Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals¹. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact^2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)^5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.     

An electrically coupled network of skeletal muscle in zebrafish distributes synaptic current

Fast and slow skeletal muscle types are readily distinguished in larval zebrafish on the basis of differences in location and orientation. Additionally, both muscle types are compact, rendering them amenable to in vivo patch clamp study of synaptic function. Slow muscle mediates rhythmic swimming, but it does so purely through synaptic drive, as these cells are unable to generate action potentials. Our patch clamp recordings from muscle pairs of zebrafish reveal a network of electrical coupling in slow muscle that allows sharing of synaptic current within and between segmental boundaries of the tail. The synaptic current exhibits slow kinetics (tau(decay) approximately 4 ms), which further facilitates passage through the low pass filter, a consequence of the electrically coupled network. In contrast to slow muscle, fast skeletal muscle generates action potentials to mediate the initial rapid component of the escape response. The combination of very weak electrical coupling and synaptic kinetics (tau(decay) <1 ms) too fast for the network low pass filter minimizes intercellular sharing of synaptic current in fast muscle. These differences between muscle types provide insights into the physiological role(s) of electrical coupling in skeletal muscle. First, intrasegmental coupling among slow muscle cells allows effective transfer of synaptic currents within tail segments, thereby minimizing differences in synaptic depolarization. Second, a fixed intersegmental delay in synaptic current transit, resulting from the low pass filter properties of the slow muscle network, helps coordinate the rostral-caudal wave of contraction.     

The Touch and Zap Method for In Vivo Whole-Cell Patch Recording of Intrinsic and Visual Responses of Cortical Neurons and Glial Cells

Whole-cell patch recording is an essential tool for quantitatively establishing the biophysics of brain function, particularly in vivo. This method is of particular interest for studying the functional roles of cortical glial cells in the intact brain, which cannot be assessed with extracellular recordings. Nevertheless, a reasonable success rate remains a challenge because of stability, recording duration and electrical quality constraints, particularly for voltage clamp, dynamic clamp or conductance measurements. To address this, we describe “Touch and Zap”, an alternative method for whole-cell patch clamp recordings, with the goal of being simpler, quicker and more gentle to brain tissue than previous approaches. Under current clamp mode with a continuous train of hyperpolarizing current pulses, seal formation is initiated immediately upon cell contact, thus the “Touch”. By maintaining the current injection, whole-cell access is spontaneously achieved within seconds from the cell-attached configuration by a self-limited membrane electroporation, or “Zap”, as seal resistance increases. We present examples of intrinsic and visual responses of neurons and putative glial cells obtained with the revised method from cat and rat cortices in vivo. Recording parameters and biophysical properties obtained with the Touch and Zap method compare favourably with those obtained with the traditional blind patch approach, demonstrating that the revised approach does not compromise the recorded cell. We find that the method is particularly well-suited for whole-cell patch recordings of cortical glial cells in vivo, targeting a wider population of this cell type than the standard method, with better access resistance. Overall, the gentler Touch and Zap method is promising for studying quantitative functional properties in the intact brain with minimal perturbation of the cell''s intrinsic properties and local network. Because the Touch and Zap method is performed semi-automatically, this approach is more reproducible and less dependent on experimenter technique.     

Effects of Nitric Oxide on Neuromuscular Properties of Developing Zebrafish Embryos

Nitric oxide is a bioactive signalling molecule that is known to affect a wide range of neurodevelopmental processes. However, its functional relevance to neuromuscular development is not fully understood. Here we have examined developmental roles of nitric oxide during formation and maturation of neuromuscular contacts in zebrafish. Using histochemical approaches we show that elevating nitric oxide levels reduces the number of neuromuscular synapses within the axial swimming muscles whilst inhibition of nitric oxide biosynthesis has the opposite effect. We further show that nitric oxide signalling does not change synapse density, suggesting that the observed effects are a consequence of previously reported changes in motor axon branch formation. Moreover, we have used in vivo patch clamp electrophysiology to examine the effects of nitric oxide on physiological maturation of zebrafish neuromuscular junctions. We show that developmental exposure to nitric oxide affects the kinetics of spontaneous miniature end plate currents and impacts the neuromuscular drive for locomotion. Taken together, our findings implicate nitrergic signalling in the regulation of zebrafish neuromuscular development and locomotor maturation.     

