We thank Colin Symons and James Wright for technical assistance. This work was supported by seed funding grant (WSU) to Y.B. and the innovation office at Western Sydney University. M.C. is supported by ARC fellowship (DECRA).

The protocol below describes the preparation of C57BL/6 and C3H/He (retinally degenerate) mouse neuronal tissue, but similar techniques can be applied to other species. All animals were healthy and handled with standard conditions of temperature, humidity, 12 h light/dark cycle, free access to food and water, and without any intended stress stimuli. All experiments were approved and performed in accordance with Western Sydney University Animal Care and Ethics committee and according to the animal use and care guidelines (Animal Research Authority #A9452, #A10396 and #A8967).
1. Brain Slice Preparation
<ol>
	<li>Anesthetize the animal by inhalation of isoflurane (5%) and decapitate using a rodent guillotine. Remove the brain quickly, as described previously9 and place it into ice-cold physiological solution (aCSF) containing (in mM): 125 NaCl, 2.5 KCl, 1 MgCl2, 1.25 NaH2PO4, 2 CaCl2, 25 NaHCO3, 25 dextrose, and saturated with carbogen (95% O2/5% CO2 mixture; 310 mOsm; pH 7.4).</li>
	<li>Cut brain slices, in the region of interest, 300 &#181;m thick with a vibrating microtome.</li>
	<li>Transfer slices to a custom built incubation system that closely monitors and controls pH levels (pH 7.2 - 7.4), carbogen flow, and temperature as previously described10,11.</li>
	<li>Set initial chamber temperature to 35 &#176;C for 15 - 30 min, and then slowly reduce to 15 - 16 &#176;C as shown in Figure 1C. Then incubate slices in the incubation system until needed, either for electrophysiology or imaging. 
	NOTE: If slices are to be used for calcium-imaging, follow steps below before cooling tissue below RT.</li>
</ol>
2. 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 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 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 eye-cup, 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 eye-cup containing the retina to a small glass jar containing 30 U/mL papain, 1 mM L-cystine, 0.5 mM EDTA and 0.005% DNase in Earl&#39;s-Balanced&#160;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 BSA (10 mg/mL) solution for 10 min in Earl&#39;s BSS. Wash tissue thoroughly with aCSF before transfer to the incubation system and reduce temperature to 15 - 16 &#176;C.</li>
	</ol>
	</li>
	<li>Maintain retinal tissue in the eye-cup until needed. For transfer to the microscope, isolate the retina from the eye-cup 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>
3. Maintaining Tissue in the Incubator
<ol>
	<li>Place tissue in the main chamber containing probes for pH and temperature measurements (Figure&#160;1A, B).</li>
	<li>Circulate aCSF through a second chamber (UVC chamber, built as previously described12) isolated from the main chamber and exposed to 1.1 W UVC light (254 nm, 5W/2P sterilizer UV lamp) in order to eradiate 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 reported12. 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>
4. Calcium Dye Loading
<ol>
	<li>Select calcium dyes based on the preference of the individual researcher. Here we use Fura-2AM, Fluo-8AM or Fluo-4AM. However, this method can be applied to other dyes as well: dissolve calcium dyes in DMSO to a 1 mM solution and 1% block copolymers (e.g., pluronic acid-127) to a final volume of 50 &#181;L and sonicate for 10 min.</li>
	<li>Add solution to aCSF to a final concentration of 10 &#181;M, 0.01% block copolymers for brain slices, and 20 &#181;M (retina), 0.02% block copolymers for the retina. In retinae (papain treated) and brain slices from young animals, load to the bath for 45 min at 37 &#176;C (Fura-2 AM) or at RT (Fluo-4 AM; Fluo-8 AM).
	<ol>
		<li>For adult animals, (&#62;12 weeks) pipette the dyes (50 &#181;L) directly onto the brain slices, and maintain for 75 min to allow better penetration of the dye into the deep layers.</li>
		<li>To ensure adequate oxygenation of the submerged slice during dye incubation, prepare a glass loading chamber with a closed lid (circular jar of diameter 2.5 cm, 3.5 cm height) with calcium dyes diluted in 2.5 mL of aCSF. Oxygenate continuously with 95% O2/5% CO2. Do not bubble.</li>
	</ol>
	</li>
	<li>Following dye loading, wash the tissue with aCSF and transfer to the incubation system, slowly reduce the temperature to 15 - 16 &#176;C until experimental use.</li>
</ol>
5. Electrophysiological Recordings and Imaging
<ol>
	<li>Place tissue in a submerged recording chamber under a microscope, and perfuse with oxygenated aCSF at a flow rate of 4 - 5 mL/min either at RT (~22 &#176;C) or physiological temperature (~35 &#176;C). Hold the tissue in place by using a custom made &#39;&#39;harp&#39;&#39;, made of nylon or gold threads stretched and glued across a U-shaped piece of gold or platinum wire.</li>
	<li>For electrophysiology:
	<ol>
		<li>Prepare recording pipettes from 1.5 mm (1.19 mm ID) borosilicate glass using a micropipette puller to achieve a final resistance of 5-6 M&#937;.</li>
		<li>Fill the pipette with 3 - 4 &#181;L of internal solution as previously described13 and visualize cells under infrared Differential Interference Contrast (IR-DIC) using a CCD camera. The intracellular solution should be carefully tailored to each experiment to achieve experimental outcomes.</li>
