Summary

<em> In Vitro</em> Подготовка к Выявление и записи программ кормления двигателя с Физиологические движения в<em> Aplysia саЩогтса</em

Published: December 05, 2012
doi:

Summary

Мы описываем технику внеклеточно записывать и стимулировать от нервов, мышц и отдельных нейронов определены<em> В пробирке</em> В то время как выявление и наблюдение за различными типами кормления поведения в кормлении аппарата<em> Aplysia</em>.

Abstract

Multifunctionality, the ability of one peripheral structure to generate multiple, distinct behaviors1, allows animals to rapidly adapt their behaviors to changing environments. The marine mollusk Aplysia californica provides a tractable system for the study of multifunctionality. During feeding, Aplysia generates several distinct types of behaviors using the same feeding apparatus, the buccal mass. The ganglia that control these behaviors contain a number of large, identified neurons that are accessible to electrophysiological study. The activity of these neurons has been described in motor programs that can be divided into two types, ingestive and egestive programs, based on the timing of neural activity that closes the food grasper relative to the neural activity that protracts or retracts the grasper2. However, in isolated ganglia, the muscle movements that would produce these behaviors are absent, making it harder to be certain whether the motor programs observed are correlates of real behaviors. In vivo, nerve and muscle recordings have been obtained corresponding to feeding programs2,3,4, but it is very difficult to directly record from individual neurons5. Additionally, in vivo, ingestive programs can be further divided into bites and swallows1,2, a distinction that is difficult to make in most previously described in vitro preparations.

The suspended buccal mass preparation (Figure 1) bridges the gap between isolated ganglia and intact animals. In this preparation, ingestive behaviors – including both biting and swallowing – and egestive behaviors (rejection) can be elicited, at the same time as individual neurons can be recorded from and stimulated using extracellular electrodes6. The feeding movements associated with these different behaviors can be recorded, quantified, and related directly to the motor programs. The motor programs in the suspended buccal mass preparation appear to be more similar to those observed in vivo than are motor programs elicited in isolated ganglia. Thus, the motor programs in this preparation can be more directly related to in vivo behavior; at the same time, individual neurons are more accessible to recording and stimulation than in intact animals. Additionally, as an intermediate step between isolated ganglia and intact animals, findings from the suspended buccal mass can aid in interpretation of data obtained in both more reduced and more intact settings. The suspended buccal mass preparation is a useful tool for characterizing the neural control of multifunctionality in Aplysia.

Protocol

1. Приготовление растворов Для подготовки хлорид магния решение, которое является изотоническим с морской водой, в которых содержатся животные (~ 1000 millosmolar), отмечают большой кувшин на уровне желаемого объема. Заполните кувшин с дистиллированной водой примерно до 80% от этого уровн?…

Representative Results

When an extracellular electrode is positioned above a neuron’s soma and used to stimulate the neuron, a one-for-one correspondence between spikes on the soma channel and on the nerve(s) the neuron projects to can be observed (Figure 6, left panel, stimulation of identified neuron B9). The soma channel (top channel) is set to stimulating mode when the current is applied (time 1 in the figure), and is then quickly switched to recording mode (time 2). By maintaining the position of the electrode, the…

Discussion

Предыдущая работа характеризуется Aplysia двигателя программ в интактных животных и в условиях ограниченной препараты, такие как изолированные ганглиев. В интактных животных, хотя записи отдельных нейронов было получено 5, такие эксперименты очень трудно, и электроды не могут …

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

Это исследование было поддержано грантом NS047073 NIH и NSF гранта DMS1010434.

Materials

Name Company Catalog Number Comments
Sodium chloride Fisher Scientific S671 Biological, Certified
Potassium chloride Fisher Scientific P217 Certified ACS
Magnesium chloride hexahydrate Acros Organics 19753 99%
Magnesium sulfate heptahydrate Fisher Scientific M63 Certified ACS
Calcium chloride dihydrate Fisher Scientifc C79 Certified ACS
Glucose (dextrose) Sigma-Aldrich G7528 BioXtra
MOPS buffer Acros Organics 17263 99%
Carbachol Acros Organics 10824 99%
Sodium hydroxide Fisher Scientific SS255 Certified
Hydrochloric acid Fisher Scientific SA49 Certified
Single-barreled capillary glass A-M Systems 6150  
Flaming-Brown micropipette puller model P-80/PC Sutter Instruments   Filament used: FT345B
Enamel coated stainless steel wire California Fine Wire   0.001D, coating h
Household Silicone II Glue GE    
Duro Quick-Gel superglue Henkel corp.    
A-M Systems model 1700 amplifier A-M Systems   Filter settings: 300-500 Hz nerves,10-500 Hz I2 muscle
Pulsemaster Multi-Channel Stimulator World Precision Instruments A300  
Stimulus Isolator World Precision Instruments A360  
AxoGraph X AxoGraph Scientific    
Veeder-Root Totalizing Counter Danaher C342-0562  
Gold Connector Pins Bulgin SA3148/1  
Gold Connector Sockets Bulgin SA3149/1  
Sylgard 184 Silicone Elastomer Dow Corning    
100 x 50 mm Crystalizing Dish Pyrex    
High Vacuum Grease Dow Corning    
Pipet Tips Fisher Scientific 21-375D  
Minutien Pins Fine Science Tools 26002-10  
Modeling Clay Sargent Art 22-4400  
Silk Sutures Ethicon K89OH  
Whisper Air Pump Tetra 77849  
Aquarium Tubing Eheim 7783 12/16 mm
Elite Airstone Hagen A962  
Vannas Spring Scissors Fine Science Tools 15000-08  
Dumont #5 Fine Forceps Fine Science Tools 11254-20  
Yaki Sushi Nori Seaweed Rhee Bros    
Kimwipes Kimberly-Clark 34155  

Referencias

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Citar este artículo
McManus, J. M., Lu, H., Chiel, H. J. An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica. J. Vis. Exp. (70), e4320, doi:10.3791/4320 (2012).

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