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...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Medium 199</td>
<td>&nbsp;</td>
<td>Sigma, St. Louis, MO</td>
<td>M3769</td>
<td>With Earle&#x2019;s salts Without L-Glutamine, Phenol Red and Sodium Bicarbonate</td>
</tr>
<tr>
<td>Sylgard</td>
<td>&nbsp;</td>
<td>Dow Corning Corp., Midland, MI</td>
<td>184 silicone elastomer kit</td>
<td>&nbsp;</td>
<tr>
<td>Insect pins used for dissection</td>
<td>&nbsp;</td>
<td>F.S.T., Foster City, CA</td>
<td>26001-30</td>
<td>&nbsp;</td>
<tr>
<td>Dumont forceps</td>
<td>&nbsp;</td>
<td>F.S.T., Foster City, CA</td>
<td>11203-23 or 11200-33</td>
<td>&nbsp;</td>
<tr>
<td>Moria MC19/B Pascheff-Wolff Spring Scissors</td>
<td>&nbsp;</td>
<td>F.S.T., Foster City, CA</td>
<td>15371-92</td>
<td>&nbsp;</td>
<tr>
<td>Pins to hold the tissue during the experiment</td>
<td>&nbsp;</td>
<td>Seirin Corp., Shizuoka, Japan</td>
<td>Seirin Spinex</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase VII</td>
<td>&nbsp;</td>
<td>Sigma, St. Louis, MO</td>
<td>C0773</td>
<td>&nbsp;</td>
<tr>
<td>Protease IX</td>
<td>&nbsp;</td>
<td>Sigma, St. Louis, MO</td>
<td>P6141</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Invitrogen, Carlsbad, CA</td>
<td>10439-016</td>
<td>&nbsp;</td>
<tr>
<td>PenStrep</td>
<td>&nbsp;</td>
<td>GIBCO, BRL</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine 100X</td>
<td>&nbsp;</td>
<td>GIBCO, BRL</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Nifedipine</td>
<td>&nbsp;</td>
<td>Sigma, St. Louis, MO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Di-4-ANEPPDHQ</td>
<td>&nbsp;</td>
<td>Molecular Probes, Eugene, OR</td>
<td>D36802</td>
<td>&nbsp;</td>
<tr>
<td>4-Channel Stimulus Generator</td>
<td>&nbsp;</td>
<td>Multi Channel System MCS GmbH, Reutlingen, Germany</td>
<td>2000 Series</td>
<td>&nbsp;</td>
<tr>
<td>Peristaltic pump</td>
<td>&nbsp;</td>
<td>Rainin, Woburn, MA</td>
<td>2-channel Dinamax RP-1</td>
<td>&nbsp;</td>
<tr>
<td>High-speed camera</td>
<td>&nbsp;</td>
<td>RedShirtImaging, LLC Decatur, GA</td>
<td>NeuroCCD-SM</td>
<td>&nbsp;</td>
<tr>
<td>1:10 demagnifier / relay lens</td>
<td>&nbsp;</td>
<td>Optem, Qioptiq Linos, Inc., Rochester, NY</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Upright microscope</td>
<td>&nbsp;</td>
<td>Olympus Optical Co., Tokyo, Japan</td>
<td>BX61TRF</td>
<td>&nbsp;</td>
<tr>
<td>Gibraltar fixed stage with motorized X-Y translator</td>
<td>&nbsp;</td>
<td>Gibraltar, Burleigh Instruments, EXFO, Quebec, Canada</td>
<td>PSSG--BX2</td>
<td>&nbsp;</td>
<tr>
<td>Linear matrix electrodes</td>
<td>&nbsp;</td>
<td>FHC, Inc., Bowdoin, ME</td>
<td>Custom made</td>
<td>&nbsp;</td>
<tr>
<td>Active vibration-isolation table</td>
<td>&nbsp;</td>
<td>Halcyonics,Inc., Menlo Park, CA</td>
<td>Micro 60</td>
<td>&nbsp;</td>
<tr>
<td>Ultra-low-ripple feedback stabilized power supply</td>
<td>&nbsp;</td>
<td>Kepco, Flushing, NY</td>
<td>ATE 75-15M</td>
<td>&nbsp;</td>
<tr>
<td>Heat filter</td>
<td>&nbsp;</td>
<td>Schott Optical Glass, Duryea, PA</td>
<td>KG-1</td>
<td>&nbsp;</td>
<tr>
<td>High-Q interference filter</td>
<td>&nbsp;</td>
<td>Chroma, Inc., Rockingham, VT</td>
<td>HQ530/50</td>
<td>&nbsp;</td>
<tr>
<td>Dichroic mirror</td>
<td>&nbsp;</td>
<td>Chroma, Inc., Rockingham, VT</td>
<td>565 nm (50% transmission)</td>
<td>&nbsp;</td>
<tr>
<td>Long-pass filter</td>
<td>&nbsp;</td>
<td>Chroma, Inc., Rockingham, VT</td>
<td>HQ572LP</td>
<td>&nbsp;</td>
<tr>
<td>Acoustic curtains</td>
<td>&nbsp;</td>
<td>Acoustical Surfaces, Inc.</td>
<td>BSC-25</td>
<td>&nbsp;</td>
<tr>
<td>Neuroplex</td>
<td>&nbsp;</td>
<td>RedShirtImaging, Inc., Decatur, GA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>IDL</td>
<td>&nbsp;</td>
<td>ITT Visual Information Solutions, Boulder, CO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>High-resolution CCD camera, with its own camera controller</td>
<td>&nbsp;</td>
<td>Hamamatsu Photonics, KK, Hamamatsu City, Japan</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Frame-grabber for high-resolution camera</td>
<td>&nbsp;</td>
<td>Data Translation, Marlboro, MA</td>
<td>DT3120K-1</td>
<td>&nbsp;</td>
<tr>
<td>Imaging software for high-resolution camera</td>
<td>&nbsp;</td>
<td>Data Translation, Marlboro, MA</td>
<td>Global Lab Image/2</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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

