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

<ol>
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None of the authors have competing interests or conflicting interests.

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.

<table><tbody><tr><td>Isolated pulse stimulus generator</td><td>A.M.P.I</td><td>Master-8</td><td></td></tr><tr><td>Isolation unit (ISO-Flex)</td><td>A.M.P.I</td><td>ISO-Flex</td><td></td></tr><tr><td>Computer controlled Amplifier&amp;nbsp;</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td></td></tr><tr><td>Digital Acquisition system</td><td>Molecular Devices</td><td>Digidata 1500</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>BX-51</td><td></td></tr><tr><td>Micromanipulator</td><td>Sutter Instruments</td><td>MPC-200</td><td></td></tr><tr><td>Chamber and in-line Heater</td><td>Warner Instruments</td><td>TC-344B</td><td></td></tr><tr><td>Vibratome Slicer</td><td>Leica&amp;nbsp;</td><td>VT1000 S</td><td></td></tr><tr><td>Micropipette Puller</td><td>Narishige</td><td>PC-10</td><td></td></tr><tr><td>Imaging Camera</td><td>Q Imaging</td><td>QIClick-F-M-12</td><td></td></tr><tr><td>Narishige pipette puller PC-10</td><td>Narishige</td><td>PC-10</td><td></td></tr><tr><td>Glass capillaries</td><td>WPI</td><td>TW150F-3</td><td></td></tr><tr><td>Slice hold-down (harp)</td><td>Warner Instruments</td><td>64-0255</td><td></td></tr><tr><td>Slice Chamber</td><td>Warner Instruments</td><td>RC-26</td><td></td></tr><tr><td>Nonmetallic syringe needle</td><td>World Precision Instruments</td><td>MF28G67-5</td><td></td></tr><tr><td>Syringe filters</td><td>Nalgene</td><td>176-0045</td><td></td></tr><tr><td>Glue Gun</td><td>Home Depot</td><td>various</td><td></td></tr><tr><td>Gas dispersion tube</td><td>Ace Glass Inc.</td><td>various</td><td></td></tr><tr><td>Decapitation scissors</td><td>Home Depot</td><td>100649198</td><td></td></tr><tr><td>Scalpel Handle #3</td><td>World Precision Instruments</td><td>500236</td><td></td></tr><tr><td>Small straight sharp tips scissors</td><td>World Precision Instruments</td><td>14218</td><td></td></tr><tr><td>Vessel canulation forceps&amp;nbsp;</td><td>World Precision Instruments</td><td>500453</td><td></td></tr><tr><td>Curved hemostatic forceps</td><td>World Precision Instruments</td><td>501288</td><td></td></tr><tr><td>Economy Tweezers #3</td><td>World Precision Instruments</td><td>501976-6</td><td></td></tr><tr><td>Spatula</td><td>Fisher Scientific</td><td>14357Q</td><td></td></tr><tr><td>Scooping spatula</td><td>Fisher Scientific</td><td>14-357Q</td><td></td></tr><tr><td>Petri dish</td><td>Fisher Scientific</td><td>08-747B</td><td></td></tr><tr><td>Filter paper</td><td>Lab Depot</td><td>CFP1-110</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Solutions&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;Cs-Gluconate internal solution (pH 7.2&amp;ndash;7.3, 280&amp;ndash;290 mOsm)&lt;/strong&gt;</td></tr><tr><td>D-gluconic acid 50%&amp;nbsp;</td><td>Sigma Aldrich/various</td><td>G1951</td><td></td></tr><tr><td>Cesium-OH (CsOH) 50%&amp;nbsp;</td><td>Sigma Aldrich/various</td><td>232041</td><td></td></tr><tr><td>NaCl, 2.8 mM</td><td>Sigma Aldrich/various</td><td>S7653</td><td></td></tr><tr><td>HEPES, 20 mM</td><td>Sigma Aldrich/various</td><td>H3375</td><td></td></tr><tr><td>EGTA, 0.4 mM</td><td>Sigma Aldrich/various</td><td>E4378</td><td></td></tr><tr><td>tetraethylammonium-Cl, 5 mM</td><td>Sigma Aldrich/various</td><td>T2265</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;GTP, 0.3 mM</td><td>Sigma Aldrich/various</td><td>G8877</td><td></td></tr><tr><td>MgATP, 2 mM</td><td>Sigma Aldrich/various</td><td>A9187</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;K-Gluconate internal solution (pH 7.2&amp;ndash;7.3, 280&amp;ndash;290 mOsm)&lt;/strong&gt;</td></tr><tr><td>K D-gluconate, 120 mM</td><td>Sigma Aldrich/various</td><td>G4500</td><td></td></tr><tr><td>KCl, 20 mM</td><td>Sigma Aldrich/various</td><td>P3911</td><td></td></tr><tr><td>HEPES, 10 mM</td><td>Sigma Aldrich/various</td><td>H3375</td><td></td></tr><tr><td>EGTA, 0.2 mM</td><td>Sigma Aldrich/various</td><td>E4378</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich/various</td><td>M8266</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;GTP, 0.3 mM</td><td>Sigma Aldrich/various</td><td>G8877</td><td></td></tr><tr><td>MgATP, 2 mM</td><td>Sigma Aldrich/various</td><td>A9187</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Standard artificial cerebrospinal fluid (ACSF, osmolarity &amp;asymp; 300-310 mOsm)&lt;/strong&gt;</td></tr><tr><td>KCl, 2.5 mM</td><td>Sigma Aldrich/various</td><td>P3911</td><td></td></tr><tr><td>NaCl, 119 mM</td><td>Sigma Aldrich/various</td><td>S7653</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;H&lt;sub&gt;2&lt;/sub&gt;O, 1 mM</td><td>Sigma Aldrich/various</td><td>S9638</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3,&lt;/sub&gt; 26.2 mM</td><td>Sigma Aldrich/various</td><td>S8875</td><td></td></tr><tr><td>Glucose, 11 mM</td><td>Sigma Aldrich/various</td><td>G8270</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;-7H&lt;sub&gt;2&lt;/sub&gt;O, 1.3 mM</td><td>Sigma Aldrich/various</td><td>230391</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;-2H&lt;sub&gt;2&lt;/sub&gt;O, 2.5 mM</td><td>Sigma Aldrich/various</td><td>C3881</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Additional compounds used for solutions preparation&lt;/strong&gt;</td></tr><tr><td>KOH</td><td>various</td><td></td><td></td></tr><tr><td>Kynurenic acid</td><td>Sigma Aldrich/various</td><td>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 ...

