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 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 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 >60 μm 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.
1. Cell Preparation
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 ≤100 pF capacitance, or cells <60 μm diameter during days 3 and 4 of the protocol. Cells without processes are optimal for recording. The following special considerations apply:
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). These sensory neurons have the advantage of a stereotyped location within the ganglia, although the right or left cluster is missing in individual animals in approximately 1% of occurrences. The BSC and PVC neuron clusters are identifiable by the dark orange color of the cell membrane and relatively small size compared to nearby cells, as shown in the large circle outline of Figure 1A.
In culture, these neurons are often without processes the day after plating (Figure 2A) and are ideal for patch clamping that day, and one day later. It is sometimes possible to achieve adequate space clamp even when the cells appear to have process outgrowth (Figure 2F), suggesting that not all sprouting that seems to emanate from the cell body is part of the cell. If handling after enzymatic digestion has been gentle, cells arrive in culture with some processes intact, and these processes grow with time (Figures 2B-2E). Such cells are not suitable for patch clamping but intracellular recording is possible.
Whole cell patch clamp currents carried by voltage gated Na+ and Ca2+ are readily recorded from PVC and BSC neurons in short term culture 6, and both cell types display a mixed K current. BSC neurons also respond to acetylcholine, serotonin and NMDA4 (Figure 3C) with excitatory currents, while both BSC and PVC neurons respond to L-Glu and D-Asp3 (Figures 3A and 3B) with excitatory currents. The pharmacological characteristics of L-Glu- and D-Asp-induced currents in these neurons have been described4.
Figure 1. Photomicrographs showing the position of the glutamatergic sensory neurons of the buccal and pleural ganglia (red circles). A. The buccal S cluster (BSC) neurons, identifiable by their dark orange color (large circle on left hemiganglion) and the corresponding position in the right hemiganglion (smaller circles). B. V-shaped, dark orange pleural ventrocaudal (PVC) cell cluster of the right pleural hemiganglion (left hemiganglion not shown).
Figure 2. Photomicrographs of glutamatergic sensory neurons in culture. A. Cells from the PVC cluster 24 hr after plating in culture dish showing 2 sensory neurons and other cells; other PVC cells from separate cultures at 24 hr are shown as insets. B, C. A BSC neuron in culture at 24 hr and the same neuron at 96 hr, respectively. D, E. A PVC neuron in culture at 24 hr and the same neuron at 96 hr, respectively. F. A PVC neuron at 48 hr in a patch clamp experiment. The patch pipette is contacting the cell from the right edge, while the picospritzer pipette is at the bottom. The large dark shadow visible to the left of the cell is the opening of the flowing bath pipette. Click here to view larger figure.
Figure 3. Glutamatergic currents in whole cell patch clamp recordings from BSC and PVC neurons. A. Whole cell current in a BSC neuron in response to pressure application of L-Glu (1 mM, 100 msec). B. Whole cell current in a PVC neuron in response to pressure application of D-Asp (1 mM, 100 msec). C. Whole cell NMDA current in the same BSC cell as in A (1 mM, 100 msec).
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 °C with slow shaking, 13 hr is sufficient for digestion of the BSC clusters while15 hr is optimal for PVC clusters. Low cell yields in the culture dish can be attributed to insufficient time in enzyme, or too much mechanical disruption during either the cell transfer step (1.11 and 1.12) or during the flicking step (1.13). Failure of the cells to stick down to the culture substrate, as well as brief survival in culture is usually due to too much mechanical disruption. Contamination of the cultures can also occur, and is best addressed by ensuring that the anesthetized animal is well rinsed, that the dissection instruments are clean, and that the ganglia of interest are put through several rinses in ASW + P/S. It may be necessary to wash and alcohol-clean instruments between different parts of the dissection procedure, or have enough instruments ready so that clean ones can be used for each step.
Aplysia neurobiological studies are most often conducted on reduced preparations that preserve nerve connections between neurons or between neurons and muscles 2, 18. These preparations facilitate the assignment of control of muscular processes to specific neurons and neuronal circuits and also permit studies of the potentiation and facilitation of repetitive reflexes. Intact preparations are not suited to certain types of voltage clamp protocols and the study of effects of agonists that rapidly desensitize their receptors.
