Work in the lab involving the culturing of Aplysia neurons is funded by list NIH R01 and NIH R21MH077921.

Preparation (see solutions section at end of protocol for composition of solutions)
<ol>
<li>Prepare culture dishes. Coat glass well of Mattek glass-bottom culture dish with poly-l-lysine (made in sodium borate). Add enough to completely cover the glass well and leave for &gt; 1hr (can be left overnight). Thoroughly remove the poly-l-lysine by rinsing in artificial seawater (ASW) 4-5 times. After removing the last rinse, add 2 mls of 50% L15 (supplemented with salts and containing L-glutamine at a final concentration of 2mM)/50% hemolymph. This culture medium must be in dish for at least one hour prior to plating cells so that the hemolymph coats the dish. </li>
<li>Clean the tools and Sylgard dishes with ethanol, rinse them thoroughly with ddH20 and then place them for &gt;1 hr under a UV light in the culture hood. </li>
<li>Prepare sharp electrodes for microdissection of neurons. Using a microelectrode puller (e.g. Sutter Flaming Brown P-97), pull glass pipette electrodes with 1.5 mm outer diameter, 0.86 to 1.12 mm inner diameter and 100 mm length (e.g. AM system catalog #628000; World Precision Instruments TW150-4, 1.55/1.12). Use parameters that produce an electrode with a long and wispy fine tip of such high resistance that there is no capillary intake of fluid when placed in solution. The presence of a fluid meniscus means that the electrode tip will damage neuron during isolation. The Sutter electrode cookbook provides excellent guidelines for setting up electrode programs. We use a box filament, and a one-step pull to generate culture electrodes. </li>
<li>Prepare anesthetic solution (0.35M MgCl2), culture medium (L15 supplemented with salts and hemolymph), and protease solution (1% protease type IX, Sigma, 1 unit/mg, to a final concentration of 10 units/ml). </li>
<li>Animals: use adult 80-100 g Aplysia for isolation of sensory neurons and of LFS motor neurons. Use juvenile 1-4 g Aplysia for isolation of L7 motor neurons. Remove the animals gently from the seawater tanks, and maintain in seawater (in beaker, bucket or plastic bag) for no more than 30 minutes before beginning anesthesia and culture procedure.</li>
</ol>
Culture procedure
A. Culturing Pleural Sensory Neurons from 80-100 g Aplysia
<ol>
<li>Anesthetize the animals for removal of ganglia: Inject 0.35 M MgCl2 into adult Aplysia (80-100 g) using a 60 ml syringe with an 18 gauge 1.5 inch needle. Enter the foot of the animal at an angle of about 35 degrees, and do not enter too deeply. The goal is to inject into the hemocoel of the animal without penetrating the internal organs. The animal should become very distended and relaxed.</li>
<li>Dissect the ganglia: Pin the anesthetized animal on a dissecting dish, with a pin (18-gauge needles work well for 80-100 g animals) at the head and the tail, and the foot facing up. Hold the foot with a toothed forceps and cut through the skin and underlying connective tissue the full length of the foot (from head to tail) with a surgical scissors. Pin the two sides down. Using a forceps and scissors, cut the esophagus and pull to the side to expose the ganglia. Using a fine forceps and fine scissors, cut out the pleural pedal ganglia (the sensory neurons are in the pleural ganglia). Leave a fair amount of nerve since this makes it easier to pin down the ganglia after protease treatment.</li>
<li>Protease digestion of ganglia: Place the ganglia in protease solution (10 mg/ml of 1 unit/mg in L15) in an incubator set at 34.5&deg;C (34-35&deg;C is okay) for 2 hrs and 15min. Using the fine tip forceps to transfer and wash the ganglia in ASW three times. Keep the ganglia in L15. Note that the protease incubation time can vary. The purpose is to allow the connective tissue to be easily removed. If it is too difficult to desheath the ganglia without damaging the neurons, increase the protease incubation time. If it is extremely easy to desheath the ganglia, and the neurons are &ldquo;soft,&rdquo; reduce the protease incubation time.</li>
<li>Desheathing the ganglia: Transfer the protease-digested ganglia to a 60 mm or 35mm dish containing sylgard and L15. Viewing under a stereo-microscope (with external halogen illumination), remove pedal ganglia and pin down the pleural ganglia in the Sylgard dish in the proper orientation using insect pins and a fine forceps. With the fine forceps and a Vanna surgical scissors, carefully lift the connective tissue and cut it to expose the sensory cluster. Be careful not to ever touch or damage the neuronal somata. Remove any excess connective tissue from the dish and add more medium, making sure to never expose the desheathed ganglion to air. &nbsp;The sensory cluster consists of approximately 200 clustered neurons of 40-50 micron diameter. Set a pin to make a &ldquo;post&rdquo; at a short distance from the ganglion (within the field of view).</li>
<li>Isolation of neurons: Using long, sharp glass culture electrode, pull out the identified neurons one by one by touching the cell body just off center (do not impale) and slowly and steadily pulling the cell soma, together with the axon, away from the ganglion. Gently tap against the pin to dislodge the neurons from the electrode. </li>
<li>Plating neurons: Use a pipetman (P10) to transfer individual neurons from the Sylgard dish to a culture dish. Before transferring the neurons, pipet culture medium into the tip so that the plastic tip is coated with hemolymph (this prevents the neurons from sticking to the plastic). Take great care to avoid exposing the neurons to any air bubbles or to any extreme forces (i.e. very gently take up and remove the neurons from the pipetteman). Distribute the neurons evenly throughout the dish. Use a sharp electrode to gently straight the processes and tap them down to the bottom. </li>
<li>Incubation: Leave the dishes on microscope stage at room temperature for no less than 3 hours. We routinely leave them overnight. Make sure that the microscope stage is unperturbed during this period. Cover the stage with aluminum foil to protect from light. Gently transfer them to an 18&deg;C incubator. </li>
<li>Cultures should adhere to the dish within approximately 3 hrs and new neurite growth should be visible within approximately 6-12 hrs. The growth of isolated sensory neurons reaches a plateau by DIV 3 or 4. Note that isolated sensory neurons doe not form autapses or chemical synapses with one another, although they do form electrical gap junctions.</li>
</ol>
B. Preparation of sensory neuron-motor neuron cocultures
