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) solutions and store in a refrigerator.</li>
	<li>Prepare 1 L of artificial cerebrospinal fluid (ACSF) (Table 4, see step 3) and keep it in the refrigerator.</li>
	<li>Prepare 1 L of Modified ACSF (Table 5) and keep it in the refrigerator.</li>
	<li>Dispense 500 &#181;L aliquots of fetal bovine serum (FBS) in 2 mL vials and store in a freezer.</li>
	<li>Dissolve 4% of agar powder in ACSF (ca. 120 mL) in a microwave and pour it in a 90 mm disposable Petri dish. The agar plate should be refrigerated before further use.</li>
	<li>Place the following items in a freezer on the day before the experiment: a surgical tray, a slicer container and an aluminum cooling block (120 x 120 x 20 mm3).</li>
	<li>Ensure that there are sufficient Plexiglass rings with membrane filters for slice handling system17,28 (see step 6.12).</li>
	<li>Dissolve 2% of agar powder in 50 mL of 3 M KCl in a microwave. Take around 85 &#181;L of the warm agar-KCl dissolvent in 200 &#181;L tips using a micropipette for the grounding electrode. Detach the tip into the still warm agar 3 M KCl gel. Repeat the step to fill about 20-40 tips with 2% agar.</li>
</ol>
2. Preparation of VSD (di-4-ANEPPS) Stock Solution
<ol>
	<li>Prepare 1 mL of 10% polyethoxylated castor oil solution with ultra-pure water.</li>
	<li>Add 1 mL of ethanol to a vial of di-4-ANEPPS (5 mg vial), vortex and sonicate for 10 min. The solution will turn into a deep red color with possible small residues of the di-4-ANEPPS crystals. 
	NOTE: The ethanol used in this step should be freshly opened.</li>
	<li>Transfer the solution to a 2 mL microtube with an O-ring. Spin down the solution and add 500 &#181;L of 10% polyethoxylated castor oil solution. 
	NOTE: The dye is highly lipophilic. DMSO and poloxamer can also be used to dissolve di-4-ANEPPS but in terms of the optical signal upon change in membrane potential, we found that the use of ethanol -polyethoxylated castor oil gives a better signal to noise ratio. This could be related to transfer rate of solvent to the cell membrane.</li>
	<li>Vortex and sonicate until the dye has completely dissolved.</li>
	<li>Avoid exposure to light and keep it in a refrigerator. Do not store in a freezer. The stock can last for a few months. 
	NOTE: On the day of the experiment, follow the steps 3-9.</li>
</ol>
3. Daily Preparation of ACSF (1 L) (Table 4)
<ol>
	<li>Weigh NaCl, NaHCO3, and glucose in a flask.</li>
	<li>Add 950 mL of distilled water to the flask and start bubbling with 95% O2/5% CO2 gas.</li>
	<li>Add 2.5 mL of stock A solution to the flask and incubate for approximately 10 min at room temperature.</li>
	<li>Add 2.5 mL of stock C solution to the flask.</li>
	<li>Add distilled water to make the solution to 1 L.</li>
</ol>
4. Daily Preparation of the Staining VSD Ssolution
<ol>
	<li>Sonicate a 500 &#181;L vial of FBS and VSD stock solution (step 2) in an ultra-sonicator for 5 min.</li>
	<li>Add 500 &#181;L of freshly prepared ACSF into the vial of FBS.</li>
	<li>Add 20 (in case of mice) or 40 (in case of rats) &#181;L of VSD stock solution to the vial.</li>
	<li>Ultra-sonicate and vortex the vial till the solution becomes pale orange.</li>
</ol>
5. Preparation for Surgery
<ol>
	<li>Take 100 mL of chilled ACSF separately in a 300 mL stainless steel container, a 300 mL beaker, and a plastic container, and place them in a freezer. Pour 150 mL of chilled modified ACSF (Table 2) in another beaker and place it in the freezer. Wait till the solutions are chilled; the time taken should be measured and determined beforehand.</li>
	<li>Fold to break a razor blade (carbon steel, industrial grade 0.13 mm thick, blade on both sides) into half for the slicer. 
	NOTE: The other half can be used for dissection with a proper blade holder.</li>
	<li>Prepare a block from the ACSF 4% agar plate with an adjusting jig (Figure 1).</li>
	<li>Prepare a moist incubation chamber (an interface type chamber; a modified 1.2 L tight sealed box with a silicone packing) for keeping the brain slices physiologically alive (Figure 2); add ACSF in a small container and carbonate with 95% O2/5% CO2 gas, and fill a 90 mm x 20 mm Petri dish with ACSF in to the top. 
	NOTE: A smaller Petri dish (60 mm x 20 mm) should be placed in the center of the 90 mm dish to support a filter paper on the dish.</li>
	<li>Put the box on a heating device and wait for 20 min to warm it up to 28 &#730;C.</li>
	<li>Add crushed ice into the container of the slicer. Place the following instruments in a stainless-steel vat (small) on ice: scalpel, blade holder, ring tweezers, agar block, and a stage of slicer. Keep frozen ACSF and modified ACSF on ice and bubble with 95% O2/5% CO2 gas (aka. carbogen).</li>
	<li>Place the following instruments in a vat (large): scissors (large, small), tweezers, a spatula, a spoon, and diagonal pliers.</li>
</ol>
6. Surgery (Mice)
<ol>
	<li>Anesthetize the mouse using isoflurane in a fume hood. Assess the level of the anesthesia by checking the pedal reflex of the animal upon toe pinch.</li>
	<li>Decapitate the mouse and immerse the head in ice-cold ACSF in a stainless-steel surgical tray.</li>
	<li>Extract the brain within 1 min and place it in a beaker containing chilled ACSF for 5 min.</li>
	<li>Take the brain out of the beaker and, using a scalpel, trim the brain block (Figure 3A).</li>
	<li>Place the brain block onto a 4% agar block (step 5.3, Figure 3B). Both hemispheres can be mounted on an agar block. Wipe the excess ACSF from the block with a filter paper.</li>
	<li>Apply thin adhesive (super glue) to the slicer table. Place the agar block on it and wipe the excess adhesive using a filter paper.</li>
	<li>Gently apply a small amount of ice-cold ACSF (~5 mL) using a pipette from the top of the brain-agar block. This will help solidify excess super glue and prevent the glue from covering the brain and disturbing the slicing.</li>
	<li>Fix the slicer table to the slicer tray (Figure 3C) and pour the modified ACSF.</li>
	<li>Set the slicer to a slow speed, with the blade frequency at maximum.</li>
	<li>Set the slice thickness to 350-400 &#181;m and start slicing (Figure 3C). Place the slices on the corner of the slice tray in a sequence, so that the depth of the slices can be easily distinguished. Usually three to five slices can be obtained from one hemisphere.</li>
	<li>Cut off the brain stem portion using a 30 G needle (Figure 3D). 
	NOTE: Microsurgery on the brain slice such as a cut between the CA3-CA1 border should be done at this stage under a binocular microscope, if necessary.</li>