The long-term neuronal co-culture is an additional tool to study potentiation and facilitation under highly controlled conditions. Long term co-cultures also lend themselves to biochemical studies on single neurons. Success of the co-culture preparation is often attributed to the addition of up to 50% Aplysia hemolymph to cultures 17. However, the success of this approach often depends on batch-to-batch variability of this hemolymph.
The dissociated cell culture preparation described here expands the use repertoire of the Aplysia model. Dissociated cell cultures are not suitable for deducing neural circuits or studying the intact synapse as the methods mentioned above do. The dissociated cell culture preparation has its greatest utility in confirming the existence of specific ionic conductances and their kinetic and pharmacological properties in single cell voltage clamp experiments. Several unique currents have been discovered using single cell preparations, including an excitatory cation current in bag cells 19 and the D-Asp receptor current in BSC neurons 4. Studies on Aplysia rarely focus on the ionic currents of individual cells, in part because of the often unwieldy size of such cells and the existence of numerous other suitable model cell types that lend themselves well to patch clamp techniques. As a result, the existence of certain currents in Aplysia, such as those activated by alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) are usually inferred from block of putative AMPA receptor (R)-related behaviors by AMPAR antagonists applied to the reduced nerve preparation, rather than directly recorded. Thus verification of the existence of certain currents is lacking. Isolation of cultured glutamatergic neurons and single cell voltage clamp methodology provides a direct test for the existence of different types of L-GluR channels in this model organism.
Another use of Aplysia neurons in single cell patch clamp experiments is in dissecting second messenger cascades. Aplysia neurons are particularly well suited to studies of second messenger induced modulation because they are stable for long periods of recording at room temperature 3, 12, 20, 21, and are sufficiently robust that individual cells can be saved after recording and recorded from again after 24 hr 5. These buccal and pleural sensory neuron clusters conveniently yield a pair of matched cultures from each ganglion, so that one dish can be designated a control culture while its mate receives a treatment.
An additional advantage of this preparation is in studies of the morphological status of individual neurons. For example, we discovered that cell bodies of Aplysia neurons from senescent animals were larger than those from younger animals, but did not elaborate cell processes after several days in culture 6. These isolated neuron preparations also facilitate studies using vital dyes to assess levels of intracellular Ca+2, pH, reactive oxygen species, mitochondrial membrane potential and other physiological traits that can then be compared with neurophysiological features of individual cells. While some of these approaches are applicable in intact or co-culture preparations, data collection is much more straightforward using isolated neurons in this easy culture system. In the future we hope to use these cells for single cell molecular profiling 13.
The dissociated short term culture provides ideal material for investigating fundamental neurophysiological processes, and may provide a particularly powerful tool when used in conjunction with studies involving reduced or co-culture preparations. The dissociated Aplysia neuron culture extends the utility of this venerable animal model to new and interesting areas of neuroscience.
The authors have nothing to disclose.
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.
Name of Reagent/Material | Company | Catalog Number | Yorumlar |
Artificial seawater ASW | Sigma-Aldrich | assorted | (mM): 417 NaCl, 10 KCl, 10 CaCl2 (2 H2O), 5MgCl2 (6H2O), 15 HEPES-NaOH, pH 7.6 |
Intracellular solution | Sigma | assorted | (mM): 450 KCl, 2.9 CaCl2 (2 H2O), 2.5 MgCl2 (6 H2O), 5 Na2ATP, 10 EGTA, and 40 HEPES-KOH, pH 7.4 |
Poly-D-lysine | Sigma | P6407 | |
