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 ...

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Neuroscience

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