We would like to thank Ryan Maneri (Oystercatcher Productions, LLC) for filming our procedure and Rodney McPhail (Department of Biological Sciences, Purdue University) for assistance with editing the dissection video. Research in the Suter lab is supported by grants from the NIH (R01 NS049233) and the Bindley Bioscience Center at Purdue University to D.M.S.

Solutions
<ul>
<li>L15-ASW cell culture medium (1l) 
<ul>
<li>1 bag of L15 powder</li>
<li>add 800 ml of H2O ultrapure</li>
<li>NaCl 400 mM</li>
<li>MgSO4&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 27 mM</li>
<li>MgCl2&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 28 mM</li>
<li>L-Glutamine&nbsp;&nbsp; 4 mM</li>
<li>Gentamicin&nbsp;&nbsp;&nbsp; 50 &micro;g/ml</li>
<li>HEPES&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 5 mM</li>
<li>Adjust to pH 7.9</li>
<li>Add drop by drop CaCl2 9.3 mM (stop if precipitates)</li>
<li>Add H2O ultrapure to 1 l</li>
<li>Check osmolarity (950-1000 mmol/kg) with osmometer (vapor pressure, Wescor # 5520)</li>
<li>Filter 0.22 &micro;m using a positive pressure filtration unit (Filters: Millipore SVGV010RS)</li>
<li>Store at 4&deg;C for up to 1 month</li>
</ul>
</li>
<li>Poly-L-lysine (70-150 kD)&nbsp; 200 &micro;g/ml stock solution in sterile H2O ultrapure kept at -20oC</li>
<li>0.5 M MgCl2 solution</li>
</ul>

Day 1: Dissection of Aplysia abdominal ganglion
Preparation of the enzymatic dissociation solution
<ol>
<li>Weigh 9-10 mg of Dispase enzyme (Worthington, Cat: LS02111) in an Eppendorf tube</li>
<li>Add 900 &micro;l of L15-ASW culture medium, and 100 &micro;l of sterile H2O ultrapure</li>
<li>Mix and place at room temperature (RT)</li>
</ol>
Dissection of abdominal ganglion
<ol>
<li>Fill a 50 ml syringe with 0.5 M MgCl2 solution.</li>
<li>Take one animal from tank, handle gently to avoid animal's inking defense response.</li>
<li>Place the Aplysia on its side on a dissection board (Styrofoam board), rostral side to the right, caudal&nbsp;side to the left.</li>
<li>Insert the needle into animal just behind the head and inject all MgCl2 solution into the body cavity.</li>
<li>Gently rub the animal to disperse the MgCl2 solution throughout body cavity, wait for a few minutes for the animal to be completely anaesthetized.</li>
<li>Pin the head and tail of the animal to the dissection board with two needles.</li>
<li>Use forceps to lift skin, cut through skin and muscle layer with large scissors towards the head and tail to make a large opening on the side.</li>
<li>Find the abdominal ganglion under the brush-like ovary. Use forceps to hold the connecting nerves about 1 cm in front of the abdominal ganglion, cut the nerves with dissection scissors in front of the forceps holding position and behind the abdominal ganglion.</li>
<li>Place ganglion into the Dispase solution and incubate for 15-16 h at 22&deg;C using a temperature-controlled water bath.</li>
</ol>
Day 2: Plating of Aplysia bag cells
Preparing poly-lysine-coated coverslips
<ol>
<li>In a flow bench, prepare three 35mm petri dishes as working dishes for dissection of the ganglion, filling them with 4 ml L15-ASW medium each. Transfer the abdominal ganglion from the dispase solution to one of the working dishes after 15.5 hours of digestion.</li>
<li>Using forceps take acid-cleaned #1.5 coverslips (22x22 mm) stored in ethanol, remove alcohol and sterilize coverslips by flaming them briefly over Bunsen burner. Let them briefly cool before placing the coverslips into the culture dishes.</li>
<li>Prepare a curved plating tip by bending a yellow tip over the Bunsen burner (flame only briefly to avoid melting of plastic!). </li>
<li>Prepare appropriate amount of 20 &micro;g/ml poly-lysine solution by diluting 200 &micro;g/ml stock solution with sterile H2O ultrapure.</li>
<li>Cover each coverslip with 500 &micro;l of 20 &micro;g/ml poly-lysine and incubate for 20 mins at RT.</li>
<li>Wash each coverslip 3-5 times with 500 &micro;l sterile H2O ultrapure each.</li>
<li>Remove water from coverslips; fill each dish with 4 ml of L15-ASW culture medium.</li>
</ol>
Dissociation of bag cells
<ol>
<li>Under a dissection microscope, cut the abdominal ganglion in half using dissection scissors and forceps previously cleaned with 95% ethanol (Figure 1 line 1).</li>
<li>On one half of the ganglion, cut between bag cell cluster and the hemiganglion cells (Figure 1 line 2), and cut away remaining nerve extensions on the other side of bag cells (Figure 1 line 3). Work on one half of ganglion at a time; leave the other half in the dish while cells from first cluster are being plated.</li>
<li>Using two forceps (or one forceps and dissection scissors), release bag cell cluster by pushing aside the connective tissue sheath surrounding the bag cell cluster. Cut away connective tissue with dissection scissors if needed. Beware not to squeeze and touch the bag cell cluster too much during this process.</li>
<li>When most of the connective tissue is removed, transfer the bag cell cluster into a new working dish with the bent yellow tip prepared earlier. Be careful not to let the bag cell cluster be trapped inside the yellow tip or stuck at the air/medium interface.</li>
<li>Triturate the cluster with yellow tip to remove individual bag cells. Do this by pipetting the cluster in and out of the tip. Start with low shear forces and increase force as needed. Always use the lowest force needed to remove neurons without killing too many cells.</li>
<li>Collect 3-5 healthy bag cells and transfer them to a dish with a poly-lysine-coated coverslip. Avoid picking up dead cells (they are black/transparent in the center) and connective tissue debris.</li>
<li>Repeat step 6. and place about 15-25 live neurons into the center of each coverslip. If dead cells or debris are transferred as well, remove the unwanted material by picking it up or blowing it away from the healthy cells. This process takes about 2-3 hours for both clusters.</li>
<li>Once finished keep the culture dishes on the flow bench for at least 2 hours at RT to allow cells to attach (keep flow bench blower off to avoid unwanted vibration).</li>
<li>Place the dishes into an incubator at 14&deg;C overnight, or until further use. Growth cones may develop in 4-10 hours.</li>
</ol>
<img src="/files/ftp_upload/old/662_2008_2_10_0/image001.jpg" alt="Figure 1. Schematic of Aplysia californica abdominal ganglion. Color lines indicate where to cut to obtain bag cell clusters. " /> Figure 1. Schematic of Aplysia californica abdominal ganglion. Color lines indicate where to cut to obtain bag cell clusters.

