1. Preparation of Electrodes and Saline Solution
<ol>
	<li>Prepare physiological saline and electrodes before the animal is dissected. Leech Ringer&#8217;s solution consists of the following: 115.3 mM NaCl, 1.8 mM CaCl2, 4.0 mM KCl, 10 mM Tris/maleic acid or HEPES. Adjust the pH to 7.4.</li>
	<li>Prepare a Ag-Cl wire by dipping a cleaned silver wire into a small amount of bleach for 20 min&#160;or until the wire has a smooth dark gray finish. Wash the wire with distilled water before using. Then place the wire into a micropipette holder that plugs into the amplifier. &#160;</li>
	<li>Prepare a sharp glass electrode of borosilicate glass using a pipette puller. The electrode resistance should be on the order of 20 M&#8486;.</li>
	<li>Backfill the pipette with 3 M potassium acetate solution and insert in into the micropipette holder.</li>
	<li>For an extracellular electrode prepare a sharp glass pipette as described above,&#160; break the tip back by rubbing another sharp pipette near the sharp edge. Then fire polish the tip so that it does not tear the connectives. Plastic extracellular electrodes will also work as long as there is good contact between the electrode and the nerve.</li>
	<li>Insert the extracellular recording electrode into a micropipette holder (with Ag-Cl wire) that is fitted with a rubber tube and syringe for suction. Use the syringe to draw saline into the electrode at least to the level of the silver wire.</li>
	<li>Set up the hardware (amplifier and stimulus isolator) and software recording parameters by following the manual for your specific signal recording unit. See Leksrisawat et al.22 to view details on equipment used in the experiments described below.&#160; &#160;</li>
</ol>
2. Ventral Ganglion Dissection
<ol>
	<li>Dissect the leech in a large silicone elastomer-lined dish with leech Ringer&#8217;s solution. If the animal is stretched longitudinally it will be easier to remove the ventral nerve cord (see Figure 1A).</li>
	<li>With #10 or #11 size scalpel, make a longitudinal incision with very shallow cuts in the ventral midline. About the middle 2/3rd of the ventral cut is shown in Figure 1B. Try not to cut through the black stocking (the ventral blood sinus) until the animal is fully opened and the blood sinus is stretched without kinks. This blood sinus runs the length of the animal along the ventral midline. The VNC is contained inside of this blood vessel.</li>
	<li>After the skin has been cut the full length of the animal use the fine iris scissors to nick the blood sinus (Figure 2). Slip one blade of the fine iris scissors under the sinus and slightly raise the blade to separate it from the nerve tissue. Cut the sinus the length of the VNC while avoiding the nerve roots, segmental nerves, or the connectives between ganglia.</li>
	<li>Next, remove part of the ventral nerve cord by cutting the roots distal to where the blood vessel joins the roots. Repeat this process for each ganglion along the length of the ventral nerve cord.</li>
	<li>Transfer a segment of one or two ganglia to a smaller Petri dish. The ganglia or the single ganglion needs to be pinned taut with some slack.</li>
	<li>Pin the connectives and the roots so the ganglion glial sheath is slightly stretched to prevent the neuron cell bodies from moving around when impaling through the glial sheath and into the neuron of interest. The pins should be placed through the black sinus for the roots and in the middle of the two connectives to minimize damage of the axons (Figure 3).</li>
	<li>Place the Petri dish with preparation under the microscope and secure it with wax at the bottom of the dish to prevent movement.</li>
	<li>Carefully focus the light beam using a mirror and light condenser to see the neurons as in Figure 4A (schematically shown in Figure 4B). Notice the two large Retzius cells in the center of the ganglion.</li>
	<li>To make an intracellular recording of the Rz, T, P, and N neurons, carefully place the tip of the electrode over the cell of interest and gently lower the tip until a dimple is seen in the glial sheath.</li>
	<li>Tap the electrode holder or manipulator (very carefully) so that the electrode tip &#8220;pops&#8221; into the cell of interest.</li>
	<li>Inject short pulses (30-40 msec) of positive current (1-10 nA) into the cell to evoke action potentials.</li>
</ol>
3. Culturing Neurons from the Ventral Ganglion Preparation
<ol>
	<li>Pin the ganglia ventral side up in a clean dish containing L-15 with fetal calf serum (FCS) (Figure 3). Pin the roots so that the ganglia are slightly stretched as done above for intracellular recordings. With fine scissors, nick the glial capsule at one end, slip one scissors blade under the capsule, then cut across the glial capsule as shown in Figure 5A.</li>
	<li>Add 100 &#181;l aliquot of Collagenase/Dispase to the dish and place on a shaker for 15 min. Examine cells by moving the culture media around the exposed cell bodies. Return the cells to the shaker for another 15 min or longer until the cells come out with minimal suction.</li>