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

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JoVE Journal

Neuroscience

59.4K Views.  University of Texas Southwestern Medical Center. 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.

Video: Whole-cell Patch-clamp Recordings in Brain Slices

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

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 ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze motor-learning induced plasticity, we trained rats using an accelerated rotor rod task. Rats performed the task 10 times at 30-s intervals for 1 or 2 days. Performance was significantly improved on the training days compared to the first trial. We then prepared acute brain slices of the primary motor cortex (M1) in untrained and trained rats. Current-clamp analysis showed dynamic changes in resting membrane potential, spike threshold, afterhyperpolarization, and membrane resistance in layer II/III pyramidal neurons. Current injection induced many more spikes in 2-day trained rats than in untrained controls.
To analyze contextual-learning induced plasticity, we trained rats using an inhibitory avoidance (IA) task. After experiencing foot-shock in the dark side of a box, the rats learned to avoid it, staying in the lighted side. We prepared acute hippocampal slices from untrained, IA-trained, unpaired, and walk-through rats. Voltage-clamp analysis was used to sequentially record miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) from the same CA1 neuron. We found different mean mEPSC and mIPSC amplitudes in each CA1 neuron, suggesting that each neuron had different postsynaptic strengths at its excitatory and inhibitory synapses. Moreover, compared with untrained controls, IA-trained rats had higher mEPSC and mIPSC amplitudes, with broad diversity. These results suggested that contextual learning creates postsynaptic diversity in both excitatory and inhibitory synapses at each CA1 neuron.
AMPA or GABAA receptors seemed to mediate the postsynaptic currents, since bath treatment with CNQX or bicuculline blocked the mEPSC or mIPSC events, respectively. This technique can be used to study different types of learning in other regions, such as the sensory cortex and amygdala.

The slice patch clamp technique is an effective method for analyzing learning-induced changes in the intrinsic properties and plasticity of excitatory or inhibitory synapses.

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze ...

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic multiplicity). Synaptic multiplicity is plastic and changes throughout development and in different physiological conditions, being an important determinant for the efficacy of synaptic transmission. Here, we outline experiments for estimating the degree of multiplicity of synapses terminating onto a given postsynaptic neuron using whole-cell patch clamp electrophysiology in acute brain slices. Specifically, voltage-clamp recording is used to compare the difference between the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The theory behind this method is that afferent inputs that exhibit multiplicity will show large, action potential-dependent sEPSCs due to the synchronous release that occurs at each synaptic contact. In contrast, action potential-independent release (which is asynchronous) will generate smaller amplitude mEPSCs. This article outlines a set of experiments and analyses to characterize the existence of synaptic multiplicity and discusses the requirements and limitations of the technique. This technique can be applied to investigate how different behavioral, pharmacological or environmental interventions in vivo affect the organization of synaptic contacts in different brain areas.

Here, we present a protocol for evaluating the functional synaptic multiplicity using whole-cell patch clamp electrophysiology in acute brain slices.

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...

Hypothalamic Kisspeptin Neurons as a Target for Whole-Cell Patch-Clamp Recordings

Kisspeptins are essential for the maturation of the hypothalamic-pituitary-gonadal (HPG) axis and fertility. Hypothalamic kisspeptin neurons located in the anteroventral periventricular nucleus and rostral periventricular nucleus, as well as the arcuate nucleus of the hypothalamus, project to gonadotrophin-releasing hormone (GnRH) neurons, among other cells. Previous studies have demonstrated that kisspeptin signaling occurs through the Kiss1 receptor (Kiss1r), ultimately exciting GnRH neuron activity. In humans and experimental animal models, kisspeptins are sufficient for inducing GnRH secretion and, consequently, luteinizing hormone (LH) and follicle stimulant hormone (FSH) release. Since kisspeptins play an essential role in reproductive functions, researchers are working to assess how the intrinsic activity of hypothalamic kisspeptin neurons contributes to reproduction-related actions and identify the primary neurotransmitters/neuromodulators capable of changing these properties. The whole-cell patch-clamp technique has become a valuable tool for investigating kisspeptin neuron activity in rodent cells. This experimental technique allows researchers to record and measure spontaneous excitatory and inhibitory ionic currents, resting membrane potential, action potential firing, and other electrophysiological properties of cell membranes. In the present study, crucial aspects of the whole-cell patch-clamp technique, known as electrophysiological measurements that define hypothalamic kisspeptin neurons, and a discussion of relevant issues about the technique, are reviewed.

Here, we present a protocol to perform a whole-cell patch-clamp on brain slices containing kisspeptin neurons, the primary modulator of gonadotrophin-releasing hormone (GnRH) cells. By adding knowledge about kisspeptin neuron activity, this electrophysiological tool has served as the basis for significant advancements in the neuroendocrinology field over the last 20 years.

Kisspeptins are essential for the maturation of the hypothalamic-pituitary-gonadal (HPG) axis and fertility. Hypothalamic kisspeptin neurons located in ...

Establishing a Whole-Cell Configuration through the Patch Clamp Method

Source: Segev, A. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54024">Whole-cell Patch-clamp Recordings in Brain Slices.</a>&#160;J. Vis. Exp.&#160;(2016)
This video demonstrates the whole-cell patch clamp technique in a mouse brain slice. This technique establishes a stable connection between micropipette and neuron membrane, which is further disrupted to establish a whole-cell configuration to study the neuron&#39;s health and physiological responses.

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

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Neurophysiology

Electrophysiology Techniques

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

Source: Engel, D. <a href="https://app-jove-com.bdigitaluss.remotexs.co/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.

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

Establishing a Whole-Cell Voltage-Clamp Configuration for Electrophysiological Recordings in Brain Slices

Source: Sunstrum, J. K., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/59461">Evaluation of synaptic multiplicity using whole-cell patch-clamp electrophysiology.</a> J. Vis. Exp. (2019)
This video demonstrates positioning a fine-pointed pipette near brain slices, forming a seal with a neuronal cell. Suction breaks the cell membrane, thereby creating a whole-cell patch clamp that enables current measurements for electrophysiology experiments.

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

Automated Image-Guided Patch Clamp Technique for Studying Neurons in Brain Slices

Source: Wu, Q., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56010">Application of automated image-guided patch clamp for the study of neurons in brain slices.</a> J. Vis. Exp. (2017).
In this video, brain slices are immobilized in a recording chamber, and cells of interest are located and focused on using a computer-controlled microscope. The automated image processing system then precisely positions a patch pipette to seal the cell membrane and monitor electrical signals.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. System Setup

	Construct ...

Whole-cell Patch-clamp Recordings in Brain Slices

Summary

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Chapters in this video

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

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

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

Hypothalamic Kisspeptin Neurons as a Target for Whole-Cell Patch-Clamp Recordings

Establishing a Whole-Cell Configuration through the Patch Clamp Method

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

Establishing a Whole-Cell Voltage-Clamp Configuration for Electrophysiological Recordings in Brain Slices

Automated Image-Guided Patch Clamp Technique for Studying Neurons in Brain Slices