Name: Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents (Video) | JoVE Journal
Uploaded: 2013-07-10T12:00:00.000+00:00
Duration: 9 min 40 s
Description: Learn to isolate sensory neurons from Aplysia californica for patch clamp recordings of glutamatergic currents. This method enables detailed electrophysiological analysis of voltage and agonist gated ion channels.

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5 minutes

Funded by NIH P40 OD010952, the Korein Foundation, a University of Miami Fellowship to SLC and a Maytag fellowship to ATK. The authors gratefully acknowledge the staff of the National Resource for Aplysia, as well as Lauren Simonitis and Hannah Peck, who provided micrographs for a figure.

1. Cell Preparation
<ol>
 <li>On Day 1, weigh and anesthetize animal.
 <ol>
 <li>Weigh a 30 g-1 kg animal. Anesthetize in 5-10 animal volumes of 1:1 seawater:isotonic MgCl . 6H20 for 1 hr with aeration such as an electric aquarium air pump with attached airstone.</li>
 </ol>
 </li>
 <li>Prepare dissection supplies.
 <ol>
 <li>Assemble clean dissection tray with stainless steel straight pins, such as fabric pins. Assemble several Petri dishes containing artificial seawater (ASW; see Tables 1 and 3) + penicillin/ streptomycin (P/S).</li>
 <li>Have ready a low power microscope. Have clean dissection instruments ready, such as ribbed nose and fine surgical forceps, and fine and large surgical scissors. Spray the instruments with 70% isopropol alcohol before use and allow to dry.</li>
 </ol>
 </li>
 <li>Position animal on dissection tray.
 <ol>
 <li>Position animal ventral side down in the dissection tray. Pin animal through edges of body wall and parapodial borders, but avoid tail and head, to expose the dorsal surface and the genital groove</li>
 <li>Rinse the dorsal surface in slow-running cold tap water. Apply a light stream of 70% isopropol alcohol from a squeeze bottle along the genital groove and over the top of the head.</li>
 </ol>
 </li>
 <li>Make the initial incision.
 <ol>
 <li>Using a low power microscope (see Table 2) and reflected light to observe the point where the genital groove originates from the anterior shell margin, tautly hold with ribbed-nose forceps a fold of tissue to the left of this origin, while snipping a shallow hole all the way through the smooth, flat area of body wall just to the right of the genital groove with fine angled scissors.</li>
 <li>Hemolymph should be observed to pour from the hole, sometimes carrying with it the abdominal ganglion and stomach.</li>
 </ol>
 </li>
 <li>Expand the incision.
 <ol>
 <li>Use large scissors to expand the incision anteriorly to a point between the rhinophores, while pulling upward with the lower blade to avoid nicking the gut. The internal organs will be observed to spill out of the incision.</li>
 </ol>
 </li>
 <li>Remove the ganglia.
 <ol>
 <li>Reposition the tray and refocus the microscope on the head region.</li>
 <li>Using clean forceps and fine scissors, remove the head ganglia by severing at one point the nerves that form the head ganglia into a ring around the esophagus to remove pleural-pedal and cerebral ganglia as a group.</li>
 <li>Remove the buccal ganglion that adheres to the ventral side of the esophagus.</li>
 <li>Remove the abdominal ganglion by cutting the 4 large nerve connectives that issue from this ganglion.</li>
 </ol>
 </li>
 <li>Isolate ganglia of interest.
 <ol>
 <li>Trim head ganglia apart, leaving a length of connectives attached to each ganglion of interest that is equal to at least the diameter of the ganglion; this will retard enzyme over-digestion of the cells of interest in the next step.</li>
 <li>Put each target ganglion through 2 rinses of ASW + P/S, moving between rinses with clean forceps.</li>
 <li>If targeting the PVC cells, leave the right pleural hemiganglion attached to the right pedal hemiganglion, and similarly leave the left pleural hemiganglion attached to the left pedal hemiganglion.</li>
 <li>Discard unwanted ganglia and the rest of the animal according to accepted research practice.</li>
 </ol>
 </li>
 <li>Enzymatically digest the ganglia.
 <ol>
 <li>For each ganglion, prepare 1 ml of enzyme solution consisting of 3.75 mg dispase, 1 mg hyaluronidase and 0.3 g collagenase type XI per ml of ASW + P/S in a 15 ml polypropylene conical tube.</li>
 <li>Place rinsed ganglia in enzyme solution and attach the cap tightly. Place the tube on its side on a rotary shaker (see Table 2) at slow speed for 13-5 hr overnight at approximately 23 &deg;C (room temperature).</li>
 </ol>
 </li>
 <li>Prepare the culture dishes.
 <ol>
 <li>Before microdissection (Day 2), prepare poly-D-lysine (PDL)-coated culture dishes in a tissue culture hood. Add ~0.5 ml PDL (0.2 mg/ml of sterile water) to the center of a 35 mm dish for ~25 min.</li>
 <li>Rinse PDL 3x with sterile water. Allow to dry. UV-sterilize the plates.</li>
 </ol>
 </li>
 <li>Day 2 Isolate neurons.
 <ol>
 <li>Fill the center of 35 mm dishes that were PDL-coated and UV sterilized with approximately 0.5 ml of ASW + P/S to create an island of media inside the otherwise dry dish. This can be done on the bench, outside the tissue culture hood. One dish should be prepared for each cell cluster originating from a hemiganglion.</li>
 <li>Have ready a 100 mm diameter dissection dish made by sylgard-coating and curing a 5 mm deep dissection surface, outfitted with fine dissection pins of several sizes to be used as pins and probes (see Table 3). Rinse this dish with 70% isopropol alcohol, then with ASW + P/S, and finally fill it with ASW + P/S.</li>
 </ol>
 </li>
 <li>Prepare microdissection of digested ganglia.
 <ol>
 <li>Pour the enzyme solution containing the digested pleural-pedal and buccal ganglia into the dissection dish.</li>
 <li>Place the dish on the stage of the microscope against a black surface under reflected light.</li>
 <li>Using the attached connective tissue, pin down each pleural-pedal hemiganglion dorsal side up. The tissues will be soft and intolerant of stretching.</li>
