transmembrane/intracellular domains of the α-subunit as the main determinants of the observed differences in receptor physiology between α1β2 and α3β2 receptors.

We have used anatomical methods and whole-cell patch-clamp recording to assess the distribution of nicotinic receptors in the tectum of Xenopus frogs and to measure effects of nicotinic ligands (carbachol, cytisine and nicotine) on glutamatergic spontaneous miniature excitatory postsynaptic currents. Our results confirm that retinotectal axons account for the majority of nicotinic receptors in the tectum and that nicotinic agonists exert presynaptic effects that increase the rate of transmitter release on to tectal cells. The nicotinic blockers mecamylamine and methyllycaconitine reduced responses to carbachol and cytisine. A small percentage of cells also showed postsynaptic responses. We have assessed whether there are developmental changes in the frequency of occurrence of spontaneous miniature excitatory postsynaptic currents. The first three months post-metamorphosis fall within the critical period for the dramatic plasticity displayed by binocular inputs during development in Xenopus. During this period, visual activity governs the formation of orderly maps relayed from the ipsilateral eye via the cholinergic projection from the nucleus isthmi to the tectum. In this study, we have found that critical-period tecta (two to 12 weeks postmetamorphosis) tend to have higher spontaneous activity than do older tecta (two to 69 weeks postmetamorphosis), and that nicotinic agonists increase that activity in both groups, with the result that the peak rates in response to nicotinic agonists are higher during the critical period than later. We also investigated the possible role of choline as an agonist of nicotinic receptors in the tectum. We have found that choline, as well as carbachol and cytisine, can cause a reversible increase in the rate of miniature excitatory postsynaptic currents. This result may help to explain how the isthmotectal projection, which accounts for the overwhelming majority of cholinergic input to the tectum, can exert effects on retinotectal terminals even though there are no morphologically identifiable synapses between the two populations. We have examined the morphology of cells filled with biocytin during the patch-clamp experiments, and we find that cells with dendrites in the stratum zonale, a layer with particularly dense input from the contralateral nucleus isthmi, have higher spontaneous activity than cells with dendrites that do not extend into that layer. Nicotinic agonists increased the activity recorded in both classes of cells. In addition, four pretectal cells were identified. Nicotinic agonists increased the rate of spontaneous activity recorded in that population. The results indicate that retinotectal transmission in the superior colliculus can be increased presynaptically by activity of the cholinergic projections of the nucleus isthmi. This modulation may be the basis for observations that blocking of cholinergic input disrupts the formation of topographic retinotectal projections. Moreover, the ability of choline to activate these receptors suggests that this metabolite of acetylcholine may permit paracrine activation of presynaptic receptors even though the tectum contains high acetylcholinesterase activity.

SARS-CoV-2 has caused a worldwide pandemic since December 2019 and the search for pharmaceutical targets against COVID-19 remains an important challenge. Here, we studied the envelope protein E of SARS-CoV and SARS-CoV-2, a highly conserved 75-76 amino acid viroporin that is crucial for virus assembly and release. E protein channels were recombinantly expressed in HEK293 cells, a membrane-directing signal peptide ensured transfer to the plasma membrane.

The NMDA receptor antagonistic effects of budipine were assessed using concentration- and patch-clamp techniques on cultured striatal, hippocampal, cortical and superior colliculus neurones. Inward current responses of striatal neurones to NMDA (200 microM) at -70 mV were antagonized by budipine in a concentration-dependent manner (50% inhibitory concentration (IC50) 59.4 +/- 10.7 microM, n = 17) with 24 times lower potency than memantine but similar potency to amantadine. In striatal neurones, budipine blocked outward currents at +70 mV with an IC50 of 827 microM, suggesting that the binding site is less deep in the channel (delta = 0.45) than for memantine. However, more detailed analysis of the fractional block by budipine 300 microM in hippocampal neurones gave a delta-value of 0.90, but revealed that 28% block is mediated at a voltage-independent site. This voltage-insensitive site was accessible in the absence of agonist. Budipine exhibited concentration-dependent open channel blocking kinetics (kappa(on) = 0.71 x 10(4) M(-1) s(-1)) whereas the fast offset rate was concentration-independent (kappa(off) = 0.63 s(-1)). Calculation of the ratio kappa(off)/kappa(on) revealed an apparent Kd value of 88.7 microM. Budipine, memantine and amantadine had similar effects against NMDA-induced currents in cultured hippocampal, cortical and superior colliculus neurones, although amantadine was somewhat more potent in cultured striatal neurones. The relevance of NMDA receptor antagonism to the anti-Parkinsonian effects of budipine remains to be established.

