Name: wide-field single-photon optical recording in brain slices using voltage-sensitive dye
Uploaded: 2019-06-20T14:00:00
Description: Watch this Scientific Journal Video about Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye at JoVE.com

TT received the JSPS KAKENHI Grant (JP16H06532, JP16K21743, JP16H06524, JP16K0038, and JP15K00413) from MEXT and grants from the Ministry of Health, Labour and Welfare (MHLW-kagaku-ippan-H27 [15570760] and H30 [18062156]). We would like to thank Editage (www.editage.jp) for English language editing.

All animal experiments were performed according to protocols approved by the Animal Care and Use Committee of Tokushima Bunri University. The following protocol for slice preparation is almost a standard procedure27 , but the modifications have been the protocols of staining and recording with VSD.
1. Preparation Before the day of Experiment
<ol>
	<li>Prepare the stock A (Table 1), stock B (Table 2), and stock C (Table 3</stro...

<ol>
	<li>Tominaga, Y., Taketoshi, M., Tominaga, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overall+Assay+of+Neuronal+Signal+Propagation+Pattern+With+Long-Term+Potentiation+(LTP)+in+Hippocampal+Slices+From+the+CA1+Area+With+Fast+Voltage-Sensitive+Dye+Imaging.">Overall Assay of Neuronal Signal Propagation Pattern With Long-Term Potentiation (LTP) in Hippocampal Slices From the CA1 Area With Fast Voltage-Sensitive Dye Imaging.</a> Frontiers in Cellular Neuroscience. 12, 389 (2018).</li><li>Tominaga, T., Kajiwara, R., Tominaga, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VSD+Imaging+Method+of+Ex+Vivo+Brain+Preparation.">VSD Imaging Method of Ex Vivo Brain Preparation.</a> Journal of Neuroscience and Neuroengineering. 2 (3), 211-219 (2013).</li><li>Homma, R., 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=Wide-field+and+two-photon+imaging+of+brain+activity+with+voltage-+and+calcium-sensitive+dyes.">Wide-field and two-photon imaging of brain activity with voltage- and calcium-sensitive dyes.</a> Methods Mol Biol. 364 (1529), 2453-2467 (2009).</li><li>Grinvald, A., Hildesheim, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VSDI:+a+new+era+in+functional+imaging+of+cortical+dynamics.">VSDI: a new era in functional imaging of cortical dynamics.</a> Nature Reviews Neuroscience. 5 (11), 874-885 (2004).</li><li>Tasaki, I., Watanabe, A., Sandlin, R., Carnay, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+fluorescence,+turbidity,+and+birefringence+associated+with+nerve+excitation.">Changes in fluorescence, turbidity, and birefringence associated with nerve excitation.</a> Proceedings of the National Academy of Sciences. 61 (3), 883-888 (1968).</li><li>Cohen, L., Keynes, R., Hille, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+Scattering+and+Birefringence+Changes+during+Nerve+Activity.">Light Scattering and Birefringence Changes during Nerve Activity.</a> Nature. 218 (5140), 438-441 (1968).</li><li>Hill, D., Keynes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opacity+changes+in+stimulated+nerve.">Opacity changes in stimulated nerve.</a> The Journal of Physiology. 108 (3), 278-281 (1949).</li><li>Waggoner, A., Salzberg, B., Davila, H., Cohen, 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+Large+Change+in+Axon+Fluorescence+that+Provides+a+Promising+Method+for+Measuring+Membrane+Potential.">A Large Change in Axon Fluorescence that Provides a Promising Method for Measuring Membrane Potential.</a> Nature New Biology. 241 (109), 159 (1973).</li><li>Salzberg, B., Davila, H., Cohen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Recording+of+Impulses+in+Individual+Neurones+of+an+Invertebrate+Central+Nervous+System.">Optical Recording of Impulses in Individual Neurones of an Invertebrate Central Nervous System.</a> Nature. 246 (5434), (1973).</li><li>Cohen, L., lzberg, B., Grinvald, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Methods+for+Monitoring+Neuron+Activity.">Optical Methods for Monitoring Neuron Activity.</a> Annual Review of Neuroscience. 1 (1), 171-182 (1978).</li><li>Ross, W. N., Salzberg, B. M., Cohen, L. B., Davila, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large+change+in+dye+absorption+during+the+action+potential.">A large change in dye absorption during the action potential.</a> Biophysical Journal. 14 (12), 983-986 (1974).</li><li>Loew, L. M., Cohen, L. B., Salzberg, B. M., Obaid, A. L., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charge-shift+probes+of+membrane+potential.+Characterization+of+aminostyrylpyridinium+dyes+on+the+squid+giant+axon.">Charge-shift probes of membrane potential. Characterization of aminostyrylpyridinium dyes on the squid giant axon.</a> Biophysical Journal. 47 (1), 71-77 (1985).</li><li>Loew, L. 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W. <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> Scientific Reports. 4 (1), srep05309 (2015).