 </ol>
 </li>
 <li>Microdissect PVC neurons.
 <ol>
 <li>Using 2 pairs of clean fine forceps, and holding a fine dissection pin in one pair of forceps as a probe, isolate the PVC cells from a pleural hemiganglion. Enzyme digestion will have removed or broken apart the connective tissue sheath covering the PVC cluster, yet the cells will adhere to one another.</li>
 <li>Use the probe to detach these cells from the pedal-pleural connective18, then let the cell cluster fall to the bottom of the dissection dish.</li>
 </ol>
 </li>
 <li>Transfer neurons to culture dish.
 <ol>
 <li>Transfer the cell cluster to a prepared 35 mm culture dish by gently sucking up the cluster into the tip of a slightly fire polished disposable glass Pasteur pipette, then dispensing it slowly into the media island in a culture dish, being careful to avoid introducing air bubbles into the pipette. Repeat isolation of the PVC cluster of the other hemiganglion and place into a separate culture dish.</li>
 </ol>
 </li>
 <li>Microdissect and transfer BSC neurons.
 <ol>
 <li>Pin out the buccal ganglion ventral side up, with care to spare the cells of interest. Repeat step 1.12 with the 2 BSC cell clusters of the buccal ganglion10.</li>
 <li>Isolation of PVC and BSC neurons will produce 4 culture dishes containing neuron clusters: 2 dishes containing BSC clusters and 2 dishes containing PVC clusters. Discard the rest of the ganglia and set aside the 100 mm dissection dish.</li>
 </ol>
 </li>
 <li>Dissociate the cell clusters.
 <ol>
 <li>Place a culture dish containing cells on the stage of the low power microscope and remove the cover.</li>
 <li>Dissociate the PVC cluster by gently flicking the bottom of the culture dish adjacent to the cell cluster with a fine dissection pin held in forceps. Flicking is digging the tip of the pin into the plastic of the bottom of the dish as though trying to dig out a fleck of plastic. When friction with the plastic releases the pin point, a percussion wave is produced through the medium that breaks apart the nearby clump of cells.</li>
 <li>5-10 flicks may be needed to nearly completely dissociate the cells, but it is better to leave small clusters of cells rather than to flick too much lest the cells be destroyed by mechanical sheer.</li>
 <li>Replace cover and repeat with other culture dishes.</li>
 </ol>
 </li>
 <li>Store cell cultures.
 <ol>
 <li>Place the culture dishes containing media islands in their centers with dissociated cells into a large container that permits air circulation, such as a 150 x 25 mm round plastic Petri dish, and place this dish in an incubator set to 17 &deg;C.</li>
 <li>Install in the chamber an open dish of water as a source of humidity. Leave undisturbed overnight. The cells will adhere to the poly-D-lysine coating in the center of the dish.</li>
 </ol>
 </li>
 <li>Flood culture dishes.
 <ol>
 <li>On Day 3, Gently flood the dishes with 2.5 ml of ASW + P/S. Examine and count the cells using an inverted cell culture microscope. Store in the 17 &deg;C incubator.</li>
 </ol>
 </li>
</ol>
2. Electrophysiology
The method is standard patch clamping that has been described in numerous texts (e.g. Sakmann and Neher, 1995)16. This protocol will work on cells &le;100 pF capacitance, or cells &lt;60 &mu;m diameter during days 3 and 4 of the protocol. Cells without processes are optimal for recording. The following special considerations apply:
<ol>
 <li>Larger cells can be accommodated with the configuration setting on the Axopatch 200B amplifier set to &beta;=0.1. Activation of very large currents may cause cells to escape voltage clamp. ASW solutions substituted for salt content (e.g. a fraction of the NaCl substituted with impermeable N-methy-D-glucamine chloride) may be used to reduce whole cell current amplitude.</li>
 <li>Fiber-filled borosilicate patch pipettes pulled on a pipette puller and backfilled halfway with intracellular solution (ICS) consisting of 450 mM KCl and other salts (see Table 1) are suitable, and should have resistance of approximately 0.5-1 MOhms. If isolation of specific ionic currents is desired, for example, Na+ currents in the absence of K currents, K+ in this solution can be replaced with other ions such as Cs+. Cl- replacement of ICS with an impermeant anion is also warranted if a more natural ICS is desired9.</li>
 <li>The cells are robust with &gt;1 hr-long recordings possible at room temperature. Recording is optimal on days 3 and 4 of the protocol, corresponding to 24-48 hr in culture. After 48 hr there is difficulty forming gigaOhm seals, perhaps because of elaboration of a glycocalyx on the cell surface, and space clamp problems caused by process outgrowth. The cells are useful, however, for non-patch clamp studies, such as toxicology, that can be completed in &lt;14 d.</li>
 <li>ASW and other solutions to be dispensed from the gravity fed solution device, as well as intracellular solution, should be filtered through a syringe-mounted 0.2 &mu;m Acrodisk filter (Table 3) to eliminate fine particles that interfere with gigaOhm seal formation.</li>
 <li>Desensitization occurs when using agonists such as D-Aspartate (D-Asp), therefore ~1-2 min between applications of agonists is recommended. Conversely, potentiation is often observed with rapid repeated application of agonists such as L-Glutamate (L-Glu).</li>
 <li>Agonist activated currents such as L-Glu can be studied by applying agonists with the picospritzer pipette. This pipette is pulled the same as the patch pipette and contains agonist in ASW. When the tip of this pipette is positioned below the outflow pipe of the gravity fed solution device, and at a 45&deg; angle from the outflow pipe (Figure 2F), agonist will be quickly washed away from the cell, and desensitization will be minimized. The agonist pipette should create an angle of 90&deg; or more relative to the patch pipette.</li>
</ol>