	<li>Use a pipette attached to a syringe to gently extract cells from the ganglion. Fire polish the pipette tip so that it is slightly larger than the cell body diameter. For Rz cells in an adult leech, the diameter should be approximately 50 &#181;m.</li>
	<li>Prepare a variety of pipettes with different diameters without a filament as this decreases the chances of the cells sticking inside the pipette. Store the pipettes in a bottle containing 80% ethanol (EtOH) using a cotton ball at the bottom to protect the tips from chipping. Remember to wash the EtOH out of the pipette with medium before aspirating cells.</li>
	<li>After the enzyme treatment, wash out the enzyme-containing medium with L-15 (with FCS) 2x. Identify the cells of interest and gently draw them into the pipette by pulling the syringe (Figure 6A). Discharge the aspirated cells into another dish which contains L-15 (with FCS).</li>
	<li>Until now, all procedures were non-sterile. Perform the following steps in a sterile hood. Place 3-4 large drops of sterile L-15 (with FCS) in different locations inside a sterile Petri dish.</li>
	<li>Pipette the cells into one of the before-mentioned drops, blowing them around in the drop to wash them. Repeat this in series in 3 or 4 drops of medium.</li>
	<li>Place cells in a clean Petri dish filled with L-15 (with FCS).</li>
	<li>Incubate the cells for 1-24 hr at room temperature. Afterwards the clean cells can be plated. By leaving the cells for a few hours or overnight the extracellular matrix comes off easily. Plate the cells immediately to generate longer processes.</li>
	<li>Use sterile substrate to coat a microwell dish and allow it to dry completely (overnight) before culturing experiments.</li>
	<li>After the dish has been coated, wash the dish gently with sterile water and then with a small amount of the L-15 medium. Remember to use the L-15 solution without the FCS added to allow the cells to adhere to the dish. The medium can be gently changed to L-15 with FCS after the cells have adhered to the substrate. If the cells are to be used a few hours after plating, then switching the medium to L-15 with FCS is not necessary.</li>
	<li>Position the cells next to each other at the time of plating to induce rapid synaptogenesis (Figure 6B). After 24 hr measure physiological activity in pairings of neurons.</li>
	<li>Use intracellular electrophysiological techniques to record RP or synaptic responses in cultured pairs of cells (stimulate 1 neuron through an intracellular electrode and record responses from the paired cell using a 2nd intracellular electrode).</li>
</ol>
4. Ganglion-body Wall Preparation
<ol>
	<li>Pin the leech dorsal side down in an elastomer-lined dish as described above.</li>
	<li>Pin the skin to one side with roots on the other side of the severed ganglion (Figure 7A, magnified in Figure 7B) or make a window on the ventral aspect of the leech over a ganglion (Figure 7C) with all the roots to the body wall intact.</li>
	<li>After obtaining a stable intracellular recording in one of the sensory cells (T, P, N) use a small glass rod that has been fire polished to lightly touch the skin in the same segment. If the neuron being recorded from is an N cell then more pressure will need to be applied to the skin. In most cases, the N cell will only respond if the skin is pinched with tweezers. For this reason these cells should be tried last as one might damage the skin or displace the intracellular recoding by disturbing the preparation in such a manner.</li>
	<li>One should be able to quickly sketch the skin and ganglion with details sufficient enough that a map of where one touched can be used to determine the receptive field for a particular neuron. After recording from as many T and P cells as possible on both sides of a ganglion try the N cells.</li>
	<li>In order to examine if the sensory neurons have processes that travel through the connectives in a rostral or caudal manner and then out the roots in the adjoining segment to the skin, the connectives between ganglia can be cut and the receptive fields reexamined in regards to the spread of detection. Basically one is examining if there has been any loss in the receptive field.</li>
</ol>
5. Injecting Fluorescent Dyes
<ol>
	<li>Prepare a leech ganglion as described above.</li>
	<li>Fill a microelectrode with a solution of Lucifer yellow-CH (3.0%) and lithium chloride (300 mM).</li>
	<li>Impale one of the sensory cells or Rz cells on the ventral side of the ganglion as described above.</li>
	<li>Inject dye into the cell by passing negative current (3nA) through the electrode for 15 min. Set the pulse duration to 100 msec and the frequency to 2 Hz.</li>
	<li>Visualize the dye-filled neurons by exciting the dye with a mercury lamp and a FITC/GFP filter set.</li>
</ol>

