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 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...

Watch this Scientific Journal Video about Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye at JoVE.com

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

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Neuroscience

7.6K Views.  Tokushima Bunri University. 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.

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

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) ...