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<li>Glansman, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Habituation+in+Aplysia%3A+The+Cheshire+Cat+of+neurobiology%5BTitle%5D)+AND+%22Neurobiol.+Learn.+Mem%22%5BJournal%5D)">Habituation in Aplysia: The Cheshire Cat of neurobiology.</a> Neurobiol. Learn. Mem. 92, 147-154 (2009).</li>
<li>Hawkins, R. D., Abrams, T. W., Carew, T. J., Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Cellular+Mechanism+of+Classical+Conditioning+in+Aplysia%3A+Activity-Dependent+Amplification+of+Presynaptic+Facilitation%5BTitle%5D)+AND+%22Science%2C+New%22%5BJournal%5D)">A Cellular Mechanism of Classical Conditioning in Aplysia: Activity-Dependent Amplification of Presynaptic Facilitation.</a> Science, New. 219 (4583), 400-405 (1983).</li>
<li>Ichinose, M., Sawada, M., Maeno, T., McAdoo, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+acetylcholine+on+ventrocaudal+sensory+neurons+in+the+pleural+ganglia+of+Aplysia%5BTitle%5D)+AND+%22Cell+Mol.+Neurobiol%22%5BJournal%5D)">Effect of acetylcholine on ventrocaudal sensory neurons in the pleural ganglia of Aplysia.</a> Cell Mol. Neurobiol. 9, 233-245 (1989).</li>
<li>Kandel, E. R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKandel%2C+ER+%22Cellular+basis+of+behavior%22">Cellular basis of behavior</a>. , W.H. Freeman. New York. (1976).</li>
<li>Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+molecular+biology+of+memory+storage%3A+a+dialog+between+nerves+and+synapses%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The molecular biology of memory storage: a dialog between nerves and synapses.</a> Science. 294 (5544), 1030-1038 (2001).</li>
<li>Kupfermann, I., Castellucci, V., Pinsker, H., Kandel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuronal+correlates+of+habituation+and+habituation+of+the+gill-withdrawal+reflex+in+Aplysia%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Neuronal correlates of habituation and habituation of the gill-withdrawal reflex in Aplysia.</a> Science. 167 (3926), 1743-1745 (1970).</li>
<li>Magoski, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+an+Aplysia+bag-cell+neuron+cation+channel+by+closely+associated+protein+kinase+A+and+a+protein+phosphatase%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Regulation of an Aplysia bag-cell neuron cation channel by closely associated protein kinase A and a protein phosphatase.</a> J. Neurosci. 24 (30), 6833-6841 (2004).</li>
<li>R, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuronal+transcriptome+of+Aplysia%3A+neuronal+compartments+and+circuitry%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Neuronal transcriptome of Aplysia: neuronal compartments and circuitry.</a> Cell. 127, 1453-1467 (2006).</li>
<li>Nick, T. A., Kaczmarek, L. K., Carew, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ionic+currents+underlying+developmental+regulation+of+repetitive+firing+in+Aplysia+bag+cell+neurons%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Ionic currents underlying developmental regulation of repetitive firing in Aplysia bag cell neurons.</a> J. Neurosci. 16 (23), 7583-7598 (1996).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Single-channel+recording%22">Single-channel recording</a>. Sakmann, B., Neher, E. , Plenum Press. NY. (1995).</li>
<li>Tam, A. K., Geiger, J. E., Hung, A. Y., Groten, C. J., Magoski, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Persistent+Ca2%2B+current+contributes+to+a+prolonged+depolarization+in+Aplysia+bag+cell+neurons%5BTitle%5D)+AND+%22J.+Neurophysiol%22%5BJournal%5D)">Persistent Ca2+ current contributes to a prolonged depolarization in Aplysia bag cell neurons.</a> J. Neurophysiol. 102 (6), 3753-3765 (2009).</li>
<li>Schacher, S., Proshansky, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurite+regeneration+by+Aplysia+neurons+in+dissociated+cell+culture%3A+modulation+by+Aplysia+hemolymph+and+the+presence+of+the+initial+axonal+segment%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Neurite regeneration by Aplysia neurons in dissociated cell culture: modulation by Aplysia hemolymph and the presence of the initial axonal segment.</a> J. Neurosci. 3 (12), 2403-2413 (1983).</li>
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</ol>

