Name: Whole-cell Currents Induced by Puff Application of GABA in Brain Slices (Video) | JoVE Journal
Uploaded: 2017-10-12T14:00:00.000+00:00
Duration: 7 min 32 s
Description: Learn the puff technique for whole-cell patch-clamp recording in brain slices. This method enables rapid drug delivery to prevent receptor desensitization during electrophysiological experiments.

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5 minutes

The authors would like to thank the following organizations: National Natural Science Foundation of China (31171018, 31171355), the Science and Technology Division of Guangdong (2013KJCX0054), the Natural Science Foundation of Guangdong Province (2014A030313418, 2014A030313440), and Guangzhou Science and Technology Bureau (201607010320).

Animal housing and all animal experiments were conducted in accordance with procedures approved by the Ethics Committee for animal research at South China Normal University, according to the Guidelines for Animal Care established by the National Institute of Health.
1. Preparation of Solutions
<ol>
	<li>Prepare 500 mL of standard artificial cerebrospinal fluid (ACSF) containing the components described in our published work7 as listed in Table 1.
	<ol>
		<li>Use clean glass beakers with deionized water to prepare a fresh ACSF solution. Clean spatulas with deionized water and dry before using to add NaCl, KCl, NaH2PO4, NaHCO3, MgSO4, D-glucose, and add the CaCl2 after having bubbled the solution with 95% O2 / 5% CO2 mixture for ~20 min (as detailed in Table 1) to 500 mL of deionized water. Stir until fully dissolved.</li>
		<li>Ensure the ACSF is completely clear without any precipitation. Then adjust the pH to 7.2-7.4. Bubble with a gas mixture composed of 95% O2 and 5% CO2 for ~20 min.</li>
	</ol>
	</li>
	<li>Prepare 250 mL of slice cutting solution containing the chemicals7 described in Table 2.
	<ol>
		<li>Add KCl, MgCl2, NaH2PO4, NaHCO3, choline&#183;Cl, D-glucose and add the CaCl2 last as indicated in 1.1.1, to 250 mL of deionized water and dissolve as described in 1.1.1 and 1.1.2.</li>
		<li>After bubbling with 95% O2/5% CO2, place the solution in a freezer for 15-20 min so that the cutting solution becomes ice-cold and partially frozen. 
		NOTE: We use this cutting solution when making slices from 1-2 month-old mice; the formula may differ for mice older than 2 months.</li>
		<li>Prepare LPS solution by dissolving in sterile endotoxin-free isotonic saline (0.9% NaCl). LPS is administered intraperitoneally (0.5 mg/kg) and begin recording 2 h later.</li>
	</ol>
	</li>
</ol>
2. Preparation of Prefrontal Cortex Slice
<ol>
	<li>Take the cutting solution out of the freezer and homogenize to obtain a slush. Place the solution on ice and bubble constantly with 95% O2/5% CO2.</li>
	<li>Using large, sharp scissors, quickly decapitate a mouse as described9 and immediately put the head into a beaker filled with ice-cold (0-4&#176;C) cutting solution.</li>
	<li>Cut the top of the skull along the midline in a caudal-to-nasal direction with fine scissors and remove the lateral skull portions. Remove the brain with a fine spatula and drop into ice-cold cutting solution. 
	NOTE: Steps 2.2-2.3 must be done rapidly (ideally &#60; 1 min).</li>
	<li>Place the brain on the lid of a 9 cm Petri dish filled with ice. Rinse the brain with ice-cold cutting solution. Cut off the cerebellum and olfactory bulb with a razor blade.</li>
	<li>Apply cyanoacrylate glue to the specimen holder of a vibratome. Carefully place the brain block on the drop of glue, rostral side up, and immediately immerse in ice-cold cutting solution.</li>
	<li>Adjust the blade holder of the vibratome to an angle of 15&#176; with reference to the horizontal plane and prepare 350-&#181;m coronal brain slices (blade vibration frequency = 85 Hz, speed = 0.1 mm/s).</li>
	<li>Using a pipette, transfer the slices into the recovery chamber filled with ACSF, incubate for 1 h (at RT) with constant bubbling of 95% O2/5% CO2.</li>
</ol>
3. Whole-cell Patch Recording
<ol>
	<li>Make glass borosilicate micropipettes for use as recording electrodes or puff micropipettes according to the specific guidelines in the Puller Operation Manual. For recording electrodes, tip resistances are in the range 4-6 M&#8486;, while puff micropipettes have tip diameters in the range 2-5 &#181;m.</li>
	<li>Add Na+ channel blocker (TTX, 1 &#181;M), to block fast Na+&#160;channel and thus action potentials, to ACSF and maintain a constant flow rate of this solution of 2-3 mL/min in the recording chamber, by peristaltic pump into the chamber and aspiration out of it. Bubble the ACSF with 95% O2/5% CO2 constantly to ensure the viability of the slices.</li>
	<li>Transfer a brain slice into the recording chamber using a modified Pasteur pipette whose fine tip was cut to fit the slice size. Cover the slice with a platinum slice anchor with parallel nylon threads spaced &#8805;2 mm apart to hold the slice on the platform.</li>
	<li>Fill the recording micropipette with internal solution7 (Table 3), and fill the puff micropipettes with GABA at 10 &#181;M, 50 &#181;M, and 100 &#181;M (dissolved in ACSF), or ACSF as vehicle control.</li>
	<li>To prevent blockage with debris, apply light positive pressure using a 1 mL syringe before immersing the recording electrode in the ACSF. Using a micromanipulator under a microscope (4x objective lens), position the recording electrode and puff micropipette so that the tips appear in the center of the video image in the monitor (Figure 1A).</li>
	<li>Adjust the microscope focus (40x objective lens) while gradually lowering the puff micropipette and place it above the recording neuron at an angle of 45&#176;. Keep the distance between the tip of the puff micropipette and the neuron recorded between 20-40 &#181;m (Figure 1B). Slowly and carefully approach the neuron with the recording electrode and then release the positive pressure. Apply a weak and brief suction through the tubing connected to the electrode holder to create a giga-ohm seal.</li>
	<li>Maintain the voltage at 0 mV. After formation of the giga-ohm seal, compensate for fast and slow capacitance manually or automatically. Then apply a brief and strong suction through the tubing mentioned above to get into whole-cell mode.</li>
	<li>Record eIPSC in voltage-clamp mode. Apply low gas pressure (nitrogen, 4 - 6 psi) to the puff micropipette using a Picospritzer controlled by a Master 8 voltage step generator to deliver single or paired GABA puffs (Figure 2).</li>
	<li>Change the puff duration to obtain the best eIPSC results (Figure 3) and measure the eIPSC in the LPS-induced depression model ( Figure 4).</li>
</ol>

