This video demonstrates the dissection and subsequent imaging of electrically stimulated enteric nervous system preparations using the natal styre perineum voltage sensitive dye D four A-N-E-P-P-D-H-Q. A small segment of the intestine is taken from a euthanized Guinea pig and dissected to first expose the submucosa and ultimately the myenteric plexus for optical recordings. Electrodes are placed on a mounted tissue stained with D for A-N-E-P-P-D-H-Q after an initial high resolution image is captured.
Optical changes indicating electrical activity are recorded with the high speed camera. During the high speed acquisition, electrical stimulation is applied. The high resolution image is then superimposed onto the pixel map of the high-speed camera for analysis using neuro plex.
Hi, I am Brian Salzberg, director of the Laboratory of Cellular Optics or loco, as we like to call it in the Department of Neuroscience at the University of Pennsylvania. My name is Analia Obide. I am also a member of the laboratory of cellular Optics.
Today we'll show you a procedure for recording electrical activity from intact mammalian neuronal networks using voltage sensitive dyes. We use this procedure to study the neuronal connectivity within and between the two networks that composed the enteric nervous system also refer to as the second brain or the brain of the gut. So let's get started.
Begin preparing sub mucus and my enteric plexus preparations by excising the small intestine of a 150 to 200 gram or two to three week old Hartley Guinea pig that has been anesthetized by isof fluorine, inhalation, and decapitated. Then remove the contents of the intestine by flushing the lumen with a warm oxygenated M1 99 DM solution. Cut an intestinal segment eight to 10 centimeters in length and making sure to preserve polarity.
Transfer it to a sard coated 10 centimeter glass dish containing room temperature M1 99 DM.Place the dish on a dissecting microscope with dark field illumination. Then using scissors. Open the mesenteric border and use insect pins to mount the intestinal segment mucosa side up.
It is extremely important to maintain even tension so that after pinning the tissue exhibits a rectangular shape with the rows of mucosal vii regularly aligned, holding fine dumont forceps with straight tips in a near horizontal position. Peel the mucosa by grabbing the VII at one end of the intestinal segment and pulling along the longitudinal axis of the tissue. Continue separating ribbons of mucosa to expose the submucosa containing the sub mucus ganglia and submucosal vasculature.
Next, using very fine scissors, make a cut in the submucosal layer following the orientation of the circular muscle fibers. Then lift the submucosa very gently with the fine forceps while pushing the circular muscle. Layer down using the scissors and cut along the longitudinal edges.
Liberate the submucosa from the rest of the preparation as the separation progresses. Now make a second cut following the orientation of the circular muscle fibers at a distance of 1.5 to three centimeters from the first one to define the final size of the submucosal preparation, the resulting submucosal segment is 30 to 50 micrometers thick and exhibits a gossamer appearance. Transfer it to another syl guard dish containing fresh M1 99 DM and pin it gently and without tension.
Then place the dish in an oxygenated chamber at room temperature for later use. The original intestinal segment should now be deprived of mucosa and submucosa and the circular muscle should be exposed. This segment is now used to yield a myenteric plexus preparation to expose the myenteric plexus.
Eliminate as much as possible of the circular muscle layer by using straight tip fine forceps to pull away bundles of fibers from each side of the preparation. The exposed myenteric plexus cannot be separated from the longitudinal layer of muscle and the underlying CI.Transfer the myenteric plexus preparation to a new sard coated dish containing oxygenated M1 99 M.Then loosely pin the sample which contains live muscle. A few minutes prior to the optical experiment, transfer the preparation to a new, smaller syl guard coated dish already filled with M1 99 DM containing two micromolar nefe to prevent muscle contraction and securely mounted carefully placing multiple very fine pins around the perimeter.
Ensure that the tension is evenly distributed and the tissue lies completely flat against the sil guard bottom. The tissue preparation is now ready for physiological experiments. Prepare a five to 50 microgram per milliliter standing solution of DAI four A-N-E-P-P-D HQ in M1 99 M with two micromolar nifedipine in a glass vial and cover it with aluminum foil.
Ethanol from the stock solution should comprise less than 0.5%of the solution to the M1 99 DM with Nifedipine that was covering the preparation and replace it with the prepared dye solution. Using enough volume of the ladder to completely cover the tissue incubate at room temperature for 10 to 30 minutes following the incubation. Wash the preparation with oxygenated M1 99 M plus NEFE by replacing the full content of the chamber.