		<li>Position pipette, while maintaining positive pressure applied through a suction port on the pipette holder, on the membrane of a cell using a micromanipulator. Since the ILM has been removed from the retina, no prior membrane scraping is required. Once the pipette is on the cell, apply gentle negative pressure to the pipette to achieve a gigaohm seal. Then rupture the cell membrane with a brief amount of negative pressure.</li>
		<li>Make whole-cell current- or voltage-clamp recordings using standard techniques as appropriate.</li>
	</ol>
	</li>
	<li>Calcium-imaging:
	<ol>
		<li>For ratiometric imaging of Fura-2, use an ultra-high speed wavelength switcher to provide excitation wavelengths of 340 nm and 380 nm. Pass emitted light from individual cells through an emission filter of 510 &#177;20 nm, and capture with a high sensitivity, high-speed digital camera.</li>
		<li>For a single excitation wavelength of Fluo-4, filter excitation light through a 460 - 490 nm band pass filter and emitted light through a 515 - 550 nm band pass filter. For the fastest and most sensitive recording use a high speed digital camera such. Acquire images as required.</li>
		<li>Measure alterations in fluorescence as a function of time either at a single wavelength (Fluo-4) or using the wavelength ratio method (Fura-2), as previously described8,14.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Colbert, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+cortical+brain+slices+for+electrophysiological+recording.">Preparation of cortical brain slices for electrophysiological recording.</a> Meth mol biol. 337, 117-125 (2006).</li><li>Gibb, A., Edwards, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+recording+from+cells+in+sliced+tissues.">Patch clamp recording from cells in sliced tissues.</a> Microelect Techniq. , 255-274 (1994).</li><li>Kantevari, S., Buskila, Y., Ellis-Davies, G. C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+characterization+of+cell-permeant+6-nitrodibenzofuranyl-caged+IP3.">Synthesis and characterization of cell-permeant 6-nitrodibenzofuranyl-caged IP3.</a> Photochem photobiol sci. 11 (3), 508-513 (2012).</li><li>Blankenship, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+and+Extrasynaptic+Factors+Governing+Glutamatergic+Retinal+Waves.">Synaptic and Extrasynaptic Factors Governing Glutamatergic Retinal Waves.</a> Neuron. 62 (2), 230-241 (2009).</li><li>Sargoy, A., Barnes, S., Brecha, N. C., Perez De Sevilla Muller, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+and+Calcium+Imaging+Methods+in+Wholemount+Rat+Retina.">Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina.</a> J Vis Exp. (92), e51396 (2014).</li><li>Kulkarni, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Ca2++dynamics+in+cone+photoreceptor+axon+terminals+of+the+mouse+retina.">Imaging Ca2+ dynamics in cone photoreceptor axon terminals of the mouse retina.</a> J Vis Exp. (99), e52588 (2015).</li><li>Velte, T. J., Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+potentials+in+the+dendrites+of+retinal+ganglion+cells.">Action potentials in the dendrites of retinal ganglion cells.</a> J neurophysiol. 81 (3), 1412-1417 (1999).</li><li>Cameron, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+imaging+of+AM+dyes+following+prolonged+incubation+in+acute+neuronal+tissue.">Calcium imaging of AM dyes following prolonged incubation in acute neuronal tissue.</a> PLoS ONE. 11 (5), (2016).</li><li>Buskila, Y., Morley, J. W., Tapson, J., van Schaik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adaptation+of+spike+backpropagation+delays+in+cortical+neurons.">The adaptation of spike backpropagation delays in cortical neurons.</a> Front Cell Neurosci. 7, (2013).</li><li>Breen, P. P., Buskila, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Braincubator:+An+incubation+system+to+extend+brain+slice+lifespan+for+use+in+neurophysiology.">Braincubator: An incubation system to extend brain slice lifespan for use in neurophysiology.</a> Eng Med Biol Soc. , 4864-4867 (2014).</li><li>Buskila, Y., Breen, P., Wright, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Device+for+storing+a+tissue+sample.">Device for storing a tissue sample.</a> WIPO Patent. , (2015).</li><li>Buskila, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extending+the+viability+of+acute+brain+slices.">Extending the viability of acute brain slices.</a> Sci Rep. 4, 4-10 (2014).</li><li>Buskila, Y., Farkash, S., Hershfinkel, M., Amitai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+reactive+nitric+oxide+production+by+astrocytes+in+mouse+neocortical+slices.">Rapid and reactive nitric oxide production by astrocytes in mouse neocortical slices.</a> Glia. 52 (3), 169-176 (2005).</li><li>Barreto-Chang, O. L., Dolmetsch, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+imaging+of+cortical+neurons+using+Fura-2+AM.">Calcium imaging of cortical neurons using Fura-2 AM.</a> J Vis Exp. (23), e3-e5 (2009).</li><li>Buskila, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+Astrocytic+Nitric+Oxide+Production+and+Neuronal+Modifications+in+the+Neocortex+of+a+NOS2+Mutant+Mouse.">Enhanced Astrocytic Nitric Oxide Production and Neuronal Modifications in the Neocortex of a NOS2 Mutant Mouse.</a> PLoS ONE. 2 (9), 9 (2007).</li><li>Russell, W. M. S., Burch, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Principles of Humane Experimental Technique. , (1959).</li></ol>

