optical recording of neuronal activity in brain slices stained with a voltage-sensitive dye

Optical Recording of Neuronal Activity in Brain Slices Stained with a Voltage-Sensitive Dye

To begin this procedure, turn on the amplifier, computer, and camera system and open the software. Pour aCSF in a 50-milliliter tube, and bubble it with carbogen. Use a peristaltic pump to circulate the aCSF, and adjust the flow rate to approximately one milliliter per minute.
Then, adjust the height of the suction pipette so that the liquid level inside the experiment chamber is always constant. Place the ground electrode in the chamber. Subsequently, fill the glass electrode with a small amount of aCSF, and place it in the electrode holder. Attach the holder to the rod of the manipulator.
Using an amplifier, ensure that the electrode resistance is approximately 1 megaohm. In the recording session, transfer a slice from the moist chamber to an experimental chamber. Press the edge of the ring firmly into the silicone O-ring. Be careful not to break the membrane or the bottom of the experiment chamber.
Under the microscope, place the tip of the stimulating electrode and the field potential recording electrode on the slice. Check the evoked response by delivering a stimulus. Confirm that the field of view covers the right neural circuit in the optical recording system.
Next, adjust the excitation light intensity to approximately 70% to 80% of the maximum capacity of the camera. Using the fluorescent light source, adjust the focus with the acquisition system. Then, start the recording, and analyze the data in an image acquisition software after the acquisition.

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Neuroscience

Neurophysiology

Imaging and Optical Techniques

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Daily Preparation of Experimental Apparatus
<ol>
	<li>Turn on the amplifier, computer, and camera system, and check that the software is running.</li>
	<li>Place artificial cerebrospinal fluid (ACSF) in a 50 mL tube and bubble with carbogen.</li>
	<li>Use a peristaltic pump to circulate the ACSF. Adjust the flow rate to approximately 1 mL/min.</li>
	<li>Adjust the height of the suction pipette so that the liquid level inside the experiment chamber is always constant. 
	NOTE: The level of the solution is important to obtain a stable recording, therefore, the adjustment should be done using a micromanipulator.</li>
	<li>Install the ground electrode made up of yellow chip filled with 3 M KCl agar (2%) into a holder with an Ag-AgCl wire with small amount of 3 M KCl solution.</li>
	<li>Fill a small amount of ACSF (approximately two-third of the volume) into the glass electrode (1 mm outer diameter, 0.78 mm inner diameter pulled with a micropipette puller) using a tapered thin tubed yellow tip and place it in the electrode holder.</li>
	<li>Attach the holder to the rod installed in the manipulator. Ensure using an amplifier that the electrode resistance is approximately 1 M&#937;. 
	NOTE: The long-shank wide opening (4-8 &#181;m opening) patch type electrode should be good for field recording and as a stimulating electrode.</li>
</ol>
2. Starting a Recording Session
<ol>
	<li>Take a slice preparation from the moist chamber with forceps.</li>
	<li>Quickly place the slice onto an experimental chamber under the microscope (Figure 1).</li>
	<li>Push the edge of the ring firmly into the silicone O-ring. Be careful not to break the membrane or the bottom of the experiment chamber. 
	NOTE: The direction of the slice should be taken into consideration with respect to the direction of the stimulating and recording electrodes in the field of view. The healthy slice should stick to the membrane filter so there is no need to use other devices to fix the slices such as weights and nylon meshes.</li>
	<li>Place the tip of the stimulating electrode and the field potential recording electrode onto the slice under the microscope with transmitted light.</li>
	<li>Use the electrophysiological recording system to check the response. Confirm the usual (non-stained) electrophysiological recording with given configuration. 
	NOTE: The recording electrode can be omitted but is useful to check the physiology of the slice.</li>
	<li>Adjust the excitation light intensity to approximately 70-80% of the maximum capacity at the camera that corresponds to 13-15 mW/cm2 at the specimen when sampling at 10 kHz with 5x water immersion objective lens and 1x PLAN APO tube lens. The excitation light wavelength is 530 nm, and the emission filter must be &#62; 590 nm. 
	NOTE: Use a shutter to minimize the amount of excitation light. Continuous light exposure may deteriorate the slice physiology. The possible harmful effect of the light depends on the intensity and duration of the light. Use electrophysiological recording to judge the effect of light. In case of the strength of 13-15 mW/cm2, about 1 s exposure should be the upper limit of the tolerance.</li>
	<li>Adjust the focus with the acquisition system using the fluorescent light source because the focus may be different depending on the wavelength and start the acquisition.</li>
	<li>Examine the data in an image acquisition software. 
	NOTE: We used original microprogramming package of numerical analysis software for detailed analysis.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>High speed image acquisition system</td>
			<td>Brainvision co. Ltd.</td>
			<td>MiCAM - Ultima</td>
			<td>Imaging system</td>
		</tr>
		<tr>
			<td>High speed image acquisition system</td>
			<td>Brainvision co. Ltd.</td>
			<td>MiCAM 02</td>
			<td>Imaging system</td>
		</tr>
		<tr>
			<td>Macroscepe for wide field imaging</td>
			<td>Brainvision co. Ltd.</td>
			<td>THT macroscope</td>
			<td>macroscope</td>
		</tr>
		<tr>
			<td>High powere LED illumination system with photo-diodode stablilizer</td>
			<td>Brainvision co. Ltd.</td>
			<td>LEX-2G</td>
			<td>LED illumination</td>
		</tr>
		<tr>
			<td>Image acquisition software</td>
			<td>Brainvision co. Ltd.</td>
			<td>BV-ana</td>
			<td>image acquisition software</td>
		</tr>
		<tr>
			<td>Multifunctional electric stimulator</td>
			<td>Brainvision co. Ltd.</td>
			<td>ESTM-8</td>
			<td>Stimulus isolator+AD/DA converter</td>
		</tr>
		<tr>
			<td>Membrane filter for slice support</td>
			<td>Merk Millipore Ltd., MA, USA</td>
			<td>Omnipore, JHWP01300, 0.45 &#181;m pores,</td>
			<td>membrane filter/ 0.45 13</td>
		</tr>
		<tr>
			<td>Numerical analysis software</td>
			<td>Wavemetrics Inc., OR, USA</td>
			<td>IgorPro</td>
			<td>analysing software</td>
		</tr>
		<tr>
			<td>Stimulation isolator</td>
			<td>WPI Inc.</td>
			<td>A395</td>
			<td>Stimulus isolator</td>
		</tr>
		<tr>
			<td>AD/DA converter</td>
			<td>Instrutech</td>
			<td>ITC-18</td>
			<td>AD/DA converter</td>
		</tr>
		<tr>
			<td>Voltage sensitive dye Di-4-ANEPPS</td>
			<td>Invitrogen, Thermo-Fisher Scientific, Waltham, MA, USA</td>
			<td>catalog number: D-1199</td>
			<td>VSD: Di-4-ANEPPS</td>
		</tr>
		<tr>
			<td>Polyethoxylated castor oil</td>
			<td>Sigma-Aldrich</td>
			<td>Cremophor EL C5135</td>
			<td>polyethoxylated castor oil</td>
		</tr>
	</tbody>
</table>