Functional neuromuscular junctions formed by embryonic stem cell-derived motor neurons

A key objective of stem cell biology is to create physiologically relevant cells suitable for modeling disease pathologies in vitro. Much progress towards this goal has been made in the area of motor neuron (MN) disease through the development of methods to direct spinal MN formation from both embryonic and induced pluripotent stem cells. Previous studies have characterized these neurons with respect to their molecular and intrinsic functional properties. However, the synaptic activity of stem cell-derived MNs remains less well defined. In this study, we report the development of low-density co-culture conditions that encourage the formation of active neuromuscular synapses between stem cell-derived MNs and muscle cells in vitro. Fluorescence microscopy reveals the expression of numerous synaptic proteins at these contacts, while dual patch clamp recording detects both spontaneous and multi-quantal evoked synaptic responses similar to those observed in vivo. Together, these findings demonstrate that stem cell-derived MNs innervate muscle cells in a functionally relevant manner. This dual recording approach further offers a sensitive and quantitative assay platform to probe disorders of synaptic dysfunction associated with MN disease.     

Preparation of Drosophila Central Neurons for in situ Patch Clamping

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker^1,2. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain^3,4 and ventral nerve cord of embryonic^5,6, larval^7,8,9,10, and adult Drosophila^11,12,13,14. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN5¹⁵), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.     

Nicotinic acetylcholine receptors (nAChRs) at zebrafish red and white muscle show different properties during development

Nicotinic acetylcholine receptors (nAChRs) are highly expressed at the vertebrate neuromuscular junction (NMJ) where they are required for muscle activation. Understanding the factors that underlie NMJ development is critical for a full understanding of muscle function. In this study we performed whole cell and outside‐out patch clamp recordings, and single‐cell RT‐qPCR from zebrafish red and white muscle to examine the properties of nAChRs during the first 5 days of development. In red fibers miniature endplate currents (mEPCs) exhibit single exponential time courses at 1.5 days postfertilization (dpf) and double exponential time courses from 2 dpf onwards. In white fibers, mEPCs decay relatively slowly, with a single exponential component at 1.5 dpf. By 2 and 3 dpf, mEPC kinetics speed up, and decay with a double exponential component, and by 4 dpf the exponential decay reverts back to a single component. Single channel recordings confirm the presence of two main conductance classes of nAChRs (～45 pS and ～65 pS) in red fibers with multiple time courses. Two main conductance classes are also present in white fibers (～55 pS and ～73 pS), but they exhibit shorter mean open times by 5 dpf compared with red muscle. RT‐qPCR of mRNA for nicotinic receptor subunits supports a switch from γ to ε subunits in white fibers but not in red. Our findings provide a developmental profile of mEPC properties from red and white fibers in embryonic and larval zebrafish, and reveal previously unknown differences between the NMJs of these muscle fibers.© 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 916–936, 2016     

A novel silicon patch‐clamp chip permits high‐fidelity recording of ion channel activity from functionally defined neurons

We report on a simple and high‐yield manufacturing process for silicon planar patch‐clamp chips, which allow low capacitance and series resistance from individually identified cultured neurons. Apertures are etched in a high‐quality silicon nitride film on a silicon wafer; wells are opened on the backside of the wafer by wet etching and passivated by a thick deposited silicon dioxide film to reduce the capacitance of the chip and to facilitate the formation of a high‐impedance cell to aperture seal. The chip surface is suitable for culture of neurons over a small orifice in the substrate with minimal leak current. Collectively, these features enable high‐fidelity electrophysiological recording of transmembrane currents resulting from ion channel activity in cultured neurons. Using cultured Lymnaea neurons we demonstrate whole‐cell current recordings obtained from a voltage‐clamp stimulation protocol, and in current‐clamp mode we report action potentials stimulated by membrane depolarization steps. Despite the relatively large size of these neurons, good temporal and spatial control of cell membrane voltage was evident. To our knowledge this is the first report of recording of ion channel activity and action potentials from neurons cultured directly on a planar patch‐clamp chip. This interrogation platform has enormous potential as a novel tool to readily provide high‐information content during pharmaceutical assays to investigate in vitro models of disease, as well as neuronal physiology and synaptic plasticity. Biotechnol. Bioeng. 2010;107:593–600. © 2010 Wiley Periodicals, Inc.     