Yossi Buskila and Paul Breen declare that they are co-owners of PAYO Scientific Pty Ltd, a company specialized in building incubation systems for acute neural tissue. All other authors report no financial interests or potential conflicts of interest related to the current study.

This article describes an incubation method to extend the viability of acute neuronal tissue for imaging and electrophysiological experiments, thereby reducing the animal numbers needed to complete experimental goals. Two main processes govern the deterioration of neuronal tissue over time: i) increased bacteria levels, and the accompanying increase in bacterial endotoxin released, and ii) excitotoxicity which follows the traumatic slicing procedure10. As acute neuronal tissue is environmentally defenseless, tight regulation of the tissue environment through close monitoring of pH, temperature and bacteria levels is essential for maintaining the cellular activity as well as network integrity. Dissection, and tissue treatment (papain digestion, calcium dye loading) can take over 2 h to complete, causing a reduction in experimental time. However, since the tissue can be kept in the incubation system for &#62;24 h without a measurable loss in viability7,8, this preparation only needs to be completed once to obtain &#62;24 h of results. As part of the 3Rs strategy (Replacement, Reduction, Refinement) aimed at providing guiding principles for ethical use of animals16, this method will have a large impact on reducing the number of animals used in these types of experiments.
Digestion of the ILM of retinal wholemount tissue allows easy targeting of cells in the GCL for patch-clamp recordings and ubiquitous calcium dye loading by bath application. These techniques are particularly useful for degenerate retinae in which the ILM is much thicker and harder to breach by scraping with a glass pipette. Furthermore, the papain preparation provides a way to perform paired-cell recordings using two electrodes to record from gap-junction coupled cells in the GCL, something that has not been previously possible. However, papain digestion likely has some effects on normal retinal physiology as K+ receptor expression has been shown to be altered in photoreceptors following dissociation with papain6. It is also possible that retinal architecture is affected. Velte and Masland showed that although some RGC somas and dendrites were damaged by papain treatment to remove the ILM, they could successfully record from RGC dendrites distal to the soma in most neurons, indicating the structural integrity of the cells and processes5. Importantly, leaving the retina attached to the retinal pigment epithelium (RPE) and sclera, as described above, isolates the outer retina from papain diffusion and may reduce any adverse effects on photoreceptor outer segments when this protocol is applied to WT retinae. In both instances, papain treatment, or scraping of the ILM, some cell damage will occur, the experimenter must choose the method appropriate to the experimental question.

Electrophysiology and functional imaging (calcium, voltage sensitive dyes) are two of the most commonly used experimental techniques in neuroscience. Brain slice preparations and retinal wholemount, which will be examined here, provide a means of examining electrophysiological properties and synaptic connectivity without contamination from anesthetics or muscle relaxants. Brain slices and retinal wholemount maintain their structural integrity, unlike cultures or cell homogenates, allowing the study of specific circuits and brain networks1. Recordings from isolated tissue have advantages over in vivo recordings as movements associated with the heartbeat and respiration are eliminated. Moreover, direct visualization allows specific classes of cells to be targeted, and local application of pharmacological tools 2,3.
Patch-clamp recordings and calcium dye-loading in retinal wholemount is complicated by the existence of the Inner Limiting Membrane (ILM), which covers the Retinal Ganglion Cell (RGC) layer and prevents direct access to the cells. Typically, this membrane is scraped away with a glass pipette to allow direct application of a patch pipette and formation of a gigaohm seal on a single cell. In addition, bath-applied calcium&#160;dyes do not cross the ILM and must either be injected beneath this membrane4, retrogradely transported following injection at the optic nerve5 or electroporated through the tissue6. Furthermore, when utilizing a rodent model of retinitis pigmentosa, the rd/rd mouse, the ILM is thicker and more impenetrable. Here, we use a technique to remove the ILM with enzymatic digestion7, to allow both ubiquitous calcium&#160;dye-loading, and direct access to retinal ganglion cells for patch-clamp recordings8.
Successful recordings from either brain slices or retinal wholemount depend on dissection and incubation of viable neuronal tissue. Typically, tissue is extracted on the morning of the experiment and incubated in artificial cerebrospinal fluid (aCSF) until it is used for recordings. Usually, tissue remains viable for 6 - 8 h, with significant degradation following this time window. However, both brain slices and wholemount retinae preparations usually produce more tissue than can be recorded from within this short time period. Consequently, tissue is often discarded at the end of the day and the dissection is completed again on subsequent days. This means another animal is utilized and ~2 h of setup and dissection/staining repeated. The following protocol describes a method for extending the life of neuronal tissue for more than 24 h, meaning fewer animals are utilized, and more experimental time is available. Tissue viability was assessed through recording electrophysiological properties and calcium dynamics, and these properties were indistinguishable between &#60;4 h and &#62;24 h postdissection.
These results indicate that not only are single cell properties intact and functional after prolonged incubation, but network activity, as assessed by calcium-imaging and electrophysiological recordings, is unchanged &#62;24 h postdissection. Moreover, we show that calcium dyes can remain in cells for prolonged periods without causing any detrimental effects. Application of this protocol demonstrates that the functional activity of neurons in acute neuronal tissue can be maintained for long periods, once the external environment is highly regulated. Moreover, as tissue viability varies greatly between laboratories due to different incubation protocols, this method establishes a gold standard for the ideal parameters that should be applied to reduce variability in the health of acute neuronal tissue.