Optical recording of neuronal activity in electrically stimulated preparations is performed using a system consisting of an upright epi fluorescence microscope equipped with an image splitter, a low resolution precision high speed camera, a high resolution black and white camera, a computer controlled stimulus generator, and a microm manipulator. The entire apparatus is mounted on an active vibration, isolation table and surrounded by heavy acoustic curtains and a Mylar roof to minimize airborne vibration. Although image acquisition from both cameras can be performed using a single computer running both neuro plex and Global Lab image two or any other image acquisition software, the setup shown here employs two computers each controlling a separate camera to begin optical experiments.
Place the chamber containing the stained panned sample on the microscope stage. Since D four A and EPPD HQ is a membrane impermanent dye that fluoresces only when in a lipid environment, healthy neurons from a stained enteric preparation should have the appearance of empty balloons under any magnification higher than 20 x when viewed. Using the filters HQ 5 35 50 for excitation HQ 5 72 LP four emission and a 5 65 nanometer diic mirror.
Next, using the micro manipulator controls position, a multi barrel linear matrix array electrode at the oral end of the tissue, making sure that the electrode is clear of the microscope objective. A linear matrix array containing 10 tungsten electrodes distributed over a one centimeter span is capable of exciting several independent neuronal pathways and is well suited for studying interconnecting ganglionic circuits. For the representative data, however, we used a homemade two lead stimulating electrode.
Use tacky wax to place a ground electrode at the opposite end of the chamber before making a functional recording acquire a high resolution black and white image of the region from which the electrical activity will be recorded to help visualization. Gray skill may be inverted so that fluorescence appears dark. To apply current stimulation using the computer controlled stimulus generator first program the device by selecting the appropriate intensity, duration, frequency, and number of stimuli, as well as delay with respect to the beginning of optical data acquisition.
To make sure that the stimulation happens within the boundaries of the optical data acquisition, configure the stimulus generator as a neuro plex controlled device. Next, set the acquisition parameters within neuro plex indicating frame rate, number of frames and camera gain. To achieve full definition of an action, potential frame rate should be set to a minimum of one kilohertz.
Since D four A and E-P-P-G-H-Q exhibits a decrease in fluorescence upon depolarization that is linearly related to the voltage change, invert the display of the optical signals within neuro plex so that the action potentials don't appear upside down. Begin acquisition. Remember to acquire a new high resolution black and white image of each field of view before performing each of the functional recordings.
Once the acquisition is complete, use neuro plex to analyze the results offline. An area of the screen called page display shows the crude image of the preparation as captured by the 80 by 80 high speed camera as an individual frame. When the page mode is absolute frame superimposed the high resolution image acquired with the second camera onto the pixel map of the crude image using neuro plex display commands.
This requires change of page mode from absolute trace to traces and selection of superimposed image from within the display command. Use the mouse to select either individual pixels or groups of pixels from the image shown in the page display. The outputs of these regions of interest will appear as traces within the trace display window revealing the magnitudes of the optical changes registered by those pixels as a function of time.
Inversion of the gray scale in the high resolution image improves visualization on the trace mode page display and it is strongly encouraged to display an analog output from the stimulator. Select the analog output channel for inclusion within the trace display window. This output indicates the precise timing of the stimuli applied during the experiment.
To view the data as a movie, use the movie commands within neuro plex to display consecutive frames within a dataset using pseudo color scales adjusted to the maximal and minimal change in fluorescence intensity. A ganglion from a myenteric plexus preparation stained with DIA four A-N-E-P-P-D-H-Q was imaged with the high resolution camera and a 60 x objective. During data acquisition, the preparation was stimulated with a train of five pulses at 40 hertz.
With each pulse having an amplitude of 15 milliamps and a duration of 0.5 milliseconds, four regions of interest or R OIS were identified and traces were generated to display the optical data averaged over the selected regions in the pseudocolor frame sequence of the first optical response regions of membrane depolarization indicating neuronal activity appear as yellow to red. These data indicate that stimulation EV oaked activity occurs in discreet regions of each ganglion, mostly carried by axons of passage. The ROIs two, three and four identify individual neurons at various distances from ROI one.
Figure two illustrates at 10 x magnification the anatomical pathways along which information may propagate the arrows indicate the direction followed by the stimulus to activate the identified myenteric ganglion. We have just shown you the components of an optical setup for recording electrical activity with voltage sensitive dyes and and how when this technology applied to the inter nerve system, it opens the possibility of studying the functional connectivity within and between two intact mammalian networks under the field of the microscope. When using optical recording technology, attention to detail is extremely important.
Sources of noise, optical, mechanical and electrical need to be carefully controlled and the health of the preparation needs to be meticulously preserved. Thus, mastery of all the individual steps of this protocol is essential for achieving success in optical recording. So that's it.
Thank you very much for watching and very good luck of your experiments.
