The pressure-polishing technique was developed in collaboration with S. R. Lockery.

Pulling pipettes
Using a filament puller such as a Sutter P-97 Flaming/Brown Microelectrode Puller, pull a set of approximately 10-20 pipettes.
<ol>
<li>Select your glass. We use borosilicate (BF150-86-10, OD = 1.5 mm, ID = 0.86 mm). Store the glass carefully so it remains clean and dust free.</li>
<li>Design a pulling program. The primary goal is a tip opening approximately the desired size, which will depend on application. Sutter's P-97 Pipette Cookbook 2 is an excellent reference.</li>
<li>Observe your pipettes under a microscope to determine opening diameter and smoothness. Discard rough, uneven, or irregular tips. For standard patch clamp recording, a tip opening that is 2-4 &mu;m across is best. A blunt tip leads to lower resistance for the same opening diameter, which is preferred. Shape and size can be modified during polishing, below.</li>
</ol>
Pressure polishing pipettes
<ol>
<li>Attach a pipette holder to a clean, compressed air line. Using a regulator, set the compressed air line pressure to ~40 PSI. Connect the compressed air line to a 4-way control valve that will allow the pipette holder pressure to be turned on and off. Connect a pipette holder to the control valve with a length of &#x215B; in Tygon&reg; tubing.</li>
<li>Use a polishing rig (microforge) with a high-pressure pipette holder, a polishing coil controlled by a foot-pedal, a microscope equipped with a low and high magnification, long working-distance (ideally, 10x and 100x) air objectives. It is critical to be able to observe the pipette opening during the protocol.</li>
<li>Optional:&nbsp;Coat the pipette with a hydrophobic insulator to decrease capacitance and improve noise characteristics (see "Pulling pipettes" for this technique).</li>
<li>Verify the pressure valve is in the off position. Place a pipette into the pipette holder and tighten the fitting to finger-tight. Safety note: If the pipette is held tightly into the pipette holder, the air pressure will cause the pipette to be ejected from the pipette holder. </li>
<li>Place the pipette and holder in the microforge. Position the pipette holder onto the microscope stage so that both the tip and the polishing coil are visible at low (10x) power on the microforge. Bring the tip ~75 microns from the filament . Keep in mind that the filament will expand when heated.</li>
<li>Switch to the high power (100x) objective. Both the pipette tip and the polishing coil should be in focus and in the field of view. If not, adjust the height of the polishing coil. (The pipette&rsquo;s position cannot be adjusted). The pipette tip and polishing coil should be close to opposite sides of the field of view. Avoid positioning the coil too close to the pipette tip because it will expand as it heats and bump into your pipette.</li>
<li>Turn the valve to direct pressurized air into the pipette, and heat the pipette tip until the taper behind the tip begins to balloon outwards.</li>
<li>Stop heating before the tip balloons too extensively. Experimentation is required to determine the appropriate distance, heat, and time for the desired shape.</li>
<li>Optional: Reduce the tip opening diameter by polishing the tip in the absence of pressure. Steps 7 and 8 can repeated to fine-tune both the shape and dimensions of the pipette tip.</li>
<li>Switch off the pressure and while pointing the pipette tip in a safe direction, remove the pipette and place it in a clean storage box where it will be protected from dust. Safety note: If the air pressure is not turned off, the pipette can be ejected from the pipette holder during removal.</li>
</ol>
Notes about the microforge
We use a microforge assembled from off-the-shelf commercially available parts. (You can also buy a pressure-polishing kit from a few companies, e.g., ALA Science, GlassWorx). Regardless of the microforge, it is essential to have optics that are good enough to see the tip opening, which is often only a few &micro;m in diameter. We use a long-working distance 100x lens for this purpose. Also, coat the polishing coil with high-melting temperature glass in order to minimize depositing vaporized metal on the pipettes.
Part 3: Anticipated Results
Moderate pressure polishing can reduce the resistance of a pipette without an appreciable change in opening diameter. If desired, this protocol can be used to make pipettes with sub-micron openings and resistances between 5-10M&Omega;.