The authors declare that they have no competing financial interests.

The dissociation techniques described here yield sensory neuron cultures containing 50-100 isolated neurons interspersed with small numbers of glia and other unidentified cells. The most critical steps in the protocol are the time the ganglia remain in enzyme solution, and flicking, the dissociation of the digested cell clusters to break apart the cluster into individual cells. Enzyme digestion (step 1.8) must be optimized at the available temperature. At 23 &deg;C with slow shaking, 13 hr is sufficient for digest...

The marine opistobranch mollusk, Aplysia, has been a useful neurobiological model for many decades. It is best known as a model of habituation and classical conditioning 7, 8. Studies on learning and memory in this model won the Nobel Prize for Physiology or Medicine in 2000 for Eric R. Kandel, in a prize he shared with Arvid Carlsson and Paul Greengard 10. Studies involving electrical recordings from reduced preparations, in which elements of the nervous system of this invertebrate are dissected from the animal with nerves and muscles left attached, have helped elucidate the roles of individual neurons in Aplysia. Identification of precise molecular mechanisms that constitute learning in Aplysia however, often employed another technique, long-term co-cultures of a sensory neuron and a motoneuron, obtained one by one from individual donor animals and allowed to form a synapse in the culture dish 21.
We and others 1, 3, 6, 14, 15, 16 have exploited the ease with which identified neurons can be targeted in this model as well as their endurance in long-term experiments to make dissociated short term cultures of clusters of nominally homogeneous neurons in which we study ionic currents under voltage clamp in the patch clamp configuration. Many Aplysia neurons stand up to repeated rounds of patch clamping to allow time for long-lasting experimental manipulations. The technique is useful for neurons such as the neurosecretory bag cells of the abdominal ganglion, and the sensory neurons of the pleural and buccal ganglia whose dissociation we describe here, but not for very large neurons &gt;60 &mu;m diameter, such as L7 or R2 of the abdominal ganglion. We do not employ Aplysia serum in our cultures, unlike the sensory-motoneuron co-cultures described elsewhere. Most neurons obtained using this procedure will be without processes for the first 48 hr in culture, facilitating whole cell voltage recording, but will then sprout and elaborate axons and other processes for approximately 14 days before dying from lack of nutrients and/or growth factors.
This technique produces primary cultures of 50-100 neurons per dish from physiologically documented regions of the buccal and pleural ganglia. This protocol is useful for researchers studying aspects of single cell physiology in experiments that require numerous experimental replicates per animal. It produces a matched pair of cultures due to the anatomical separation of the target cells into left and right hemiganglia, permitting studies that benefit from matched treatment and control cultures.
The protocol targets buccal S cluster (BSC) neurons of the buccal ganglion, and pleural ventrocaudal (PVC) neurons of the pleural ganglion. These cells are an appropriate size for whole cell voltage recordings and display robust glutamatergic responses. The discussed methodology is appropriate for most ganglia in the Aplysia nervous system.