<table><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&amp;nbsp;</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>Sodium phosphate monobasic</td><td>Sigma Aldrich</td><td>S2554</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma Aldrich</td><td>M9272&amp;nbsp;</td><td></td></tr><tr><td>Calcium chloride&amp;nbsp;</td><td>Sigma Aldrich</td><td>223506</td><td></td></tr><tr><td>Papain Dissociation System</td><td>Worthington&amp;nbsp;</td><td>LK003150</td><td></td></tr><tr><td>Dimethyl sulfoxide anhydrous</td><td>Sigma Aldrich</td><td>276855</td><td></td></tr><tr><td>Pluronic F-127 Low UV absorbance*</td><td>Life technologies</td><td>P-6867</td><td>20% solution in DMSO&amp;nbsp;</td></tr><tr><td>Fura-2 AM&amp;nbsp;&amp;nbsp;</td><td>Anaspec</td><td>84017</td><td></td></tr><tr><td>Fluo-4 AM&amp;nbsp;&amp;nbsp;</td><td>Life Technologies</td><td>F23917</td><td></td></tr><tr><td>Fluo-8 AM</td><td>Abcam</td><td>Ab142773</td><td></td></tr><tr><td>Vibrating blade microtome&amp;nbsp;</td><td>Leica Biosystem</td><td>VT1200 S</td><td>Equiped with Leica&amp;rsquo;s Vibrocheck&amp;trade; measurement device&amp;nbsp;</td></tr><tr><td>Antivibration table</td><td>Kinetic Systems Vibraplane&amp;nbsp;</td><td>Vibraplane 9100/9200&amp;nbsp;</td><td></td></tr><tr><td>Fixed Stage Upright Microscope</td><td>Olympus</td><td>BX51WI&amp;nbsp;</td><td></td></tr><tr><td>Microscope Objective</td><td>Olympus</td><td>XLUMPlanFLN 20x/1.00w</td><td>20X&amp;nbsp;</td></tr><tr><td>Microscope Objective</td><td>Olympus</td><td>LUMPlanFLN 60x/1.00w</td><td>60X</td></tr><tr><td>CCD Camera</td><td>Andor</td><td>Ixon +</td><td></td></tr><tr><td>High speed wavelength switcher</td><td>Sutter instrument</td><td>Lambda DG-4&amp;nbsp;</td><td></td></tr><tr><td>Patch clamp amplifier</td><td>Molecular Devices</td><td>MultiClamp 700B</td><td></td></tr><tr><td>Data acquisition system</td><td>Molecular Devices</td><td>Digidata 1440A</td><td>Axon Digidata&amp;reg; System</td></tr><tr><td>Borosilicate glass capillaries</td><td>Sutter Instrument Co</td><td>BF150-86-10</td><td></td></tr><tr><td>Microelectrode puller</td><td>Sutter Instrument Co</td><td>P-97</td><td>Flaming/Brown type micropipette puller</td></tr><tr><td>Recording/perfusion chamber</td><td>Warner Instruments</td><td>RC-40LP</td><td>Low Profile Open Bath Chambers</td></tr><tr><td>Software</td><td>Molecular Devices</td><td>pClamp 10.2</td><td></td></tr><tr><td>Andor iQ Live Cell Imaging Software</td><td>Andor</td><td>Andor iQ3</td><td></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>

