VSR was supported by an IIF Marie Curie Grant (WOUND IN EARTH, acronym for: Wound induced electrical signals in Arabidopsis thaliana).

1. Aphid Rearing
Note: The choice of plant and aphid species for EPG recordings depends on the research aim. For studies on Arabidopsis thaliana, the aphid Brevicoryne brassicae is appropriate.
<ol>
	<li>Rear B. brassicae aphids in a greenhouse on Brassica oleracea. Keep the plants used for aphid rearing in cages, in order to avoid contaminating other plants. Keep aphid-rearing plants and experimental plants (in our case B. oleracea and A. thaliana) in separate rooms, in order to avoid contamination of experimental plants with aphids.</li>
	<li>Transfer aphids to fresh plants about every 2 weeks, before causing significant plant damage, or reaching overpopulation. Transfer 10-20 adult aphids to a fresh rearing plant to initiate a new colony.</li>
	<li>Monitor rearing plants regularly for contamination by unwanted aphid species, other insect herbivores, aphid parasitoids, and fungi that may affect the health of the aphid colony.</li>
	<li>Collect adult, wingless aphids up to one week after their final molt for EPG recording.</li>
	<li>After the experiments, return the experimental plants that were not used to the growth chamber, as they often have some offspring that has been produced during the recording, which could inadvertently contaminate other plants.</li>
</ol>
2. Insect Wiring for EPG Recording
<ol>
	<li>To make insect electrodes, obtain brass connector pins (nails, &#216; 1.2 mm), thin copper wire (&#216; 0.2 mm), very thin gold wire (&#216; ca. 20 &#181;m), water-based silver glue, a simple small soldering bolt with soldering fluid and resin-cored soldering wire, stereomicroscope with 10X magnification, small scissors or scalpel, two fine forceps, and a Styrofoam sheet or box. Note: A vortex mixer might be useful. Note: The step-by-step protocol to make electrodes is indicated in Figure 2.</li>
	<li>For aphid handling and glue application, obtain: a small and soft watercolor camelhair brush (size 2 or smaller) and insect pins such as those used for insect collections, although a fine sewing needle or toothpick may work as well. Step 4 shows how to start EPG recording.</li>
	<li>Step 1. 
	&#8203;Note: Steps 1 and 2 below show how to prepare insect electrodes minus the aphid. Step 3 shows how to connect an aphid to the electrode. Vacuum fixation of the aphid is recommended during wiring, but not always required for slow moving species (e.g., B. brassicae).
	<ol>
		<li>Switch on the soldering bolt and melt some soldering wire at its tip (Figure&#160;2A). Moisten the head of the brass connector pin with some soldering fluid (Figure&#160;2B) and dip it into the melted soldering metal (Figure&#160;2C).</li>
		<li>Apply a sheath of melted solder metal on one end of a 1-2 cm long piece of the thin copper wire (Figure 2D). Then bring the pin and copper wire together against the hot bolt (Figure 2E) and move them together away to cool and solidify (Figure 2F).</li>
	</ol>
	</li>
	<li>Step 2.
	<ol>
		<li>Thoroughly shake (or vortex) the vial with silver glue for several minutes until a smooth emulsion is shown. Cut (scissors or scalpel) a few pieces of the gold wire (of approximately 1.5 cm in length) on the object plate of the stereomicroscope (Figure&#160;2G).</li>
		<li>Take a brass pin with soldered copper wire (made in section 2.3) and dip the free end of the copper wire into the small silver glue reservoir that will have gathered at inside the lid of the vial after opening it (Figure&#160;2H). Note: Only a small droplet is needed.</li>
		<li>Move the glue-dipped end of the copper wire to the piece of gold wire, while lifting one end to avoid smearing the glue onto the stereomicroscope object plate. Try to overlap copper and gold wire for a few mm (Figure&#160;2I), distributing the glue along the overlap of the two wires.</li>
		<li>Wait until the glue has been dried enough to keep the wires united. Check the glue contact after drying and add some fresh glue with a small pin or other piece of copper wire if some parts of the joined wires show glue-free parts.</li>
		<li>After the insect electrode is ready, store it, for example inserted into a piece of Styrofoam. 
		Note: The length of the gold wire will determine the freedom of movement of the aphid: if it is too short (less than 5 mm), the aphid may feel constrained and will not behave normally; if it is too long (&#62;2 cm), the aphid will move freely. Aphids tend to move to the adaxial side of leaves, if allowed to. If the gold wire touches the leaf, the signal will be short-circuited.</li>
	</ol>
	</li>
	<li>Step 3.
	<ol>
		<li>The aphid may be kept in place by means of light suction, using a vacuum; in this case, install the suction device under the stereomicroscope. Place the suction opening in the center of the field.</li>
		<li>Thoroughly shake the vial with silver glue for several minutes (or vortex) until a smooth emulsion is formed. Collect an aphid with the small brush.</li>
		<li>Switch on the suction device and mount the aphid on the suction opening (Figure&#160;2J), with the back of the abdomen turned to the experimenter. With the fine brush, remove any surface wax from the abdomen (abundant in cabbage aphids).</li>
		<li>Open the glue vial and wet a pin with a very small droplet of silver glue (Figure&#160;2K). Apply the droplet of the silver glue onto the back of the aphid&#39;s abdomen (Figure&#160;2L-M). Let this droplet completely dry during several minutes, vigorously shake the glue vial again and add a second droplet of silver glue on top of the first. Note: While the silver glue is an electrical conductor, it does not cause significant damage to the insect&#8217;s cuticle.</li>
