light-evoked postsynaptic responses in neurons of mouse retinal slices

Light-Evoked Postsynaptic Responses in Neurons of Mouse Retinal Slices

For patch-clamp recordings, prime the perfusion tubes with Ames&#39; medium and allow all the bubbles to pass from the tubing. After making recording pipettes with a glass puller, backfill the tip with pipette solution. Next, use a micropipette filler to fill about one-third of the pipette. Once filled, store each pipette in a moist pipette box.
Next, turn on the equipment for patch-clamp recordings, including the computer, amplifier, CCD camera, and microscope. Working in dark conditions place a retinal slice preparation onto the microscope stage with the aid of an infrared viewer. After it is immobilized, begin continuous perfusion.
Set the perfusion temperature to 33 to 37 degrees Celsius. View the slice surface with a CCD camera. Focus on the target location where the target cell types reside. Select a healthy-looking cell for patch-clamp recording. Once a healthy cell is identified, place a recording pipette in a pipette holder, and advance the pipette to the slice preparation.
When it is close to the slice preparation, find the tip of the pipette with a microscope. Once the pipette tip is visible under the microscope, move the tip down towards the target cell. Next, set the amplifier, and adjust the pipette at picoamp 5 volts per nanoamp. Start two continuous electric pulse of approximately 5 millivolts and approximately 10 hertz and check the pipette resistance.
An ideal resistance is between 5 and 12 megaohms for most retinal neurons. When ready, use a mouthpiece or syringe to blow out the internal solution, until the tip of the pipette is on the surface of the target cell. When the positive pressure makes a small dimple on the cell surface, advance the tip slightly and stop blowing out.
If the pipette resistance is continuously increasing, leave it and monitor the resistance until it reaches greater than 1 gigaohm. If the resistance does not spontaneously increase, gently apply negative pressure until it becomes a gigaseal. After the gigaseal is achieved, change the holding potential to negative 70 millivolts.
Then, intermittently apply negative pressure to rupture the membrane inside the pipette tip. When the whole-cell configuration is made, the pipette resistance can be between 500 megaohms and one gigaohm, and the capacitive current is observable. Record the I-V relationship from negative 80 to 40 millivolts. Different types of voltage-gated channels will be activated depending on the cell type. Lastly, record light-evoked synaptic currents or voltages.

light-evoked-postsynaptic-responses-in-neurons-of-mouse-retinal-slices

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Imaging and Optical Techniques

