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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.
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.
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°C. Note that hemolymph stored at −20°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.
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, although it is possible to prepare them on any laboratory bench or desk, as long as the microscope is unperturbed during the culturing (any vibrations or mechanical disturbances prevent the neurons from adhering). Since the neurons grow in seawater at 18°C, bacterial and fungal contaminations are significantly less common than in mammalian cell culture. The single most important variable in culture preparation is the quality of the animal. Aplysia can either be purchased from vendors who collect them directly from the Pacific Ocean (Alacrity, or Marinus), or from the University of Miami Mariculture facility, which collects breeding pairs from the Pacific and then grows Aplysia through one breeding cycle. As such, the animals are genetically heterogeneous, and, in the case of the animals collected from the ocean, they are also heterogenous in terms of their environmental history. There is some seasonality to the quality of neurons obtained from Aplysia, with the worst period usually occurring in August, and another lower quality period around December. Another important variable is the hemolymph. It is wise to test different batches of hemolymph for their growth-promoting capacity, and to use the same batch of hemolymph for a set of experiments.
Despite the difficulty of preparing Aplysia neuronal cultures, they possess a number of unique and valuable advantages to the study of synapse formation and synaptic plasticity. They form monosynaptic connections in culture that can be monitored by sharp electrode recording for periods of days. Well characterized protocols exist that elicit forms of plasticity that have clear parallels in the behavior of the animal. Stimuli can be applied to the bath or to subsets of synapses6,7, and the geometry of cultures can be varied so that one sensory neuron contacts a single motor neuron, or one sensory neuron with a bifurcated axon contacts two motor neurons, or two sensory neurons contact an individual motor neuron. Molecular and cell biological pathways can be altered in individual neurons by microinjection of DNA, RNA, siRNA and other reagents, and the effects on neuronal structure and function, synaptic transmission and plasticity can be monitored with temporal and spatial resolution. The NIH and Broad Institute are nearing completion of the sequencing of the Aplysia genome, which should
Work in the lab involving the culturing of Aplysia neurons is funded by list NIH R01 and NIH R21MH077921.