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

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye  DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan  DIGE technology and analysis of protein expression changes using DeCyder  2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

A simple and specific method was demonstrated for fluorescent labeling and enhanced detection of cell surface proteins without a fractionation step. Differential abundance in cell surface proteins was analyzed using two-dimensional (2-D) electrophoresis and Ettan&#x2122; DIGE technology.

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of ...

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

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 ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...

Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential application in molecular biology studies, including gene expression analyses. In the context of eye growth regulation and myopia, the RPE likely plays a role as a cellular relay for growth modulatory signals, as it is located between the retina and the two walls of the eye, such as the choroid and sclera. While protocols for isolating the RPE have been developed for both chicks and mice, these protocols have proven not to be directly translatable to the guinea pig, which has become an important and widely used mammalian myopia model. In this study, molecular biology tools were used to examine the expression of specific genes to confirm that the samples were free of contamination from the adjacent tissues. The value of this protocol has already been demonstrated in an RNA-Seq study of RPE from young pigmented guinea pigs exposed to myopia-inducing optical defocus. Beyond eye growth regulation, this protocol has other potential applications in studies of retinal diseases, including myopic maculopathy, one of the leading causes of blindness in myopes, in which the RPE has been implicated. The main advantage of this technique is that it is relatively simple and&#160;once perfected, yields high-quality RPE samples suitable for molecular biology studies, including RNA analysis.

We describe a simple and efficient method for isolating cells of the retinal pigment epithelium (RPE) cells from the eyes of young pigmented guinea pigs. This procedure allows for follow-up molecular biology studies on the isolated RPE, including gene expression analyses.

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential ...