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The authors have nothing to disclose.

The purpose of this set of experiments was 1) to teach and exhibit the fundamental principles of intracellular recordings from identifiable neurons and 2) to recognize the characteristic shapes of electrical signals that can arise from these particular neuronal types using adult leeches. There is a rich history of investigations using the leech for neurophysiological investigations and research is ongoing in addressing questions of neural circuitry and gene regulation during development as well as during synaptic repair and regeneration10. Neuromodulation, particularly through biogenic amine pathways, has also been a successful research direction in the leech. By adding dopamine and other pharmacological agents to the nervous system through the saline solution, it has been shown that dopamine modulates neural networks involved in decision-making23 and motor patterns for crawling behavior24,25. This experimental approach is a simple (and inexpensive) way to incorporate modulation into the teaching laboratory, and adjusting the ionic makeup of the saline solution is an effective way to teach the Nernst and Goldman-Hodgkin-Katz equations.
To be successful with the ganglion preparations it is important to have the ganglion taut prior to attempting to poke a soma with an intracellular electrode. Otherwise the soma will move when pushing down on the glial packet. In addition, when desheathing the ganglion for removing neurons for culture, avoid the region of interest when cutting across the ganglion so somas are not damaged as they are removed. Also, excessive incubation in the enzymes may result in the neurons being too fragile to be removed. It requires some trial and error to achieve an ideal incubation time because each batch of enzymes is slightly different in their actions.
Further investigation to optimize culture media could improve this technique. Various factors, such as the presence of insulin, sugars, or growth factors could be examined for longer maintenance of cultures. To determine if cells maintain their capacity to synthesize transmitters, such as serotonin synthesis in Rz cells, neutral red staining can be applied to label serotonergic processes. Also microglial invasion to injured and regenerating tissue can be quantified by nuclear stains days after an injury.
Although this preparation has many benefits to offer in examining regeneration and repair as well as neural circuitry, the pharmacological and molecular identification of the various receptor subtypes at synapses have not been fully characterized. An expressed sequence tag library is available26, and the medicinal leech genome is currently being assembled27, but the main limitation in leech compared to some model preparations is the ability to modify the genome for selective genes in specific cell types. In embryos this limitation has been addressed by injecting DNA or dsRNA to regulate gene expression28. The gene gun technique has recently been used to express invertebrate connexin (innexin) proteins in leech neurons. Expression of an innexin from Hirudo verbana (Hve-inx6) is sufficient for the formation of ectopic gap junctions in the medicinal leech29. The medicinal leech&#8212;or H. verbena as it were30 &#160;is sure to advance our understanding of the physiology and evolution of electrical synapses over the next few years.&#160;

