We would like to thank D.A. Coulter (CHOP) for allowing us to use his 2-photon microscope to acquire the 2-photon images shown at the beginning of the video, and H. Takano for sharing with us his technical expertise.

Part 1: Tissue preparation
<ol>
<li>The submucous and myenteric plexus preparations are isolated by sequential dissection from the small intestines of 150-200g (2-3 week old) Hartley guinea pigs that have been anesthetized by isoflurane inhalation and decapitated. After euthanasia, the small intestine is excised and its contents removed by flushing the lumen with a warm, oxygenated solution. We use Medium 199 supplemented with 25 mM HEPES, 5 mM NaHCO3 and 2 mM Glutamine, and pH adjusted to 7.4 (referred to, from now on, as M199-DM), which has been pre-warmed at 37 &deg;C. All the remaining steps of the protocol, however, will be carried out at room temperature.</li>
<li>An intestinal segment, 8-10 cm in length, is transferred to a Sylgard dish containing M199-DM, opened along the mesenteric border, and mounted mucosa side-up using insect pins. It is extremely important to maintain even tension, so that after pinning the tissue exhibits a rectangular shape with the rows of mucosal villi regularly aligned.</li>
<li>Using fine Dumont forceps with straight tips, in an almost horizontal position, it is easy to peel the mucosa by grabbing the villi at one end of the preparation and pulling away along the longitudinal axis of the tissue. When the sample is evenly pinned, ribbons of mucosa can be separated in a few individual strokes, exposing the submucosa that contains the submucous ganglia and the submucosal vasculature. </li>
<li> For the fine dissection of the plexuses, we recommend using a dissecting microscope with dark-field illumination. Indeed, the pseudo three-dimensional effect created by this type of illumination helps to distinguish the individual layers, thereby preventing or minimizing instrument-inflicted damage.</li>
<li>To make a submucosal preparation, we follow the classical procedure of Hirst and McKirdy 4 that requires: a) making a cut in the submucosal layer following the orientation of the circular muscle fibers; this can be achieved using very fine scissors (such as a Moria MC19 or its equivalent); b) lifting the submucosa very gently with the fine forceps, while pushing the circular muscle layer down using the fine scissors; c) cutting along the longitudinal edges as the separation progresses to liberate the submucosa from the rest of the prep; d) making a second cut following the orientation of the circular muscle fibers at a distance from the first one, to define the final size of the submucosal preparation. The resulting submucosal segment, which is only 30 to 50 &mu;m thick and has a gossamer appearance, can be transferred to another Sylgard dish containing fresh M199-DM, pinned gently and without tension, and kept in an oxygenated chamber for later use.</li>
<li>The original gut-segment, now deprived of mucosa and submucosa, and with the circular muscle exposed, can still be used to yield a myenteric plexus preparation. To expose the myenteric plexus, one must eliminate as much as possible of the circular muscle layer. This can be achieved by using the same straight-tip fine forceps employed in previous steps. Bundles of fibers can be pulled gently away, first from one, and then from the other side of the preparation. The exposed myenteric plexus cannot be separated from the longitudinal layer of muscle and the underlying serosa. Therefore, what is called the myenteric plexus preparation is the segment that can be cut along the pins. Because it contains live muscle, it is advisable to pin it very loosely in the dish containing oxygenated M199-DM until the physiological experiment begins.</li>
<li>Since the mucosa is the main ENS sensor, and all the effectors are also embedded in the gut wall, it is possible to dissect semi-intact preparations that preserve entire reflex pathways. These custom-designed preparations require combinations of the steps described in 1.3. to 1.6., applied to adjacent areas of the isolated intestinal segment.</li>
<li>A few minutes prior to the actual optical experiment, the preparation of choice is mounted in the experimental chamber, making sure that the tension is evenly distributed and the tissue lies completely flat against the Sylgard bottom. This is achieved by holding the specimen with multiple very fine pins that can be positioned all around the perimeter without inflicting tissue damage.</li>
</ol>
Part 2: Preparation of tissue samples for optical recording
<ol>
