Подготовка Aplysia Сенсомоторной нейронов клеточных культур

Summary

Первичные культуры<em> Aplysia</em> Сенсорно-моторные нейроны обеспечивают модели подготовки для изучения образование синапсов и синаптической пластичности<em> В пробирке</em>. Это видео демонстрирует идентификации и микродиссекции сенсорных и моторных нейронов из<em> Aplysia</em> Ганглиях, а также методы создания и поддержания сенсорно-моторных нейронов в культуре.

Abstract

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 animal^2,3. 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 culture^4,5. 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.

Protocol

Подготовка (см. раздел решения в конце протокола состав растворов) Подготовка культуры блюд. Пальто из стекла и Маттек со стеклянным дном культуры блюдо с поли-L-лизин (пр-во борат натрия). Добавьте достаточно, чтобы полностью покрыть стекло хорошо и оставить на> 1 час (можно остави…

Discussion

Успешной подготовки Aplysia сенсомоторной культуры имеет несколько медленно обучения, так как она включает в себя развитие мелкой моторики связано с микродиссекции и манипулирование отдельными нейронами рассматривается через стерео-микроскопа. По нашему опыту, это занимает около 1…

Materials

Solutions needed for culture

1. 0.35 M MgCl₂, stored at room temperature, used for anesthesia.
2. Poly-L-lysine solution, Sigma: P-1524, MW >300,000, made in 0.1M Sodium Borate pH8.2 to 0.5mg/ml solution. Vortex well and filter sterilize through a 0.22 μm filter, store it at 4C°. Do not freeze-thaw.
3. L-15 Medium powder (Leibovitz) (Sigma: L4386) supplemented with the salts as below to make 1 liter
   

   L-15 powder	13.8g
   NaCl	15.4 g
   D-Glucose	6.24 g
   MgSO4•7H20	6.45g
   KCl	350 mg
   NaHCO3	170 mg
   MgCl2•6H2O	5.49 g
   CaCl2•2H2O	1.43g
   HEPES	3.53g

   Add ddH20 to 1 liter.  The pH should be about 7.4-7.5, add 10ml of 100X Pen/Strep solution, and filter-sterilize through a 0.22 µm filter. Store at 4C0 for no longer than 1 month.
4. 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 µm 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).
5. Culture medium.  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 μl of 200mM L- Glutamine, mix well and use for preparing cultures. This medium should be prepared fresh each time cultures are made.
6. 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.