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents. The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.Download video file.^{(217M, mp4)}     

Integration of Sensory Quanta in Cuneate Nucleus Neurons In Vivo

Discriminative touch relies on afferent information carried to the central nervous system by action potentials (spikes) in ensembles of primary afferents bundled in peripheral nerves. These sensory quanta are first processed by the cuneate nucleus before the afferent information is transmitted to brain networks serving specific perceptual and sensorimotor functions. Here we report data on the integration of primary afferent synaptic inputs obtained with in vivo whole cell patch clamp recordings from the neurons of this nucleus. We find that the synaptic integration in individual cuneate neurons is dominated by 4–8 primary afferent inputs with large synaptic weights. In a simulation we show that the arrangement with a low number of primary afferent inputs can maximize transfer over the cuneate nucleus of information encoded in the spatiotemporal patterns of spikes generated when a human fingertip contact objects. Hence, the observed distributions of synaptic weights support high fidelity transfer of signals from ensembles of tactile afferents. Various anatomical estimates suggest that a cuneate neuron may receive hundreds of primary afferents rather than 4–8. Therefore, we discuss the possibility that adaptation of synaptic weight distribution, possibly involving silent synapses, may function to maximize information transfer in somatosensory pathways.     

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously^1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.     

Blind patch clamp recordings in embryonic and adult mammalian brain slices

To obtain electrophysiological recordings in brain slices, sophisticated and expensive pieces of equipment can be used. However, costly microscope equipment with infrared differential interference contrast optics is not always necessary or even desirable. For instance, obtaining a randomized unbiased sample in a given preparation would be better accomplished if cells were not directly visualized before recording. In addition, some preparations require thick slices, and direct visualization is not possible. Here we describe a protocol for the 'blind patch clamp method' that we developed several years ago to perform electrophysiological recordings in mammalian brain slices using a standard patch clamp amplifier, dissecting microscope and recording chamber. Overall, it takes approximately 3-4 h to set up this procedure.     

Intracellular Neural Recording with Pure Carbon Nanotube Probes

The computational complexity of the brain depends in part on a neuron’s capacity to integrate electrochemical information from vast numbers of synaptic inputs. The measurements of synaptic activity that are crucial for mechanistic understanding of brain function are also challenging, because they require intracellular recording methods to detect and resolve millivolt- scale synaptic potentials. Although glass electrodes are widely used for intracellular recordings, novel electrodes with superior mechanical and electrical properties are desirable, because they could extend intracellular recording methods to challenging environments, including long term recordings in freely behaving animals. Carbon nanotubes (CNTs) can theoretically deliver this advance, but the difficulty of assembling CNTs has limited their application to a coating layer or assembly on a planar substrate, resulting in electrodes that are more suitable for in vivo extracellular recording or extracellular recording from isolated cells. Here we show that a novel, yet remarkably simple, millimeter-long electrode with a sub-micron tip, fabricated from self-entangled pure CNTs can be used to obtain intracellular and extracellular recordings from vertebrate neurons in vitro and in vivo. This fabrication technology provides a new method for assembling intracellular electrodes from CNTs, affording a promising opportunity to harness nanotechnology for neuroscience applications.     

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of Förster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.     

MicroRNA-8 promotes robust motor axon targeting by coordinate regulation of cell adhesion molecules during synapse development

Neuronal connectivity and specificity rely upon precise coordinated deployment of multiple cell-surface and secreted molecules. MicroRNAs have tremendous potential for shaping neural circuitry by fine-tuning the spatio-temporal expression of key synaptic effector molecules. The highly conserved microRNA miR-8 is required during late stages of neuromuscular synapse development in Drosophila. However, its role in initial synapse formation was previously unknown. Detailed analysis of synaptogenesis in this system now reveals that miR-8 is required at the earliest stages of muscle target contact by RP3 motor axons. We find that the localization of multiple synaptic cell adhesion molecules (CAMs) is dependent on the expression of miR-8, suggesting that miR-8 regulates the initial assembly of synaptic sites. Using stable isotope labelling in vivo and comparative mass spectrometry, we find that miR-8 is required for normal expression of multiple proteins, including the CAMs Fasciclin III (FasIII) and Neuroglian (Nrg). Genetic analysis suggests that Nrg and FasIII collaborate downstream of miR-8 to promote accurate target recognition. Unlike the function of miR-8 at mature larval neuromuscular junctions, at the embryonic stage we find that miR-8 controls key effectors on both sides of the synapse. MiR-8 controls multiple stages of synapse formation through the coordinate regulation of both pre- and postsynaptic cell adhesion proteins.     