Source: Tominaga, Y., et al. <a href="https://app.jove.com/v/59692">Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye.</a> J. Vis. Exp. (2019).
This video demonstrates a procedure for observing changes in neuronal activity in a brain slice stained with a voltage-sensitive dye. The dye reflects changes in the neuron&#39;s membrane potential by altering its fluorescence intensity. Through the application of electrical stimuli and advanced imaging techniques, this process enables visual tracking of real-time changes in neuronal membrane potential based on VSD fluorescence.

<img alt="Figure 1" src="/files/ftp_upload/22736/22736_Figure1.jpg" /> 
Figure 1. Recording system for optical signals from slice preparations. (A) A photograph of the microscope used to image the slices in the current manuscript. The optics consist of an objective lens (5x NA0.60), a mirror box for dichroic filter (580 nm), and a projection (tube) lens (PLAN APO x1.0). A high-speed camera is attached on the top of the projection lens through a c-mou...

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Daily Preparation of ...

Take an immobilized mouse brain slice stained with a voltage-sensitive dye or VSD, fluorescent molecules that integrate into the neuronal membrane and respond to membrane potential changes.
Place the slice in a recording chamber.
Install the ground electrode for a baseline voltage reference.
Next, position the stimulating and recording electrodes on the slice.
Apply an electrical pulse.
The pulse stimulates the neurons, triggering voltage-gated sodium channel opening and sodium ion influx, leading to a membrane potential change.
This induces an action potential, measured by the recording electrode, and alters the VSD&#39;s fluorescent properties.
Expose the brain slice to a specific wavelength to excite the VSD and trigger fluorescence emission.
Post-stimulation, the sodium channels close while the voltage-gated potassium channels open, promoting potassium ion efflux.
This changes the membrane potential again and consequently alters the VSD&#39;s fluorescence.
Record the fluorescence in real-time to visually represent neuronal activity.

21 조회수. 전압 민감성 염료로 염색된 뇌 절편의 신경 활동에 대한 광학 기록

비디오: 전압 민감성 염료로 염색된 뇌 절편의 신경 활동에 대한 광학 기록 - 실험

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

고립 된 뮤린 심실 근세포의 광학 이미징

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

연구

JoVE Journal

생체공학

High-resolution Optical Mapping of the Mouse Sino-atrial Node

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

Here, we present a protocol for optical mapping of electrical activity from the mouse right atrium and especially the sino-atrial node, at a high spatial and temporal resolution.

마우스 중국 - 심방 노드의 고해상도 광학 매핑

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The ...

생물학

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

비 트랜스제닉 무척추 동물의 단일 신경 해상도를 가진 네트워크 역학을 기록하기위한 포토다이오드 기반 광학 이미징

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...

Optical Recording of Neuronal Activity in Brain Slices Stained with a Voltage-Sensitive Dye

내레이션 대본

Optical Recording of Neuronal Activity in Brain Slices Stained with a Voltage-Sensitive Dye