The leech preparation is useful for investigating the function of identifiable (sensory, motor and inter-neurons within the animal&#39;s central nervous system (CNS). The useful property of leech neurons is that the shape of their action potentials and biophysical properties are characteristic for a given cell type (i.e. P, N, T, Rz). Moreover neurons can be removed from the animal and cultured in a suitable medium in which the cells maintain their electrical characteristics for as long as 45 days1,2.
A distinct advantage of the leech ganglion for learning how to make electrophysiological recordings is that cells are characteristically and reliably arranged in the ganglion in a pattern that allows identification of distinct cell types from ganglion to ganglion and from animal to animal. The unique location and morphology of neurons in leech ganglia were noted by Retzius as early as 18913. Knowledge of the identity and function of a neuron is advantageous for investigating neural circuits and for making experiments with a given cell type. Due to the relatively large somata of the neurons within a ganglion they are visible with a dissecting microscope, and the somata can be selectively impaled with microelectrodes for recording their electrical properties.
Dyes and large molecules can be injected into individual cells and channel properties studied by patch clamp. Ionic currents that give rise to the various shapes of the characteristic action potentials in various neurons have been identified4-7. From investigations in the pattern of innervation and branching of individual neurons within the CNS, the synaptic connections have been reproduced in culture with identified neurons2,8. Studies in the leech ganglia have provided insight into the development of functional circuits that can be measured with electrophysiology as well as with whole animal behavior studies. The leech CNS has also served as a valuable preparation for investigating mechanisms involved with neuronal repair and regeneration in the whole animal and in culture4-6,9-12.
In mammalian brains it is a daunting task to explain behavior in terms of the properties and connections of individual identified neurons. In principle one can hope that the study of less complex systems such as the leech could help to define basic mechanisms used in higher animals. The connections that are used which underlie a swim pattern13,14 and rhythmicity of the heart15 have been well characterized in the leech. The ability of some leech neurons to form both electrical and chemical communication is intriguing. Some connections are bidirectional while others are unidirectional chemically but bidirectional electrically7. Understanding the synaptic connections within the animal as well as recreating the same type of connections in a culture dish are powerful tools for investigating synaptogenesis16-18 as well as pharmacological profiling of synapses19. The intracellular recording and stimulation techniques described here provide a foundation for pharmacological assays and research in neuroethology and development.
In addition, the segmental ventral nerve cord can be dissected with a patch of innervated skin. With the ability to keep a patch of skin and the neurons alive, sensory receptive fields can be probed by recording electrical signals from the cell body (i.e. soma) of sensory neurons within respective abdominal ganglion in the CNS. Thereby one can assess the sensitivity of the sensory endings by applying varying degrees of force on the skin and map receptive fields: light touch, pressure, and noxious (painful) stimuli20,21.
An advantage of this leech ganglion-skin preparation is that one can draw anatomically the receptive field map and trace the neuronal processes to the identified neuron once the electrical responses are being recorded. The three leech experiments described below can all be completed multiple times with a single specimen, which makes this a cost effective teaching module for academic laboratories.