Day 3: Setting up chamber for live imaging of Aplysia bag cells
Assembly of imaging chamber
<ol>
<li>Before using bag cells for live cell imaging experiments, inspect cells on a low power microscope. If cells have not developed healthy growth cones, supply fresh medium and leave at RT for 1-2 hours.</li>
<li>To supply plates with fresh L15-ASW medium, remove half (~2ml) of old medium with a vacuum line, and add 2 ml of new L15-ASW medium per petri dish. It is important not to completely remove all medium from the dish all at once, as this will remove cells from the coverslip.</li>
<li>For bag cell imaging, we assemble two coverslips and two plastic spacers in a sandwich-like imaging chamber. The top coverslip in the sandwich is cut shorter to allow easy exchange of medium and reagents. During assembly, coverslips with bag cells attached should be submerged in L15-ASW medium at all times to prevent cells from dislodging.</li>
<li>To prepare the chamber, cut off ~2mm from right side of a #1 coverslips 22x22 mm with diamond pen (glass cutter) and the ruler.</li>
<li>Using a 1 ml syringe, apply high vacuum grease to two sides of the shorter coverslip, and attach plastic spacers to each side. Apply vacuum grease to the top of both spacers.</li>
<li>Invert the coverslip and spacer assembly, and place into petri dish containing bag cells on a coverglass. Align and gently press down to stick top coverslip to bottom coverslip with the reverse end of two Q-tips.</li>
<li>Using forceps, gently lift sandwich assembly out of the petri dish without the medium leaking out. Align the two coverslips, if needed, and wipe off excess medium with a Kimwipe. Place the sandwich onto the bottom part of the microscope slide holder and attach the cover with care using clips.</li>
<li>Clean the center of the top side of sandwich with Q-tips dabbed in glass cleaner (Sparkle is used as glass cleaner). Quickly dry off the glass cleaner with a second, dry Q-tip to avoid streak lines. Do the same for the bottom coverslip. Clean coverslips free of streak lines are important for high quality imaging.</li>
</ol>
Bag cells on the microscope
Since Aplysia bag cell neuronal cultures are at low density, it is best if your microscope setup has stage positioning functions to facilitate switching between cells. Bag cells can be easily kept at RT for several hours on stage during imaging experiments. Exchange the medium periodically to avoid evaporation.