</li><li>Tanemura, K., 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=Neurodegeneration+with+Tau+Accumulation+in+a+Transgenic+Mouse+Expressing+V337M+Human+Tau.">Neurodegeneration with Tau Accumulation in a Transgenic Mouse Expressing V337M Human Tau.</a> Journal of Neuroscience. 22 (1), 133-141 (2002).</li><li>Tominaga, Y., Ichikawa, M., Tominaga, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+potential+response+profiles+of+CA1+pyramidal+cells+probed+with+voltage-sensitive+dye+optical+imaging+in+rat+hippocampal+slices+reveal+the+impact+of+GABAA-mediated+feed-forward+inhibition+in+signal+propagation.">Membrane potential response profiles of CA1 pyramidal cells probed with voltage-sensitive dye optical imaging in rat hippocampal slices reveal the impact of GABAA-mediated feed-forward inhibition in signal propagation.</a> Neuroscience Research. 64 (2), 152-161 (2009).</li><li>Suh, J., Rivest, A. J., Nakashiba, T., Tominaga, T., Tonegawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entorhinal+Cortex+Layer+III+Input+to+the+Hippocampus+Is+Crucial+for+Temporal+Association+Memory.">Entorhinal Cortex Layer III Input to the Hippocampus Is Crucial for Temporal Association Memory.</a> Science. 334 (6061), 1415-1420 (2011).</li><li>Juliandi, B., 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=Reduced+Adult+Hippocampal+Neurogenesis+and+Cognitive+Impairments+following+Prenatal+Treatment+of+the+Antiepileptic+Drug+Valproic+Acid.">Reduced Adult Hippocampal Neurogenesis and Cognitive Impairments following Prenatal Treatment of the Antiepileptic Drug Valproic Acid.</a> Stem cell reports. 5 (6), 1-14 (2016).</li><li>Stepan, J., Dine, J., Eder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+optical+probing+of+the+hippocampal+trisynaptic+circuit+in+vitro:+network+dynamics,+filter+properties,+and+polysynaptic+induction+of+CA1+LTP.">Functional optical probing of the hippocampal trisynaptic circuit in vitro: network dynamics, filter properties, and polysynaptic induction of CA1 LTP.</a> Frontiers in Neuroscience. 9, 160 (2015).</li><li>Tominaga, Y., Taketoshi, M., Tominaga, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overall+Assay+of+Neuronal+Signal+Propagation+Pattern+With+Long-Term+Potentiation+(LTP)+in+Hippocampal+Slices+From+the+CA1+Area+With+Fast+Voltage-Sensitive+Dye+Imaging.">Overall Assay of Neuronal Signal Propagation Pattern With Long-Term Potentiation (LTP) in Hippocampal Slices From the CA1 Area With Fast Voltage-Sensitive Dye Imaging.</a> Frontiers in Cellular Neuroscience. 12, 389 (2018).</li><li>Kajiwara, R., Tominaga, Y., Tominaga, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+Plasticity+Involved+in+the+Spread+of+Neural+Activity+Within+the+Rhinal+Cortices+as+Revealed+by+Voltage-Sensitive+Dye+Imaging+in+Mouse+Brain+Slices.">Network Plasticity Involved in the Spread of Neural Activity Within the Rhinal Cortices as Revealed by Voltage-Sensitive Dye Imaging in Mouse Brain Slices.</a> Frontiers in Cellular Neuroscience. 13, 20 (2019).</li><li>Popovic, M., Gao, X., Zecevic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-sensitive+dye+recording+from+axons,+dendrites+and+dendritic+spines+of+individual+neurons+in+brain+slices.">Voltage-sensitive dye recording from axons, dendrites and dendritic spines of individual neurons in brain slices.</a> Journal of visualized experiments. , (2012).</li><li>Sakmann, B., Stuart, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-Channel Recording. , (1995).</li><li>Tominaga, T., Tominaga, Y., Ichikawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+long-lasting+depolarization+on+burst+stimulation+in+area+CA1+of+rat+hippocampal+slices.">Optical imaging of long-lasting depolarization on burst stimulation in area CA1 of rat hippocampal slices.</a> Journal of neurophysiology. 88 (3), 1523-1532 (2002).</li><li>Mennerick, S., 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=Diverse+Voltage-Sensitive+Dyes+Modulate+GABAAReceptor+Function.">Diverse Voltage-Sensitive Dyes Modulate GABAAReceptor Function.</a> The Journal of Neuroscience. 30 (8), 2871-2879 (2010).</li><li>Canitano, R., Pallagrosi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autism+Spectrum+Disorders+and+Schizophrenia+Spectrum+Disorders:+Excitation/Inhibition+Imbalance+and+Developmental+Trajectories.">Autism Spectrum Disorders and Schizophrenia Spectrum Disorders: Excitation/Inhibition Imbalance and Developmental Trajectories.</a> Frontiers in Psychiatry. 8, 69 (2017).</li><li>Anticevic, A., Murray, J. 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The slice physiology is vital for collecting the right signal. The use of the ring-membrane filter system in this protocol ensures that the slice remains healthy and un-distorted throughout the procedure2,16,17. Other systems can be used to retain slice physiology during the recording, but the slice should not get deformed at any time as the imaging needs every part of the slice to be healthy. The ring-membrane filter system is ...

Wide-field single photon voltage-sensitive dye (VSD) imaging of bulk-stained brain slice preparations has become a useful quantitative tool to assess the dynamics of neural circuits1,2,3,4. After the analysis of the changes in optical properties due to membrane excitation5,6,7, VSD imaging was first described in the early 1970s by Cohen and others6,8,9.; it is a suitable method to monitor the brain functions in real-time as the dye directly probes the membrane potential changes (i.e., the primary signal of the neurons).
The earliest VSDs possessed the desirable characteristics to understand the brain system, such as a fast time-constant to follow the rapid kinetics of neuronal membrane potential events, and linearity with the change in membrane potential9,10,11,12,13,14,15. Similar to other imaging experiments, this technique requires a wide range of specific tunings, such as the cameras, optics, software, and slice physiology, to accomplish the desired results. Because of these technical pitfalls, the expected benefits during initial efforts did not necessarily materialize for most of the laboratories that did not specialize in this technique.
The primal cause of the technical difficulty was the low sensitivity of the VSD toward the membrane potential change when applied to bulk staining of slice preparations. The magnitude of the optical signal (i.e., the fractional change in fluorescence) is usually 10-4-10-3 of the control (F0) signal under physiological conditions. The time scale of membrane potential change in a neuron is approximately milliseconds to few hundreds of milliseconds. To measure the changes in the membrane potential of the neuron, the camera being used for the recording should be able to acquire images with high speed (10 kHz to 100 Hz). The low sensitivity of VSD and the speed needed to follow the neural signal requires a large amount of light to be collected at the camera at a high speed, with a high signal-to-noise ratio (S/N)2,16.
The optics of the recording system are also a critical element to ensure collection of sufficient light and to improve S/N. The magnification achieved by the optics is often excessively low, such as 1X to 10X, to visualize a local functional neural circuit. For example, to visualize the dynamics of the hippocampal circuit, a magnification of approximately 5 would be suitable. Such low magnification has low fluorescence efficiency; therefore, advanced optics would be beneficial for such recording.
In addition, the slice physiology is also essential. Since the imaging analysis requires the slices to be intact, careful slice handling is needed17. Furthermore, measures taken to maintain the slice viability for a longer time are important18.
The present article describes the protocol for preparation of slices, VSD staining, and measurements. The article also outlines the improvements to the VSDs, imaging device, and optics, and other additional refinements to the experimental system that have enabled this method to be used as a straightforward, powerful, and quantitative assay for visualizing the modification of the brain functions19,20,21,22,23,24,25. The technique can also be widely used for long-term potentiation in the CA1 area of hippocampal slices1. Moreover, this technique is also useful in optical recording of membrane potentials in a single nerve cell26.

<table border="0" cellpadding="0" cellspacing="0" style="width:940px;" width="942">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">High speed image acquisition system</td>
			<td style="width:195px;">Brainvision co. Ltd.</td>
			<td style="width:353px;">MiCAM - Ultima</td>
			<td style="width:171px;">Imaging system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">High speed image acquisition system</td>
			<td>Brainvision co. Ltd.</td>
			<td>MiCAM 02</td>
			<td>Imaging system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Macroscepe for wide field imaging</td>
			<td>Brainvision co. Ltd.</td>
			<td>THT macroscope</td>
			<td>macroscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">High powere LED illumination system with photo-diodode stablilizer</td>
			<td>Brainvision co. Ltd.</td>
			<td style="width:353px;">LEX-2G</td>
			<td style="width:171px;">LED illumination</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Image acquisition software</td>
			<td>Brainvision co. Ltd.</td>
			<td>BV-ana</td>
			<td>image acquisition software</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Multifunctional electric stimulator</td>
			<td>Brainvision co. Ltd.</td>
			<td>ESTM-8</td>
			<td>Stimulus isolator+AD/DA converter</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Slicer</td>
			<td>Leica</td>
			<td>VT-1200S</td>
			<td>slicer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Slicer</td>
			<td>Leica</td>
			<td>VT-1000</td>
			<td>slicer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Blade for slicer</td>
			<td>Feather Safety Razor Co., Ltd.</td>
			<td>#99027</td>
			<td>carbon steel razor blade</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">Numerical analysis software</td>
			<td>Wavemetrics Inc., OR, USA</td>
			<td>IgorPro</td>
			<td>analysing software</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Stimulation isolator</td>
			<td>WPI Inc.</td>
			<td>A395</td>
			<td>Stimulus isolator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AD/DA converter</td>
			<td>Instrutech</td>
			<td>ITC-18</td>
			<td>AD/DA converter</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">poloxamer</td>
			<td>Invitrogen, Thermo-Fisher Scientific, Waltham, MA, USA</td>
			<td>Pluronic F-127 P30000MP</td>
			<td>poloxamer / Pluronic F-127 (20% solution in DMSO)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">polyethoxylated castor oil</td>
			<td>Sigma-Aldrich</td>
			<td>Cremophor EL C5135</td>
			<td>polyethoxylated castor oil</td>
		</tr>
	</tbody>
</table>

wide-field single-photon optical recording in brain slices using voltage-sensitive dye

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

Figure 5&#160;shows the representative optical signal upon electrical stimulation of the Schaffer collateral in area CA1 of a mouse hippocampal slice. The consecutive images in Figure 5A show the optical signal before any spatial and temporal filters were applied, while Figure 5B shows the same data after applying a 5 x 5 x 5 cubic filter (a Gaussian Kernel convolution, 5 x 5 spatial- and 5 to tempor...

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Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...

The optical recording method with voltage sensitive dye is thought to be an ideal technology to visualize how the brain works. This method allowed us to record a neuropsychic dynamics stably and reliably over 12 hours, given us a view on psychic activity. We are trying to expand the application to detect circuit changes caused by chemicals on the all stage of development, such as valuable acid application to mothers.The technique is already well established. Please try to reproduce studies by following the method presented here. To begin this procedure, immerse the decapitated head in ice cold ACSF in a stainless steel surgical tray.Extract the brain within one minute and place it in a beaker containing chilled ACSF for five minutes. Next, divide the brain section at the midline with a scalpel and place both hemispheres onto a 4%agar block. Then, place the brain block with both hemispheres onto a 4%agar block.Wipe the excess ACSF from the block with a filter paper. Subsequently, apply superglue to the vibratome platform. Place the agar block on it and wipe the excessive adhesive with a filter paper.Gently apply a small amount of ice cold ACSF from the top of the brain agar block to help solidify excess superglue and to prevent the glue from covering the brain and disturbing the slicing. Afterward, fix the platform to the vibratome tray and pour the modified ACSF. Set the vibratome to a slow speed and have the blade frequency at its maximum setting.Then start slicing. Place the slices in the corner of the slice chamber in sequence so that the order of the slices can be easily distinguished. Usually three to five slices can be obtained from one hemisphere.Next, cut off the brain stem portion using a 30 gauge needle. Using a small tipped paint brush, place the slice on the membrane filter held with a plexiglass ring. Place the ring in a moist recovery chamber and secure the cover to keep the inner pressure high.When placing the slice onto the membrane filter, adjust the direction and position of the slice within the ring to ensure it is well centered and has a consistent direction. Leave the specimen at 28 degrees Celsius for 30 minutes and then at room temperature for at least 10 to 30 minutes for recovery. To stain the slices, gently apply 100 microliters of the staining solution onto each slice and incubate at 20 minutes for room temperature.Next, prepare 50 to 100 milliliters of ACSF in a container. Place the ring with the specimen in it to rinse the staining solution. Then, transfer the rinsed slices to another incubation chamber.Wait more than one hour for recovery before the experiment. 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 one 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 at 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.This figure shows the representative optical signal upon electrical stimulation of the Schaffer collaterol in area CA1 of a mouse hippocampal slice. These consecutive images show the optical signal before any spacial and temporal filters were applied while these images show the same data after applying a five by five by five cubic filter twice. Due to the high frame rate and high spatial resolution, the application of the filter did not change the signal, but filtered out the noise which can also be observed in the time course recorded in pixels in a single trial.Here is the comparison of the typical responses in area CA1 of the hippocampal slices between mouse and rat. The small hyperpolarizing response is observed in the distal side of the CA1 after 24 milliseconds in the mouse hippocampal slice, but more massive hyperpolarizing response overtook the depolarizing response in the rat hippocampal slice. Once the slices are stained, they should last for over 12 hours and you can perform other electrophysiological recordings on them.

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Neuroscience

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various heterologous genes could thus be investigated easily while maintaining many aspects of in vivo biology. There has been increasing interest to transfect terminally differentiated neurons for which conventional transfection methods have been fraught with difficulties such as low yields and significant losses in viability. Biolistic transfection can circumvent many of these difficulties yet only recently has this technique been modified so that it is amenable for use in mammalian tissues.
New modifications to the accelerator chamber have enhanced the gene gun&#39;s firing accuracy and increased its depths of penetration while also allowing the use of lower gas pressure (50 psi) without loss of transfection efficiency as well as permitting a focused regioselective spread of the particles to within 3 mm. In addition, this technique is straight forward and faster to perform than tedious microinjections. Both transient and stable expression are possible with nanoparticle bombardment where episomal expression can be detected within 24 hr and the cell survival was shown to be better than, or at least equal to, conventional methods. This technique has however one crucial advantage: it permits the transfection to be localized within a single restrained radius thus enabling the user to anatomically isolate the heterologous gene&#39;s effects. Here we present an in-depth protocol to prepare viable adult organotypic slices and submit them to regioselective transfection using an improved gene gun.

Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices.

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various ...

A Large Lateral Craniotomy Procedure for Mesoscale Wide-field Optical Imaging of Brain Activity

The craniotomy is a commonly performed procedure to expose the brain for in vivo experiments. In mouse research, most labs utilize a small craniotomy, typically 3 mm x 3 mm. This protocol introduces a method for creating a substantially larger 7 mm x 6 mm cranial window exposing most of a cerebral hemisphere over the mouse temporal and parietal cortices (e.g., bregma 2.5 - 4.5 mm, lateral 0 - 6 mm). To perform this surgery, the head must be tilted approximately 30&#176; and much of the temporal muscle must be retracted. Due to the large amount of bone removal, this procedure is intended only for acute experiments with the animal anesthetized throughout the surgery and experiment.
The main advantage of this innovative large lateral cranial window is to provide simultaneous access to both medial and lateral areas of the cortex. This large unilateral cranial window can be used to study the neural dynamics between cells, as well as between different cortical areas by combining multi-electrode electrophysiological recordings, imaging of neuronal activity (e.g., intrinsic or extrinsic imaging), and optogenetic stimulation. Additionally, this large craniotomy also exposes a large area of cortical blood vessels, allowing for direct manipulation of the lateral cortical vasculature.

This protocol presents a method for creating a large unilateral craniotomy over the temporal and parietal regions of the mouse cerebral cortex. This is especially useful for real time imaging over an expansive area of a cortical hemisphere.

The craniotomy is a commonly performed procedure to expose the brain for in vivo experiments. In mouse research, most labs utilize a small craniotomy, ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...