<ol><li>Neher, E., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-channel+currents+recorded+from+membrane+of+denervated+frog+muscle+fibres%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Single-channel currents recorded from membrane of denervated frog muscle fibres.</a> Nature. 260 (5554), 799-802 (1976).</li>
<li>Sakmann, B., Edwards, F., Konnerth, A., Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patch+clamp+techniques+used+for+studying+synaptic+transmission+in+slices+of+mammalian+brain%5BTitle%5D)+AND+%22Q+J+Exp+Physiol%22%5BJournal%5D)">Patch clamp techniques used for studying synaptic transmission in slices of mammalian brain.</a> Q J Exp Physiol. 74 (7), 1107-1118 (1989).</li>
<li>Eyo, U. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuronal+hyperactivity+recruits+microglial+processes+via+neuronal+NMDA+receptors+and+microglial+P2Y12+receptors+after+status+epilepticus%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and microglial P2Y12 receptors after status epilepticus.</a> J Neurosci. 34 (32), 10528-10540 (2014).</li>
<li>Dickinson, G. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Factors+determining+the+recruitment+of+inositol+trisphosphate+receptor+channels+during+calcium+puffs%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Factors determining the recruitment of inositol trisphosphate receptor channels during calcium puffs.</a> Biophys J. 105 (11), 2474-2484 (2013).</li>
<li>Lee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Columnar+distribution+of+activity+dependent+gabaergic+depolarization+in+sensorimotor+cortical+neurons%5BTitle%5D)+AND+%22Mol+Brain%22%5BJournal%5D)">Columnar distribution of activity dependent gabaergic depolarization in sensorimotor cortical neurons.</a> Mol Brain. 5, 33(2012).</li>
<li>Glykys, J., Mody, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+main+source+of+ambient+GABA+responsible+for+tonic+inhibition+in+the+mouse+hippocampus%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">The main source of ambient GABA responsible for tonic inhibition in the mouse hippocampus.</a> J Physiol. 582, (Pt 3) 1163-1178 (2007).</li>
<li>Chen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((APP+modulates+KCC2+expression+and+function+in+hippocampal+GABAergic+inhibition%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">APP modulates KCC2 expression and function in hippocampal GABAergic inhibition.</a> Elife. 6, (2017).</li>
<li>Dunn, A. J., Swiergiel, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+interleukin-1+and+endotoxin+in+the+forced+swim+and+tail+suspension+tests+in+mice%5BTitle%5D)+AND+%22Pharmacol+Biochem+Behav%22%5BJournal%5D)">Effects of interleukin-1 and endotoxin in the forced swim and tail suspension tests in mice.</a> Pharmacol Biochem Behav. 81 (3), 688-693 (2005).</li>
<li>Engel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subcellular+Patch-clamp+Recordings+from+the+Somatodendritic+Domain+of+Nigral+Dopamine+Neurons%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons.</a> J Vis Exp. (117), (2016).</li>
<li>Wondolowski, J., Frerking, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subunit-dependent+postsynaptic+expression+of+kainate+receptors+on+hippocampal+interneurons+in+area+CA1%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Subunit-dependent postsynaptic expression of kainate receptors on hippocampal interneurons in area CA1.</a> J Neurosci. 29 (2), 563-574 (2009).</li>
<li>Yang, L., Wang, Z., Wang, B., Justice, N. J., Zheng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amyloid+precursor+protein+regulates+Cav1.2+L-type+calcium+channel+levels+and+function+to+influence+GABAergic+short-term+plasticity%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Amyloid precursor protein regulates Cav1.2 L-type calcium channel levels and function to influence GABAergic short-term plasticity.</a> J Neurosci. 29 (50), 15660-15668 (2009).</li>
<li>Huo, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prefrontal+Cortical+GABAergic+Dysfunction+Contributes+to+Aberrant+UP-State+Duration+in+APP+Knockout+Mice%5BTitle%5D)+AND+%22Cereb+Cortex%22%5BJournal%5D)">Prefrontal Cortical GABAergic Dysfunction Contributes to Aberrant UP-State Duration in APP Knockout Mice.</a> Cereb Cortex. , (2016).</li>
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The authors have nothing to disclose.