<ol>
	<li>Goodman, M. B., Lockery, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure+polishing:+a+method+for+re-shaping+patch+pipettes+during+fire+polishing. .">Pressure polishing: a method for re-shaping patch pipettes during fire polishing. .</a> J Neurosci Methods. 100, 13-15 (2000).</li></ol>

We originally developed this method to make pipettes useful for patch-clamp recording from 2-3 &micro;m neurons in C. elegans 1. It is in routine use in the Goodman laboratory. We pressure-polish pipettes not only for recording from tiny C. elegans neurons, but also for obtaining macropatch and single-channel recordings of ion channels expressed in Xenopus oocytes. For a given tip-opening diameter, the blunt shape produces a lower-resistance pipette. Lower pipette resistance leads to lower se...

<table><tbody><tr><td>Glass Capillaries</td><td>Consumable</td><td>Sutter Instrument Co.</td><td>BF150-85-10 (or similar)</td><td>The method works with any type of capillary except those made from quartz. However, we recommend using capillaries with a filament.</td></tr><tr><td>Inverted Microscope</td><td>Microscope</td><td>Leica Microsystems</td><td>DM IL (or similar)</td><td>Any simple inverted microscope will do. However, it is essential to have sufficient magnification to see pipettes during polishing. We use a 100x metallurgical lens with a long working distance for this purpose.</td></tr><tr><td>Microforge Kit</td><td>Tool</td><td>ALA Science</td><td>CPM-2</td><td>Supplies polishing coil, controller and foot pedal.</td></tr><tr><td>Air pressure source</td><td>Tool</td><td>ALA Science</td><td>PR-60</td><td>Accessory for providing high pressure, includes a suitable valve. Alternatively, compressed air from the building can be used together with a filter/regulator (set to 40 PSI) and a 4-way valve (see below)</td></tr><tr><td>4-way valve</td><td>Tool</td><td>Cole-Parmer</td><td>A-98150-01</td><td>Fittings will be needed to couple valve to tubing</td></tr><tr><td>Pipette Holder with Luer Connector</td><td>Tool</td><td>World Precision Instruments, Inc.</td><td>MPH-6S20</td><td>Use a male luer-to-barb fitting to connect pipette holder to high-pressure tubing</td></tr><tr><td>Tygon&amp;#174; tubing</td><td>Tool</td><td>Cole-Parmer</td><td>06408-62</td><td>Use this tubing to connect pipette holder to 4-way valve</td></tr></tbody></table>

pressure-polishing pipettes for improved patch-clamp recording

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (&lt;3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (&lt;5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.

Watch this Scientific Journal Video about Pressure-polishing Pipettes for Improved Patch-clamp Recording at JoVE.com

Pressure-polishing Pipettes for Improved Patch-clamp Recording

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

pressure-polishing-pipettes-for-improved-patch-clamp-recording

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

12.9K Views.  Stanford University School of Medicine. This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

Video: Pressure-polishing Pipettes for Improved Patch-clamp Recording

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades. Today, such pipettes are made from glass capillaries using a programmable puller. Such instruments heat the capillary using either a metal filament or a laser and draw out the glass using gravity, a motor or both. Pipettes for patch-clamp recording are formed using only heat and gravity, while sharp electrodes for intracellular recording use a combination of heat, gravity, and a motor. The procedure used to make intracellular recording pipettes is similar to that used to make injection needles for a variety of applications, including cRNA injection into Xenopus oocytes. In general, capillary glass &lt;1.2 mm in diameter is used to make pipettes for patch clamp recording, while narrower glass is used for intracellular recording (outer diameter = 1.0 mm). For each tool, the puller is programmed slightly differently. This video shows how to make both kinds of recording pipettes using pre-established puller programs.

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades.  Today, such pipettes are made from glass capillaries ...

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

Neuroscience

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers easy identification and characterization of these cell types, prerequisites to study their specific functions. This article describes the multiplex single cell reverse transcription polymerase chain reaction (RT-PCR) protocol, which allows, after patch-clamp recording in slices, to detect simultaneously the expression of tens of genes in a single cell. This simple method can be implemented with morphological characterization and is widely applicable to determine the phenotypic traits of various cell types and their particular cellular environment, such as in the vicinity of blood vessels. The principle of this protocol is to record a cell with the patch-clamp technique, to harvest and reverse transcribe its cytoplasmic content, and to detect qualitatively the expression of a predefined set of genes by multiplex PCR. It requires a careful design of PCR primers and intracellular patch-clamp solution compatible with RT-PCR. To ensure a selective and reliable transcript detection, this technique also requires appropriate controls from cytoplasm harvesting to amplification steps. Although precautions discussed here must be strictly followed, virtually any electrophysiological laboratory can use the multiplex single cell RT-PCR technique.