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Experimental Solution
<ol>
	<li>Prepare the dissecting solution 1 day to 1 week before the actual experiment. Use a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution for retinal dissection because of its strong buffering ability at lower temperatures16. Mix all chemicals as follows (in mM): 115 NaCl, 2.5 KCl, 2.5 CaCl2, 1.0 MgCl2, 10 HEPES, 28 glucose. Adjust pH to 7.4 with NaOH. Keep the solution in a refrigerator up to 1 week.</li>
	<li>The recording solution is Ames&#39; medium, which is an artificial cerebral spinal fluid (aCSF) designed for retinal preparations. By the day of the experiment, weigh out Ames&#39; powder (4.4 g for 500 ml solution) in a tube. Also, weigh out NaHCO3 (0.95 g for 500 ml solution) and mix with the Ames&#39; powder.</li>
	<li>Prepare the intracellular pipette solution for patch clamp recordings by the day of the experiment. Mix all chemicals as follows (in mM): 111 K-gluconate, 1.0 CaCl2, 10 HEPES, 1.1 ethylene glycol tetraacetic acid (EGTA), 10 NaCl, 1.0 MgCl2, 5 ATP-Mg, and 1.0 GTP-Na. Adjust pH to 7.2 with KOH. Filter the pipette solution with a conventional syringe filter. Make ~500 &#181;l aliquots and store them in a freezer or a deep freezer.</li>
</ol>
2. Preparation for the Day of Experiment
<ol>
	<li>The night before the experiment, place a mouse in a cage (C57BL/6J or similar background, 4 - 8 weeks old, male) in a dark space for overnight (O/N) dark adaptation.</li>
	<li>Preparation for dissection:
	<ol>
		<li>Fill dissecting solution (100 - 200 ml) in a glass beaker, place it on ice, and bubble with oxygen for at least 10 min.</li>
		<li>Start oxygenation of the dark box for retinal preparation storage.</li>
		<li>Prepare plastic coverslips with grease rails for retinal slice preparations and place each of them in a 35 mm plastic dish.</li>
		<li>Attach a new razor blade to a chopper (tissue slicer).</li>
		<li>Cut a filter membrane into two halves. This is for retinal slicing.</li>
		<li>Align the dissecting tools and transfer pipettes near the dissecting microscope. The work area must be well organized so that each tool can be easily accessed in the dark.</li>
	</ol>
	</li>
	<li>Preparation for patch clamp recordings:
	<ol>
		<li>Dissolve Ames&#39; powder and NaHCO3 in distilled water (Ames 4.4 g and NaHCO3 0.95g/500 ml ddH2O). Bubble the Ames&#39; medium with mixed oxygen (95% O2 and 5% CO2) for at least 30 min while heating to a recording temperature level (~35 &#176;C). Adjust the pH to 7.4 with NaHCO3 while still bubbling.</li>
		<li>Thaw the internal pipette solution during the dissection. After it is thawed, add 0.01% sulforhodamine B for intracellular staining.</li>
	</ol>
	</li>
</ol>
3. Patch Clamp Recordings from Retinal Slice Preparation
<ol>
	<li>Recording preparation:
	<ol>
		<li>Prime the perfusion tubes with Ames&#39; medium. Allow all the bubbles to pass through when filling the tubing.</li>
		<li>Make patch clamp recording pipettes with a puller. To fill a pipette with the pipette solution, dip in the backside of a pipette for 1 min until the pipette tip is backfilled. Next, fill ~1/3 of the pipette using a micro-pipette filler. Store each pipette in a moist pipette box.</li>
		<li>Turn on the equipment for patch clamp recordings, including the computer, amplifier, charge-coupled device (CCD) camera, and microscope.</li>
	</ol>
	</li>
	<li>Shut off the room light. Place a retinal slice preparation onto the microscope stage chamber using an infrared viewer. After it is immobilized, begin continuous perfusion. Set the perfusion temperature at 33 to 37 &#176;C.</li>
	<li>View the slice surface with the CCD camera. Focus on the target location where the target cell types reside. Select a healthy-looking cell for patch clamp recording (i.e., it should have smooth-looking surface and a good cell shape).</li>
	<li>Place a recording pipette in a pipette holder. Advance the pipette to the slice preparation. When it is close to the slice preparation (~2 mm above), find the tip of the pipette with the microscope. Once the pipette tip is visible under the microscope, move the tip down towards the target cell.</li>
	<li>Set the amplifier. Adjust the pipette at 0 mV/0 pA (zeroing) and start a continuous electric pulse of ~5 mV at ~10 Hz. Check the pipette resistance; ideal resistance is between 5 and 10 M&#8486; for most retinal neurons.</li>
	<li>Start blowing out from the tip. Use a mouthpiece, or use a syringe, to apply positive pressure when the pipette dips into the bath solution. Continuously blow out the internal solution until the tip is on the surface of the target cell.
	<ol>
		<li>When the positive pressure makes a small dimple on the cell surface, advance the tip slightly and stop blowing out. Check the pipette resistance, which should increase. If it is continuously increasing, leave it and monitor the resistance until it reaches &#62;1 G&#8486; (gigaseal). If the resistance does not spontaneously increase, gently apply negative pressure until it becomes a gigaseal.</li>
	</ol>
	</li>
	<li>After the gigaseal is achieved, change the holding potential to -70 mV. Then, intermittently apply negative pressure to rupture the membrane inside the pipette tip. When the whole-cell configuration is made, the pipette resistance can be between 500 M&#8486; and 1 G&#8486;, and the capacitive current is seen. Sometimes spontaneous postsynaptic currents can be observed.</li>
	<li>Record the current&#8211;voltage (I-V) relationship from -80 to +40 mV. Different types of voltage-gated channels are activated depending on the cell type.</li>
	<li>Record light-evoked synaptic currents or voltages.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>mice (28-60 days old, male)</td>
			<td>Jackson laboratory</td>
			<td>C57BL/6J strain</td>
			<td></td>
		</tr>
		<tr>
			<td>Ames&#39; medium powder</td>
			<td>Sigma</td>
			<td>A1420</td>
			<td>excellent</td>
		</tr>
		<tr>
			<td>infrared viewer</td>
			<td>Night Owl Optics</td>
			<td>NOBG1</td>
			<td>It shows bright view. Focusing small objects is an issue.</td>
		</tr>
		<tr>
			<td>infrared pocket scopes</td>
			<td>B.E. Meyers</td>
			<td>OWL Gen 3 NV pocketscope</td>
			<td>excellent view</td>
		</tr>
		<tr>
			<td>puller</td>
			<td>Sutter</td>
			<td>P-1000</td>
			<td>excellent. Make consistent size pipettes.</td>
		</tr>
		<tr>
			<td>dark box</td>
			<td>Pelican</td>
			<td>dark box</td>
			<td>excellent</td>
		</tr>
		<tr>
			<td>patch clamp system</td>
			<td>Scientifica</td>
			<td>slice scope 2000</td>
			<td>Excellent setup. Most key components are included in one package. Micromanipulators are excellent.</td>
		</tr>
		<tr>
			<td>amplifier</td>
			<td>Molecular Devices</td>
			<td>multiclamp 700B</td>
			<td>Excellent and easy control.</td>
		</tr>
		<tr>
			<td>acquiring software</td>
			<td>Molecular Devices</td>
			<td>pClamp software</td>
			<td>Excellent and easy control.</td>
		</tr>
		<tr>
			<td>light source (LED)</td>
			<td>Cool LED</td>
			<td>pE-2 4 channel system</td>
			<td>Excellent</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Q-imaging</td>
			<td>Retiga 2000</td>
			<td>Excellent</td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td>handmade</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Hellmer, C. B., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/52422">Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques.</a> J. Vis. Exp. (2015).
This video demonstrates the recording of light-evoked post-synaptic responses in a dark-adapted mouse retinal tissue slice preparation. The retinal slice is placed in a recording chamber, and a recording pipette is used to establish electrical continuity with a retinal ganglion cell. Light pulses are applied to generate excitatory post-synaptic potentials in the ganglion cells, which are recorded by the pipette.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Experimental ...