Sensory neurons form synapse with target motor neurons in culture. The most commonly used motor neurons are the LFS motor neurons and the L7 motor neuron, from the abdominal ganglion. The LFS motor neurons are isolated from adult (80-100 g) Aplysia, and there are approximately 20 LFS motor neurons per abdominal ganglion. The L7 motor neuron is isolated from juvenile (1-4 g) animals, and there is only one L7 per abdominal ganglia. LFS motor neurons are 40-50 microns in diameter, are interspersed among the LE sensory neurons close to the root of siphon nerve on the ventral surface of the abdominal ganglia, and are characterized by a subtle dark pigment spot in each soma. The L7 motor neuron is 100-150 microns in diameter and is present on the dorsal surface of the ganglion, on the middle edge of the left side of the ganglion. While it is more economical to use LFS motor neurons, the large size of the L7 motor neuron is advantageous for some experiments. The L11 motor neuron, also on the dorsal left surface of the abdominal ganglion, caudal to L7, is a nontarget motor neuron and can be effectively used as a control with which the sensory neuron fasciculates but does not form chemical synapses.
<ol>
<li>Anesthetize the animals for removal of ganglia. For LFS motor neurons, do as described for pleural sensory neurons. For L7 motor neurons, inject 0.35 M MgCl2 into juvenile Aplysia (1-4 g) using a 10 ml syringe with a 21-gauge needle.</li>
<li>Dissect the abdominal ganglia: Pin the anesthetized animal on a dissecting dish as described above (using 21 gauge needles for juvenile animals). Using a fine forceps and fine scissors, cut out the abdominal ganglia. </li>
<li>Protease digestion of ganglia: This is done as described for pleural sensory neurons, except that the digestion time is reduced to 1 hr 45 min for ganglia from juvenile (1-4 g) animals. </li>
<li>Desheathing the ganglia: Transfer the protease-digested ganglia to a 35 or 60 mm dish containing sylgard and L15. Viewing under a stereo-microscope (with external halogen illumination), in the ganglia down in the Sylgard dish in the proper orientation using insect pins and a fine forceps. With the fine forceps and a Vanna surgical scissors, carefully lift the connective tissue and cut it to expose the neuronal cell bodies. To isolate the LFS motor neurons, pin the ganglion so that the ventral surface is facing up, and with a forceps pull the connective tissue sheath back to expose the entire half of the ganglion containing the LFS motor neurons (to your right), desheath the remaining parts of the ganglion, remove any connective tissue from the dish and add more L15. To isolate the L7 or L11 motor neuron, pin the ganglion so that the dorsal surface is facing up, and with a forceps pull the connective tissue sheath back to expose the half of the ganglion containing both L7 and L11 (to your left). Be careful not to ever touch or damage the neuronal somata. Place a pin to make a &ldquo;post&rdquo; at a short distance from the ganglion (within the field of view).</li>
<li>Isolation of neurons: Remove neurons with a sharp electrode as described for the sensory neurons. The LFS neurons are differentiated from neighboring LE sensory neurons on the basis of a small dark pigment spot in their somata. The L7 neuron can be differentiated from the L11 neuron first because the L11 is caudal to L7, because the L11 cell body is more oblong in shape while the L7 cell body is rounder and because the L11 axon tends to branch into two close to the cell body. </li>
<li>Plating neurons: Use a pipetman (P10) to transfer individual neurons from the Sylgard dish to a culture dish as described for the sensory neurons.</li>
<li>Pairing with sensory neurons: Let the motor neurons adhere to the culture dish for at least 1 hr. Then add the isolated sensory neurons with a pipetman, delivering each sensory neuron adjacent to a plated motor neuron. Using a sharp glass electrode carefully straighten out the sensory neuron axon, and coax the sensory cell body to a position next to the motor neuron, at a distance equal to or slightly less than the length of the sensory cell axon. Using the glass electrode, coax the sensory axon to come into contact with the motor neuron, very gently using the side of the tapered electrode to finally have the sensory axon physically contact the axon of the motor neuron. Do not lift the culture dish, but rather gently glide the dish along the microscope stage.</li>
<li>Leave the dishes on microscope stage at room temperature for no less than 3 hrs and transfer to an 18&deg;C incubator as described for the sensory neurons.</li>
<li>Cultures should adhere to the dish within approximately 3 hrs and new neurite growth should be visible within approximately 6-12 hrs. Sensory neurons form glutamatergic synapses with motor neurons very quickly--as soon as the cell are adherent enough to perform sharp electrode recording (3-5 hrs), an excitatory post-synaptic potential can be recorded. Synaptic connectivity continues to increase until DIV 3, and is then stable until DIV7. The neurons should show elaborate neurite outgrowth during the first one to three days in culture.</li>
</ol>
C. Preparation of Hemolymph
Hemolymph is used as a growth factor in Aplysia culture (analogous to the use of fetal calf serum in mammalian cell culture). It is collected from large (500 g-1 kg) animals, and the best time to collect hemolymph (anecdotally) is during the spring (mid-March to June). Swaddle the animal in a disposable underpad so that only a small portion of the animal is exposed, clean that portion of the skin with ethanol, and then hold it while another person uses a sterile razor blade to make an incision in the exposed area. Squeeze the animal so that the hemolymph squirts into a clean beaker, making sure that it does not contact the dirty skin of the animal. The hemolymph fills the hemocoel of the animal (i.e., the animal is basically a sac of hemolymph). Rewrap the animal once or twice, make a new incision and squeeze hard to collect as much hemolymph as possible (collect in a new beaker so that if it becomes contaminated, the first collection is still usable). Hemolymph from each animal should be kept separately (i.e., do not pool hemolymph from different animals). Spin the hemolymph at 2000 x g for 10 min to remove blood cells. Aliquot the supernatant in 10 ml aliquots, label by animal and store at -80&deg;C. Note that hemolymph stored at -20&deg;C forms a precipitate in medium. A new aliquot of hemolymph must be used every time culture medium is prepared, and the aliquot should be thawed just prior to preparing the medium. Do not refreeze after thawing.