Quantifying Mitophagy in &lt;em&gt;C. elegans&lt;/em&gt; and a Liver Cancer Cell Line

Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, regulated cell death, and the generation of reactive oxygen species (ROS). ROS are vital for key biological processes. However, when uncontrolled, they can lead to oxidative injury, including mitochondrial damage. Damaged mitochondria release more ROS, thereby intensifying cellular injury and the disease state. A homeostatic process named mitochondrial autophagy (mitophagy) selectively removes damaged mitochondria, which are then replaced by new ones. There are multiple mitophagy pathways, with the common endpoint being the breakdown of the damaged mitochondria in lysosomes. 
Several methodologies, including genetic sensors, antibody immunofluorescence, and electron microscopy, use this endpoint to quantify mitophagy. Each method for examining mitophagy has its advantages, such as specific tissue/cell targeting (with genetic sensors) and great detail (with electron microscopy). However, these methods often require expensive resources, trained personnel, and a lengthy preparation time before the actual experiment, such as for creating transgenic animals. Here, we present a cost-effective alternative for measuring mitophagy using commercially available fluorescent dyes targeting mitochondria and lysosomes. This method effectively measures mitophagy in the nematode Caenorhabditis elegans and human liver cells, which indicates its potential efficiency in other model systems.

Author Spotlight: Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes  

Exploring mitophagy through electron microscopy, genetic sensors, and immunofluorescence requires costly equipment, skilled personnel, and a significant time investment. Here, we demonstrate the efficacy of a commercial fluorescence dye kit in quantifying the mitophagy process in both Caenorhabditis elegans and a liver cancer cell line.

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, ...

Rapid, Cost-Efficient, Enzyme-Free Passaging of Human Pluripotent Stem Cells on Feeder Cells by Ethylenediaminetetraacetic Acid-Mediated Dis-Adhesion

Human pluripotent stem cells (human embryonic stem cells, hESCs, and human induced pluripotent stem cells, hiPSCs) were originally cultured on different types of feeder cells for maintenance in an undifferentiated state in long-term culture. This approach has been supplanted to a large extent by feeder-free culture protocols, but these involve more costly reagents and can promote a transition to a primed state, which restricts the cells&#39; differentiation capacity. In both feeder and feeder-free conditions, the harvesting of hESC or hiPSC colonies for passaging is a necessary procedure for expanding the cultures.
To provide an easy and high-yield procedure for passaging hESCs/hiPSCs cultured on feeder cells, we have established a harvesting method using dis-adhesion elicited by the calcium chelator ethylenediaminetetraacetic acid (EDTA). We have assessed the yield and quality of the resultant passaged cells by comparing this approach to the original mechanical harvesting approach, in which colonies are isolated with a scalpel under a microscope (mechanical harvesting was chosen as a comparator to avoid the reagent variability associated with enzymatic harvesting).
In one set of experiments, two different hESC lines were maintained on a feeder cell layer of human foreskin fibroblasts. Each line was subjected to multiple passages using EDTA-based or mechanical harvesting and assessed for&#160;colony size and morphology, cell density, stemness marker expression, differentiation to the three germ layers in embryoid bodies, and genomic aberrations. In another set of experiments, we used EDTA-based harvesting on two different hiPSC lines and obtained similar results. EDTA-induced dis-adhesion saved time and gave a higher yield of colonies of a more favorable size and more uniform morphology compared to mechanical harvesting. It was also faster than enzymatic harvesting and not prone to enzyme batch variability. The EDTA-induced dis-adhesion method also facilitates the transfer of hESC/hiPSC lines from feeder cell-based culture to feeder-free conditions if desired for downstream use and analysis.

To avoid the limitations associated with the enzymatic or mechanical passaging of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) cultured on feeder cells, we have established a fast, effective, cost-efficient, high-yield method for harvesting hESC or hiPSC colonies maintained on a feeder cell layer of human foreskin fibroblasts using EDTA-mediated dis-adhesion.

Human pluripotent stem cells (human embryonic stem cells, hESCs, and human induced pluripotent stem cells, hiPSCs) were originally cultured on different ...