<li>(Optional) To prevent background fluorescence from dye binding to residual smooth muscle and connective tissue, both isolated plexuses may be incubated for 30-60 min in M199-DM containing collagenase VII (&le;50 U/ml) and Protease IX (&le;0.5 &mu;g/ml). Following the enzyme treatment the preparations should be washed with, and maintained overnight in, M199-DM plus 10% animal serum (e.g., equine or bovine) and antibiotics (penicillin, 100U/ml; streptomycin, 100 &mu;g/ml), in a chamber saturated with 95% O2 and 5% CO2. This procedure, which we have used for many years because it noticeably enhances the signal-to-noise ratio of the optical recordings, may not be helpful for beginners and should be adopted with caution. Indeed, a preparation damaged during dissection runs the risk of disintegrating during the enzyme treatment. However, even in the absence of the enzyme treatment, we recommend that the preparations be maintained overnight in M199-DM with serum and antibiotics. This step promotes tissue healing after acute dissection damage, and increases the transparency of the sample.</li>
<li>Optical recordings of electrical activity cannot be achieved in a preparation in which the muscle is contracting. Therefore, to prevent muscle contraction when recording from the myenteric plexus, a few minutes prior to the optical experiment we add 2 &mu;M nifedipine to the M199-DM bathing the tissue.</li>
</ol>
Part 3: Staining with di-4-ANEPPDHQ
<ol><li>Di-4-ANEPPDHQ is commercially available in lyophilized form. We recommend dissolving it in pure ethanol, making a concentrated stock solution [e.g., 15-20 mg/ml]. This stock, when kept at -20&deg;C, is extremely stable. Indeed, in our laboratory, we just finished the last drop of a stock solution of this dye that we had made almost 6 years ago.</li>
<li>The staining solution should be prepared in M199-DM, and should contain 2 &mu;M nifedipine if the protocol requires it. The final concentration of dye can be as low as 5 &mu;g/ml and should not be higher than 50 &mu;g/ml. However, depending upon the concentration of the stock, a certain amount of ethanol will be present in the staining solution and should be taken into account. For example, a staining solution with 50 &mu;g/ml of dye will contain ~0.3% ethanol if made from a 15 mg/ml stock, but close to 1% ethanol if the stock had a concentration of only 5 mg/ml. </li>
<li>For staining, the preparation is submerged for 10-30 minutes in the dye solution. After that period, the dye is washed away and replaced by fresh, oxygenated M199-DM. Since di-4-ANEPPDHQ is a membrane-impermeant dye that fluoresces only when in a lipid environment, healthy neurons from a stained enteric preparation should have the appearance of empty balloons.</li>
</ol>
Part 4: Electrical stimulation
<ol>
<li>Stimulation can be driven by voltage or by current. We use current stimulation, delivered by a computer controlled, 4-channel stimulus generator with a maximum output of 16 mA per channel.</li>
<li>The electrode model to be used in a given experiment is determined by the experimental objective. Intracellular pipettes or patch electrodes can be used when the goal is the stimulation of a particular neuron. When the stimulation is intended to activate more than one neuron in a given ganglion, an extracellular electrode touching a connective may be used. To study interconnecting ganglia, large, low-resistance electrodes and/or multi-element electrode arrays can be employed; however, when using current stimulation as we generally do, multi-element arrays become the only option. Many models of extracellular electrodes, as well as some linear electrode arrays, are readily available from commercial sources.</li>
<li>Electrodes can be positioned using different types of micromanipulators. To avoid the vibration induced by long and fine barrels, we favor using small micromanipulators on magnetic bases that can be positioned adjacent to the experimental chamber, and supporting the flexible barrels of the tungsten electrodes with thin but very rigid metal bars.</li>
</ol>
Part 5: Perfusion
We use a 2-channel peristaltic pump with one channel for input of oxygenated solutions, and the other for output. To eliminate mechanical disturbances induced by the perfusion, the pump is turned-off during the acquisition of optical data.
Part 6: Apparatus for multiple site optical recording of membrane potential
<ol>
<li>Optical components: 
 The apparatus for multiple site optical recording of membrane potential comprises a NeuroCCD-SM camera (RedShirtImaging) and a 1:10 demagnifier / relay lens mounted on the trinocular tube of an upright microscope. The microscope moves on an X-Y translator, independently of a stage that is rigidly fixed to the top of an active vibration isolation table. To minimize airborne vibration, the entire apparatus is surrounded by heavy acoustic curtains and a mylar roof. Epi-illumination is provided by a 100W tungsten-halogen lamp powered by an ultra-low-ripple feedback stabilized power supply. The incident light is made quasi-monochromatic using a heat filter, a high-Q interference filter (HQ530/50) and a 565 nm (50% transmission) dichroic mirror. Fluorescence emission is separated using the dichroic mirror and a long-pass filter (HQ572LP). The intensity of the incident light is adjusted using neutral density filters. Trans-illumination, for bright-field viewing of the preparation is provided by a second 100 W tungsten-halogen lamp driven by a 0-55 V, 1-10 A power supply that is not required to possess any special attributes. </li>
<li>High-speed camera specifications: 
 The electronic design and the performance characteristics of the NeuroCCD-SM camera have been described in detail 3. Briefly, it is a cooled, low resolution, precision high-speed camera that uses the back-thinned, back-illuminated Marconi CCD39-01 chip (80x80 pixels). Digitization is 14-bit, and the full frame rate is 2 kHz. However, for specific purposes, one may increase the frame-rate up to a maximum of 10 kHz by using various binning combinations or reducing the number of functional pixels. The camera has the important advantage of extremely low read noise (23 electrons at 2 kHz; 9 electrons at 1 kHz). In addition, its pixels have a relatively large well depth (215,000 electrons), permitting moderate light intensities at high frame rates. </li>
<li>Data Acquisition and Analysis Software: 
 The optical experiment is controlled by Neuroplex, a software package from RedShirtImaging, that operates as an application within the IDL platform (Interactive Data Language) and includes multiple features necessary for acquisition, display and analysis of the optical data. </li>
<li>Additional camera: 
 As an integral part of the apparatus, a beam splitter on the trinocular head of the microscope relays an image of the preparation onto a second, high-resolution CCD camera connected to a frame-grabber. The image, which is acquired and stored using Global Lab Image/2 software, can be superimposed onto the display of the sub-millisecond time-resolved optical signals captured by the NeuroCCD-SM camera to generate an accurate map of the spatial origin of the optically recorded voltage signals. In our lab, the frame grabber for this camera resides in a different computer than the one responsible for functional data acquisition. Therefore, the image file has to be transferred from one computer to another, and small adjustments in size and position of the high-resolution image are required before perfect registration of the two cameras is achieved.</li>
 </ol>
Part 7: Optical experiments
<ol>
<li>Prior to each functional recording, we acquire the high-resolution black and white image of the region from which we plan to record electrical activity by optical means (as indicated in 6.4). This image, when superimposed onto the pixel-map of the high-speed camera, helps us to identify the structures that are the source of the optical signals. </li>
<li>In the appropriate system configuration, Neuroplex (see 6.3) also controls the shutter, and triggers the electrical stimulation. Since the high-speed data acquisition requires considerable computer power, it is advisable, if at all possible, to use a dedicated computer with a large amount of memory and a high capacity hard drive. That, however, is not trivial. Our own system, e.g., already several years old, employs data acquisition boards that are too large to fit in state-of-the-art computers. As a compromise, we adopted and dedicated to our optical system a DELL Precision 390 equipped with a 500 GB hard drive and 4 GB of RAM. This computer allowed us to keep our older, but adequate, A/D and D/A boards, while still providing sufficient power to run our experiments.</li>
<li>As soon as the acquisition is over, Neuroplex can display the results in multiple complementary formats, some of which are detailed below:
<ol class="lalpha">
<li>An area of the screen called page display shows the crude image of the preparation as captured by the 80X80 high-speed camera as an individual frame. Superposition onto this pixel-map of the high-resolution image acquired with the second camera (as explained in 6.4 and 7.1) refines the operator s ability to identify areas of interest.</li>
<li>When pixels of this page display are selected under mouse control, Neuroplex automatically converts their optical outputs into traces that show the magnitude of the optical change registered by those pixels as a function of time. (e.g., &Delta;F/F vs. time). The operator can decide whether these traces should reflect the output of individual pixels, or, rather, the space-averaged output of multiple loci over a given region of the preparation.</li>
<li>The data can also be displayed in movie-format. 
 To ensure that all the information contained in individual experimental runs is fully extracted, however, we favor off-line analysis of optical data. Representative results of this analysis are available.</li>