<table><tbody><tr><td>Sylgard</td><td>Dow Corning</td><td>182 silicone kit</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C5670</td><td></td></tr><tr><td>Tris/maleic acid</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Bleach&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td>To chloride silver wire</td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>221465</td><td>To adjust pH</td></tr><tr><td>HCl</td><td>Sigma-Aldrich</td><td>H1758</td><td>To adjust pH</td></tr><tr><td>Collagenase/Dispase</td><td>Boehringer Mannheim</td><td>269-638</td><td></td></tr><tr><td>Leibovitz L-15 with L-Glutamine</td><td>Gibco</td><td>041-01415H</td><td></td></tr><tr><td>Fetal calf serum</td><td>Ready Systems Ag</td><td></td><td></td></tr><tr><td>04-001</td><td></td><td></td><td></td></tr><tr><td>D-Glucose-Monohydrate</td><td>Merck</td><td>8342</td><td></td></tr><tr><td>Gentamycin sulfate&amp;nbsp;</td><td>any supplier</td><td></td><td>10 mg/ml</td></tr><tr><td>Glutamine</td><td>Gibco</td><td>043-0503H</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>3375</td><td>Free acid, crystalline</td></tr><tr><td>Concanavalin A Type IV</td><td>Sigma</td><td>C 2010</td><td></td></tr><tr><td>Poly-L-Lysine Hydrobromide</td><td>Sigma</td><td>P 7890</td><td>MW 15-30,000</td></tr><tr><td>&amp;nbsp;Microwell plate, 60 x 10 &amp;mu;l</td><td>Falcon</td><td>3034</td><td>Flat bottom, 10 in a bag</td></tr><tr><td>Dissecting tools</td><td>World Precision Instruments</td><td>assortment</td><td></td></tr><tr><td>Intracellular electrode probe</td><td></td><td></td><td></td></tr><tr><td>Faraday cage</td><td></td><td></td><td></td></tr><tr><td>Insect Pins</td><td>Fine Science Tools, Inc</td><td>26001-60</td><td></td></tr><tr><td>Dissecting microscope (100X)</td><td></td><td></td><td></td></tr><tr><td>Fiber optic lamp</td><td></td><td></td><td></td></tr><tr><td>Small adjustable mirror</td><td></td><td></td><td>To direct light beam toward the preparation.</td></tr><tr><td>Glass electrodes</td><td>Sigma-Aldrich</td><td>CLS7095B5X</td><td>Box of 200, Suction electrodes</td></tr><tr><td>Micromanipulator</td><td>World Precision Instruments</td><td>MD4-M3-R</td><td>&amp;nbsp;Can fix for base or on a metal rod</td></tr><tr><td>Raised preparation stand</td><td></td><td></td><td></td></tr><tr><td>Silver wire (10/1,000 inch)</td><td>A-M Systems</td><td>782500</td><td></td></tr><tr><td>Computer</td><td>any company</td><td></td><td></td></tr><tr><td>AC/DC differential amplifier</td><td>A-M Systems</td><td>Model 3000</td><td></td></tr><tr><td>PowerLab 26T</td><td><a href="http://www.adinstruments.com/" target="_blank"><img src="/img/new_window.png"> AD Instruments</a></td><td>27T</td><td></td></tr><tr><td>Head stage</td><td><a href="http://www.adinstruments.com/" target="_blank"><img src="/img/new_window.png"> AD Instruments</a></td><td></td><td>Comes with AC/DC amplifier</td></tr><tr><td>LabChart7</td><td><a href="http://www.adinstruments.com/" target="_blank"><img src="/img/new_window.png"> AD Instruments</a></td><td></td><td></td></tr><tr><td>Electrical leads</td><td>any company</td><td></td><td></td></tr><tr><td>Glass tools&amp;nbsp;</td><td>make yourself</td><td></td><td>For manipulating nerves</td></tr><tr><td>Cable and connectors</td><td>any company</td><td></td><td></td></tr><tr><td>Pipettes with bulbs&amp;nbsp;</td><td>Fisher Scientific</td><td>13-711-7</td><td>Box of 500</td></tr><tr><td>Beakers</td><td>any company</td><td></td><td></td></tr><tr><td>Wax or modeling clay</td><td>any company or local stores</td><td></td><td></td></tr><tr><td>Stimulator</td><td>Grass Instruments</td><td>SD9 or S88</td><td></td></tr><tr><td>Plastic tip for suction electrode</td><td>local hardware store (Watt&amp;rsquo;s brand)</td><td>&amp;frac14; inch OD x 0.170 inch ID</td><td>Cut in small pieces. Pull out over a flame and cut back the tip to the correct size.</td></tr></tbody></table>