豚鼠不同部位微动脉平滑肌细胞电生理学特性的比较

本研究应用电生理技术在豚鼠离体小脑前下动脉(anterior inferior cerebellar artery,AICA)、肠系膜动脉(mesenteric artery,MA)和耳蜗螺旋动脉(spiral modiolar artery,SMA)分支(直径小于100μm)上比较微动脉平滑肌细胞电生理学特性的异同。结果显示:(1)应用细胞内微电极记录技术测得AICA、MA和SMA细胞静息膜电位分别为(-68±1.8)(n=65)、(-71±2.4)(n=80)和(-66±2.9)mV(n=58),各微动脉间无统计学差异。(2)一段血管微动脉标本全细胞膜片钳记录的平滑肌细胞膜电容和膜电导都远大于单个细胞标本,且微动脉间存在统计学差异,大小顺序为MAAICASMA。应用缝隙连接阻断剂2-APB(100μmol/L)后记录一段微动脉平滑肌细胞膜电容和膜电导与单个细胞十分接近。(3)AICA、MA和SMA单个平滑肌细胞膜电流I/V关系呈明显的外向整流特性,都对1mmol/L4-AP和10mmol/LTEA敏感。当指令电压为+40mV时,AICA、MA和SMA血管平滑肌细胞电流密度分别为(26±2.0)、(24±1.7)和(18±1.3)pA/pF,SMA和AICA、MA间存在统计学差异。上述结果提示,豚鼠不同部位微动脉平滑肌细胞在缝隙连接耦联力和电流密度等电生理特性存在差异。     

Micromolded PDMS planar electrode allows patch clamp electrical recordings from cells

The patch clamp method measures membrane currents at very high resolution when a high-resistance 'gigaseal' is established between the glass microelectrode and the cell membrane (Pflugers Arch. 391 (1981) 85; Neuron 8 (1992) 605). Here we describe the first use of the silicone elastomer, poly(dimethylsiloxane) (PDMS), for patch clamp electrodes. PDMS is an attractive material for patch clamp recordings. It has low dielectric loss and can be micromolded (Annu. Rev. Mat. Sci. 28 (1998) 153) into a shape that mimics the tip of the glass micropipette. Also, the surface chemistry of PDMS may be altered to mimic the hydrophilic nature of glass (J. Appl. Polym. Sci. 14 (1970) 2499; Annu. Rev. Mat. Sci. 28 (1998) 153), thereby allowing a high-resistance seal to a cell membrane. We present a planar electrode geometry consisting of a PDMS partition with a small aperture sealed between electrode and bath chambers. We demonstrate that a planar PDMS patch electrode, after oxidation of the elastomeric surface, permits patch clamp recording on Xenopus oocytes. Our results indicate the potential for high-throughput patch clamp recording with a planar array of PDMS electrodes.     

Reliability in the identification of midbrain dopamine neurons

Brain regions typically contain intermixed subpopulations of neurons with different connectivity and neurotransmitters. This complicates identification of neuronal phenotypes in electrophysiological experiments without using direct detection of unique molecular markers. A prime example of this difficulty is the identification of dopamine (DA) neurons in the midbrain ventral tegmental area (VTA). Although immunocytochemistry (ICC) against tyrosine hydroxylase (TH) is widely used to identify DA neurons, a high false negative rate for TH ICC following ex vivo electrophysiology experiments was recently reported, calling into question the validity of comparing DA and non-DA VTA neurons based on post-hoc ICC. However, in whole cell recordings from randomly selected rat VTA neurons we have found that TH labeling is consistently detected in ∼55% of neurons even after long recording durations (range: 2.5–150 min). This is consistent with our prior anatomical finding that 55% of VTA neurons are TH(+). To directly estimate a false negative rate for our ICC method we recorded VTA neurons from mice in which EGFP production is driven by the TH promoter. All 12 EGFP(+) neurons recorded with a K-gluconate internal solution (as used in our rat recordings) were strongly labeled by TH ICC (recording duration 16.6±1.8 min). However, using recording electrodes with an internal solution with high Cl⁻ concentration reduced the intensity of TH co-labeling, in some cases to background (recording duration 16.7±0.9 min; n = 10). Thus TH is a highly reliable molecular marker for DA neurons in VTA patch clamp recordings provided compatible microelectrode solutions are used.     

Methods for patch clamp capacitance recordings from the calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.