		<li>After closing the glue vial, insert the free end of the gold wire into the wet droplet and keep the wire still while allowing the glue to dry completely (Figure&#160;2N). Avoid smearing glue onto legs or antennae and discard an aphid if this has happened.</li>
		<li>Switch off the suction fixation device and carefully lift the insect (Figure&#160;2O). If needed, use a fine brush to assist in the lifting of the aphid from the suction device. 
		&#8203;Note: Wiring B. brassicae does not require a vacuum, as they can be wired on a piece of a precision laboratory tissue, the rough surface of which provides the aphid with enough grip so that it will not be lifted after applying a drop of wet glue to its abdomen. After glue drying one can lift the aphid from the tissue with the help of a fine brush.</li>
		<li>Insert the brass pin with the wired insect into the Styrofoam and if needed, continue wiring all other insects to be used for the EPG recording session. 
		Note: These protocols for wiring aphids work well for us. The user may find his/her own method for wiring aphids.</li>
	</ol>
	</li>
	<li>Step 4.
	<ol>
		<li>Put plants in the Faraday cage (Figure&#160;2P) on a non-conductive support: use Petri dishes or a plate of glass or plastic.</li>
		<li>Insert a plant electrode into the soil of each pot. Insert the brass pin of the wired insect into the input connector of the EPG pre-amplifier (Figure&#160;2Q). Note: the soil electrode does not correspond to the ground electrode used in other electrophysiological techniques. It has the offset voltage needed to adjust and compensate for electrode polarization voltages.</li>
		<li>On the interface of the acquisition software Stylet+, with fixed sample frequency of 100 Hz, enter a filename, specify the recording time, and write text to specify details of the experiment (treatment, plant/insect species, etc.) in Comment lines 2 and 3.</li>
		<li>Lower the insects onto a suitable landing area of the plant and start the recording session by clicking on the Start button of the acquisition software (Stylet+) interface. 
		Note 1: a maximum of 8 channels can be used simultaneously in an EPG set up. One EPG-electrode or several EPG-electrodes per plant can be used. 
		Note 2: when the focus of the study is the aphid behavior, start the recording before plant access of the aphids to avoid missing the first plant penetration activities.</li>
		<li>For studying the electrophysiological responses of SEs to stimuli, wait at least 10 min after the aphid has entered into phloem phase, in order to ensure that the aphid is in a sustained phloem ingestion phase, and that the signal baseline is stable. Only then, start any plant stimulation experiment.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/52826/52826fig2.jpg" /> 
Figure 2. Making EPG-electrodes with aphids or other hemipteran insects forelectrical penetration graph (EPG) recordings. <a href="https://www.jove.com/files/ftp_upload/52826/52826fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Panels A-I, steps required to prepare EPG-electrodes minus the aphid. First, melt a piece of soldering metal on the tip of a soldering bolt (A). Then, dip the head of the brass pin into a drop of soldering fluid (B), and contact it with the melted metal at the soldering bolt tip (C). Immediately after this step, contact the end of a copper wire to the tip of the soldering bolt, in order to glue it to the head of the brass pin (E-F). With a scalpel or a blade, cut a piece of the gold wire (G). Dip the free end of the copper wire (joined at the other end to the brass pin) on the silver glue (H), and quickly join the gold wire to it (I) before the silver dries up. The gold wire is an excellent conductor, and can be polarized. In reality, in the majority of the cases the polarization is too small to be detected, and if so, it can be compensated for with the offset voltage (V).
Panels J-O, steps needed to connect an aphid (or other hemipteran insect) to the electrode. First, carefully lift an aphid with a fine watercolor brush and place it on the opening of the vacuum suction device (J). Turn on the vacuum pump and cover the air valve hole with a piece of paper to apply suction. Dip the tip of the insect pin into the silver glue (K), and put a small glue droplet on top of the aphid&#39;s abdomen, under a stereomicroscope (L-M). Within the next ~20 sec, before the silver glue droplet on the aphid dries, insert the end of the gold wire of the insect electrode into the wet droplet of silver glue, and keep it in place for 1-3 min, until the silver glue has completely air-dried (N). At this point, disable suction by removing the piece of paper that covers the air valve hole of the suction device and carefully remove the aphid, from the suction device: lifting the aphid after wiring often requires some help by a fine brush (O).
Panel P shows an overview of the entire EPG set up inside the Faraday cage, and Panel Q shows an overview of the plant-aphid combination for EPG. See section 2 above for a more detailed explanation of this process.
Small letters are labels referring to the items that one needs to make EPG-electrodes: a: soldering bolt; b: melted soldering metal; c: soldering fluid; d: brass connector pin (nail); e: copper wire; f: &#216; 18&#181;m gold wire; g: suction device; h: aphid; i: water-based silver glue; j: Faraday cage; k: plant electrode; l: input connector (BNC) of the EPG pre-amplifier.