intracellular recording, sensory field mapping, and culturing identified neurons in the leech, hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular recordings from sensory neurons in the leech ganglion are characterized by their distinct voltage-time traces shown in Figure 4C. The resting cell membrane potential in healthy leech ganglion cells is -45 to -60 mV. If potentials are smaller (less negative) one should change the glass electrode, or if the potential is still low, move on to another segment of the nerve cord. Spontaneous action potentials with small amplitude may indicate that the electrode is in a motor neuron. If this happens in the ganglion-body wall a current injection can be used to determine if the neuron innervates any of the muscles still attached to the skin. The annulus erector motor neuron (AE) is on the ventral side of the ganglion (Figure 4B) and it will cause the annuli (ridges or segments) to rise by activating the annulus erector muscle.
Injecting current into the sensory cells should evoke action potentials with the characteristic waveforms shown in Figure 4C. If the waveforms look different it could be due to an unbalanced bridge or offset capacitance compensation. One should consult the manual that accompanies the signal acquisition unit for details on how to balance the bridge inside a cell. Notice the stimulus artifacts at the beginning and end of the current pulses in Figure 4C. This is how the artifacts look when the bridge is balanced.
Primary cultures of neurons from the leech ganglion form electrical and chemical synapses in the dish. Initially the electrical properties of the cultured cells are not exactly the same as they are in vivo. However after a few days the impulses are nearly indistinguishable from those recorded in the ganglion. This is demonstrated in Figure 6C with a pair of Rz cells. After 7 hr in culture the amplitude of synaptic potentials was 1-2 mV. After 56 hr in culture synaptic potentials were larger than 10 mV (top traces in Figure 6C). The shape of the action potential also changes after 56hr in culture. Under the conditions described these cells are viable for several days.
Intercellular communication can be demonstrated by stimulating the nerve roots or connectives and recording from neurons in the ganglion. Stimulating these nerves generates robust synaptic potentials and action potentials in different ganglion cells. Traces in Figure 8B were recorded from a Retzius cell in a ganglion that had been removed from the animal and pinned out in a dish. The black trace was evoked by a subthreshold stimulus applied with an extracellular electrode to the connective. Increasing the stimulus voltage caused the cell to fire an action potential (red trace). Fluorescent dyes or horse radish peroxidase can be injected into these cells to study their morphology. Lucifer yellow was injected into an Rz neuron by passing a negative current through the electrode. An alternative is to inject the dye using puffs of air pressure. Figure 9A shows the dye shortly after the injection began. Thirty minutes later the dye already had diffused into the nerve roots (Figure 9B). The type of dye depends on the application, e.g. small molecular weight dyes that can pass through gap junctions are used to identify electrical synapses.
<img alt="Pinned specimen in petri dish for anatomical study; focus on external and internal features." fo:content-width="5in" src="/files/ftp_upload/50631/50631fig1.jpg" width="600px" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Ventral leech dissection. (A) The animal is thoroughly pinned ventral side up in an elastomer-lined dish filled with leech saline. (B) A shallow incision spanning the anterior: posterior axis is made just lateral to the midline to avoid damaging the nerve cord. The black blood sinus (which contains the ventral nerve cord) can easily be seen. Note the one exposed ganglion that is white compared to the sheath. <a href="https://www.jove.com/files/ftp_upload/50631/50631fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Microscope image of nerve fiber; tool needle points; neuroanatomy study." fo:content-width="5in" src="/files/ftp_upload/50631/50631fig2.jpg" width="600px" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Dissection of the black sinus. The blood vessel (arrow) should be carefully cut on one side to expose the ventral nerve cord (double arrow). Leave segments of the blood vessel intact around the segmental roots for use as a handle to manipulate the ventral nerve cord when transferring between dissection and recording dishes. <a href="https://www.jove.com/files/ftp_upload/50631/50631fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Starfish regeneration process, microscope image, studying limb growth and cellular regeneration mechanisms." fo:content-width="3in" src="/files/ftp_upload/50631/50631fig3.jpg" width="400px" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Isolated section of the ventral nerve cord. Pinning the ventral nerve cord by the blood vessel fragments remaining on the segmental roots and at the ends of the segmental connectives exposes the ganglia for electrophysiological recordings. A taut stretching of the ganglia allows one to see the identifiable cell bodies and will allow an easier penetration of the glial sheath with a sharp glass electrode. <a href="https://www.jove.com/files/ftp_upload/50631/50631fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Neural structure diagrams and action potentials; data chart depicts voltage over time." fo:content-width="5in" src="/files/ftp_upload/50631/50631fig4.jpg" width="600px" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. Cellular anatomy of the leech ganglion. (A) Ideally the Retzius cells (R) and other large cell bodies (P- pressure, T- touch, N-nociceptive, AE- annulus erector) will be clearly visible if the microscope and light condenser are set up properly. (B) Schematic