<table><tbody><tr><td>Artificial seawater ASW</td><td>Sigma-Aldrich</td><td>assorted</td><td>(mM): 417 NaCl, 10 KCl, 10 CaCl&lt;sub&gt;2&lt;/sub&gt; (2 H&lt;sub&gt;2&lt;/sub&gt;O), 5MgCl&lt;sub&gt;2&lt;/sub&gt; (6H&lt;sub&gt;2&lt;/sub&gt;O), 15 HEPES-NaOH, pH 7.6</td></tr><tr><td>Intracellular solution</td><td>Sigma</td><td>assorted</td><td>(mM): 450 KCl, 2.9 CaCl&lt;sub&gt;2&lt;/sub&gt; (2 H&lt;sub&gt;2&lt;/sub&gt;O), 2.5 MgCl&lt;sub&gt;2&lt;/sub&gt; (6 H&lt;sub&gt;2&lt;/sub&gt;O), 5 Na&lt;sub&gt;2&lt;/sub&gt;ATP, 10 EGTA, and 40 HEPES-KOH, pH 7.4</td></tr><tr><td>Poly-D-lysine</td><td>Sigma</td><td>P6407</td><td>&amp;nbsp;</td></tr><tr><td>penicillin/streptomycin added to ASW at 1:100</td><td>Lonzo Walkersville, Inc.</td><td>17-603E</td><td>5,000 Units/ml penicillin plus 5,000 mg/ml streptomycin</td></tr><tr><td>Neutral dispase II</td><td>Roche Diagnostics</td><td>10165859001</td><td>&amp;nbsp;</td></tr><tr><td>hyaluronidase</td><td>Sigma-Aldrich</td><td>H4272</td><td>&amp;nbsp;</td></tr><tr><td>collagenase type XI</td><td>Sigma-Aldrich</td><td>C9407</td><td>&amp;nbsp;</td></tr><tr><td>L-Glutamate (L-Glu)</td><td>Sigma-Aldrich</td><td>49601-100G</td><td>&amp;nbsp;</td></tr><tr><td>D-Aspartate (D-Asp)</td><td>Sigma-Aldrich</td><td>11200-10G</td><td>&amp;nbsp;</td></tr><tr><td>N-methyl-D-aspartate (NMDA)</td><td>Biomol</td><td>100002-268</td><td>&amp;nbsp;</td></tr><tr><td>L-Asp</td><td>Sigma</td><td>A6683-25G</td><td>&amp;nbsp;</td></tr><tr><td>alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA)</td><td>Sigma</td><td>A6816-5MG</td><td>&amp;nbsp;</td></tr><tr><td>L-Glu R antagonists</td><td>various</td><td>various</td><td>&amp;nbsp;</td></tr><tr><td>agar</td><td></td><td></td><td>&amp;nbsp;</td></tr><tr><td>kynurenate</td><td>Sigma-Aldrich</td><td>61250</td><td>&amp;nbsp;</td></tr><tr><td>APV</td><td>Sigma-Aldrich</td><td>A5282</td><td>&amp;nbsp;</td></tr><tr><td>DL-2-Amino-5-phosphonopentanoic acid (NMDAR antagonist)</td><td></td><td></td><td>&amp;nbsp;</td></tr><tr><td>2-propanol</td><td>VWRSP</td><td>BDH1133</td><td>&amp;nbsp;</td></tr><tr><td>Chloriding solution</td><td>Sigma</td><td>assorted</td><td>25 g FeCl&lt;sub&gt;3&lt;/sub&gt; + 25 ml concentrated HCl + 50 ml H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Sylgard silicone 2-part polymer</td><td>World Precision Instruments (WPI)</td><td>SYL184</td><td>Provides pin-out surface for small dissection dishes</td></tr><tr><td>0-40x zoom magnification microscope for dissections</td><td>Wild</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Techniquip 150 Watts Fiber Optic Illuminator</td><td>Microoptics of Florida</td><td>TQ FOI-150</td><td>&amp;nbsp;</td></tr><tr><td>RotoMix 50800 orbital mixer</td><td></td><td></td><td>&amp;nbsp;</td></tr><tr><td>Nikon Diaphot inverted phase-contrast microscope with 4x, 20x (optional) &amp;amp; 40x objectives</td><td>SR Research Ltd.</td><td>Eyelink II</td><td>&amp;nbsp;</td></tr><tr><td>Tektronix digital oscilloscope</td><td>SR Research Ltd.</td><td></td><td>&amp;nbsp;</td></tr><tr><td>pClamp 10 data acquisition and analysis software</td><td>Molecular Devices</td><td></td><td>&amp;nbsp;</td></tr><tr><td>PC with Windows XP or higher operating system</td><td>PC Solutions</td><td></td><td>Thinkserver with solid state hard drives (80GB) and low noise monitors</td></tr><tr><td>Flaming/Brown P87 micropipette puller</td><td>Sutter Instruments, Novato, CA</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Axon Instruments Axopatch 200B clamp amplifier with a capacitance compensation range of 1-1000 pF; preamplifier</td><td>Molecular Devices, Sunnyvale, CA</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Axon instruments electrode holder assembly for Axopatch 200B preamplifier</td><td>Molecular Devices, Sunnyvale, CA</td><td>CV203BU</td><td>&amp;nbsp;</td></tr><tr><td>Digidata 1200 A/D