<ol>
	<li>Salvador-Recatal&#224;, V., Tjallingii, W. F., Farmer, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time,+in+vivo.+intracellular+recordings+of+caterpillar-induced+depolarization+waves+in+sieve+elements+using+aphid+electrodes.">Real-time, in vivo. intracellular recordings of caterpillar-induced depolarization waves in sieve elements using aphid electrodes.</a> New Phytologist. 203 (2), 674-684 (2014).</li><li>Van Bel, A. J., Knoblauch, M., Furch, A. C., Hafke, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(Questions)n+on+phloem+biology.+1.+Electropotential+waves,+Ca2++fluxes+and+cellular+cascades+along+the+propagation+pathway.">(Questions)n on phloem biology. 1. Electropotential waves, Ca2+ fluxes and cellular cascades along the propagation pathway.</a> Plant Science. 181 (3), 210-218 (2011).</li><li>Rhodes, J. D., Thain, J. F., Wildon, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathway+for+electrical+signal+conduction+in+the+wounded+tomato+plant.">The pathway for electrical signal conduction in the wounded tomato plant.</a> Planta. 200, 50-57 (1996).</li><li>Fromm, J., Eschrich, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+ionic+movements+with+phloem+unloading+and+loading+in+barley+leaves.">Correlation of ionic movements with phloem unloading and loading in barley leaves.</a> Plant Physiology and Biochemistry. 27, 577-585 (1989).</li><li>Brette, R., Destexhe, A., Brette, R., Destexhe, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Recordings.">Intracellular Recordings.</a> Handbook of Neural Activity Measurement. , 44-91 (2012).</li><li>Umrath, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Untersuchungen+&#252;ber+Plasma+und+Plasamstromung+an+Characeen.+IV.+Potentialmessungen+an+Nitella+mucronata.+mit+besonderer+Ber&#252;cksichtingung+der+Erregungserscheinungen.">Untersuchungen &#252;ber Plasma und Plasamstromung an Characeen. IV. Potentialmessungen an Nitella mucronata. mit besonderer Ber&#252;cksichtingung der Erregungserscheinungen.</a> Protoplasma. 9, 576-597 (1930).</li><li>Umrath, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Der+Erregungsvorgang+bei+Nitella+mucronata.">Der Erregungsvorgang bei Nitella mucronata.</a> Protoplasma. 17, 258-300 (1932).</li><li>Carden, D. E., Walker, D. J., Flowers, T. J., Miller, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+measurements+of+the+contribution+of+cytosolic+Na++and+K++to+salt+tolerance.">Single-cell measurements of the contribution of cytosolic Na+ and K+ to salt tolerance.</a> Plant Physiology. 131 (2), 676-683 (2003).</li><li>Miles, P. W., McLean, D. L., Kinsey, M. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electronic+recording+of+penetration+behaviour+by+aphids.">Electronic recording of penetration behaviour by aphids.</a> Entomologia Experimentalis et Applicata. 24, 721-730 (1978).</li><li>Tjallingii, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+potentials+as+an+indication+for+plant+cell+penetration+by+aphid+stylets.">Membrane potentials as an indication for plant cell penetration by aphid stylets.</a> Entomologia Experimentalis et Applicata. 38, 187-193 (1985).</li><li>Alvarez, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+Solanum+stoloniferum.+responses+to+probing+by+the+green+peach+aphid+Myzus+persicae.+and+the+potato+aphid+Macrosiphum+euphorbiae.">Comparative analysis of Solanum stoloniferum. responses to probing by the green peach aphid Myzus persicae. and the potato aphid Macrosiphum euphorbiae.</a> Insect Science. 20 (2), 207-227 (2013).</li><li>Carmo-Sousa, M., Moreno, A., Garzo, E., Fereres, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-persistently+transmitted+virus+induces+a+pull-push+strategy+in+its+aphid+vector+to+optimize+transmission+and+spread.">A non-persistently transmitted virus induces a pull-push strategy in its aphid vector to optimize transmission and spread.</a> Virus Research. 186, 38-46 (2014).</li><li>Jacobson, A. 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WFT launched EPG Systems as a retirement activity, and is affiliated with it financially.

This article provides a detailed protocol for making Electrical Penetration Graph (EPG) recordings. The EPG technique is well established, with 100-200 active users worldwide, and it has been implemented for many studies on different topics, for example: a) host plant resistance to aphids and other stylet-bearing insects13; b) plant virus and pathogen transmission mechanisms14; c) insecticide mode of action, (toxicity and behavior changes)15; d) EPGs have even been useful to demonstrate t...

The ability to produce long-distance electrical signals is an advantageous trait of multi-cellular organisms that allows for efficient responses to external stimuli. This trait has evolved independently in plants and animals, and thus represents a case of convergent evolution. Given that electrical signals are coupled with important functions in animals such as neural transmission and muscle contraction, the molecular basis, mechanism of transmission, and function of stimulus-induced electrical signals in animals are subjects of intensive research. In contrast, stimulus-induced electrical signaling in plants has received little research attention. Although plants have no nerves or muscles, there seems to be enough evidence to assume that stimulus-induced electrical signals in plants play a key role in their responses to environmental factors.
The phloem, the living component of the plant vasculature, has been postulated as a major substrate for the transmission of stimulus-induced electrical signals, from stimulated/damaged to non-stimulated/undamaged areas2. The main cells in the phloem are the sieve elements (SEs), relatively simple, elongated cells. The ends of SEs are connected to other SEs, forming a continuous, low-resistance, sieve tube system that is spread throughout the plant. There are, however, very few studies on the electrical properties of these highly specialized cells. In these previous studies, researchers accessed SEs with either glass micro-electrodes3 or with glass electrodes that were coupled to plant-inserted stylets of aphids, after stylectomy (cutting)4. Glass microelectrodes are made from glass capillaries that are pulled at one end with heat into a fine tip of less than 1 &#181;m in diameter, and then filled with a KCl solution. A Ag/AgCl or platinum wire, inserted into the KCl-filled glass electrode is then connected to the amplifier input, and a referent electrode is inserted into the bath surrounding the cell of interest, completing the circuit. This setup records the difference in potential between the extracellular referent electrode and the intracellular measuring electrode, i.e., the membrane potential of the cell5. With this method, Umrath made the first intracellular recording from a plant cell, using the algae Nitella6,7. Nitella is a relatively simple organism with large cells, and therefore amenable to intracellular electrophysiology experiments. In contrast, the insertion of intracellular glass electrodes into the small cells of multi-cellular, three-dimensional terrestrial plants is technically demanding, requires a highly skilled researcher, as well as sophisticated visualization, micromanipulation, and anti-vibration equipment. Although glass electrodes are suitable to record from superficial cells in plants, such as root epidermal cells8, intracellular recordings from cells deeply embedded in the plant&#39;s tissue, such as SEs, very likely cause damage-induced responses, confusing the results. In 1989, Fromm and Eschrich reported the use of an alternative method, called the &#39;aphid method&#39;, in which glass electrodes are coupled to aphid stylets after stylectomy4. The aphid method is minimally invasive, because flexible stylets do not cause tissue or cell damage as glass electrodes do. Aphid stylets are nature&#39;s great invention for plant penetration, and aphids are considerably more skilled than humans in finding SEs. Unfortunately, this aphid method is also highly demanding in terms of technical expertise and equipment. In addition, the success of each experiment that implements this technique depends entirely on the aphid being in feeding mode &#8212; with the stylet stably inserted into a SE, at the time of stylectomy. Thinking in retrospective, one can see that the odds of success of this technique could have been improved by adding to the experimental setup an instrument that allows identifying whether or not the aphid stylet is in the SE when applying stylectomy.
