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. tha...

<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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+two+species+of+aphid+ingest+food+through+an+open+stylet+sheath.">Evidence that two species of aphid ingest food through an open stylet sheath.</a> Experientia. 20 (10), 582 (1964).</li><li>McLean, D. L., Kinsey, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+electronically+recording+aphid+feeding+and+salivation.">A technique for electronically recording aphid feeding and salivation.</a> Nature. 202, 1358-1359 (1965).</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=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. L., Kennedy, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+Penetration+Graph+studies+to+investigate+the+effects+of+cyantraniliprole+on+feeding+behavior+of+Myzus+persicae.+(Hemiptera:+Aphididae)+on+Capsicum+annuum.">Electrical Penetration Graph studies to investigate the effects of cyantraniliprole on feeding behavior of Myzus persicae. (Hemiptera: Aphididae) on Capsicum annuum.</a> Pest Management Science. 70 (5), 836-840 (2014).</li><li>Morris, G., Foster, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duelling+aphids:+electrical+penetration+graphs+reveal+the+value+of+fighting+for+a+feeding+site.">Duelling aphids: electrical penetration graphs reveal the value of fighting for a feeding site.</a> Journal of Experimental Biology. 211 (9), 1490-1494 (2008).</li></ol>

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Brass connector pins</td>
			<td>EPG Systems/hardw.shop</td>
			<td></td>
			<td>&#934; 1.2 mm</td>
		</tr>
		<tr>
			<td>Thin copper wire</td>
			<td>EPG Systems/hardw.shop</td>
			<td></td>
			<td>approx. &#934; 0.2 mm</td>
		</tr>
		<tr>
			<td>Thin gold wire</td>
			<td>EPG Systems</td>
			<td></td>
			<td>&#934; 18 &#181;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><a href="http://www.epgsystems.eu/">recipe in: www.epgsystems.eu</a></td>
		</tr>
		<tr>
			<td>Paper wipes</td>
			<td>Kimberly-Clark</td>
			<td>5511</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></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 x10</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 100Hz(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&#160;</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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14.6K 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. 