Puff application is widely used to evaluate postsynaptic receptor function3,4,7, but requires precise control in each experiment. We describe here a procedure involving whole-cell patch clamping, which demonstrates GABA-puff induced IPSCs (i.e., eIPSCs) in prefrontal cortical brain slices. The resistance of the recording electrode is about 5 M&#937;, while the tip diameter of puff micropipettes is about 2-5 &#181;m. Puff pressur...

The patch-clamp technique, a primary tool for investigating electrical signals in neurons, was developed in the 1970s1,2. A major advantage of this technique is that it provides knowledge on how specific treatments (e.g., pharmacological) may alter neuronal functions or channels in real time3. Pharmacological evaluation of neuronal function during whole-cell recording in a brain slice requires the application of drugs, such as agonists or antagonists of specific receptors, to the neurons being recorded. This method allows the identification of neuronal alterations that occur following application of a specific drug, thus leading to a better understanding of the physiological and pathological properties of the neurons4. Although pharmacological administration can be conducted via either perfusion5 or puff6, the latter is the superior technique. In particular: (i) puff application rapidly increases drug concentration around the recorded neuron to a level such that desensitization of the membrane receptors is prevented; (ii) the volume of drug puffed is extremely low, so that there is little effect on the bath solution, which therefore reduces any undesirable effects of the administered chemicals on brain slices; (iii) the puff protocol can be set and saved, making the experiment very precisely reproducible; (iv) puff application represents economical use of agonists/antagonists, particularly where such reagents are expensive or difficult to obtain.
Here, we will focus on recording whole-cell currents induced by puffing GABA in acutely prepared brain slices, a preparation that has the advantage of relatively well-preserved brain circuits. How we conduct puff-induced inhibitory currents7 will be described in this article. By using cesium (Cs+)-based internal solutions, and holding the neurons at 0 mV, we will introduce GABA-puff evoked inhibitory postsynaptic currents (eIPSCs) with appropriate technical details. Using a mouse model of depression induced by lipopolysaccharide (LPS) injection8, we show that the eIPSC amplitude evoked by a GABA puff is significantly reduced in slices of LPS-injected mice compared to vehicle controls. Our intention is that this article should show how the puff technique is widely applicable to studies aimed at evaluating the effect of any chemicals, compounds or drugs on neuronal activities in brain slices.