Take a dark-adapted mouse retinal slice inside a recording chamber in a dark room. The retina sits on a supportive artificial base set over a cover slip. Perfuse the slice with aCSF.
The retinal neural network includes photoreceptors synaptically linked to bipolar cells that synapse with ganglion cells.
Position a recording pipette near the retina.
Under a microscope, apply positive pressure while approaching a target ganglion cell.
Switch to negative pressure to pull in a membrane patch, then rupture it to establish continuity with the cytoplasm.
In the dark, the photoreceptor membrane remains depolarized, leading to neurotransmitter release.
Neurotransmitters binding to bipolar cell receptors trigger cation channel closure, inhibiting signal transmission.
Apply a light pulse to hyperpolarize the photoreceptors. The decreased neurotransmitter release triggers cation channel opening in bipolar cells.
Cation influx induces neurotransmitter release that binds to ganglion cell receptors,&#160; generating an excitatory post-synaptic potential recorded by the pipette.

13 ビュー. マウス網膜切片のニューロンにおける光誘発シナプス後応答

ビデオ: マウス網膜切片のニューロンにおける光誘発シナプス後応答 - 実験

Direct-Coupled Electroretinogram (DC-ERG) for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a specialized monolayer of cells strategically located between the retina and the choriocapillaris that maintain the overall health and structural integrity of the photoreceptors. The RPE is polarized, exhibiting apically and basally located receptors or channels, and performs vectoral transport of water, ions, metabolites, and secretes several cytokines.
In vivo noninvasive measurements of RPE function can be made using direct-coupled ERGs (DC-ERGs). The methodology behind the DC-ERG was pioneered by Marmorstein, Peachey, and colleagues using a custom-built stimulation recording system and later demonstrated using a commercially available system. The DC-ERG technique uses glass capillaries filled with Hank&#8217;s buffered salt solution (HBSS) to measure the slower electrical responses of the RPE elicited from light-evoked concentration changes in the subretinal space due to photoreceptor activity. The prolonged light stimulus and length of the DC-ERG recording make it vulnerable to drift and noise resulting in a low yield of useable recordings. Here, we present a fast, reliable method for improving the stability of the recordings while reducing noise by using vacuum pressure to reduce/eliminate bubbles that result from outgassing of the HBSS and electrode holder. Additionally, power line artifacts are attenuated using a voltage regulator/power conditioner. We include the necessary light stimulation protocols for a commercially available ERG system as well as scripts for analysis of the DC-ERG components: c-wave, fast oscillation, light peak, and off response. Due to the improved ease of recordings and rapid analysis workflow, this simplified protocol is particularly useful in measuring age-related changes in RPE function, disease progression, and in the assessment of pharmacological intervention.

Direct-Coupled Electroretinogram DC-ERG for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium

Here, we present a method for recording light-evoked electrical responses of the retinal pigment epithelium (RPE) in mice using a technique known as DC-ERGs first described by Marmorstein, Peachey, and colleagues in the early 2000s.

マウス網膜色素上皮の光誘発電気応答を記録するための直接結合電解電解(DC-ERG)

The retinal pigment epithelium (RPE) is a specialized monolayer of cells strategically located between the retina and the choriocapillaris that maintain ...

JoVE Journal

化学

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using 128-channel high-density electroencephalography (EEG). The specific aim of the described stimuli and analyses is to examine whether it is feasible to replicate previously reported CVEP morphological patterns elicited by an apparent motion stimulus, designed to simultaneously stimulate both ventral and dorsal central visual networks, using object and motion stimuli designed to separately stimulate ventral and dorsal visual cortical networks.&#160; Four visual paradigms are presented: 1. Randomized visual objects with consistent temporal presentation. 2. Randomized visual objects with inconsistent temporal presentation (or jitter).&#160; 3. Visual motion via a radial field of coherent central dot motion without jitter.&#160; 4. Visual motion via a radial field of coherent central dot motion with jitter.&#160; These four paradigms are presented in a pseudo-randomized order for each participant.&#160; Jitter is introduced in order to view how possible anticipatory-related effects may affect the morphology of the object-onset and motion-onset CVEP response.&#160; EEG data analyses are described in detail, including steps of data exportation from and importation to signal processing platforms, bad channel identification&#160;and removal, artifact rejection, averaging, and categorization of average CVEP morphological pattern type based upon latency ranges of component peaks. Representative data show that the methodological approach is indeed sensitive in eliciting differential object-onset and motion-onset CVEP morphological patterns and may, therefore, be useful in addressing the larger research aim. Given the high temporal resolution of EEG and the possible application of high-density EEG in source localization analyses, this protocol is ideal for the investigation of distinct CVEP morphological patterns and the underlying neural mechanisms which generate these differential responses.&#160; &#160; &#160;&#160;

In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined.&#160;&#160;

刺激特異的皮質視覚誘発潜在的形態パターン

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using ...

神経科学

Microtransplantation of Synaptic Membranes to Reactivate Human Synaptic Receptors for Functional Studies

Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal communication. Therefore, alterations in their abundance, function, and relationships with other synaptic elements have been observed as a major correlate of alterations in brain function and cognitive impairment in neurodegenerative diseases and mental disorders. Understanding how the function of excitatory and inhibitory synaptic receptors is altered by disease is of critical importance for the development of effective therapies. To gain disease-relevant information, it is important to record the electrical activity of neurotransmitter receptors that remain functional in the diseased human brain. So far this is the closest approach to assess pathological alterations in receptors' function. In this work, a methodology is presented to perform microtransplantation of synaptic membranes, which consists of reactivating synaptic membranes from snap frozen human brain tissue containing human receptors, by its injection and posterior fusion into the membrane of Xenopus laevis oocytes. The protocol also provides the methodological strategy to obtain consistent and reliable responses of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and &#947;-aminobutyric acid (GABA) receptors, as well as novel detailed methods that are used for normalization and rigorous data analysis.

The protocol demonstrates that by performing microtransplantation of synaptic membranes into Xenopus laevis oocytes, it is possible to record consistent and reliable responses of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and &#947;-aminobutyric acid receptors.

機能研究のためのヒトシナプス受容体を再活性化するためのシナプス膜の微小移植

Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal ...