Name: whole-cell patch-clamp recordings in brain slices
Uploaded: 2016-06-15T14:00:00
Description: Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings in Brain Slices at JoVE.com

This research was supported by UT Southwestern startup funds (SK).

All experiments were carried out in accordance with protocols approved by the UT Southwestern Institutional Animal Care and Use Committee, and were chosen so as to minimize stress, discomfort, and pain experienced by the experimental animals.
1. Solutions
Note: Prepare micropipette internal solutions in advance. For most basic experimental purposes, two kinds of solutions should suffice: Cs+-based and K+-based solutions.
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	<li>Use Cs+-based solutions (e.g., Cs...

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This protocol describes the basic procedure for performing whole-cell patch-clamp experiments on neurons in brain slices. However, the complexity, potential and sensitivity of this technique cannot be fully described in this article. Here, we have tried to delineate the most basic steps and underscore important parameters that must be controlled for achieving successful and rigorous whole-cell recordings. For further theoretical learning, many books and articles have been published on both whole-cell patch-clamp recordin...

The patch-clamp technique, an electrophysiological technique that has been developed in the late 1970s1,2, is a primary tool for studying single or multiple ion channel functions in live tissue. Among the different patch configurations that can be achieved, whole-cell patch-clamp recordings allow the study of the electrical behavior of a substantial part of the neuron. Classically, this technique is performed in vitro either on brain slices, freshly dissociated neurons, or on cell culture models3. When performed on neurons in brain slices, this technique presents several advantages. In particular: (i) neurons are recorded in relatively preserved brain circuits that to some extent, and compared to cell culture preparations, provide an environment that is physiologically relevant3. This allows capturing early, or even monitoring in real time, cellular and molecular events that are triggered by any type of acute pharmacological manipulations &#8212; a temporal resolution that cannot be achieved using classical in vivo conditions; (ii) capability to visually identify brain regions in brain slices allows high regional specificity3 both for the brain region studied and for specific neurons when they express fluorescent markers; (iii) access to the intracellular space of the cell by opening a significant portion of the plasma membrane (in contrast to puncturing the membrane with a sharp micropipette for intracellular recordings)4. In turn, this allows the content or concentration of specific ions composing the internal solution to be modified so molecular targets or cellular mechanisms can be studied under different conditions. For example, upon establishing whole-cell configuration, any specific pharmacological agent (e.g., antagonists) that one can add to the recording micropipette (patch pipette) solution will directly diffuse into the cytoplasm and act on its putative intracellular targets without altering the target function in neighboring cells. Additionally, compared to sharp micropipette recording, the large opening at the tip of the patch clamp electrode provides lower resistance, less competing noise, and thus better electrical access to the inside of the cell4. However, note that the large opening at the pipette tip may lead to cell dialysis, and thereby the loss of intracellular molecular machinery that may be critical for the expression of the biological phenomena that are under study5,6. In this case, sharp electrode recordings may be more suitable. This type of recordings requires micropipettes with a pore that is much smaller than those used for whole-cell recordings, thereby preventing most of the ion exchange between intracellular space and the internal pipette solution.