converter</td><td>Molecular Devices, Sunnyvale, CA</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Picospritzer, powered by N2 adjustable for pressure and duration</td><td>Parker Hannifin, Cleveland, OH</td><td></td><td>&amp;nbsp;</td></tr><tr><td>TMC Micro-G Vibration isolation table</td><td>Ametek</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Faraday cage</td><td></td><td></td><td>custom manufacture</td></tr><tr><td>Burleigh Piezoelectric Clamshell Micromanipulators</td><td>Burleigh Instruments; Thorlabs</td><td></td><td>presently PCS-5000; -6000 series + mounts</td></tr><tr><td>Narishige M-152 manual manipulators (for perfusion system and picospritzer)</td><td>Narishige USA</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Filament pipette glass,1.5 mm OD, 0.84 mm ID -</td><td>WPI</td><td>1B150-3</td><td>&amp;nbsp;</td></tr><tr><td>3 inch length</td><td></td><td></td><td>&amp;nbsp;</td></tr><tr><td>Ag/AgCl half cell</td><td>WPI</td><td>EP4</td><td>&amp;nbsp;</td></tr><tr><td>15 ml centrifuge tubes, 35-2097 BD Falcon* Centrifuge Tubes</td><td>VWRSP</td><td>21008-918</td><td>&amp;nbsp;</td></tr><tr><td>Angled Scissors</td><td>Fine Science Tools</td><td>15006-09</td><td>&amp;nbsp;</td></tr><tr><td>Dumostar Fine forceps</td><td>Fine Science Tools</td><td>11295-00</td><td>&amp;nbsp;</td></tr><tr><td>35 mm falcon tissue culture dishes</td><td>VWRSP</td><td>25382-064</td><td>&amp;nbsp;</td></tr><tr><td>falcon 150 x 25 mm tissue culture dishes; 1013</td><td>VWRSP</td><td>1013</td><td>also can be made into small dissection dishes with sylgard</td></tr><tr><td>sylgard</td><td>WPI</td><td>SYL184</td><td>&amp;nbsp;</td></tr><tr><td>animal dissection tray</td><td>various</td><td></td><td>&amp;nbsp;</td></tr><tr><td>15 ml centrifuge tubes, 35-2097 BD Falcon</td><td>VWRSP</td><td>21008-918</td><td>For 6-bore gravity-fed perfusion system</td></tr><tr><td>Aluminum clips with screw hole ends</td><td>hardware store</td><td></td><td>For perfusion system</td></tr><tr><td>23 gauge needles (manually file off points)</td><td>VWRSP</td><td></td><td>For perfusion system</td></tr><tr><td>Polyethylene tubing 0.022&quot;ID x 0.042&quot;OD; 427411</td><td>Becton-Dickinson</td><td></td><td>For perfusion system</td></tr><tr><td>H-7 pipette stand/holder for microcap perfusion array</td><td>Narishige USA</td><td></td><td>For perfusion system</td></tr><tr><td>one-way valves</td><td></td><td></td><td>For perfusion system</td></tr><tr><td>Drummond Microcaps 1 &amp;mu;l</td><td>VWRSP</td><td></td><td>For perfusion system</td></tr><tr><td>18 gauge needles for suction (filed off points)</td><td></td><td></td><td>&amp;nbsp;</td></tr><tr><td>Polyethylene tubing</td><td>Cole Parmer</td><td>4.27436E+11</td><td>&amp;nbsp;</td></tr><tr><td>fine dissection pins</td><td>Fine Science Tools</td><td>26002-20</td><td>&amp;nbsp;</td></tr><tr><td>capillary tubes</td><td>Kimble 71900-100</td><td></td><td>fire-polished and U-shaped in a Bunsen burner flame and filled with 3% agar in ECS</td></tr><tr><td>modeling clay</td><td>craft store</td><td></td><td>&amp;nbsp;</td></tr><tr><td>dish holder for microscope stage with isolated ground bath</td><td></td><td></td><td>Custom manufacture</td></tr><tr><td>pasteur pipettes</td><td>VWRSP</td><td>14672-412</td><td>&amp;nbsp;</td></tr><tr><td>pipette bulbs</td><td>VWRSP</td><td>53283-911</td><td>&amp;nbsp;</td></tr><tr><td>acrodisk syringe filters</td><td>VWRSP</td><td>28144-040</td><td>&amp;nbsp;</td></tr><tr><td>thick-walled 1.5 mm diameter borosilicate filament glass</td><td>WPI</td><td>1B150F-3</td><td>&amp;nbsp;</td></tr><tr><td>High purity nitrogen cylinder and bifurcating regulator</td><td></td><td></td><td>&amp;nbsp;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;&lt;strong&gt;Tables 1-3. Lists of Reagents, Materials, and Equipment.&lt;/strong&gt;&lt;/p&gt;</td></tr></tbody></table>