In 1964, McLean and Kinsey described an &#39;electronic monitoring system&#39; for the study of the feeding behavior of aphids in real time9,10. In this system, the aphid and the stylet-penetrated plant were integrated into an electrical circuit. Later, in 1978, Tjallingii devised a modified version of the system, called the &#39;Electrical Penetration Graph&#39; (EPG) system11,12. Whereas the original electronic monitoring system was sensitive to the resistance-originated potentials only, with the EPG system, the electromotive force (emf) originated potentials, i.e., generated in the plant or in the insect, could be recorded in addition to potentials arising from resistance (R) in the insect. This represents an important improvement, because both signal components, emf and R, provide biological relevant information on events during plant penetration by aphids. What makes the EPG pre-amplifier sensitive to the R-components is its relatively low input resistance of 1 G&#937;, which is close to the average of the plant/aphid resistance. A small offset voltage (Figure&#160;1, V) of approximately +100 mV is applied to the plant, which then is divided across plant and insect on one side, and the input resistance on the other. The voltages and their changes are measured at a point (Figure 1A,B) between the insect and the input resistor. Therefore, the R-components represent plant-aphid resistance modulations of the offset voltage, whereas the emf-components are a certain fraction of plant potentials at the stylet tip and potentials caused in the insect. The plant potentials &#8212; most relevant here &#8212; are mainly membrane potentials of the plant cells punctured by the aphid stylets. The insect potentials appear to be mainly streaming potentials caused by fluid movements within the two stylet canals, i.e., the food and the salivary canals; no internal nerve or muscle potentials are recorded in the EPG. In practice, the stylet tip functions as an electrode tip. All plant cells are negatively charged inside relative to the positive outside of the cell. The electrical current (i.e., the movement of charged ions in watery solution) flowing from the inside to the outside and vice versa is very limited due to the high resistance of the cell membrane. Normally the resting potential is kept constant. However, when negative ions move out or positive ions move in through the cell membrane, the membrane potential is reduced, i.e., it &#39;depolarizes&#39;. Depolarization occurs in case of cell excitation. Ions then move in or out when specific ion channels in the membrane are opened or when the membrane is damaged and ions leak in and out. All cells have ion channels and pumps in the plasma membrane that bring the membrane potential to its resting level by restoring the original concentration of various ions inside the cell. The resting potential and its changes are emf components, and therefore, the EPG technique is suitable to measure them.
<img alt="Figure 1" src="/files/ftp_upload/52826/52826fig1.jpg" /> 
Figure 1. EPG-electrodes. The EPG-electrode is a living aphid integrated into the Electrical Penetration Graph (EPG) circuit, whose stylet is inserted into a sieve element (SE) in stable feeding mode. If the stylet-impaled SE is at rest (panel A), the voltage in the circuit, recorded by EPG, is stable and at the resting potential level (Panel C, Rest). If the SE is excited, its membrane depolarizes (panel B), which is visualized in the EPG as a gradual increase in voltage (panel C, Depolarization). As the ionic balance in the SE returns to rest, i.e., it repolarizes, the voltage recorded by EPG gradually decreases to the rest potential level (Panel C, Repolarization). In panel C, &#8220;A&#8221; and &#8220;B&#8221; refer to the scenarios shown in panels A and B, respectively. V = Adjustable offset voltage source. Ri = Input resistor. In parallel to the 1 G&#8486; external resistor, the amplifier has an internal (in the OpAmp) high 1.5&#160;T&#8486; resistor (panels A and B, in gray). By remote control of the switch the EPG pre-amp can be changed from normal to emf-mode, which allows obtaining highly accurate voltage values. <a href="https://www.jove.com/files/ftp_upload/52826/52826fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In the next section, we provide the reader with a basic protocol for performing EPG experiments that is valid for both insect-focused and plant-focused studies.