In a previous study, we demonstrated that inflammation suppressed inward rectifying K(+) (Kir) currents in satellite glial cells (SGCs) from the trigeminal ganglia (TRGs) and that this impairment of glial potassium homeostasis in the trigeminal ganglion (TRG) contributed to trigeminal pain. The aim of the present study was to investigate whether activation of GABAB receptors modulates the Kir current in SGCs using in vivo patch-clamp and immunohistochemical techniques. Immunohistochemically, we found that immunoreactivity for glial-specific Kir channel subunit Kir4.1 and the GABAB receptor was co-expressed in SGCs from the TRGs. In vivo whole-cell recordings were made using SGCs from the TRGs of urethane-anesthetized rats. Application of baclofen, a GABAB receptor agonist, significantly increased the mean peak amplitude of Kir currents in a concentration-dependent and reversible manner. Baclofen-induced potentiation of the Kir current was abolished by co-application of 3-amino-2-(4-chlorophenyl)-2-hydroxyprophylsulfonic acid (saclofen). In addition, baclofen significantly potentiated the density of the Ba(2+)-sensitive Kir current, and resulted in hyperpolarization of the mean membrane potential. These results suggest that activation of GABAB receptors potentiates the Kir current in SGCs and that GABA released from the TRG neuronal soma could contribute to buffering of extracellular K(+) concentrations following excitation of TRG neurons during the processing of sensory information, including the transmission of noxious stimuli.

Single-cell transcriptomic approaches are revolutionizing neuroscience. Integrating this wealth of data with morphology and physiology, for the comprehensive study of neuronal biology, requires multiplexing gene expression data with complementary techniques. To meet this need, multiple groups in parallel have developed "Patch-seq," a modification of whole-cell patch-clamp protocols that enables mRNA sequencing of cell contents after electrophysiological recordings from individual neurons and morphologic reconstruction of the same cells. In this review, we first outline the critical technical developments that enabled robust Patch-seq experimental efforts and analytical solutions to interpret the rich multimodal data generated. We then review recent applications of Patch-seq that address novel and long-standing questions in neuroscience. These include the following: (1) targeted study of specific neuronal populations based on their anatomic location, functional properties, lineage, or a combination of these factors; (2) the compilation and integration of multimodal cell type atlases; and (3) the investigation of the molecular basis of morphologic and functional diversity. Finally, we highlight potential opportunities for further technical development and lines of research that may benefit from implementing the Patch-seq technique. As a multimodal approach at the intersection of molecular neurobiology and physiology, Patch-seq is uniquely positioned to directly link gene expression to brain function.

To resolve the subcellular distribution of endolysosomal ion channels, we have established a novel experimental approach to selectively patch clamp Rab5 positive early endosomes (EE) versus Rab7/LAMP1-positive late endosomes/lysosomes (LE/LY). To functionally characterize ion channels in endolysosomal membranes with the patch-clamp technique, it is important to develop techniques to selectively enlarge the respective organelles. We found here that two small molecules, wortmannin and latrunculin B, enlarge Rab5-positive EE when combined but not Rab7-, LAMP1-, or Rab11 (RE)-positive vesicles. The two compounds act rapidly, specifically, and are readily applicable in contrast to genetic approaches or previously used compounds such as vacuolin, which enlarges EE, RE, and LE/LY. We apply this approach here to measure currents mediated by TRPML channels, in particular TRPML3, which we found to be functionally active in both EE and LE/LY in overexpressing cells as well as in endogenously expressing CD11b+ lung-tissue macrophages.

The patch clamp method is a widely applied electrophysiological technique used to understand ion channel activity and cellular excitation. The formation of a high resistance giga-ohm seal is required to obtain high-quality recordings but can be challenging due to variables including operator experience and cell preparation. Therefore, the identification of methods to promote the formation and longevity of giga-ohm seals may be beneficial. In this report, we describe our observation that the application of reducing agents (DTT and TCEP) to the external bath solution during whole-cell patch clamp recordings of heterologous cells (HEK and LM) and cultured primary cells (DRG neurons) enhanced the success of giga-ohm seal formation. Reducing agents also maintained the integrity of the seal for longer periods of time at strong hyperpolarizing voltages, whereas an oxidizing agent (H2O2) appeared to have the opposite effect. In summary, we report a useful tool to improve the quality of patch clamp recordings that may be helpful in certain experimental contexts.