Department of Biology, University of Kentucky
Leksrisawat, B., Cooper, A. S., Gilberts, A. B., Cooper, R. L. Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception . J. Vis. Exp. (45), e2323, doi:10.3791/2323 (2010).
The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive information for an animal. An additional objective of this experiment is to learn anatomy of the preparation by staining, dissection and viewing of neurons and sensory structures under a dissecting microscope. This is performed by using basic neurophysiological equipment to record the electrical activity from a joint receptor organ and staining techniques. The muscle receptor organ (MRO) system in the crayfish is analogous to the intrafusal muscle spindle in mammals, which aids in serving as a comparative model that is more readily accessible for electrophysiological recordings. In addition, these are identifiable sensory neurons among preparations. The preparation is viable in a minimal saline for hours which is amenable for student laboratory exercises. The MRO is also susceptible to neuromodulation which encourages intriguing questions in the sites of modulatory action and integration of dynamic signals of movements and static position along with a gain that can be changed in the system.
Proprioceptors are neurons that detect joint position, direction, speed, and muscle stretch. Proprioception is a unique sensory modality, because proprioceptors are interoceptors and sense stimuli within the body instead of from the outside world.
In the vertebrate system, it appears that many of the joint and tension receptors are not necessary to detect gross proprioceptive information. The annulospiral and flowerspray (sensory nerve endings) receptors on muscle fibers have been shown by ablation as well as vibratory and anesthetic studies to be the two essential receptor groups needed for proprioception (Burgess et al. for a review, 1982). However, it is notable that there is redundant information gathered by other receptors, such as those in the joints, that are used for fine control of movements. Arthropods like vertebrates have articulated appendages. Therefore, it is not surprising that the proprioceptors described for vertebrates have their counterparts in arthropod limbs and joints.
The anatomical arrangement of chordotonal organs in crabs allows the analysis of each individual neuron according to function. In addition, developmental questions can be addressed as the animal grows or when the animal regenerates a limb (Cooper and Govind, 1991; Hartman and Cooper, 1993). Some joint chordotonal organs in crabs contain hundreds of primary sensory neurons (Cooper, 2008) and these neurons monitor aspects in the range fractionation in movements and positions of the joint. A less complex proprioceptive system of monitoring joint movements and positions is the muscle receptor organs (MROs) in the abdomen of crayfish (Eckert, 1961a,b; McCarthy and MacMillan, 1995). The mechanoreceptors in crayfish abdomen MROs transduce a stretch stimulus in the sensory endings, embedded in a muscle, into a graded receptor potential. When potential exceeds a threshold, an action potential will result at the axon base. This is what is defined and known as "the site of spike initiation" in neurobiology. In this system the cell body resides in close apposition to the muscle it monitors. Two distinct types of stretch receptors exist in this sensory system: a slowly-adapting and a rapidly-adapting receptor. The activity is dependent on the strength of the mechanical stretch. The MRO system in the crayfish is analogous to the intrafusal muscle spindle in mammals and the muscles also have efferent control to maintain the taut nature of the muscles as known for intrafusal muscles in mammals.
The muscle spindle sensory neurons in mammals are challenging to investigate electrophysiologically because of the small nature of the sensory endings. It is also difficult to track the location of the cell bodies in the dorsal root ganglion to their peripheral endings. In comparison, the MRO neurons in crayfish are readily accessible for extracellular and intracellular electrodes for long term recordings. The cell bodies of the MRO sensory neurons are relatively large (50-100 μm in diameter). Sensory neurons have also served as a model in addressing how "stretch activated" channels in neurons function, ionic flow, channel distribution, and density of sensory neurons (Brown et al., 1978; Edwards et al., 1981; Erxleben, 1989; Hunt et al., 1978; Purali and Rydqvist, 1992; Rydqvist and Purali, 1991; Rydqvist and Swerup, 1991; Cooper et al., 2003). The integration of the sensory input from the MRO in one segment can influence other adjoining segments (Eckert, 1961a,b). There are a few reports on modulation of the sensory input from the MRO (Pasztor and Macmillan, 1990; Cooper et al., 2003) . Modulation of neural circuits is a rich area for future investigations of basic science and this preparation can serve as a foundation in mammals for future applications, potentially in the spinal cords of vertebrates (Rossignol et al., 2001, 2002; Donnelan, 2009)
1.1) Learning outcomes
In this laboratory experiment, one will dissect a crayfish abdomen and learn the associated anatomy and physiology of the MRO. One will learn how to monitor neuronal activity with extracellular recordings and to use common electrophysiological equipment. One will graph and interpret the data obtained based on the sensory stimulation provided. The sensory stimulation will range from static positions as well as dynamic movements of the segment being monitored. One will address the concept of proprioception in this sensory system and its significance. Sensory adaptation will be observed in a series of experiments. The significance as well as the potential mechanism behind sensory adaption will be addressed by the students.
Figure 1: The equipment set up
Each abdominal segment has two sets of the rapidly and slowly-adapting MROs on the right and left hemisegments. The associated nerve bundles run along the lateral edge next to the cuticle. This is the nerve bundle that one will be recording from. One will not be able to view the MROs because they are located under the DEL1 and 2 muscles (Appendix Figures 1 & 2). Figure 8 provides an overview of the dissections to be made in order to isolate the abdomen.