<ol>
	<li>Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+biology+of+memory+storage:+a+dialogue+between+genes+and+synapses.">The molecular biology of memory storage: a dialogue between genes and synapses.</a> Science. 294 (5544), 1030-1038 (2001).</li><li>Rayport, S. G., Schacher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+plasticity+in+vitro:+cell+culture+of+identified+Aplysia+neurons+mediating+short-term+habituation+and+sensitization.">Synaptic plasticity in vitro: cell culture of identified Aplysia neurons mediating short-term habituation and sensitization.</a> J Neurosci. 6 (3), 759-763 (1986).</li><li>Goldberg, D. J., Schacher, S., Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , 213-236 (1998).</li><li>Glanzman, D. L., Kandel, E. R., Schacher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identified+target+motor+neuron+regulates+neurite+outgrowth+and+synapse+formation+of+aplysia+sensory+neurons+in+vitro.">Identified target motor neuron regulates neurite outgrowth and synapse formation of aplysia sensory neurons in vitro.</a> Neuron. 3 (4), 441-450 (1989).</li><li>Lyles, V., Zhao, Y., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapse+formation+and+mRNA+localization+in+cultured+Aplysia+neurons.">Synapse formation and mRNA localization in cultured Aplysia neurons.</a> Neuron. 49 (3), 349-356 (2006).</li><li>Martin, K. C., Casadio, A., Zhu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapse-specific,+long-term+facilitation+of+aplysia+sensory+to+motor+synapses:+a+function+for+local+protein+synthesis+in+memory+storage.">Synapse-specific, long-term facilitation of aplysia sensory to motor synapses: a function for local protein synthesis in memory storage.</a> Cell. 91 (7), 927-938 (1997).</li><li>Guan, Z., Giustetto, M., Lomvardas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+long-term-memory-related+synaptic+plasticity+involves+bidirectional+regulation+of+gene+expression+and+chromatin+structure.">Integration of long-term-memory-related synaptic plasticity involves bidirectional regulation of gene expression and chromatin structure.</a> Cell. 111 (4), 483-493 (2002).</li></ol>