<table><tbody><tr><td>Glass Borosilicate micropipettes</td><td>Shutter Instruments</td><td>BF150-86-10</td><td>1.50 mm outer diameter; 0.86 mm inner diameter</td></tr><tr><td>Micropipette Puller</td><td>Shutter Instruments</td><td>MODEL P-97&amp;nbsp;</td><td>Flaming/Brow Micropipette Puller</td></tr><tr><td>Micromanipulators</td><td>Shutter Instruments</td><td>MP-285</td><td></td></tr><tr><td>Computer controlled Amplifier&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td></td></tr><tr><td>Digital Acquisition system&amp;nbsp;</td><td>Molecular Devices</td><td>Digidata 1440A</td><td></td></tr><tr><td>Imaging Camera</td><td>Nikon&amp;nbsp;</td><td>2115001</td><td>Inspection equipment</td></tr><tr><td>Microscopy&amp;nbsp;</td><td>Nikon&amp;nbsp;</td><td>Eclipse FN1&amp;nbsp;</td><td></td></tr><tr><td>Master 8</td><td>A.M.P.I.</td><td>Master-8 Pulse stimulator</td><td></td></tr><tr><td>Vibratome Slicer</td><td>Leica</td><td>VT 1000S</td><td></td></tr><tr><td>Razor blade</td><td>Gillette</td><td>74-S</td><td>FLYING EAGLE</td></tr><tr><td>Video monitor</td><td>Panasonic</td><td>WV-BM 1410</td><td></td></tr><tr><td>502 Glue</td><td>Deli</td><td>7146</td><td>Cyanoacrylate Glue</td></tr><tr><td>Peristaltic pump</td><td>Shanghai JIA PENG Corporation</td><td>BT100-1F</td><td></td></tr><tr><td>Video Camera</td><td>Olympus America Medical</td><td>OLY-150</td><td></td></tr><tr><td>Transfer Pipets</td><td>Biologix</td><td>30-0138A1</td><td></td></tr></tbody></table>

whole-cell currents induced by puff application of gaba in brain slices

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug application during electrophysiological recording is the puff technique, which can be used to study the effect of pharmacological reagents on neuronal activities in brain slices. The greatest advantage of puff application is that the drug concentration around the recording site increases rapidly, thus preventing desensitization of membrane receptors. Successful use of puff application involves careful attention to the following elements: the concentration of the drug, the parameters of the puff micropipette, the distance between the tip of a puff micropipette and the neuron recorded, and the duration and pressure driving the puff (pounds per square inch, psi). This article describes a step-by-step procedure for recording whole-cell currents induced by puffing gamma-aminobutyric acid (GABA) onto a neuron of a prefrontal cortical slice. Notably, the same procedure can be applied with minor modifications to other brain areas such as the hippocampus and the striatum, and to different preparations, such as cell cultures.

The puff technique allows researchers to study the effect of drugs on particular receptors on the surface of a given neuron. In this article, we focus on currents mediated by GABA receptors. Figure 1 shows the puff and recording micropipettes. To eliminate any effect on recorded neurons of the physical pressure generated by the puff, ACSF and sucrose puffs (Figure 2A) are used as controls. The response induced by...

We describe the puff technique, by which pharmacological reagents can be administered during whole-cell patch-clamp recording, and highlight various aspects of the features that are crucial for its success.

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

whole-cell-currents-induced-puff-application-gaba-brain

Research

JoVE Journal

Neuroscience

We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons.  Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4.

GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

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Recording Whole-Cell Currents with GABA Puffing in a Mouse Brain Slice

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

Oscillations are fundamental network properties and are modulated by disease and drugs. Studying brain-slice oscillations allows characterization of isolated networks under controlled conditions. Protocols are provided for the preparation of acute brain slices for evoking CA1 γ oscillations.

Generation of Local CA1 &#947; Oscillations by Tetanic Stimulation

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of 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 Recordings in Brain Slices

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Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

Acute seizure models are important for studying the mechanisms underlying epileptiform events. Furthermore, the ability to generate epileptiform events on-demand provides a highly efficient method to study the exact sequence of events underlying their initiation. Here, we describe the acute 4-aminopyridine cortical seizure models established in mouse and human tissue.

Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

Horizontal Hippocampal Slices of the Mouse Brain

Learn the puff technique for whole-cell patch-clamp recording in brain slices. This method enables rapid drug delivery to prevent receptor desensitization during electrophysiological experiments.

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

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