isolation of sensory neurons of aplysia californica for patch clamp recordings of glutamatergic currents

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.

The locations of the sensory neurons within the ganglia that are targeted in this protocol, the BSC and PVC neurons are shown in Figure 1. The BSC neurons are located in 2 symmetrical oval clusters on the ventral side of the buccal ganglion, the surface that faces away from the buccal mass in the intact ganglion (Figure 1A). The PVC neurons form bilateral, V-shaped clusters that wrap around the dorsal surface of the pleural ganglion toward the central axis (Figure 1B). T...

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 ...

The overall goal of this procedure is to isolate individual sensory neurons of plegia in short-term culture. For patch clamp studies, this is accomplished by first dissecting the ganglia containing sensory cells from an anesthetized animal. The second step is to digest the ganglia in enzyme solution overnight.Next, the sensory neurons of interest are dissected from the digested ganglia. The final step is the dissociation of the prepared culture dishes for adhering to the culture substrate overnight. Ultimately, voltage clamping is used to study the voltage and agonist gated ion currents expressed in these neurons.The main advantage of this technique over existing methods, such as in situ cell recording, is that isolating the neurons allows the use of a single electrode voltage clamping technique. This method can help identify key unknowns in the field of aple neurobiology, such as the identity of ion currents activated by neurotransmitters implicated in learning and memory. Generally, individuals new to this method will struggle due to the sensitivity of the neurons to enzymatic digestion and dispersal onto culture dishes.We first had the idea for this method when we realized that certain glutamatergic currents important in learning and memory would benefit from the cell isolation that this method provides. Demonstrating the procedure will be graduate students, Andy Kesel and Justin Greer. Begin this procedure by placing an anesthetized animal ventral side down in the dissection tray.Pin the animal through the edges of the body wall and para poal borders, but avoid the tail and head to expose the dorsal surface of the genital groove. Next, rinse the dorsal surface in slow running cold tap water. Then apply a light stream of 70%isopropyl alcohol from a squeeze bottle along the genital groove and over the top of the head.To make the initial incision tautly hold a fold of tissue to the left of the base of the genital groove with the rib nose forceps while snipping a shallow hole all the way through the smooth flat area of body wall just to the right of the genital groove with a pair of fine angled scissors. Then expand the incision anteriorly to a point between the ROF fours while pulling upward with the lower blade to avoid nicking the gut. Next, use clean forceps and fine scissors to remove the head ganglia by severing the point where the nerves forming the head ganglia turn into a ring around the esophagus in order to remove pleural petal and cerebral ganglia as a group.After that, remove the buccal ganglion that adheres to the ventral side of the esophagus. To isolate the ganglia of interest, trim the head ganglia apart, leaving a length of connective attached to each ganglion of interest that is equal to at least the diameter of the ganglion. This will retard the enzyme from over digesting the cells of interest in the next step.Then rinse each target ganglion twice in dishes of artificial sea water with penicillin streptomycin. If targeting the PVC cells, leave the right pleural hemi ganglion attached to the right petal hemi ganglion, and similarly, leave the left plural hemi ganglion attached to the left petal hemi ganglion. Then place the rinsed ganglia in enzyme solution and attach the cap tightly.Subsequently, place the tube on its side on a rotary shaker and set at slow speed for 13 to 15 hours overnight at approximately 23 degrees Celsius. In this procedure, fill the center of the 35 millimeter dishes that were PDL coated and UV sterilized with approximately 0.5 millimeters of artificial seawater and penicillin streptomycin to create an island of media inside. To prepare the microdissection of digested ganglia, pour the enzyme solution containing the digested pleural pedal and buccal ganglia into the dissection dish.Next, place the dish on the stage of the microscope against a black surface under the reflected light. Then using the attached connective tissue pinned down each pleural petal hemi ganglion dorsal side up the tissues will be soft and intolerant of stretching. Then isolate the PVC cells from a pleural hemi ganglion using two pairs of forceps while holding a fine dissection pin with one of them as a probe.Use the probe to detach these cells from the petal, plural, connective. Then