The successful preparation of Aplysia sensory-motor cultures has a somewhat slow learning curve, since it involves the development of fine motor skills associated with microdissection and manipulation of individual neurons viewed through a stereo-microscope. In our experience, it takes approximately 1-3 weeks of practice to obtain healthy isolated sensory neurons in culture and an additional 1-3 weeks to learn how to pair sensory neurons with motor neurons. We routinely prepare cultures in a Labconco Clean Bench...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
			<div>
			Solutions needed for culture 
<ol>
 <li>0.35 M MgCl2, stored at room temperature, used for anesthesia.</li>
 <li>Poly-L-lysine solution, Sigma: P-1524, MW &gt;300,000, made in 0.1M Sodium Borate pH8.2 to 0.5mg/ml solution. Vortex well and filter sterilize through a 0.22 &mu;m filter, store it at 4C&deg;. Do not freeze-thaw.</li>
 <li>L-15 Medium powder (Leibovitz) (Sigma: L4386) supplemented with the salts as below to make 1 liter 
<table border="1" cellspacing="0" cellpadding="0">
 <tr>
 <td>L-15 powder</td>
 <td>13.8g</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>15.4 g </td>
 </tr>
 <tr>
 <td>D-Glucose </td>
 <td>6.24 g </td>
 </tr>
 <tr>
 <td>MgSO4&#149;7H20 </td>
 <td>6.45g </td>
 </tr>
 <tr>
 <td>KCl </td>
 <td>350 mg </td>
 </tr>
 <tr>
 <td>NaHCO3</td>
 <td>170 mg </td>
 </tr>
 <tr>
 <td>MgCl2&#149;6H2O </td>
 <td>5.49 g </td>
 </tr>
 <tr>
 <td>CaCl2&#149;2H2O </td>
 <td>1.43g </td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>3.53g</td>
 </tr>
</table>

 Add ddH20 to 1 liter.&nbsp; The pH should be about 7.4-7.5, add 10ml of 100X Pen/Strep solution, and filter-sterilize through a 0.22 &#181;m filter. Store at 4C0 for no longer than 1 month.</li>
<li>Protease digestion solution: 1% Protease IX (1unit/mg) is made in L15 (supplemented with salts as above) or in ASW immediately before use, filter-sterilized through a 0.22 &#181;m Millipore. 5mls should be enough for the ganglia from two animals (make sure the ganglia are completely immersed in protease solution). Sigma has reported that they will discontinue selling Protease IX. A substitute protease is: Dispase II (Roche Applied Science catalog # 04942078001).</li>
<li>Culture medium.&nbsp; Immediately prior to preparing cultures, thaw a 10 ml aliquot of hemolymph and mix it with 10 mls of L15 (supplemented with salts as above) to make 20mls culture medium. Add 200 &mu;l of 200mM L- Glutamine, mix well and use for preparing cultures. This medium should be prepared fresh each time cultures are made.</li>
<li>Artificial Seawater can be made from Instant Ocean (Aquarium Systems, Mentor, OH) or as follows: 450 mM NaCl, 10 mM KCl, 30 mM MgCl2(6H2O), 20 mM MgSO4, 10 mM CaCl2(2H2O), 10 mM HEPES, with pH adjusted to 7.4.</li></ol>		</div>

preparation of aplysia sensory-motor neuronal cell cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Watch this Scientific Journal Video about Preparation of Aplysia Sensory-motor Neuronal Cell Cultures at JoVE.com

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

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

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

preparation-of-aplysia-sensory-motor-neuronal-cell-cultures

Research

JoVE Journal

Biology

15.9K Aufrufe.  University of California, Los Angeles. Primäre Kulturen von Aplysia Senso-motorischen Neuronen ein Modell Vorbereitung auf das Studium Synapsenbildung und synaptische Plastizität In-vitro-. Dieses Video zeigt die Identifikation und Mikrodissektion der sensorischen und motorischen Neuronen aus Aplysia Ganglien sowie die Methoden zur Festlegung und Aufrechterhaltung senso-motorischen Neuronen in Kultur.

Serie: Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...