<table><tbody><tr><td>Brass connector pins</td><td>EPG Systems/hardw.shop</td><td></td><td>&amp;Phi; 1.2 mm</td></tr><tr><td>Thin copper wire</td><td>EPG Systems/hardw.shop</td><td></td><td>approx. &amp;Phi; 0.2 mm</td></tr><tr><td>Thin gold wire</td><td>EPG Systems</td><td></td><td>&amp;Phi; 18 &amp;micro;m</td></tr><tr><td>Soldering fluid</td><td>hardware shop</td><td></td><td>matching the soldering wire</td></tr><tr><td>Resin-cored soldering wire</td><td>hardware shop</td><td></td><td></td></tr><tr><td>Styrofoam</td><td>any</td><td></td><td></td></tr><tr><td>Water-based silver glue</td><td>EPG Systems</td><td></td><td>&lt;a href=&quot;http://www.epgsystems.eu/&quot;&gt;recipe in: www.epgsystems.eu&lt;/a&gt;</td></tr><tr><td>Paper wipes</td><td>Kimberly-Clark</td><td>5511</td><td></td></tr><tr><td>Soldering bolt</td><td>any</td><td></td><td></td></tr><tr><td>Stereomicroscope</td><td>Hund Wetzlar</td><td></td><td>minimum magnification is 10X</td></tr><tr><td>Small scissors</td><td>Fine Science Tools</td><td>14088-10</td><td></td></tr><tr><td>Scalpel</td><td>Fine Science Tools</td><td>10050-00</td><td></td></tr><tr><td>Fine forceps</td><td>Fine Science Tools</td><td>11231-20</td><td></td></tr><tr><td>Vortex</td><td>A. Hartenstein</td><td>L46</td><td></td></tr><tr><td>Watercolor brushes</td><td>any</td><td></td><td>Number 1 or 2</td></tr><tr><td>Air suction device</td><td></td><td></td><td>see description in: www.epgsystems.eu</td></tr><tr><td>Insect pins</td><td>any</td><td></td><td>No. 1 or 2</td></tr><tr><td>Solid table</td><td></td><td></td><td></td></tr><tr><td>Faraday cage</td><td></td><td></td><td>Hand made</td></tr><tr><td>Computer</td><td>Fujitsu Siemens</td><td></td><td></td></tr><tr><td>Data acquisition software</td><td>EPG Systems</td><td></td><td>Stylet+d</td></tr><tr><td>Giga-4 (-8) Complete System</td><td>EPG Systems</td><td></td><td></td></tr><tr><td>includes the following:</td><td></td><td></td><td></td></tr><tr><td>Main control box with USB output</td><td></td><td>Di155/Di710</td><td>12/14 bit, rate 100&amp;nbsp;Hz (softw. fixed)</td></tr><tr><td>EPG probes 4 (8)</td><td></td><td></td><td>50x DC pre-amplifier</td></tr><tr><td>Swivel clamps on rod</td><td></td><td></td><td></td></tr><tr><td>DC power adaptor</td><td></td><td></td><td>bipolar, 230/115 VAC to -/+8 VDC</td></tr><tr><td>Plant electrodes and cables</td><td></td><td></td><td></td></tr><tr><td>Additional test and ground cables&amp;nbsp;</td><td></td><td></td><td></td></tr></tbody></table>

a new application of the electrical penetration graph (epg) for acquiring and measuring electrical signals in phloem sieve elements

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of electrical transmission between distant cells in an organism requires in vivo, artifact-free recordings of cells embedded within their native environment. The transmission of electrical signals from wounded to unwounded areas in a plant has since long piqued the interest of botanists. The phloem, the living part of the plant vasculature that is spread throughout the plant, has been postulated as a major tissue in electrical transmission in plants. The lack of suitable electrophysiological methods poses many challenges for the study of the electrical properties of the phloem cells in vivo. Here we present a novel approach for intracellular electrophysiology of sieve elements (SEs) that uses living aphids, or other phloem-feeding hemipteran insects, integrated in the electrical penetration graph (EPG) circuit. The versatility, robustness, and accuracy of this method made it possible to record and study in detail the wound-induced electrical signals in SEs of central veins of the model plant Arabidopsis thaliana1. Here we show that EPG-electrodes can be easily implemented for intracellular electrophysiological recordings of SEs in marginal veins, as well as to study the capacity of SEs to respond with electrical signals to several external stimuli. The EPG approach applied to intracellular electrophysiology of SEs can be implemented to a wide variety of plant species, in a large number of plant/insect combinations, and for many research aims.