let the cell cluster fall to the bottom of the dissection dish. Transfer the cell cluster to a prepared 35 millimeter culture dish by gently sucking up the cluster into the tip of a slightly fire polished disposable glass pasture pipette.Then dispense it slowly into the media island in a culture dish, being careful to avoid introducing air bubbles into the pipette. Repeat the isolation of the PVC cluster of the other hemi ganglion and place it into a separate culture dish. Here is a photo micrograph of the V-shaped dark orange PVC cell cluster of the right pleural hemi ganglion.To dissect the BSC neurons. First pin out the buccal ganglion with the ventral side up with care to spare. The cells of interest subsequently isolate the BSC cells using two pairs of forceps while holding a fine dissection pin with one of them as a probe.Next, use the probe to detach these cells from the buccal ganglion. Then let the cells fall to the bottom of the dissection dish. This photo micrograph shows the position of the glutamatergic cells of the buccal ganglion.The BSC neurons are identifiable by their dark orange color. The isolation of PVC and BSC neurons produces four culture dishes containing neuron clusters. Two dishes containing BSC clusters and two dishes containing PVC clusters.Discard the rest of the ganglia and set aside the 100 millimeter dissection dish to dissociate the cell clusters. Place a culture dish containing cells on the stage of the low power microscope and remove the cover. Gently flick the bottom of the culture dish adjacent to the cell cluster.With a fine dissection pin held in forceps five to 10 flicks may be needed to completely dissociate the cells. Then repeat with other culture dishes. Next, place the culture dishes containing media islands with the dissociated cells in a large container that permits air circulation, such as a 150 by 25 millimeter round plastic Petri dish.And place this dish in an incubator set to 17 degrees Celsius. Afterward, place an open dish of water as a source of humidity in the chamber and leave them undisturbed overnight to allow the cells to adhere to the polylysine coating in the center of the dish. On the next day, gently flood the dishes with 2.5 milliliters of artificial seawater and penicillin streptomycin, examine and count the cells using an inverted cell culture microscope.Here are live BSC neurons after one day in culture, the BSC cells are generally larger than PVC cells at 40 microns. Shown here is a live PVC neuron after one week in culture showing process outgrowth. Here is an excitatory current carried by sodium ions in response to a pulse of AL glutamate, and here is a current in response to D aspartate.The currents activated by NMDA are generally smaller in amplitude than those activated by the other agonists. While attempting this procedure, it's important to remember to not damage the neurons during dissociation. One advantage of this technique is that it creates two cultures from each ganglion, so that one may be used as a match control to the experimental culture.This technique can be used to create primary cultures of neurons from plegia or other marine invertebrates. After watching this video, you should have a good understanding of how to isolate sensory neurons of plegia by enzymatic digestion of the ganglia, followed by careful dissociation of the cells of interest.

isolation-sensory-neurons-aplysia-californica-for-patch-clamp

Research

JoVE Journal

Neuroscience

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro.  This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

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

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

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia.  We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We describe a technique to extracellularly record and stimulate from nerves, muscles, and individual identified neurons in vitro while eliciting and observing different types of feeding behaviors in the feeding apparatus of Aplysia.

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

This video demonstrates the procedure to map synaptic connections by securing a brain slice, positioning pipettes to achieve whole-cell configurations, and stimulating presynaptic neurons to record postsynaptic responses, thereby elucidating neuronal connectivity.

Paired Patch Clamp Recording of the Neuronal Connections in Acute Brain Slices

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.

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

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Learn to isolate sensory neurons from Aplysia californica for patch clamp recordings of glutamatergic currents. This method enables detailed electrophysiological analysis of voltage and agonist gated ion channels.

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

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