<ol>
	<li>Forscher, P., Kaczmarek, L. K., Buchanan, J. A., Stephen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+induces+changes+in+distribution+and+transport+of+organelles+within+growth+cones+of+Aplysia+bag+cell+neurons.">Cyclic AMP induces changes in distribution and transport of organelles within growth cones of Aplysia bag cell neurons.</a> J. Neurosci. 7, 3600-3611 (1987).</li><li>Suter, D. M., Errante, L. D., Belotserkovsky, V., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ig+superfamily+cell+adhesion+molecule,+apCAM,+mediates+growth+cone+steering+by+substrate-cytoskeletal+coupling.">The Ig superfamily cell adhesion molecule, apCAM, mediates growth cone steering by substrate-cytoskeletal coupling.</a> J. Cell. Biol. 141, 227-240 (1998).</li><li>Schaefer, A. W., Kabir, N., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filopodia+and+actin+arcs+guide+the+assembly+and+transport+of+two+populations+of+microtubules+with+unique+dynamic+parameters+in+neuronal+growth+cones.">Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones.</a> J. Cell. Biol. 158, 139-152 (2002).</li><li>Suter, D. M., Schaefer, A. W., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+dynamics+are+necessary+for+SRC+family+kinase-dependent+growth+cone+steering.">Microtubule dynamics are necessary for SRC family kinase-dependent growth cone steering.</a> Curr. Biol. 14, 1194-1199 (2004).</li></ol>

Aplysia bag cell neurons provide a serum-free neuronal cell culture system with very few non-neuronal cells. These neurons form very large growth cones suitable to address a number of important cell biological questions. Bag cell neurons can easily be manipulated and imaged at room temperature over several hours. Using Fluorescent Speckle Microscopy (FSM) one can quantitatively analyze the various parameters of F-actin and microtubule polymerization and translocation dynamics. These imaging tools together with the recent...

<!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>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>#1 glass coverslips 22x22 mm</td>
<td>Tool</td>
<td>VWR</td>
<td>48366067</td>
<td>&nbsp;</td>
<tr>
<td>#1.5 glass coverslips 22x22 mm</td>
<td>Tool</td>
<td>Corning</td>
<td>2870-22</td>
<td>&nbsp;</td>
<tr>
<td>35 mm Petri dishes</td>
<td>Tool</td>
<td>Falcon</td>
<td>353001</td>
<td>&nbsp;</td>
<tr>
<td>Filters for medium filtration</td>
<td>Tool</td>
<td>Millipore</td>
<td>SVGV010RS</td>
<td>&nbsp;</td>
<tr>
<td>High vacuum grease</td>
<td>Tool</td>
<td>Dow Corning</td>
<td>1597418</td>
<td>&nbsp;</td>
<tr>
<td>Osmometer</td>
<td>Tool</td>
<td>Wescor</td>
<td>5520</td>
<td>&nbsp;</td>
<tr>
<td>Plastic shims</td>
<td>Tool</td>
<td>Small Parts Inc </td>
<td>SHSP-200</td>
<td>&nbsp;</td>
<tr>
<td>Calcium chloride</td>
<td>Reagent</td>
<td>Fisher</td>
<td>C79-500</td>
<td>&nbsp;</td>
<tr>
<td>Gentamicin</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15750-060</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H4030</td>
<td>&nbsp;</td>
<tr>
<td>L15 medium</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>41300-039</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>G8540</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium chloride</td>
<td>Reagent</td>
<td>Mallinckrodt</td>
<td>5958-04</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium sulfate</td>
<td>Reagent</td>
<td>Mallinckrodt</td>
<td>6070-12</td>
<td>&nbsp;</td>
<tr>
<td>Poly-L-lysine (70-150 kD) </td>
<td>Reagent</td>
<td>Sigma</td>
<td>P6282</td>
<td>&nbsp;</td>
<tr>
<td>Sodium chloride</td>
<td>Reagent</td>
<td>Mallinckrodt </td>
<td>7581</td>
<td>&nbsp;</td>
<tr>
<td>Neutral Protease (Dispase)</td>
<td>Reagent</td>
<td>Worthington</td>
<td>LS02111</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones at JoVE.com

Neuronal Cell Cultures from Aplysia for High-Resolution 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 ...

neuronal-cell-cultures-from-aplysia-for-high-resolution-imaging

Research

JoVE Journal

Biology

11.3K Views.  Purdue University. Aplysia californica neuroni sviluppare grandi coni di crescita nella cultura che sono eccellenti per imaging ad alta risoluzione della motilità del cono di crescita e di orientamento. Qui, vi presentiamo un protocollo per la dissezione e la placcatura dei neuroni borsa cellule Aplysia così come per la creazione di una camera per l'imaging cellulare dal vivo.

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

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 ...

Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization 3. Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro 3. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. 
Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later andexcess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20&deg;C.

We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. ...

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 ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

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.

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.

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

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...