a technique for the prolonged incubation of neuronal tissue of the retina

A Technique for the Prolonged Incubation of Neuronal Tissue of the Retina

For retinal whole mount preparation, immediately enucleate the eyes from a euthanized animal. Make a small cut along the ora serrata, and place the eyes in either Ames Media or aCSF with carbogen supply at room temperature. After that, immediately remove the cornea, lens, and vitreous by cutting along the ora serrata with small scissors, and remove them with forceps. Place the tissue in the incubation system at room temperature.
To remove the inner limiting membrane, transfer the eye cup containing the retina to a small glass jar containing papain, L-cysteine, EDTA, and DNAse at 37 degrees Celsius for 20 minutes. Apply carbogen to the solution through the lid, but do not bubble. Stop the enzymatic digestion by placing the tissue in an ovomucoid and BSA solution for 10 minutes in Earl&#39;s BSS. Then, wash the tissue thoroughly with aCSF.
Subsequently, transfer the tissue to the incubation system and reduce the temperature to about 15 to 16 degrees Celsius. Following that, transfer the retinal tissue to the microscope; isolate the retina from the eye cup, and cut it into four pieces with a razor blade. If the entire retina is required, make four small cuts in the periphery of the retina to allow it to lie flat.
This tissue can now be transferred to the microscope for electrophysiology, or maintained in the Braincubator. The tissue is maintained in the main chamber containing the probes for pH and temperature measurements. A second chamber is isolated from the main chamber and is exposed to 1.1 watts UVC light in order to irradiate bacteria floating in the solution. Use a peristaltic pump to circulate aCSF through the two chambers, and a Peltier thermoelectric cold plate to either cool or heat the main chamber.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Retinal Wholemount Preparation and Inner Limiting Membrane Removal
<ol>
	<li>Prepare retinal wholemounts under either normal laboratory lighting conditions or dim red/infra-red light.</li>
	<li>Euthanize animal by cervical dislocation and immediately enucleate eyes. Make a small cut along the ora serrata, and place in either Ames media, or artificial cerebrospinal fluid (aCSF) containing (mM): 125 NaCl, 25 NaHCO3, 3 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, 0.5 L-glutamine, and saturate with carbogen (95% O2/5% CO2 mixture; ~300 mOsm; pH 7.4), at room temperature (RT).</li>
	<li>Immediately remove the cornea, lens and vitreous by cutting along the ora serrata with small scissors and removing the lens and vitreous with forceps. Place tissue in the incubation system at RT. 
	NOTE: Retinal tissue can be maintained in the eyecup, after slow temperature reduction to 15 - 16 &#176;C, for &#62;24 h in the incubation system until needed.</li>
	<li>To remove the ILM, transfer the eyecup containing the retina to a small glass jar containing 30 U/mL papain, 1 mM L-cystine, 0.5 mM ethylenediaminetetraacetic acid (EDTA) and 0.005% deoxyribonuclease (DNase) in Earl&#39;s Balanced Salt Solution (BSS) at 37 &#176;C for 20 min. Apply 95% O2/5% CO2 to the solution through the lid but do not bubble. If using tissue from young animals (&#60;6 weeks), dilute the solution to half-strength.
	<ol>
		<li>Stop enzymatic digestion by placing the tissue in an ovomucoid (10 mg/mL) and bovine serum albumin (BSA, 10 mg/mL) solution for 10 min in Earl&#39;s BSS. Wash tissue thoroughly with aCSF before transferring to the incubation system and reduce temperature to 15 - 16 &#176;C.</li>
	</ol>
	</li>
	<li>Maintain retinal tissue in the eyecup until needed. For transfer to the microscope, isolate the retina from the eyecup and cut into 4 pieces with a razorblade. If the entire retina is required, make four small cuts in the periphery of the retina to allow it to lie flat. 
	NOTE: For imaging experiments, load with calcium-dyes before transfer to the incubation system, see below.</li>
</ol>
2. Maintaining Tissue in the Incubator
<ol>
	<li>Place tissue in the main chamber containing probes for pH and temperature measurements (Figure 1A, B).</li>
	<li>Circulate aCSF through a second chamber (UVC chamber, built as previously described) isolated from the main chamber and exposed to 1.1 W ultraviolet C (UVC) light (254 nm, 5W/2P sterilizer UV lamp) in order to eradicate bacteria floating in the solution (Figure 1A). Control UVC light timing via a programmable timer using a random feature that turns ON at times varying between 15 and 26 min every 15 - 30 min to avoid excessive heating of the aCSF.</li>
	<li>Set the flow rate in the UVC chamber to 12 mL/min as previously reported. Cover the UVC chamber with aluminum foil to prevent UVC illumination outside the chamber, which can damage neuronal tissue. 
	NOTE: For dark-adapted retinal tissue, apply a custom-made cover to the main chamber to exclude light.</li>
	<li>Use a peristaltic pump to circulate the solution (aCSF) through the two chambers and a Peltier thermoelectric cold plate cooler to either cool or heat the main chamber to the desired temperatures in the range of 0 - 50 &#176;C. For optimal tissue incubation, use 15 - 16 &#176;C for long-term viability.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich VETEC</td>
			<td>V800372</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Methyl-D-glucamine</td>
			<td>Sigma Aldrich</td>
			<td>M2004</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma Aldrich</td>
			<td>G5767</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma Aldrich</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma Aldrich</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain Dissociation System</td>
			<td>Worthington</td>
			<td>LK003150</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixed Stage Upright Microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Objective</td>
			<td>Olympus</td>
			<td>XLUMPlanFLN 20x/1.00w</td>
			<td>20X</td>
		</tr>
		<tr>
			<td>Microscope Objective</td>
			<td>Olympus</td>
			<td>LUMPlanFLN 60x/1.00w</td>
			<td>60X</td>
		</tr>
		<tr>
			<td>Braincubator</td>
			<td>Payo Scientific</td>
			<td>BR26021976</td>
			<td>www.braincubator.com.au</td>
		</tr>
		<tr>
			<td>Peltier-Thermoelectric Cold Plate Cooler</td>
			<td>TE Technology</td>
			<td>CP-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature Controller</td>
			<td>TE Technology</td>
			<td>TC-36-25 RS232</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Cameron, M. A., et al. <a href="https://app.jove.com/v/55396">Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging.</a> J. Vis. Exp. (2017).
This video demonstrates a method for prolonged incubation of neuronal tissue of the retina. Eyecups containing retina are obtained from a mouse eye; the inner limiting membrane (ILM) is enzymatically digested to expose the neuronal cells of the retina, and either the treated eyecup or the isolated retina is transferred to a specialized incubator for prolonged incubation at a low temperature.

<img alt="Figure 1" src="/files/ftp_upload/22379/22379_Figure1.jpg" /> 
Figure 1. An Incubation System That Enables Tight Regulation of Tissue Environment. A, Schematic diagram of the incubation system composed of a cooling Peltier plate, main chamber, peristaltic pump and UVC chamber. B, Side view of the main chamber showing the inlet and outlet points of the aCSF, as well as the carbogen inlet, temperature and pH probes. Tissue is placed on the mes...

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

Take an intact mouse eye in artificial cerebrospinal fluid, or aCSF, and incise to isolate the eyecup containing the retina.
Treat with an enzymatic solution to remove the inner limiting membrane, exposing the neuronal cells.
Immerse the tissue in an inhibition solution to stop digestion, and wash to remove excess solution.
Place the tissue in a specialized incubator at a low temperature to slow cellular metabolism, preserving the tissue.
This incubator comprises a main chamber with a mesh for mounting tissues. The chamber is filled with aCSF, and its temperature and pH are controlled to preserve tissue functionality.
To eliminate microbial contamination, the aCSF is pumped into another chamber and treated with ultraviolet radiation to kill any microbes.
The sterilized aCSF is then pumped back into the main chamber.
To isolate the retina, remove the eyecup, separate the retina from the surrounding tissue, and return it to the incubator.&#160;

17 Views. A Technique for the Prolonged Incubation of Neuronal Tissue of the Retina

Video: A Technique for the Prolonged Incubation of Neuronal Tissue of the Retina - Experiment

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in ...

JoVE Journal

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.

We present the preparation of acute pancreatic tissue slices and their use in confocal laser scanning microscopy to study calcium dynamics simultaneously in a large number of live cells, over long time periods, and with high spatiotemporal resolution.

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails ...

A Technique for the Prolonged Incubation of Neuronal Tissue of the Retina

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A Technique for the Prolonged Incubation of Neuronal Tissue of the Retina