auditory nerve root stimulation and postsynaptic current recordings in a mouse brain wedge slice

Auditory Nerve Root Stimulation and Postsynaptic Current Recordings in a Mouse Brain Wedge Slice

To set up the wedge slice for electrophysiology analysis, place the sample into a recording chamber that is being continuously perfused with 35 degrees Celsius aCSF and stabilize the slice. Using DIC optics, focus on the auditory nerve root on the thick side of the slice, and use a micromanipulator to move the bipolar tungsten stimulating electrode to the auditory nerve root and gently into the surface of the tissue.
Move the field of view to the ventral nucleus of the trapezoid body on the thin side, and select a medial olivocochlear neuron as the target for patch-clamp electrophysiology under epifluorescence using a 561-nanometer emission filter. Fill a recording pipette with the appropriate internal solution for the proposed experiment, and under DIC optics, patch, and record from the medial olivocochlear neuron in the whole-cell configuration.
Adjust the electrical stimulation amplitude of the auditory nerve root to obtain consistent postsynaptic events in the medial olivocochlear neuron. Then, run appropriate stimulation protocols to observe evoked synaptic currents in medial olivocochlear neurons.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Electrophysiology set-up and recording
<ol>
	<li>Place the wedge slice in the recording chamber and secure the slice with a harp or stabilizing system. Perfuse the tissue continuously at a rate of 7-10 mL/min with warm (35 &#176;C) ACSF bubbled with carbogen.</li>
	<li>Identify genetically labeled medial olivocochlear MOC neurons in the ventral nucleus of the trapezoid body VNTB using epifluorescence with 561 nm emission filters for patch-clamp recordings. Flip slice if there are no potentially patchable cells.</li>
	<li>Using DIC optics, focus on the auditory nerve root on the thick side of the slice and use a micromanipulator to move the bipolar tungsten stimulating electrode down to the auditory nerve root and gently into the surface of the tissue. 
	NOTE: Suction electrodes have been used in auditory nerve stimulation experiments in other labs. Theta glass electrodes or optical stimulation methods can be employed if applicable to other specific preparations.</li>
	<li>Move the field of view back to the VNTB to choose a MOC neuron to target for patch clamp electrophysiology.</li>
	<li>Fill a recording pipette with an appropriate internal solution for the proposed experiment.</li>
	<li>Patch and record from the MOC neuron in the whole-cell configuration. Compensate membrane capacitance and series resistance if required.</li>
	<li>Adjust the electrical stimulation amplitude of the auditory nerve root to obtain consistent postsynaptic events in the MOC neuron. 
	NOTE: It may be necessary to move the stimulation electrode.</li>
	<li>Run appropriate stimulation protocols to observe evoked synaptic currents in MOC (voltage clamp). 
	NOTE: The wedge slice preparation can be used with any typical patch-clamp tools such as loose patch recordings, pharmacology, optogenetics, calcium imaging, neurotransmitter uncaging, etc.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A1R Upright Confocal Microscope</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td>Electrophysiology and imaging microscope, can be any microscope compatible with electrophysiology</td>
		</tr>
		<tr>
			<td>Electrode Borosilicate glass w/ Filament OD 1.5mm, ID 1.1mm, 10 cm long</td>
			<td>Sutter Instrument</td>
			<td>BF150-110-10</td>
			<td>Patch clamping pipette glass</td>
		</tr>
		<tr>
			<td>Electrode Filler MicroFil</td>
			<td>WPI</td>
			<td>CMF20G</td>
			<td>Patch electrode pipette filler</td>
		</tr>
		<tr>
			<td>In-line solution heater</td>
			<td>Warner Instruments (GSAdvantage)</td>
			<td>SH-27B</td>
			<td>Slice perfusion system heater</td>
		</tr>
		<tr>
			<td>Multi-Micromanipulator Systems</td>
			<td>Sutter Intruments</td>
			<td>MPC-200 with MP285</td>
			<td>Micromanipulators for patch clamp and stimulation electrode placement</td>
		</tr>
		<tr>
			<td>P-1000 horizontal pipette puller for glass micropipettes</td>
			<td>Sutter instruments</td>
			<td>FG-P1000</td>
			<td>Patch clamp pipetter puller</td>
		</tr>
		<tr>
			<td>Patch-clamp amplifier and Software</td>
			<td>HEKA</td>
			<td>EPC-10 / Patchmaster Next</td>
			<td>Can be any amplifier/software</td>
		</tr>
		<tr>
			<td>Recording Chamber</td>
			<td>Warner Instruments</td>
			<td>RC26G</td>
			<td>Slice &#34;bath&#34; during recording</td>
		</tr>
		<tr>
			<td>Recording Chamber Harp</td>
			<td>Warner Instruments</td>
			<td>640253</td>
			<td>Stablizes slice during electrophysiology recording</td>
		</tr>
		<tr>
			<td>Slice Incubation Chamber</td>
			<td>Custom Build</td>
			<td></td>
			<td>Heated, oxygenated holding chamber for slices during recovery after slicing</td>
		</tr>
		<tr>
			<td>Stimulus isolation unit</td>
			<td>A.M.P.I.</td>
			<td>Iso-Flex</td>
			<td>Stimulus isolation unit for electrophysiology</td>
		</tr>
		<tr>
			<td>Syringe 60CC</td>
			<td>Fischer Scientific (Intramalls)</td>
			<td>14-820-11</td>
			<td>Electrophysiology perfusion fluid handling</td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>Warner Instruments (GSAdvantage)</td>
			<td>TC-324C</td>
			<td>Slice perfusion system temperature controller</td>
		</tr>
		<tr>
			<td>Tubing 1/8 OD 1/16 ID</td>
			<td>Fischer Scientific (Intramalls)</td>
			<td>14-171-129</td>
			<td>Electrophysiology perfusion fluid handling</td>
		</tr>
		<tr>
			<td>Tugsten concentric bipolar microelectrode</td>
			<td>WPI</td>
			<td>TM33CCINS</td>
			<td>Stimulating electrode for electrophysiology</td>
		</tr>
	</tbody>
</table>

Source: Fischl, M. J., et al. <a href="https://app.jove.com/v/61664">In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity.</a> J. Vis. Exp.(2020)
This video demonstrates a protocol for stimulating the auditory nerve root and recording postsynaptic currents in medial olivocochlear neurons in mouse brain wedge slices.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Electrophysiology set-up ...

Secure a transgenic mouse brain wedge slice in a recording chamber perfused with oxygenated aCSF.
The thicker wedge side contains the auditory nerve root and cochlear nucleus, including T-stellate and globular bushy cells.
The thinner side contains the medial and ventral nucleus of the trapezoid body with fluorescent medial olivocochlear neurons.
Microscopically locate the auditory nerve root and position a stimulating electrode on it.
Identify a fluorescent medial olivocochlear neuron and advance a recording pipette toward it.
Patch the neuron to achieve a whole-cell configuration and record its electrical activity.
Apply electrical pulses to the auditory nerve root, activating cochlear nucleus cells.
Activated T-stellate cells release excitatory neurotransmitters to the medial olivocochlear neurons.
However, globular bushy cells activate inhibitory pathways, releasing inhibitory neurotransmitters to the medial olivocochlear neurons.
The integration of the excitatory and inhibitory signals results in postsynaptic currents, confirming neuronal activity within the slice.

17 ビュー. マウス脳ウェッジスライスにおける聴神経根刺激とシナプス後電流記録

ビデオ: マウス脳ウェッジスライスにおける聴神経根刺激とシナプス後電流記録 - 実験

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

パッチクランプ技術を用いたダーク適応、マウス網膜スライス標本におけるニューロンにおける記録光誘発シナプス後応答

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

JoVE Journal

神経科学

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

無傷の神経上皮におけるマウス嗅細胞の穿孔パッチクランプ記録：同定された嗅覚受容体を発現するニューロンの機能解析

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

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.

膠様質ニューロンの細胞パッチク ランプ記録のため急性脊髄スライスの準備

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