	<li>Using a small tipped paint brush, place the slice on the center of the membrane filter (0.45 &#181;m pores, PTFE-membrane, 13 mm diameter) held with the Plexiglass ring17 (15 mm outer diameter, 11 mm inner diameter, 1 mm thickness, Figure 3E). Place the ring in the moist recovery chamber (Figure 2) and secure the cover to keep the inner pressure high. 
	NOTE: The slices will stick to the membrane within 30 min and can be handled with the rings in the subsequent steps in the recording chamber. There is no need to use weights or other measures to keep the slice in place.</li>
	<li>Adjust the direction and position of the slice in the ring to ensure it is well centered and has a consistent direction (see step 9.3).</li>
	<li>Leave the specimen at 28 &#176;C for 30 min, and then at room temperature for at least 10 to 30 min for the recovery of slices. 
	NOTE: The slices can now retain good physiological condition at least for 15 h.</li>
</ol>
7. Staining and Rinsing of the Slices (Mice)
<ol>
	<li>Gently apply 100-110 &#181;L of the staining solution (Step 4) onto each slice on the ring using a micropipette. Eight to nine slices can be stained with one staining solution prepared in step 4. Leave the slices for 20 min for staining.</li>
	<li>Prepare 50-100 mL of ACSF in a container and put the ring with sliced specimen in it to rinse the staining solution.</li>
	<li>Store the rinsed slice to another incubation chamber. Wait more than 1 h for recovery before the experiment. 
	NOTE: The incubation chamber can be detached from the gas and moved to the place of recording in a tight sealed condition. The slice can remain alive for at least 20 min without gas supply. This is useful in case you need to move the slice to another place for recording.</li>
</ol>
8. Daily Preparation of Experimental Apparatus
<ol>
	<li>Turn on the amplifier, computer, and camera system, and check that the software is running.</li>
	<li>Place ACSF in a 50 mL tube and bubble with carbogen.</li>
	<li>Use a peristaltic pump to circulate the ACSF. Adjust the flow rate to approximately 1 mL/min.</li>
	<li>Adjust the height of the suction pipette so that the liquid level inside the experiment chamber is always constant. 
	NOTE: The level of the solution is important to obtain a stable recording, therefore, the adjustment should be done using a micromanipulator.</li>
	<li>Install the ground electrode made up of yellow chip filled with 3 M KCl agar (2%) (step 1.8) into a holder with an Ag-AgCl wire with small amount of 3 M KCl solution.</li>
	<li>Fill a small amount of ACSF (approximately two-third of the volume) into the glass electrode (1 mm outer diameter, 0.78 mm inner diameter pulled with a micropipette puller) using a tapered thin tubed yellow tip and place it in the electrode holder.</li>
	<li>Attach the holder to the rod installed in the manipulator. Ensure using an amplifier that the electrode resistance is approximately 1 M&#937;. 
	NOTE: The long-shank wide opening (4-8 &#181;m opening) patch type electrode should be good for field recording and as a stimulating electrode.</li>
</ol>
9. Starting a Recording Session
<ol>
	<li>Take a slice preparation from the moist chamber with forceps.</li>
	<li>Quickly place the slice onto an experimental chamber under the microscope (Figure 4).</li>
	<li>Push 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. 
	NOTE: The direction of the slice should be taken into consideration with respect to the direction of the stimulating and recording electrodes in the field of view. The healthy slice should stick to the membrane filter so there is no need to use other devices to fix the slices such as weights and nylon meshes.</li>
	<li>Place the tip of the stimulating electrode and the field potential recording electrode onto the slice under the microscope with transmitted light.</li>
	<li>Use the electrophysiological recording system to check the response. Confirm the usual (non-stained) electrophysiological recording with given configuration. 
	NOTE: The recording electrode can be omitted but is useful to check the physiology of the slice.</li>
	<li>Adjust the excitation light intensity to approximately 70-80% of the maximum capacity at the camera that corresponds to 13-15 mW/cm2 at the specimen when sampling at 10 kHz with 5x water immersion objective lens and 1x PLAN APO tube lens. The excitation light wavelength is 530 nm, and the emission filter must be &#62; 590 nm. 
	NOTE: Use a shutter to minimize the amount of excitation light. Continuous light exposure may deteriorate the slice physiology. The possible harmful effect of the light depends on the intensity and duration of the light. Use electrophysiological recording to judge the effect of light. In case of the strength of 13-15 mW/cm2, about 1 s exposure should be the upper limit of the tolerance.</li>
	<li>Adjust the focus with the acquisition system using the fluorescent light source because the focus may be different depending on the wavelength and start the acquisition.</li>
	<li>Examine the data in an image acquisition software. 
	NOTE: We used original microprogramming package of numerical analysis software for detailed analysis.</li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rebalancing+Altered+Computations:+Considering+the+Role+of+Neural+Excitation+and+Inhibition+Balance+Across+the+Psychiatric+Spectrum.">Rebalancing Altered Computations: Considering the Role of Neural Excitation and Inhibition Balance Across the Psychiatric Spectrum.</a> Biological Psychiatry. 81 (10), 816-817 (2017).</li><li>Busche, M., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairments+of+neural+circuit+function+in+Alzheimer&#8217;s+disease.">Impairments of neural circuit function in Alzheimer&#8217;s disease.</a> Phil. Trans. R. Soc. B. 371 (1700), 20150429 (2016).</li><li>Kn&#246;pfel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+optical+indicators+for+the+analysis+of+neuronal+circuits.">Genetically encoded optical indicators for the analysis of neuronal circuits.</a> Nature Reviews Neuroscience. 13 (10), 687 (2012).</li><li>Kn&#246;pfel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+toolbox+for+remote+control+of+neuronal+circuits.">Expanding the toolbox for remote control of neuronal circuits.</a> Nature Methods. 5 (4), 293 (2008).</li><li>Tominaga, T., 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=A+new+nonscanning+confocal+microscopy+module+for+functional+voltage-sensitive+dye+and+Ca2++imaging+of+neuronal+circuit+activity.">A new nonscanning confocal microscopy module for functional voltage-sensitive dye and Ca2+ imaging of neuronal circuit activity.</a> Journal of Neurophysiology. 110 (2), 553-561 (2013).</li></ol>

The authors have nothing to disclose.

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><tbody><tr><td>High speed image acquisition system</td><td>Brainvision co. Ltd.</td><td>MiCAM - Ultima</td><td>Imaging system</td></tr><tr><td>High speed image acquisition system</td><td>Brainvision co. Ltd.</td><td>MiCAM 02</td><td>Imaging system</td></tr><tr><td>Macroscepe for wide field imaging</td><td>Brainvision co. Ltd.</td><td>THT macroscope</td><td>macroscope</td></tr><tr><td>High powere LED illumination system with photo-diodode stablilizer</td><td>Brainvision co. Ltd.</td><td>LEX-2G</td><td>LED illumination</td></tr><tr><td>Image acquisition software</td><td>Brainvision co. Ltd.</td><td>BV-ana</td><td>image acquisition software</td></tr><tr><td>Multifunctional electric stimulator</td><td>Brainvision co. Ltd.</td><td>ESTM-8</td><td>Stimulus isolator+AD/DA converter</td></tr><tr><td>Slicer</td><td>Leica</td><td>VT-1200S</td><td>slicer</td></tr><tr><td>Slicer</td><td>Leica</td><td>VT-1000</td><td>slicer</td></tr><tr><td>Blade for slicer</td><td>Feather Safety Razor Co., Ltd.</td><td>#99027</td><td>carbon steel razor blade</td></tr><tr><td>Membrane filter for slice support</td><td>Merk Millipore Ltd., MA, USA</td><td>Omnipore, JHWP01300, 0.45 &amp;micro;m pores,</td><td>membrane filter/0.45 13</td></tr><tr><td>Numerical analysis software</td><td>Wavemetrics Inc., OR, USA</td><td>IgorPro</td><td>analysing software</td></tr><tr><td>Stimulation isolator</td><td>WPI Inc.</td><td>A395</td><td>Stimulus isolator</td></tr><tr><td>AD/DA converter</td><td>Instrutech</td><td>ITC-18</td><td>AD/DA converter</td></tr><tr><td>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><td>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><td>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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Research

JoVE Journal

Neuroscience

7.7K Views.  Tokushima Bunri University. De optische opname methode met spanning gevoelige kleurstof wordt beschouwd als een ideale technologie om te visualiseren hoe de hersenen werken. Deze methode stelde ons in staat om een neuropsychische dynamiek stabiel en betrouwbaar op te nemen gedurende 12 uur, gaf ons een visie op psychische activiteit. We proberen de toepassing uit te breiden om circuitveranderingen veroorzaakt door chemische stoffen in het hele ontwikkelingsstadium te detecteren, zoals waardevolle zuurtoepassing voor moe...

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

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

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 ...

Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons (10 to 25 &mu;m in diameter) are arranged in plexuses that are confined to distinct planes of the gut wall 1; the myenteric plexus can be found between the longitudinal and circular muscle layers, and the submucous plexus between the circular muscle layer and the mucosa. Since the effector systems for these plexuses (transporting epithelium, endocrine cells, immune elements, blood vessels and smooth muscle) are also contained within the gut wall, semi-intact preparations can be dissected that preserve individual components of different reflex pathways. The behavior of the effector systems is controlled by the submucous and myenteric plexuses acting in concert. Therefore, detailed knowledge of synaptic interactions within and between ganglia, and of communication between the plexuses, is essential for understanding normal gastrointestinal function. The ENS, as an intact nervous system, is a unique experimental model in which one can correlate molecular and cellular events with the electrical behavior of the neuronal network and its physiological outputs. Because of the quasi-two-dimensional organization of its plexuses, the ENS is particularly well suited for the study of neural networks using multiple site optical recording techniques that employ voltage-sensitive dyes 2,7,8,9. We will illustrate here the use of a relatively new naphthylstyryl-pyridinium dye (di-4-ANEPPDHQ) 3 that offers multiple advantages over its predecessors, including very low phototoxicity, slow rate of internalization, and remarkable chemical stability. When used in conjunction with a camera that permits sub-millisecond time resolution, this dye allows us to monitor the electrical activity of all the neurons in the field of view with a maximal spatial resolution of ~ 2.5 &mu;m at 100X magnification. At lower magnification (10X or 20X), the sacrifice of single-cell resolution is compensated by a gain in perspective, revealing the intricacies of the inter-ganglionic circuitry.

This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons ...

Biology

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

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+ ...

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

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/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

Optical Recording of Neuronal Activity in Brain Slices Stained with a Voltage-Sensitive Dye

Source: Tominaga, Y., et al. <a href="https://app.jove.com/v/59692">Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye.</a> J. Vis. Exp. (2019).
This video demonstrates a procedure for observing changes in neuronal activity in a brain slice stained with a voltage-sensitive dye. The dye reflects changes in the neuron&#39;s membrane potential by altering its fluorescence intensity. Through the application of electrical stimuli and advanced imaging techniques, this process enables visual tracking of real-time changes in neuronal membrane potential based on VSD fluorescence.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Daily Preparation of ...

Simultaneous Optical and Electrophysiological Monitoring of Neuronal Cells in Brain Slices

Source:&#160; Murphy, C. A., et al. <a href="https://app.jove.com/v/61333">Optogenetic activation of afferent pathways in brain slices and modulation of responses by volatile anesthetics.</a> J. Vis. Exp. (2020)
This video outlines a method for simultaneously imaging and recording electrical activity from transgenic brain slices that contain neuron-specific fluorescent proteins. The procedure involves maintaining the brain slice in a perfusion chamber with aerated artificial cerebrospinal fluid, using a light source to excite fluorescent-labeled cells, and recording the electrical signals with a multichannel probe and patch clamp.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.&#160;&#160;&#160;&#160;&#160;
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Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

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