This protocol describes the critical steps and precautions required to perform single cell multiplex reverse transcription polymerase chain reaction after patch-clamp. This technique is a simple and effective method to analyze the expression profile of a predetermined set of genes from a single cell characterized by patch-clamp recordings.

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...

Recording Electrical Currents across the Plasma Membrane of Mammalian Sperm Cells

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for electrophysiologists for many decades. The method known as "spermatozoan patch clamp" was introduced in 2006. It has enabled the direct recording of ion channel activity in whole-cell and cell-attached configurations and has been instrumental in describing sperm cell physiology and the molecular identity of various calcium, potassium, sodium, chloride, and proton ion channels. However, recording from single spermatozoa requires advanced skills and training in electrophysiology. This detailed protocol summarizes the step-by-step procedure and highlights several 'tricks-of-the-trade' in order to make it available to anyone who wishes to explore the fascinating physiology of the sperm cell. Specifically, the protocol describes recording from human and murine sperm cells but can be adapted to essentially any mammalian sperm cell of any species. The protocol covers important details of the application of this technique, such as isolation of sperm cells, selection of reagents and equipment, immobilization of the highly motile cells, formation of the tight (Gigaohm) seal between a recording electrode and the plasma membrane of the sperm cells, transition into the whole-spermatozoan mode (also known as break-in), and exemplary recordings of the sperm cell calcium ion channel, CatSper, from six mammalian species. The advantages and limitations of the sperm patch clamp method, as well as the most critical steps, are discussed.

This protocol describes how to perform electrical recordings from mammalian sperm cells in a whole-cell configuration, with the goal of directly recording ion channel activity. The method has been instrumental in describing the electrophysiological profiles of several sperm ion channels and helped to reveal their molecular identity and regulation.

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for ...

Patch-Clamp Techniques for Single Endolysosomal Vesicle Analysis

Endolysosomal ion channels are critical for endolysosomal ion and pH homeostasis, membrane potential regulation, and vesicle trafficking. However, electrophysiologically accessing these channels within small intracellular vesicles has been a challenge. The development of endolysosomal patch-clamp techniques has been instrumental in overcoming this barrier, allowing for the direct measurement of ion channel activity in endolysosomal membranes.
Compared to existing planar patch-clamp techniques, endolysosomal patch-clamp can simultaneously record multiple cells and easily combine with other measurement methods. Manual operation offers the advantage of visualizing targeted vesicles. It also addresses the limitation of the indispensable presence of Ca2+ on one side of the endolysosomal membrane, increasing the flexibility of experimental design. Utilizing endolysosomal patch-clamp techniques enables the direct measurement and analysis of ion channel activity within endolysosomes.
Given the close link between aberrant endolysosomal ion channel function and diseases such as neurodegenerative diseases and metabolic disorders, investigating and modulating these channels may unveil new drug targets. By restoring intracellular ion balance, we may alleviate or cure related diseases. Therefore, this technique is pivotal for discovering new drug targets and developing relevant medications.

This protocol details a method for directly measuring ion channel activity on intracellular vesicles using a manual endolysosomal patch-clamp system. We illustrate the method that involves enlarging endolysosomes and manually isolating these vesicles. This approach ensures that researchers can accurately replicate and apply the procedure.

Endolysosomal ion channels are critical for endolysosomal ion and pH homeostasis, membrane potential regulation, and vesicle trafficking. However, ...

Patch-Clamp Recordings from the Dendrite of a Dopaminergic Neuron in a Brain Slice

Source: Engel, D. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54601">Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons.</a> J. Vis. Exp. (2016).
This video demonstrates a technique for patch-clamp recording from the dendritic membrane surface. This helps to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels in dopaminergic neurons.

Nagrania patch-clamp z dendrytu neuronu dopaminergicznego w wycinku mózgu

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...