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Neuroscience

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

Published: March 25th, 2013

DOI:

10.3791/50189

1Department of Biology, Case Western Reserve University , 2Department of Neurosciences, Case Western Reserve University , 3Department of Biomedical Engineering, Case Western Reserve University

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.

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.

1. Preparation of Recording Dish

  1. 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.
  2. 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 .......

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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.......

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

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Name Company Catalog Number Comments
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: 10-500 Hz for the I2 nerve/muscle; 300-500 Hz for all the other nerves
Pulsemaster Multi-Channel Stimulator World Precision Instruments A300
Stimulus Isolator World Precision Instruments A360
AxoGraph X AxoGraph Scientific Software for recordings
Gold Connector Pins Bulgin SA3148/1
Gold Connector Sockets Bulgin SA3149/1
Sylgard 184 Silicone Elastomer Dow Corning
100 x 15 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
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
Kimwipes Kimberly-Clark 34155

  1. McCrohan, C. R., Benjamin, P. R. Synaptic relationships of the cerebral giant cells with motoneurones in the feeding system of Lymnaea stagnalis. J. Exp. Biol. 85, 169-186 (1980).
  2. Benjamin, P. R., Rose, R. M. Central generation of bursting in the feeding system of the snail, Lymnaea stagnalis. J. Exp. Biol. 80, 93-118 (1979).
  3. Peters, M., Altrup, U. Motor organization in pharynx of Helix pomatia. J. Neurophysiol. 52 (3), 389-409 (1984).
  4. Church, P. J., Cohen, K. P., Scott, M. L., Kirk, M. D. Peptidergic motoneurons in the buccal ganglia of Aplysia californica: immunocytochemical, morphological, and physiological characterizations. J. Comp. Physiol. A. 168 (3), 323-336 (1991).
  5. Lu, H., Chestek, C. A., Shaw, K. M., Chiel, H. J. Selective extracellular stimulation of individual neurons in ganglia. J. Neural. Eng. 5 (3), 287-309 (2008).
  6. Church, P. J., Lloyd, P. E. Expression of diverse neuropeptide cotransmitters by identified motor neurons in Aplysia. J. Neurosci. 11 (3), 618-625 (1991).
  7. Church, P. J., Lloyd, P. E. Activity of multiple identified motor neurons recorded intracellularly during evoked feedinglike motor programs in Aplysia. J. Neurophys. 72 (4), 1794-1809 (1994).
  8. 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 (2012).
  9. Cullins, M. J., Chiel, H. J. Electrode fabrication and implantation in Aplysia californica for multi-channel neural and muscular recordings in intact, freely behaving animals. J Vis. Exp. (40), e1791 (2010).
  10. Zhurov, Y., Weiss, K. R., Brezina, V. Tight or loose coupling between components of the feeding neuromusculature of Aplysia. J. Neurophysiol. 94 (1), 531-549 (2005).
  11. Hurwitz, I., Goldstein, R. S., Susswein, A. J. Compartmentalization of pattern-initiation and motor functions in the B31 and B32 neurons of the buccal ganglia of Aplysia californica. J. Neurophysiol. 71 (4), 1514-1527 (1994).
  12. Morton, D. W., Chiel, H. J. The timing of activity in motor neurons that produce radula movements distinguishes ingestion from rejection in Aplysia. J. Comp. Physiol. A. 173 (5), 519-536 (1993).