In a previous study, we implemented the EPG-electrode technique with the purpose of characterizing the electrical signals produced in SEs of the midvein during caterpillar attack1. The midvein is a preferred insertion site for conventional glass electrodes, as well as for glass-stylet electrodes, because it is SE-dense, and relatively robust, hence amenable to the fixation needed for implementing these techniques. Here, we took advantage of the versatility of the EPG electrode with the purpose of gathering ele...

Watch this Scientific Journal Video about A New Application of the Electrical Penetration Graph EPG for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements at JoVE.com

A New Application of the Electrical Penetration Graph EPG for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrical Penetration Graph (EPG) is a well-established technique for studying the feeding behavior of stylet-bearing insects. Here we show a new application of EPG as a non-invasive tool for the acquisition of intracellular electrophysiology recordings of sieve elements (SEs), the cells that form the phloem vasculature in plants.

A New Application of the Electrical Penetration Graph (EPG) for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of ...

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Research

JoVE Journal

Environment

14.7K Views.  University of Würzburg. Electrical Penetration Graph (EPG) is a well-established technique for studying the feeding behavior of stylet-bearing insects. Here we show a new application of EPG as a non-invasive tool for the acquisition of intracellular electrophysiology recordings of sieve elements (SEs), the cells that form the phloem vasculature in plants. 

Video: A New Application of the Electrical Penetration Graph EPG for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Measurement of Bioelectric Current with a Vibrating Probe

Electric fields, generated by active transport of ions, are present in many biological systems and often serve important functions in tissues and organs. For example, they play an important role in directing cell migration during wound healing. Here we describe the manufacture and use of ultrasensitive vibrating probes for measuring extracellular electric currents. The probe is an insulated, sharpened metal wire with a small platinum-black tip (30-35 &mu;m), which can detect ionic currents in the &mu;A/cm2 range in physiological saline. The probe is vibrated at about 200 Hz by a piezoelectric bender. In the presence of an ionic current, the probe detects a voltage difference between the extremes of its movement. A lock-in amplifier filters out extraneous noise by locking on to the probe's frequency of vibration. Data are recorded onto computer. The probe is calibrated at the start and end of experiments in appropriate saline, using a chamber which applies a current of exactly 1.5 &mu;A/cm2. We describe how to make the probes, set up the system and calibrate. We also demonstrate the technique of cornea measurement, and show some representative results from different specimens (cornea, skin, brain).

The manufacture, calibration and use of non-invasive vibrating probes to measure bioelectric current in various biological systems is described.

Electric fields, generated by active transport of ions, are present in many biological systems and often serve important functions in tissues and organs. ...

Bioengineering

Development of a 3D Graphene Electrode Dielectrophoretic Device

The design and fabrication of a novel 3D electrode microdevice using 50 &#181;m thick graphene paper and 100 &#181;m double sided tape is described. The protocol details the procedures to construct a versatile, reusable, multiple layer, laminated dielectrophoresis chamber. Specifically, six layers of 50 &#181;m x&#160;0.7 cm x&#160;2 cm graphene paper and five layers of double sided tape were alternately stacked together, then clamped to a glass slide. Then a 700 &#956;m diameter micro-well was drilled through the laminated structure using a computer-controlled micro drilling machine. Insulating properties of the tape layer between adjacent graphene layers were assured by resistance tests. Silver conductive epoxy connected alternate layers of graphene paper and formed stable connections between the graphene paper and external copper wire electrodes. The finished device was then clamped and sealed to a glass slide. The electric field gradient was modeled within the multi-layer device. Dielectrophoretic behaviors of 6 &#956;m polystyrene beads were demonstrated in the 1 mm deep micro-well, with medium conductivities ranging from 0.0001 S/m to 1.3 S/m, and applied signal frequencies from 100 Hz to 10 MHz. Negative dielectrophoretic responses were observed in three dimensions over most of the conductivity-frequency space and cross-over frequency values are consistent with previously reported literature values. The device did not prevent AC electroosmosis and electrothermal flows, which occurred in the low and high frequency regions, respectively. The graphene paper utilized in this device is versatile and could subsequently function as a biosensor after dielectrophoretic characterizations are complete.

A microdevice with high throughput potential is used to demonstrate three-dimensional (3D) dielectrophoresis (DEP) with novel materials. Graphene nanoplatelet paper and double sided tape were alternately stacked; a 700 &#956;m micro-well was drilled transverse to the layers. DEP behavior of polystyrene beads was demonstrated in the micro-well.