Any form of experience (acute or chronic), including learning7-10, exposure to drugs of abuse11,12, stress13,14, etc., can alter various aspects of neuronal function in specific brain regions. Because these alterations often require time to develop (hours to days), whole-cell recordings in brain slices from animals that have undergone a specific experience allow researchers to identify these changes. Basically, many (if not all) components that participate in neuronal functions (e.g., ligand-activated ion channels, voltage-gated ion channels, neurotransmitter transporters), and thereby brain circuit activity and behavior, can be altered by experience (experience-dependent plasticity)10,15-17. At the neuronal level, brain circuit activity emerges from constant interactions between synaptic (e.g., glutamate transmission) and intrinsic cellular excitability factors (e.g., axosomato-dendritic ion channels: sodium, Na+; potassium, K+; and calcium, Ca2+). Under specific conditions using whole-cell patch-clamp electrophysiological techniques, signal alterations originating specifically from changes in synaptic vs. intrinsic excitability can be isolated.
In most cases, synaptic excitability is assessed using the whole-cell voltage-clamp technique. This recording mode allows the measurement of ion currents [e.g., mediated by &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA receptors) and N-Methyl-D-aspartic acid receptors (NMDA receptors)] through the neuronal plasma membrane while holding the membrane potential at a set voltage. Here, experimenters use internal micropipette solutions that contain cesium (Cs+), a broad blocker of K+ channels (key intrinsic excitability factors). Upon establishment of whole-cell configuration, the diffusion of Cs+ in intracellular space will block K+ channels, and thereby will allow both a relatively efficient space-clamp and prevent influence of intrinsic excitability factors on other measurements. Space-clamp issues, i.e., the difficulty to voltage-clamp the whole cell, arise when recording irregular shaped cells (e.g., neurons), and particularly neurons with a vast and complex dendritic arbor18,19. Because somatic voltage clamp poorly controls voltage in the dendritic tree of neurons, various aspects of dendritic electrical signals under study are distorted in a dendritic distance-dependent manner. Combined with pharmacological tools such as picrotoxin (gamma-Aminobutyric acid, GABAA receptor antagonist) or kynurenic acid (broad blocker of glutamate receptors) dissolved in the extracellular solution (artificial cerebro-spinal fluid, ACSF), this technique allows the measurement of glutamate receptor- and GABAAR-mediated currents respectively.
In contrast, intrinsic excitability is usually assessed in current-clamp recording mode. As opposed to voltage-clamp recording, this recording mode allows the measurement of variations in membrane potentials induced by ion currents flowing through the neuronal plasma membrane. Typically, alteration in intrinsic excitability is assessed through changes in the capability for neurons to generate action potentials, which requires both Na+ and K+ channels. Therefore, when performing current-clamp recordings, micropipettes are filled with an internal solution that contains K+ instead of Cs+. Combined with pharmacological agents that block glutamate and GABAA receptor-mediated currents dissolved in the ACSF, this experimental design allows the measurement of the contribution of intrinsic factors (e.g., K+ channels) to neuronal firing without being contaminated by potential changes in synaptic excitability factors.
This article will describe the basic necessary procedural steps to (i) prepare healthy brain slices; (ii) achieve whole-cell configuration, and (iii) monitor basic parameters to assess synaptic and intrinsic excitability.