view of cells in the ganglion. Slight differences in the location of the somas will occur in each ganglion due to how the ganglion is stretched and pinned taut. (C) Action potentials recorded from the soma of these neurons have characteristic waveforms. These spikes were evoked by injecting positive current into the cell (square pulse overlaying the voltage-time traces). <a href="https://www.jove.com/files/ftp_upload/50631/50631fig4highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Neuronal surgery diagram showing cutting process and intact neuron for scientific analysis." fo:content-width="5in" src="/files/ftp_upload/50631/50631fig5.jpg" width="600px" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5. Desheathing the ganglion to remove individual cell bodies. (A) The glial sheath should be carefully cut across the ganglion to avoid damaging cells of interest. The red line indicates a cut to avoid hitting the soma of the Rz neurons. Opening the sheath gives the digestive enzymes access to the connective tissue matrix. (B) Healthy cell bodies bulging out of the ganglion after the glial sheath has been opened. <a href="https://www.jove.com/files/ftp_upload/50631/50631fig5highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Neuroscience experiment microscopy; cell structure analysis and voltage recordings; data graph." fo:content-width="5in" src="/files/ftp_upload/50631/50631fig6.jpg" width="600px" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6. Culturing neurons from the leech ganglion. (A) Cells are aspirated from the ganglion using a micropipette and transferred to a dish containing culture media. (B) Placing multiple cells near one another will increase the rate of synaptogenesis between identifiable neurons. Here the stumps of two neurons have formed a synapse. (C) Neurons in culture exhibit slightly different electrical properties. The top traces show synaptic potentials evoked by stimulating the adjacent cell 7 hr and 56 hr after the cells were plated. The bottom traces were evoked by injecting current directly into one of the Rz cells. Note the increase in synaptic potential amplitude and change in the action potential waveform after 56 hr in culture. <a href="https://www.jove.com/files/ftp_upload/50631/50631fig6highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Neurophysiology experiment diagrams; nerve response to stimuli; electrophysiological recordings." fo:content-width="5in" src="/files/ftp_upload/50631/50631fig7.jpg" width="600px" data-altupdatedat="1747911167000" data-alt="Figure 7"> 
Figure 7. The leech ganglion-body wall preparation for mapping receptive fields. (A) One approach is to remove an entire section of the body wall along with 1-3 ganglia. Here the roots on one side have been cut and the ganglion has been pinned out to the side. (B) A pinned out ganglion at higher magnification. (C) Another approach is to create a small window in the body wall and record from the intact ganglia while mapping a receptive field. (D-G) Each of the sensory neurons exhibits a characteristic response to a mechanical stimulus. (D) A light touch evokes activity in T cells but not in P or N cells. (E) The T cell response shuts off in response to stronger stimuli, giving way to activation of the P cell. As the stimulus gets stronger the N cells becomes activated (F-G). <a href="https://www.jove.com/files/ftp_upload/50631/50631fig7highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Neuron microscopy and graph; action potential measurement; electrophysiology experiment; voltage vs. time chart." fo:content-width="5in" src="/files/ftp_upload/50631/50631fig8.jpg" width="600px" data-altupdatedat="1747911167000" data-alt="Figure 8"> 
Figure 8. Evoked activity in the leech ganglion. Extracellular stimulation of the connectives can be used to drive synaptic transmission in the nerve cord. (A) This is done by attaching a suction electrode to one of the connectives and applying a small voltage. The sharp glass electrode (arrow) can be seen impaling a Retzius cell in the ganglion. (B) Voltage-time traces showing the stimulus artifact followed by a synaptic potential (black) and an action potential (red). <a href="https://www.jove.com/files/ftp_upload/50631/50631fig8highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Microscopy images of green fluorescence in optical excitation study, showing cellular processes." fo:content-width="5in" src="/files/ftp_upload/50631/50631fig9.jpg" width="600px" data-altupdatedat="1747911167000" data-alt="Figure 9"> 
Figure 9. Injecting dye into leech neurons. (A) Lucifer yellow can be injected into the soma by passing current through the recording microelectrode. A mercury lamp and FITC filter set can then be used to visualize the fluorescence. (B) Dye has diffused into the Rz axons about 30 min after the injection. <a href="https://www.jove.com/files/ftp_upload/50631/50631fig9highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis at JoVE.com

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

intracellular-recording-sensory-field-mapping-culturing-identified

Research

JoVE Journal

Neuroscience

19.6K Views.  University of Kentucky. This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

Video: Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

A technique is described for implanting four in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia californica.

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB.&#160;The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi&#160;channel wire electrodes is demonstrated. Pairwise differential amplification of the micro&#160;wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long&#160;term recordings over many hours up to days, which is a clear advantage compared to conventional extra&#160;and intracellular in vivo recording techniques.

Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

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