prolonged incubation of acute neuronal tissue for electrophysiology and calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Tight regulation of the bacterial load and temperature of the aCSF during incubation is essential to maintain neuronal tissue viability. This can be optimized through irradiation with UVC light and maintaining the temperature of the aCSF at 15 - 16 &#176;C (Figure 1). Furthermore, the aCSF parameters stamp (APS; Figure 1C) provides the experimenter with a record of the environmental conditions (pH and temp.), thus offering a gold standard for parameters when incubating neuronal tissue, which, if followed, will reduce variability between experiments.
<img alt="Figure 1" src="/files/ftp_upload/55396/55396fig1.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 mesh as indicated. All measuring probes are attached to the lid to allow structural stability. C, aCSF Parameters Stamp describing the temperature and pH of the aCSF during incubation. D, Top view of the main chamber showing the mounting holes for the measuring probes. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55396/55396fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Tissue viability can be measured by means of network activity (Figure 2), as well as various imaging methods, including calcium responses (Figure&#160;2B, C). To monitor the network activity, we applied aCSF containing a high concentration of potassium chloride (KCl; 30 mM) locally, which led to depolarization of neurons and glia within the vicinity of the applied K+. This depolarization can be observed by the increase in calcium transients (Figure&#160;2B, C) and spiking activity (Figure&#160;2D), which also serves as a physiological indicator for the cell viability. Our results show that spiking activity in slices that were incubated for &#62;24 h in the incubation system was similar to &#34;fresh&#34; slices incubated for shorter periods (&#62;4 h; Figure 2D). Furthermore, individual neuron viability, which was monitored through intracellular electrophysiological recordings of both passive and active membrane properties, including the resting membrane potential, input resistance, time constant, and action potential was comparable to parameters reported in the literature15 (Figure&#160;2E, F).
<img alt="Figure 2" src="/files/ftp_upload/55396/55396fig2.jpg" /> 
Figure 2. Electrophysiological and Calcium Properties of Brain Slices after Prolonged Incubation. A, Image of a brain slice taken 28 h after slicing with two extracellular recording electrodes used for measuring the network activity. A third electrode (marked with asterisk) is used to puff 30 mM of KCl. B, Time lapse images of slices loaded with Fluo-4AM for &#62;24 h, depicting an increase in intracellular calcium transients following bath application of KCl. C, Intracellular calcium transients following brief application of KCl (30 mM; 1 s). Red - average signal, Grey - calcium traces in single cells as shown in B. D, Extracellular physiological recording of the network activity before and after the application of KCl (red arrows) were largely unchanged between &#34;fresh&#34; slices (&#60;4 h postdissection) and those that were incubated for &#62;24 h in the incubator. Note the increase in spiking activity immediately after KCl application indicating viable neurons that respond to increase in [K+] with depolarization. E&#38;F, Passive (E), and active (F) membrane properties of pyramidal neuron following 2 h (blue traces) or 26 h (black traces) of incubation in the incubation system. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55396/55396fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To provide direct access to the cells of the ganglion cell layer of the wholemount retina of retinally degenerate rd/rd mice, the ILM was digested by the enzyme papain7,8. After digestion, RGCs and displaced amacrine cells can be clearly visualized under DIC illumination (Figure 3A), and cells can be targeted for patch-clamp recordings without any prior scraping of the ILM. Representative current-clamp recordings from a RGC are shown in Figure 3B. Depolarization of the cell via the patch pipette caused dose-dependent action potential generation, indicating the viability of the cells following papain treatment.
Removal of the ILM also allowed ubiquitous staining of the ganglion cell layer (GCL) with Fura-2AM (Figure 3D) and cells responded to 30 mM KCl application with a large increase in 340/380 nm ratio relative to F0, signifying a large increase in intracellular calcium concentration (Figure 3E). Calcium levels returned to baseline following stimulation and cells could be subsequently stimulated to produce a similar amplitude response. Moreover, these responses were indistinguishable between retinae recorded at &#60;4 h and &#62;24 h postdissection (Figure&#160;3F, G).
<img alt="Figure 3" src="/files/ftp_upload/55396/55396fig3.jpg" /> 
Figure 3: Electrophysiological and Calcium Recordings in Retinal Wholemount. A, Differential interference contrast image of a patch-clamp recording from a retinal ganglion cell in the GCL after papain digestion. B, Cells retain dose dependent spiking responses when depolarized with 50, 100 and 150 pA respectively. C&#38;D, Removal of the inner limiting membrane before bath application of Fura-2AM allows ubiquitous loading of retinal ganglion cells, image taken with 380 nm excitation. E, When 30 mM KCl ASCF is applied, 85% of stained cells responded with an increase in 340/380 ratio. F&#38;G, The increase in 340/380 ratio returns to baseline after 30 mM KCl application (red bars), and cells respond to subsequent stimulation both &#60;4 h and &#62;24 h following dissection. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55396/55396fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging at JoVE.com

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

prolonged-incubation-acute-neuronal-tissue-for-electrophysiology

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

11.9K Views.  Western Sydney University. Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for >24 h.

Video: Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.

We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

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.

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

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

Establishing a Whole-Cell Configuration for Two-Photon Calcium Imaging of Brain Slices

Source: Camir&#233;, O., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56776">Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices.</a> J. Vis. Exp. (2018).
This video demonstrates a protocol for whole-cell patch-clamp recording and two-photon calcium imaging in brain hippocampal slices. This method allows to study the dynamics of local Ca2+ transients (CaTs) in dendrites of different neuronal types in acute brain slices.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review.
1. Preliminary Preparation (Optional: ...

JoVE Encyclopedia of Experiments

Neurophysiology

Imaging and Optical Techniques

Measuring Neural Activity in a Mouse Brain Slice after Prolonged Incubation

Source: Cameron, M. A., et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55396">Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging.</a> J. Vis. Exp. (120), (2017)
This video demonstrates a technique for measuring neuronal activity in mouse brain slices using calcium imaging and electrophysiological recordings. By perfusing the tissue with potassium ions and recording intracellular calcium changes through high-speed digital imaging, this method reveals real-time neuronal responses.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Calcium Dye Loading

	...

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

Source: Cameron, M. A., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/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.

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

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Summary

Explore More Videos

Chapters in this video

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Establishing a Whole-Cell Configuration for Two-Photon Calcium Imaging of Brain Slices

Measuring Neural Activity in a Mouse Brain Slice after Prolonged Incubation

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