 </ol>
 </li>
 </ol>
Part 8: How to achieve technical quality in an optical experiment
The technical success of an optical experiment is measured, ultimately, by the signal-to noise ratio of the recordings. Furthermore, a good signal-to-noise ratio stems not from an epiphany but from a journey. As in an opera performance, the final outcome is determined by a multiplicity of factors that interact synergistically. Thus, to achieve a technically sound optical experiment, it is essential to refine, independently, the optical apparatus and the skills required for each individual step of the protocol. What follows is a list of potential trouble-areas, and some suggestions for avoiding problems not always anticipated 5:
<ol>
<li>Noise sources 
 Mechanical and optical stability are crucial when recording electrical activity with voltage-sensitive dyes, because the fractional change (&Delta;F/F) reflecting physiological responses is relatively small (&lt; 10% / 100 mV in most of the gut recordings) and a noisy trace always augurs a bad outcome. Therefore, to optimize the signal-to-noise-ratio, it is imperative to eliminate, or, at least, to minimize, the contributions of the two worst offenders: a) mechanical vibrations, and b) light source fluctuation. 
 <ol class="lalpha">
 <li>Mechanical vibrations are generated by a variety of sources as diverse as setup configuration, building stability, proximity of motorized equipment, air-currents, acoustic noise, fluid motion, or particles floating in the bathing solution. A good vibration-isolation system, and acoustic curtains for extreme cases (see, e.g., description in 5.1), make a big difference and are well worth their cost. In addition, common-sense approaches, such as filtration of solutions used in the experiment, or the use of magnetic bases for small, moveable parts of the setup, are quite effective.</li>
 <li>Light sources are most stable at quasi-maximal intensity. Therefore, one should set a current-limit for the power supply, and, when necessary, reduce the light intensity by inserting neutral-density filters in the light path instead of by reducing the voltage. Arc lamps are intrinsically noisy. Tungsten-halogen bulbs or light-emitting diodes are much more stable and should be the preferred choice 6. </li>
 </ol>
 </li>
<li>Animal age 
 Dissections should be done using the gut of young animals because, as the connective tissue within the gut toughens with age, it becomes more difficult to separate individual tissue layers without inflicting major damage on the neurons of the two plexuses, whose afferent and efferent processes may cross layer boundaries and/or project for quite a distance along the longitudinal axis of the gut. This advisory, which can be disregarded when studying intra-ganglionic connections, is of paramount importance when the experimental objective is the analysis of inter-ganglionic circuits or the activation of reflex pathways. </li>
<li>Dye internalization 
 It is important to use as low a dye concentration as possible, and to minimize unnecessary light exposure. Although di-4-ANEPPDHQ has proven to be much less phototoxic than other dyes, it will get internalized, eventually, as the health of the preparation deteriorates. Dye internalization diminishes the signal-to-noise ratio by increasing dramatically the background fluorescence 3. </li>
<li>Electrode maintenance 
<ol class="jove_lalpha">
<li>Electrodes should be washed carefully and thoroughly after each experiment. Organic deposits at the tips of tungsten electrodes increase electrode resistance and prevent stimulation.</li>
<li>The insulation of commercially available tungsten electrode is very delicate and prone to damage. A leak in the insulation will shunt current, compromise the effectiveness of the stimulation and jeopardize the success of the experiment. </li>
</ol>
</li>
<li>Perfusion perils 
 Never leave the set-up unattended while the perfusion is on. If the in-flow and out-flow of the peristaltic pump are not properly balanced, two catastrophic events may take place:
<ol class="lalpha">
<li>A flood may cause irreversible damage to the microscope.</li>
<li>The preparation may dry out during the experiment. </li>
</ol>
</li>
<li>Software hassles 
 It is essential to familiarize oneself with the software that controls acquisition as well as analysis. Fast data-acquisition and/or the creation of very large files tend to drive the system to the brink of breakdown, and Neuroplex, which is quite unforgiving, will crash relentlessly unless its boundary conditions are respected. Subtleties within the records, easily missed when the analysis is confined to limited regions of interest, often reveal themselves in movie displays of the data.</li>
</ol>

<ol>
	<li>Costa, M. a. r. c. e. l. l. o., Furness, J. o. h. n. . B., Llewellyn-Smith, I. J., Johnson, L. e. o. n. a. r. d. . R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> in Physiology of the Gastrointestinal Tract, edited by Leonard R. , 1-1 (1987).</li><li>Obaid, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+patterns+of+activity+in+an+intact+mammalian+network+with+single-cell+resolution:+optical+studies+of+nicotinic+activity+in+an+enteric+plexus.">Spatiotemporal patterns of activity in an intact mammalian network with single-cell resolution: optical studies of nicotinic activity in an enteric plexus.</a> J Neurosci. 19 (8), 3073-3073 (1999).</li><li>Obaid, A. L., Loew, L. M., Wuskell, J. P., Salzberg, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+naphthylstyryl-pyridinium+potentiometric+dyes+offer+advantages+for+neural+network+analysis.">Novel naphthylstyryl-pyridinium potentiometric dyes offer advantages for neural network analysis.</a> J. Neurosci. Methods. 134 (2), 179-179 (2004).</li><li>Hirst, G. D., McKirdy, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+potentials+recorded+from+neurones+of+the+submucous+plexus+of+guinea-pig+small+intestine.">Synaptic potentials recorded from neurones of the submucous plexus of guinea-pig small intestine.</a> J Physiol (Lond). 249 (2), 369-369 (1975).</li><li>Salzberg, B. M., Barker, J., McKelvy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Methods+in+Cellular+Neurobiology.">Current Methods in Cellular Neurobiology.</a> , 139-139 (1983).</li><li>Salzberg, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ultra-stable+non-coherent+light+source+for+optical+measurements+in+neuroscience+and+cell+physiology.">An ultra-stable non-coherent light source for optical measurements in neuroscience and cell physiology.</a> Journal of Neuroscience Methods. 141, 165-16 (2005).</li><li>Obaid, A. L., Nelson, M. E., Lindstrom, J., Salzberg, B. 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+studies+of+nicotinic+acetylcholine+receptor+subtypes+in+the+guinea-pig+enteric+nervous+system.">Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system.</a> J Exp Biol. 208 (Pt 15), 2981-2981 (2005).</li><li>Obaid, A. L., Zou, D. J., Rohr, S., Salzberg, B. 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+recording+with+single+cell+resolution+from+a+simple+mammalian+nervous+system:+Electrical+activity+in+ganglia+from+the+submucous+plexus+of+the+guinea-pig+ileum.">Optical recording with single cell resolution from a simple mammalian nervous system: Electrical activity in ganglia from the submucous plexus of the guinea-pig ileum.</a> Biol. Bull. 183, 344-344 (1992).</li><li>Salzberg, B. M., Davila, H. V., Cohen, L. B. <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), 508-508 (1973).</li></ol>