Figure 8: Overview of general dissection to isolate abdomen. A, B, and C are the series of steps in dissecting the crayfish.
Generalized responses obtained from the slowly and rapidly-adapting MROs while stretching and maintaining a stretch are depicted in Figure 9. In this exercise one will be recording from both MROs together as their axons are contained in the same nerve bundle.
Figure 9: The crayfish has two types of neurons in the MRO. The phasic, which are innervated by fast motor axons, and the tonic which are innervated by slow motor axons. (a) When a tonic receptor is stimulated it slowly adapts to the stimulus and continues a steady firing pattern of action potentials. (b) When a phasic receptor is stimulated it rapidly adapts to the stimulus and fires only a short pattern of action potentials.
The whole nerve which contains motor and sensory neurons is recorded from (Figure 10). However one will only detect the sensory neurons as the motor drive has been severed from the ventral nerve cord of the animal.
Figure 10: The nerve bundle to be sucked up into the recording electrode. (A) The free nerve is shown floating over the dissected abdomen. (B) outlines the nerve bundle and the plastic suction electrode close by the nerve. (C) The segmental nerve is pulled into the suction electrode, which is outlined in blue.
One is now ready to record the electrical responses from the MROs.
|Angle(°)||# of Action Potentials in 1 second after 3 seconds||# of Action Potentials in 1 second after 10 seconds|
Questions to think about while conducting these experiments are: Is there a pattern and consistent response to the extension and flexion movements of the joint? What sort of responses are evoked by pinning or holding the telson at various fixed positions? Is that response consistent when repeated?
Make careful notes of the kinds of responses observed. After you are satisfied with your observations, make permanent records of this activity by saving the data files. Once satisfied with the observations that you have made in segment 3, move on to further recordings from nerves in segments 4 or 5 or on the other side of segment 3 to observe the activity.
One may wish to determine if neuromodulators (octopamine, serotonin, and proctolin) or other compounds or an altered composition in the ionic nature of the saline produces varied responses from those obtained in the defined saline.
3.2) Staining with methylene Blue
One may be able to dissect out the muscle (See appendix) to view the MROs with a staining technique. Take the preparation and pour the crayfish saline out. Place approximately 5 mLs of methylene blue solution into the preparation and gently swirl the dish for a few minutes. Then pour the excess methylene blue into the waste container and pour fresh saline onto the preparation. Now place the dish under the microscope to start dissecting the muscle to view the MROs. Cut the segment along the rib (lateral to midsagittal) by placing one part of the scissors under the muscle and pulling up as you cut along the muscle. Once the DEL 1 and 2 muscles are cut then peel the muscle back and a thin layer of muscle (SEM) should be observed. The MROs are the last two medial fibers lying parallel to the helix muscle (Figure 11).
Figure 11: The schematic of an abdominal segment illustrates the muscle groups (A) and a stained preparation with methylene blue (B) helps to delineate the muscle groups in an intact preparation. The outlined area in A is shown in B with an enlarged view. In B, the DEL1 and 2 muscle groups are not cut away as shown in the lower half of the schematic as shown in A.
For student exercises one might wish to have the students answer the following questions:
The details provided in the associated movie and text have provided key steps in order to sufficiently record the activity in the MRO of the crayfish in situ. One goal of our report is to increase the awareness in the potential for this preparation in student run investigative laboratories to teach fundamental concepts in sensory physiology. The preparations are very robust in viability while being bathed in a minimal saline.
The motor control on the MRO muscles has been identified but the regulation in transmission and the potential of synaptic plasticity as well as efficacy of transmission for the excitatory and inhibitory neurons remains an open area for investigation (Elekes and Florey, 1987a,b; Florey and Florey, 1955; Kuffler, 1954; Kuffler and Eyzaguirre, 1955).
This preparation can be used to investigate a number of experimental conditions as well as the natural range of locomotion within the animal to better understand the biology of the MRO for primary research as well as demonstrative purposes. The biophysical properties of these sensory neurons has in part been addressed in their nature of adaptation in the neural activity with a maintained stimulus (Brown et al, 1978; Edwards et al., 1981; Purali, 1997; Rydqvist and Purali,1991; Rydqvist and Swerup, 1991). However, only a few reports address neuromodulation on these sensory receptors and associated muscle fibers (Cooper et al., 2003; Pasztor and Macmillan, 1990). The reports deal with only a few of the many compounds that are known to be present in the hemolymph. Many modulators and cocktails of modulators remain to be examined on the MRO complex (muscles and neurons). Pasztor and Macmillan (1990) did examine the neuromodulators 5-HT and octopamine on the activity of MROs among various crustacean species and noted that there are species differences. They did not examine in detail the long-term influences of these neuromodulators nor the effects on activity at different static positions of the MRO.
This type of preparation can aid in understanding the basis of sensory perception and regulation of neural processing which is important in rehabilitation and disease management for humans with motor unit abnormities (Patel et al., 2009; Rabin et al., 2009; Marino et al., 2010). The various types of input and firing patterns for monitoring joint movements in accessible invertebrate preparations can be used in robotics/prosthesis (Macmillan and Patullo, 2001). There are still many questions awaiting answers in this preparation that can be beneficial in a number of ways.
No conflicts of interest declared.
Supported by University of Kentucky, Department of Biology, Office of Undergraduate Studies and College of Arts & Sciences.
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