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			<td height="21" style="height:21px;width:215px;">Isolated pulse stimulus generator</td>
			<td style="width:183px;">A.M.P.I</td>
			<td style="width:139px;">Master-8</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isolation unit (ISO-Flex)</td>
			<td>A.M.P.I</td>
			<td>ISO-Flex</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer controlled Amplifier&#160;</td>
			<td>Molecular Devices</td>
			<td>Multiclamp 700B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital Acquisition system</td>
			<td>Molecular Devices</td>
			<td>Digidata 1500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td>Olympus</td>
			<td>BX-51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>MPC-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chamber and in-line Heater</td>
			<td>Warner Instruments</td>
			<td>TC-344B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome Slicer</td>
			<td>Leica&#160;</td>
			<td>VT1000 S</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Micropipette Puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaging Camera</td>
			<td>Q Imaging</td>
			<td>QIClick-F-M-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Narishige pipette puller PC-10</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
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			<td height="21" style="height:21px;">Glass capillaries</td>
			<td>WPI</td>
			<td>TW150F-3</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Slice hold-down (harp)</td>
			<td>Warner Instruments</td>
			<td>64-0255</td>
			<td></td>
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			<td height="21" style="height:21px;">Slice Chamber</td>
			<td>Warner Instruments</td>
			<td>RC-26</td>
			<td></td>
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			<td height="21" style="height:21px;">Nonmetallic syringe needle</td>
			<td>World Precision Instruments</td>
			<td>MF28G67-5</td>
			<td></td>
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			<td height="21" style="height:21px;">Syringe filters</td>
			<td>Nalgene</td>
			<td>176-0045</td>
			<td></td>
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			<td height="21" style="height:21px;">Glue Gun</td>
			<td style="width:183px;">Home Depot</td>
			<td style="width:139px;">various</td>
			<td></td>
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			<td height="21" style="height:21px;">Gas dispersion tube</td>
			<td>Ace Glass Inc.</td>
			<td>various</td>
			<td></td>
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			<td height="21" style="height:21px;">Decapitation scissors</td>
			<td>Home Depot</td>
			<td>100649198</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Handle #3</td>
			<td>World Precision Instruments</td>
			<td>500236</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Small straight sharp tips scissors</td>
			<td>World Precision Instruments</td>
			<td>14218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vessel canulation forceps&#160;</td>
			<td>World Precision Instruments</td>
			<td>500453</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved hemostatic forceps</td>
			<td>World Precision Instruments</td>
			<td>501288</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Economy Tweezers #3</td>
			<td>World Precision Instruments</td>
			<td>501976-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spatula</td>
			<td>Fisher Scientific</td>
			<td>14357Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scooping spatula</td>
			<td>Fisher Scientific</td>
			<td>14-357Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dish</td>
			<td>Fisher Scientific</td>
			<td>08-747B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter paper</td>
			<td>Lab Depot</td>
			<td>CFP1-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;"></td>
			<td style="width:183px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">Cs-Gluconate internal solution (pH 7.2&#8211;7.3, 280&#8211;290 mOsm)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-gluconic acid 50%&#160;</td>
			<td>Sigma Aldrich/various</td>
			<td>G1951</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cesium-OH (CsOH) 50%&#160;</td>
			<td>Sigma Aldrich/various</td>
			<td>232041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl, 2.8 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES, 20 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA, 0.4 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tetraethylammonium-Cl, 5 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>T2265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2GTP, 0.3 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgATP, 2 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">K-Gluconate internal solution (pH 7.2&#8211;7.3, 280&#8211;290 mOsm)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">K D-gluconate, 120 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>G4500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl, 20 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES, 10 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA, 0.2 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td>Sigma Aldrich/various</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2GTP, 0.3 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgATP, 2 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">Standard artificial cerebrospinal fluid (ACSF, osmolarity &#8776; 300-310 mOsm)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl, 2.5 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl, 119 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaH2PO4-H20, 1 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3, 26.2 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>S8875</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose, 11 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4-7H2O, 1.3 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>230391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2-2H20, 2.5 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>C3881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Additional compounds used for solutions preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KOH</td>
			<td>various</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kynurenic acid</td>
			<td>Sigma Aldrich/various</td>
			<td style="width:139px;">K3375</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Temperature, a factor that is easily controlled by the experimenter, influences the biophysical properties of ion channels and receptors, and thereby the waveform of post-synaptic currents (PSCs) (EPSC and IPSCs) and the capability of neurons to elicit spikes. Figure 3 and Figure 4 show the effect of temperature on neuronal firing and the slope of evoked EPSCs (eEPSCs) respectively. The firing pattern (Figure 3) (i.e., latency to...

Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings in Brain Slices at JoVE.com

Whole-cell Patch-clamp Recordings in Brain Slices

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 ...

The overall goal of this procedure is to describe how to perform whole-cell patch-clamp recordings in freshly dissected brain slices. This method can help answer key questions in the field of neuroscience, such has how experimental control manipulations alter the activity of specific neurons in specific brain regions. The main advantage of this technique is that it provides the medium to identify an X vivo preparation long lasting changes in neuron functions that have developed in intact awake animals.Though this method can provide insight into neuronal functions in brain slices, it can also be applied to other systems, such as cells in culture and neurons in vivo. Demonstrating the procedure will be Francisco Garcia-Oscos, a student from my lab. To begin this procedure, using a plastic trim-tipped transfer pipette, gently draw up one brain slice from the recovery chamber.Place the transfer pipette in the recording chamber, and gently squeeze the slice out of the pipette on to the cover slip lining at the bottom of the chamber. Next, using the microscope 4x objective, position the slice so that the desired area is placed exactly in the center of the recording chamber. After the desired position has been achieved, secure the brain slice position with the slice hold down, also known as a harp.After that, switch to the 40x objective, and lower the lens gently until it contacts the ACSF in the chamber. Then, use the fine adjustment wheel to bring the tissue into focus. When the focus is at tissue level, observe the cells in the targeted region.Now, look for a target cell. Mark it on the computer screen in order to help guide the recording micropipette. Raise the objective lens to allow for sufficient space for the placement of the recording micropipette.In this step using a one milliliter syringe, a non-metallic microsyringe needle, and a dedicated filter, fill a micropipette with the internal solution prepared in advance. Make sure there are no air bubbles in the micropipette. Then place the micropipette in an electrode holder, so the solution comes in contact with the silver chloride coated wire electrode.Tighten the pipette cap, so that the cone washer forms a seal around the micropipette. Next, apply positive pressure with the air filled syringe connected to the pipette holder before immersing he micropipette in the ACSF to prevent debris from entering it. Using the micro-manipulator, guide the pipette down to the chamber so that it is roughly under the center of the immersed objective.While moving the micropipette with the micro-manipulator at medium to high speed, locate the micropipette on the computer screen, and guide the micropipette toward the location of the cell on the XY axis. In the mean time, measure the micropipette resistance by applying a voltage step. Apply positive pressure in order to remove any air bubbles or other foreign objects blocking the micropipette.After clearing the micropipette, perform a voltage offset to reduce pipette current to zero. Using the fine focus wheel of the microscope, start focusing down while lowering the micropipette gradually. Always focus down first and then lower the micropipette to the plane of focus to ensure that the micropipette tip does not abruptly penetrate into the slice.When the micropipette comes in contact with the surface of the slice, slow down the micro-manipulator speed to medium low mode. Gently apply light positive pressure to clear any debris on the path. Then approach the cell, either by alternating with the XYZ control knobs or by approaching diagonally where both XZ axis are changed with rotation of the Z-axis knob.When the micropipette is close enough to the cell, a dimple will appear on the cell surface. Now apply a weak and brief suction through the tube that is connected to the pipette holder suction tube in order to create a seal. While a giga-seal is forming, use the computer controlled amplifier commander to bring the cells holding potential to the physiological resting potential in order to prevent sudden changes, once the membrane is ruptured.After the giga-seal has formed, compensate for the fast or slow capasitons. Here it is very important that the suction applied is not too strong. Otherwise the membrane may rupture before establishing the seal.If the seal remains stable and above one gigaom, apply a brief and strong suction to rupture the plasma membrane. The suction must be brief and stronger than the pressure applied when establishing a seal. In order to properly rupture the membrane, and achieve a stable whole-cell configuration.Switching to cell mode in the membrane test, view different parameters of the cell, such as input resistance, series resistance, and membrane capacitons. After achieving the whole-cell configuration, continue to monitor these parameters during recording. Shown here, is an example of the evoked EPSCs slope from a single nucleus accumbens shell MSN.Increasing the temperature from 24 to 28 and to 32 degrees Celsius, increases the slope of evoked EPSCs. Here the evoked EPSCs amplitude is assessed in the voltage clamp mode at minus 80 millivolts. When series resistance increased, the amplitude of evoked EPSCS decreases.And here, is a example of the traces from two neurons showing the effect of input resistance on the neuron capability to generate spikes. Neurons are current clamped and held at minus 80 millivolts. When input resistance increases, the number of action potentials increases as well.While attempting this procedure, it's important to remember that generating healthy brain slices is critical. It is also important to monitor the parameters that may influence the electrical signal waveform, such as the series resistance, input resistance, and the temperature. After watching this video, you should have a good understanding of the basic aspect of whole-cell recording technique in brain slices.

whole-cell-patch-clamp-recordings-in-brain-slices

Research

JoVE Journal

Neuroscience

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 previously1-4 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.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

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 intact2,3,4. 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.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

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

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

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.

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

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

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

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.

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...