penicillin/streptomycin added to ASW at 1:100 | Lonzo Walkersville, Inc. | 17-603E | 5,000 Units/ml penicillin plus 5,000 mg/ml streptomycin |
Neutral dispase II | Roche Diagnostics | 10165859001 | |
hyaluronidase | Sigma-Aldrich | H4272 | |
collagenase type XI | Sigma-Aldrich | C9407 | |
L-Glutamate (L-Glu) | Sigma-Aldrich | 49601-100G | |
D-Aspartate (D-Asp) | Sigma-Aldrich | 11200-10G | |
N-methyl-D-aspartate (NMDA) | Biomol | 100002-268 | |
L-Asp | Sigma | A6683-25G | |
alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) | Sigma | A6816-5MG | |
L-Glu R antagonists | various | various | |
agar | |||
kynurenate | Sigma-Aldrich | 61250 | |
APV | Sigma-Aldrich | A5282 | |
DL-2-Amino-5-phosphonopentanoic acid (NMDAR antagonist) | |||
2-propanol | VWRSP | BDH1133 | |
Chloriding solution | Sigma | assorted | 25 g FeCl3 + 25 ml concentrated HCl + 50 ml H2O |
Sylgard silicone 2-part polymer | World Precision Instruments (WPI) | SYL184 | Provides pin-out surface for small dissection dishes |
0-40x zoom magnification microscope for dissections | Wild | ||
Techniquip 150 Watts Fiber Optic Illuminator | Microoptics of Florida | TQ FOI-150 | |
RotoMix 50800 orbital mixer | |||
Nikon Diaphot inverted phase-contrast microscope with 4x, 20x (optional) & 40x objectives | SR Research Ltd. | Eyelink II | |
Tektronix digital oscilloscope | SR Research Ltd. | ||
pClamp 10 data acquisition and analysis software | Molecular Devices | ||
PC with Windows XP or higher operating system | PC Solutions | Thinkserver with solid state hard drives (80GB) and low noise monitors | |
Flaming/Brown P87 micropipette puller | Sutter Instruments, Novato, CA | ||
Axon Instruments Axopatch 200B clamp amplifier with a capacitance compensation range of 1-1000 pF; preamplifier | Molecular Devices, Sunnyvale, CA | ||
Axon instruments electrode holder assembly for Axopatch 200B preamplifier | Molecular Devices, Sunnyvale, CA | CV203BU | |
Digidata 1200 A/D converter | Molecular Devices, Sunnyvale, CA | ||
Picospritzer, powered by N2 adjustable for pressure and duration | Parker Hannifin, Cleveland, OH | ||
TMC Micro-G Vibration isolation table | Ametek | ||
Faraday cage | custom manufacture | ||
Burleigh Piezoelectric Clamshell Micromanipulators | Burleigh Instruments; Thorlabs | presently PCS-5000; -6000 series + mounts | |
Narishige M-152 manual manipulators (for perfusion system and picospritzer) | Narishige USA | ||
Filament pipette glass,1.5 mm OD, 0.84 mm ID – | WPI | 1B150-3 | |
3 inch length | |||
Ag/AgCl half cell | WPI | EP4 | |
15 ml centrifuge tubes, 35-2097 BD Falcon* Centrifuge Tubes | VWRSP | 21008-918 | |
Angled Scissors | Fine Science Tools | 15006-09 | |
Dumostar Fine forceps | Fine Science Tools | 11295-00 | |
35 mm falcon tissue culture dishes | VWRSP | 25382-064 | |
falcon 150 x 25 mm tissue culture dishes; 1013 | VWRSP | 1013 | also can be made into small dissection dishes with sylgard |
sylgard | WPI | SYL184 | |
animal dissection tray | various | ||
15 ml centrifuge tubes, 35-2097 BD Falcon | VWRSP | 21008-918 | For 6-bore gravity-fed perfusion system |
Aluminum clips with screw hole ends | hardware store | For perfusion system | |
23 gauge needles (manually file off points) | VWRSP | For perfusion system | |
Polyethylene tubing 0.022″ID x 0.042″OD; 427411 | Becton-Dickinson | For perfusion system | |
H-7 pipette stand/holder for microcap perfusion array | Narishige USA | For perfusion system | |
one-way valves | For perfusion system | ||
Drummond Microcaps 1 μl | VWRSP | For perfusion system | |
18 gauge needles for suction (filed off points) | |||
Polyethylene tubing | Cole Parmer | 4.27436E+11 | |
fine dissection pins | Fine Science Tools | 26002-20 | |
capillary tubes | Kimble 71900-100 | fire-polished and U-shaped in a Bunsen burner flame and filled with 3% agar in ECS | |
modeling clay | craft store | ||
dish holder for microscope stage with isolated ground bath | Custom manufacture | ||
pasteur pipettes | VWRSP | 14672-412 | |
pipette bulbs | VWRSP | 53283-911 | |
acrodisk syringe filters | VWRSP | 28144-040 | |
thick-walled 1.5 mm diameter borosilicate filament glass | WPI | 1B150F-3 | |
High purity nitrogen cylinder and bifurcating regulator |
Tables 1-3. Lists of Reagents, Materials, and Equipment.