  13. Iles, J. F. Structure and synaptic activation of the fast coxal depressor motoneurone of the cockroach. Periplaneta americana. J. Exp. Biol. 56 (3), 647-656 (1972).
  14. Westerfield, M., McMurray, J. V., Eisen, J. S. Identified motoneurons and their innervation of axial muscles in the zebrafish. J. Neurosci. 6 (8), 2267-2277 (1986).
  15. Susswein, A. J., Rosen, S. C., Gapon, S., Kupfermann, I. Characterization of buccal motor programs elicited by a cholinergic agonist applied to the cerebral ganglion of Aplysia californica. J. Comp. Physiol. A. 179 (4), 509-524 (1996).
  16. Hurwitz, I., Neustadter, D., Morton, D. W., Chiel, H. J., Susswein, A. J. Activity patterns of the B31/B32 pattern initiators innervating the I2 muscle of the buccal mass during normal feeding movements in Aplysia californica. J. Neurophys. 75 (4), 1309-1326 (1996).
  17. Morton, D. W., Chiel, H. J. In vivo buccal nerve activity that distinguishes ingestion from rejection can be used to predict behavioral transitions in Aplysia. J. Comp. Physiol. A. 172 (1), 17-32 (1993).
  18. Warman, E. N., Chiel, H. J. A new technique for chronic single-unit extracellular recording in freely behaving animals using pipette electrodes. J. Neurosci. Methods. 57 (2), 161-169 (1995).
  19. Nargeot, R. N., Baxter, D. A., Byrne, J. H. Contingent-dependent enhancement of rhythmic motor patterns: an in vitro analog of operant conditioning. J. Neurosci. 17 (21), 8093-8105 (1997).
  20. Kandel, E. R. . Behavioral biology of Aplysia. , (1979).
  21. Scott, M. L., Govind, C. K., Kirk, M. D. Neuromuscular organization of the buccal system in Aplysia californica. J. Comp. Neurol. 312 (2), 207-222 (1991).
  22. Rosen, S. C., Miller, M. W., Cropper, E. C., Kupfermann, I. Outputs of radula mechanoafferent neurons in Aplysia are modulated by motor neurons, interneurons, and sensory neurons. J. Neurophysiol. 83 (3), 1621-1636 (2000).
  23. Rosen, S. C., Miller, M. W., Evans, C. G., Cropper, E. C., Kupfermann, I. Diverse synaptic connections between peptidergic radula mechanoafferent neurons and neurons in the feeding system of Aplysia. J. Neurophysiol. 83 (3), 1605-1620 (2000).
  24. Weiss, K. R., Chiel, H. J., Koch, U., Kupfermann, I. Activity of an identified histaminergic neuron, and its possible role in arousal of feeding behavior in semi-intact Aplysia. J. Neurosci. 6 (8), 2403-2415 (1986).
  25. Rosen, S. C., Teyke, T., Miller, M. W., Weiss, K. R., Kupfermann, I. Identification and characterization of cerebral-to-buccal interneurons implicated in the control of motor programs associated with feeding in Aplysia. J. Neurosci. 11 (11), 3630-3655 (1991).
  26. Jing, J., Weiss, K. R. Generation of variants of a motor act in a modular and hierarchical motor network. Curr. Biol. 15 (19), 1712-1721 (2005).
  27. Azizi, F., Lu, H., Chiel, H. J., Mastrangelo, C. H. Chemical neurostimulation using pulse code modulation (PCM) microfluidic chips. J. Neurosci. Methods. 192 (2), 193-198 (2010).
  28. Zhurov, Y., Proekt, A., Weiss, K. R., Brezina, V. Changes of internal state are expressed in coherent shifts of neuromuscular activity in Aplysia feeding behavior. J. Neurosci. 25 (5), 1268-1280 (2005).
  29. Baker, B. J., Kosmidis, E. K., Vucinic, D., Falk, C. X., Cohen, L. B., Djurisic, M., Zecevic, D. Imaging brain activity with voltage- and calcium-sensitive dyes. Cell. Mol. Neurobiol. 25 (2), 245-282 (2005).
  30. Fejtl, M., Stett, A., Nisch, W., Boven, K. -. H., Möller, A., Baudry, M., Taketani, M. On Micro-Electrode Array Revival. Advances in Network Electrophysiology Using Multi-Electrode Arrays. , 24-37 (2006).

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