This protocol has been written with two goals in mind. The first one is to persuade other investigators that, thanks to the many technological advances of the last decade, optical recording of electrical activity using voltage-sensitive dyes has become one of the most powerful, reliable and affordable methodologies for studying intact neuronal networks; indeed, it could be easily implemented even in a laboratory having modest resources. The second goal is to promote the enteric nervous system as a unique experimental mod...

<table><tbody><tr><td>Medium 199</td><td>Sigma-Aldrich</td><td>M3769</td><td>With Earle&amp;rsquo;s salts Without L-Glutamine, Phenol Red and Sodium Bicarbonate</td></tr><tr><td>Sylgard</td><td>Dow Corning</td><td>184 silicone elastomer kit</td><td></td></tr><tr><td>Insect pins used for dissection</td><td>F.S.T.</td><td>26001-30</td><td></td></tr><tr><td>Dumont forceps</td><td>F.S.T.</td><td>11203-23 or 11200-33</td><td></td></tr><tr><td>Moria MC19/B Pascheff-Wolff Spring Scissors</td><td>F.S.T.</td><td>15371-92</td><td></td></tr><tr><td>Pins to hold the tissue during the experiment</td><td>Seirin Corp.</td><td>Seirin Spinex</td><td></td></tr><tr><td>Collagenase VII</td><td>Sigma-Aldrich</td><td>C0773</td><td></td></tr><tr><td>Protease IX</td><td>Sigma-Aldrich</td><td>P6141</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Invitrogen</td><td>10439-016</td><td></td></tr><tr><td>PenStrep</td><td>GIBCO, by Life Technologies</td><td>15140</td><td></td></tr><tr><td>L-Glutamine 100X</td><td>GIBCO, by Life Technologies</td><td>25030</td><td></td></tr><tr><td>Nifedipine</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Di-4-ANEPPDHQ</td><td>Molecular Probes, Life Technologies</td><td>D36802</td><td></td></tr><tr><td>4-Channel Stimulus Generator</td><td>Multi Channel System MCS GmbH</td><td>2000 Series</td><td></td></tr><tr><td>Peristaltic pump</td><td>Rainin</td><td>2-channel Dinamax RP-1</td><td></td></tr><tr><td>High-speed camera</td><td>RedShirtImaging, LLC</td><td>NeuroCCD-SM</td><td></td></tr><tr><td>1:10 demagnifier / relay lens</td><td>Optem, Qioptiq Inc.</td><td></td><td></td></tr><tr><td>Upright microscope</td><td>Olympus Corporation</td><td>BX61TRF</td><td></td></tr><tr><td>Gibraltar fixed stage with motorized X-Y translator</td><td>Gibraltar, Burleigh Instruments, EXFO, Quebec, Canada</td><td>PSSG--BX2</td><td></td></tr><tr><td>Linear matrix electrodes</td><td>FHC, Inc.</td><td>Custom made</td><td></td></tr><tr><td>Active vibration-isolation table</td><td>Halcyonics,Inc., Menlo Park, CA</td><td>Micro 60</td><td></td></tr><tr><td>Ultra-low-ripple feedback stabilized power supply</td><td>Kepco, Flushing, NY</td><td>ATE 75-15M</td><td></td></tr><tr><td>Heat filter</td><td>Schott AG</td><td>KG-1</td><td></td></tr><tr><td>High-Q interference filter</td><td>Chroma Inc.</td><td>HQ530/50</td><td></td></tr><tr><td>Dichroic mirror</td><td>Chroma Inc.</td><td>565 nm (50% transmission)</td><td></td></tr><tr><td>Long-pass filter</td><td>Chroma Inc.</td><td>HQ572LP</td><td></td></tr><tr><td>Acoustic curtains</td><td>Acoustical Surfaces, Inc.</td><td>BSC-25</td><td></td></tr><tr><td>Neuroplex</td><td>RedShirtImaging, LLC</td><td></td><td></td></tr><tr><td>IDL</td><td>ITT Visual Information Solutions</td><td></td><td></td></tr><tr><td>High-resolution CCD camera, with its own camera controller</td><td>Hamamatsu Corp.</td><td></td><td></td></tr><tr><td>Frame-grabber for high-resolution camera</td><td>Data Translation</td><td>DT3120K-1</td><td></td></tr><tr><td>Imaging software for high-resolution camera</td><td>Data Translation</td><td>Global Lab Image/2</td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes at JoVE.com

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

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 ...

optical-recording-electrical-activity-guinea-pig-enteric-networks

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.9K Views.  University of Pennsylvania-School of Medicine. 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.

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

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Bioengineering

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 ...

Neuroscience

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

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 ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

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.

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 ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...

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

Source: Tominaga, Y., et al. <a href="https://appjove.unicartagenaproxy.elogim.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 ...

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

Summary

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Chapters in this video

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

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

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

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

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

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Optical Imaging of Isolated Murine Ventricular Myocytes

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

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

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