Name: extracellularly identifying motor neurons for a muscle motor pool in aplysia californica
Uploaded: 2013-03-25T12:00:00
Description: Watch this Scientific Journal Video about Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica at JoVE.com

This research was supported by NIH grant NS047073 and NSF grant DMS1010434.

1. Preparation of Recording Dish
<ol>
 <li>During the force transducer experiments, the buccal ganglia, cerebral ganglion, and buccal mass are placed in a round Pyrex dish that is specialized for force studies.</li>
 <li>To induce ingestive-like patterns in the experiments, we need to apply the non-hydrolyzable cholinergic agonist carbachol to the cerebral ganglion15. To avoid direct contact from carbachol onto the buccal ganglia and buccal mass, separate chambers are needed to isolate the cerebral ganglion ...

<ol>
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Biol. 80, 93-118 (1979).</li><li>Peters, M., Altrup, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+organization+in+pharynx+of+Helix+pomatia.">Motor organization in pharynx of Helix pomatia.</a> J. Neurophysiol. 52 (3), 389-409 (1984).</li><li>Church, P. J., Cohen, K. P., Scott, M. L., Kirk, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidergic+motoneurons+in+the+buccal+ganglia+of+Aplysia+californica:+immunocytochemical,+morphological,+and+physiological+characterizations.">Peptidergic motoneurons in the buccal ganglia of Aplysia californica: immunocytochemical, morphological, and physiological characterizations.</a> J. Comp. Physiol. A. 168 (3), 323-336 (1991).</li><li>Lu, H., Chestek, C. A., Shaw, K. M., Chiel, H. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+synaptic+activation+of+the+fast+coxal+depressor+motoneurone+of+the+cockroach.+Periplaneta+americana.">Structure and synaptic activation of the fast coxal depressor motoneurone of the cockroach. Periplaneta americana.</a> J. Exp. Biol. 56 (3), 647-656 (1972).</li><li>Westerfield, M., McMurray, J. V., Eisen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identified+motoneurons+and+their+innervation+of+axial+muscles+in+the+zebrafish.">Identified motoneurons and their innervation of axial muscles in the zebrafish.</a> J. Neurosci. 6 (8), 2267-2277 (1986).</li><li>Susswein, A. J., Rosen, S. 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In animals with large identified neurons, such as mollusks (for example, Lymnaea, Helix, and Aplysia), analysis of motor pools is typically done using intracellular recording1,2,3,4. In this protocol, we describe a process for uniquely identifying the motor neurons for a motor pool using an extracellular technique. We used the force measurements as an illustration of this process. One could also use EMG to measure muscle innervations. Briefly, to do so, the protocol needs to be ...

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 <td>Name </td>
 <td>Company</td>
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 <td>Comments</td>
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 <td>Sodium chloride</td>
 <td>Fisher Scientific</td>
 <td>S671</td>
 <td>Biological, Certified</td>
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 <td>Potassium chloride</td>
 <td>Fisher Scientific</td>
 <td>P217</td>
 <td>Certified ACS</td>
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 <td>Magnesium chloride hexahydrate</td>
 <td>Acros Organics</td>
 <td>19753</td>
 <td>99% </td>
 </tr>
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 <td>Magnesium sulfate heptahydrate</td>
 <td>Fisher Scientific</td>
 <td>M63</td>
 <td>Certified ACS </td>
 </tr>
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 <td>Calcium chloride dihydrate</td>
 <td>Fisher Scientifc</td>
 <td>C79</td>
 <td>Certified ACS </td>
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 <td>Glucose (dextrose)</td>
 <td>Sigma-Aldrich</td>
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 <td>MOPS buffer</td>
 <td>Acros Organics</td>
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 <td>Carbachol</td>
 <td>Acros Organics</td>
 <td>10824</td>
 <td>99%</td>
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 <td>Sodium hydroxide</td>
 <td>Fisher Scientific</td>
 <td>SS255</td>
 <td>Certified</td>
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 <td>Hydrochloric acid</td>
 <td>Fisher Scientific</td>
 <td>SA49</td>
 <td>Certified</td>
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 <td>Single-barreled capillary glass</td>
 <td>A-M Systems</td>
 <td>6150</td>
 <td></td>
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 <td>Flaming-Brown micropipette puller model P-80/PC</td>
 <td>Sutter Instruments</td>
 <td></td>
 <td>Filament used: FT345B</td>
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 <td>Enamel coated stainless steel wire</td>
 <td>California Fine Wire </td>
 <td> </td>
 <td>0.001D, coating h </td>
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 <td>Household Silicone II Glue </td>
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 <td></td>
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 <td>Duro Quick-Gel superglue </td>
 <td>Henkel corp. </td>
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 <td>A-M Systems model 1700 amplifier </td>
 <td>A-M Systems</td>
 <td></td>
 <td>Filter settings: 10-500 Hz for the I2 nerve/muscle; 300-500 Hz for all the other nerves </td>
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 <td>Pulsemaster Multi-Channel Stimulator</td>
 <td>World Precision Instruments</td>
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 <td>World Precision Instruments</td>
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 <td>AxoGraph X</td>
 <td>AxoGraph Scientific </td>
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 <td>Sylgard 184 Silicone Elastomer</td>
 <td>Dow Corning</td>
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 <td>100 x 15 mm Crystalizing Dish</td>
 <td>Pyrex</td>
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 <td>High Vacuum Grease</td>
 <td>Dow Corning</td>
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 <td>Fisher Scientific</td>
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 <td>Minutien Pins</td>
 <td>Fine Science Tools</td>
 <td>26002-10</td>
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 <td>Modeling Clay</td>
 <td>Sargent Art</td>
 <td>22-4400</td>
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 <td>Whisper Air Pump</td>
 <td>Tetra</td>
 <td>77849</td>
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 <td>Aquarium Tubing</td>
 <td>Eheim</td>
 <td>7783</td>
 <td>12/16 mm</td>
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 <td>Elite Airstone</td>
 <td>Hagen</td>
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 <td>Vannas Spring Scissors</td>
 <td>Fine Science Tools</td>
 <td>15000-08</td>
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 <td>Dumont #5 Fine Forceps</td>
 <td>Fine Science Tools</td>
 <td>11254-20 </td>
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 <td>Kimwipes</td>
 <td>Kimberly-Clark</td>
 <td>34155</td>
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extracellularly identifying motor neurons for a muscle motor pool in aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Figures 4 and 5 show typical results used to identify two I1/I3 motor neurons. Figure 4 shows the soma recordings of a large motor neuron, B3, during egestive-like and ingestive-like patterns (Figures 4C, 4D). The one-for-one corresponding spikes on the soma channel and the ipsilateral BN2 channel (Figure 4E) show that the specificity of B3 soma recording was maintained during patterns. B3 fires during the middle-to-late retraction phase...

Watch this Scientific Journal Video about Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica at JoVE.com

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

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An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

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Multifunctionality, the ability of one peripheral  structure to generate multiple, distinct behaviors1, allows animals  to rapidly adapt their behaviors ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

A Minimally Invasive Lesion Technique for Muscles Intrinsic to the Odontophore of Aplysia californica

A Minimally Invasive Lesion Technique for Muscles Intrinsic to the Odontophore of Aplysia californica

Aplysia californica is a model system for studying the neural control of learning and behavior. This animal has a semi-open circulatory system, making it ...