The design and fabrication of a novel 3D electrode microdevice using 50 &#181;m thick graphene paper and 100 &#181;m double sided tape is described. The ...

Engineering

Characterizing Electron Transport through Living Biofilms

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under physiologically relevant conditions.1 These measurements are performed on living biofilms in aqueous medium using source and drain electrodes patterned on a glass surface in a specialized configuration referred to as an interdigitated electrode (IDA) array. A biofilm is grown that extends across the gap connecting the source and drain. Potentials are applied to the electrodes (ES and ED) generating a source-drain current (ISD) through the biofilm between the electrodes. The dependency of electrical conductivity on gate potential (the average of the source and drain potentials, EG = [ED + ES]/2) is determined by systematically changing the gate potential and measuring the resulting source-drain current. The dependency of conductivity on gate potential provides mechanistic information about the extracellular electron transport process underlying the electrical conductivity of the specific biofilm under investigation. The electrochemical gating measurement method described here is based directly on that used by M. S. Wrighton2,3 and colleagues and R. W. Murray4,5,6 and colleagues in the 1980&#39;s to investigate thin film conductive polymers.

A protocol for measuring electrical conductivity of living microbial biofilms under physiologically relevant conditions is presented.

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under ...

Chemistry

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 &#181;m in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in ...

Neuroscience

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

Evaluating Electroporation Effects on Genetically Engineered Spiking HEK Cells

Monitoring Electroporation-Induced Changes in Action Potential Generation in Genetically Engineered Tet-On Spiking HEK cells

Excitable cells such as&#160;neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be affected by electric pulses even in therapies where they are not the primary targets, and this can cause adverse side effects. Therefore, to optimize the electroporation-based treatments of excitable and non-excitable tissues, there is a need to study the effects of electric pulses on excitable cells, their ion channels, and excitability in vitro. For this purpose, a protocol was developed for optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells. With the use of a fluorescent potentiometric dye, the changes in transmembrane voltage were monitored under a fluorescence microscope, and relevant parameters of cell responses were extracted automatically with a MATLAB application. This way, the excitable cell responses to different electric pulses and the interplay between excitation and electroporation could&#160;be efficiently evaluated.

For studying responses of excitable cells in vitro, the protocol describes optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells as well as changes in transmembrane voltage with automated extraction of relevant parameters.

Excitable cells such as&#160;neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be ...

TEEI Method for Electroporation Analysis and Delivery Prediction in A431 Cells

Porous Substrate-Based Electroporation with Transepithelial Electrical Impedance Monitoring

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other types of intracellular delivery, PSEP relies heavily on fluorescent markers and fluorescent microscopy to determine successful delivery. To gain insight into the intermediate steps of the electroporation process, a PSEP platform with integrated transepithelial electrical impedance (TEEI) monitoring was developed. Cells are cultured in commercially available inserts with porous membranes. After a 12 h incubation period to allow for the formation of a fully confluent cell monolayer, the inserts are placed in transfection media located in the wells of the PSEP device. The cell monolayers are then subjected to a user-defined waveform, and delivery efficiency is confirmed through fluorescent microscopy. This workflow can be significantly enhanced with TEEI measurements between pulsing and fluorescent microscopy to collect additional data on the PSEP process, and this additional TEEI data is correlated with delivery metrics such as delivery efficiency and viability. This article describes a protocol for performing PSEP with TEEI measurements.

Author Spotlight: Optimizing Porous Substrate Electroporation Through Micro and Nanochannels for Enhanced Monitoring and Intermediate Stage Characterization

Porous substrate electroporation (PSEP) pairs consistent, high throughput delivery with high cell viability. Introduction of transepithelial electrical impedance (TEEI) measurements provides insight into the intermediate processes of PSEP and allows for label-free delivery. This article discusses a method for performing PSEP delivery experiments and TEEI measurement analysis simultaneously.

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

A New Application of the Electrical Penetration Graph (EPG) for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Summary

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Chapters in this video

Measurement of Bioelectric Current with a Vibrating Probe

Development of a 3D Graphene Electrode Dielectrophoretic Device

Characterizing Electron Transport through Living Biofilms

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Monitoring Electroporation-Induced Changes in Action Potential Generation in Genetically Engineered Tet-On Spiking HEK cells

Porous Substrate-Based Electroporation with Transepithelial Electrical Impedance Monitoring

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique