Name: proprioception and tension receptors in crab limbs: student laboratory exercises
Uploaded: 2013-10-24T17:00:00
Duration: 12 min 58 s
Description: Watch this Scientific Journal Video about Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises at JoVE.com

The authors are grateful for the artistic contributions of Hyewon Cooper.

1. Dissection and Recording Electrical Activity from the Propodite-dactylopodite (PD) Nerve
<ol>
	<li>Hold the crab across the carapace from behind, and avoiding the claws, pinch the proximal part of the meropodite with forceps. The leg will autotomize to prevent the animal from bleeding to death. Use caution when handling blue crabs as they are rather aggressive and very fast. 
	<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig1.jpg" width="500px" /> 
	Figure 1. First walking leg of a crab. The anatomical location of chordotonal organs (hatched regions) are superimposed on this schematic. The double arrow head indicates where to transect the leg for the PD nerve experiments. <a href="https://www.jove.com/files/ftp_upload/51050/51050fig1highres.jpg" target="_blank">Click here to view larger image</a>.</li>
	<li>Make a cut between the propodite and carpodite. Discard the carpopodite and the attached meropodite (Figure 1).</li>
	<li>Cut a large window in the cuticle on the pigmented (lateral) side of the propodite with a scalpel with a #11 blade (Figure 2 and 3). Note: Do not cut deeply.</li>
	<li>Remove the cuticle layer by sliding the scalpel blade beneath and parallel to the cuticle. This severs the muscle fibers attached to the cuticle.</li>
	<li>Using the same technique cut a smaller window on the pigmentless (medial) side of the propodite, but leave the condyle (the socket joint or hinge between segments) attachment intact. 
	<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig2.jpg" width="500px" /> 
	Figure 2. Cut along the dotted line on the propodite. <a href="https://www.jove.com/files/ftp_upload/51050/51050fig2highres.jpg" target="_blank">Click here to view larger image</a>. 
	<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig3.jpg" width="500px" /> 
	Figure 3. Expose the nerve in the window (the arrows outline the nerve bundle). <a href="https://www.jove.com/files/ftp_upload/51050/51050fig3highres.jpg" target="_blank">Click here to view larger image</a>.</li>
	<li>Prepare a Sylgard-lined dish containing crab saline to pin the preparation down. Note: See Table 1 for species specific saline recipes.</li>
	<li>Locate the PD organ by carefully probing with the fire-polished glass needles. The elastic strand spanning the joint has a silver appearance.</li>
	<li>Remove muscle fibers that obscure your view of the organ from both sides of the tendon. Be very careful not to injure the PD organ or its nerve.</li>
	<li>Once this has been accomplished, firmly reattach the preparation to the dish with the pigment side (lateral) facing up (Figure 4). 
	<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig4.jpg" width="500px" /> 
	Figure 4. Exposed PD organ and nerve. <a href="https://www.jove.com/files/ftp_upload/51050/51050fig4highres.jpg" target="_blank">Click here to view larger image</a>.</li>
	<li>Follow the PD organ nerve in the propodite as far proximally as possible in order to free-up a long length of nerve (1.5 cm) for recording purposes. This is best done while the PD nerve is still attached to the main leg nerve.</li>
	<li>After separating the PD nerve from the main leg nerve with the aid of glass needles, sever the PD nerve proximally with the iris scissors. Note: Do not stretch or pull on the nerve during the dissection.</li>
	<li>Move the dactylopodite to an extended and fully flexed position. Take note about where the extreme flexion and extension positions are and a half-way point for later use.</li>
	<li>Place a ground wire inside the saline bath.</li>
	<li>Turn on the electrophysiology recording hardware/software. Note:&#160;Our setup has been described previously23.</li>
	<li>Position the microscope so that it is overlooking the microscope stage. Once the prep dish is placed on the stage, you will need to adjust the position of the high intensity illuminator beam to best visualize the preparation.</li>
	<li>Position the micromanipulator so the attached suction electrode assembly will have easy access to the saline bath and preparation. The suction electrode is constructed as shown in an online video24.</li>
	<li>To detect neural activity, draw the cut end of the PD nerve into the suction electrode.</li>
	<li>Move the dactyl throughout extended and flexed positions for several cycles with the aid of a glass probe or wooden dowel.</li>
	<li>Next observe activity when the dactyl is pinned in the extended, flexed, and mid positions.</li>
</ol>
2. Recording Electrical Activity from the Tension Nerve while Monitoring Force Generation
<ol>
	<li>Determine the lateral and medial side of the leg. The medial side has a soft texture that can be felt by pinching gently in the meropodite region with a fingernail.</li>
	<li>Place this soft cuticle side up in the dish. The excitor motor nerve that innervates the opener muscle also innervates the stretcher muscle in the carpopodite.</li>
	<li>In order to stimulate the opener muscle the stretcher motor nerve in the carpodite region is isolated and stimulated with a suction electrode. The proximal part of the leg is removed by transecting the meropodite with scissors.</li>
	<li>Remove a section of the cuticle in the carpus region on the inner side (medial side, Figure 5A).</li>
	<li>Cut the apodeme of the bender muscle and remove the muscle carefully as not to pull the main leg nerve out of the leg cavity (Figure 5B, C note the arrows where to bender apodeme is separated). The main leg nerve and a branch to the stretcher muscle can then be observed (Figure 5D).</li>
	<li>Find the nerve branching from the main nerve bundle to the stretcher muscle (Figure 5E, at arrow). This can be cut close to the muscle and pulled into a suction electrode to stimulate (Figures 5F and&#160;G, arrow depicts the branch).</li>
	<li>Tease out a section of the nerve that projects towards the opener without pinching the nerve. Transect the stretcher/opener motor nerve.</li>
	<li>Pull the motor nerve into the suction electrode and then stimulate it.</li>
	<li>To expose the opener muscle and tension nerve remove the closer muscle and the ventral region of the propodite. Cut off the closer muscle with a scalpel with a #11 blade in the propodite (Figure 6). Note: Do not cut deeply in the proximal region because it might damage the opener motor nerve. 
	<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig5.jpg" width="500px" /> 
	Figure 5. Dissection steps for exposing the motor neuron for stimulating the opener muscle. <a href="https://www.jove.com/files/ftp_upload/51050/51050fig5highres.jpg" target="_blank">Click here to view larger image</a>. 
	<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig6.jpg" width="500px" /> 
	Figure 6. Cut the cuticle on the ventral half of the propodite to expose the closer muscle so it can be removed. <a href="https://www.jove.com/files/ftp_upload/51050/51050fig6highres.jpg" target="_blank">Click here to view larger image</a>.</li>
	<li>Exchange the saline with fresh cooled saline throughout the dissection process to keep the neurons alive. For further dissection, place the preparation dish under a dissecting microscope and use fiber-optic illumination.</li>
	<li>Carefully cut the closer tendon from its attachment to the dactyl using sharp-pointed medium-size scissors.</li>
	<li>Be very careful not to disturb the branches to the opener muscle from the main leg nerve which should be clearly visible.</li>
	<li>Remove and discard the closer muscle and tendon.</li>
	<li>Make a hole in the dactyl with a dissection pin. This hole will be used later to hook the metal pin on the tension (force) transducer, but it is necessary to make it at this stage of the procedure.</li>
	<li>Locate the nerve branch that projects to the opener muscle and apodeme in the distal region of the opener muscle. Carefully probe the nerve with the fire-polished glass tool.</li>
	<li>Observe the tension nerve that arises from the distal end of the apodeme and proceeds to the motor nerve bundle.</li>
	<li>To detect neural activity, fill a suction electrode with crab saline and draw the cut end of the opener tension nerve into the electrode. Ensure that the suction electrode fits tightly on the nerve.</li>
	<li>Examine for the neural correlate of passive tension on the opener apodeme. While recording electrical activity from the tension nerve, rotate the dactyl rapidly into an extended joint (i.e. stretching the opener muscle).</li>
	<li>Next, test active force development in relation to stimulation frequency.</li>
	<li>Firmly attach a metal hook so that the tip of the hook goes through a small hole in the dactyl.</li>
	<li>Attach the other end of the metal hook to the force transducer. Note: Make sure the transducer is at a 90&#176; angle each time so that the pin is perpendicular to the transducer for maximal detection of the force generated.</li>
	<li>Pull the hook and dactyl so that it is at about a 45&#176; angle of the opener joint.</li>
	<li>Now stimulate the motor nerve at 100 Hz for 250 msec and measure the force as well as the firing frequency of the tension nerve.</li>
	<li>Place the joint into a fully extended position so that the opener muscle fibers are flaccid.</li>
	<li>Stimulate the motor nerve at 100 Hz for 250 msec and measure the force as well as the firing frequency of the tension nerve.</li>
	<li>Bend/flex the joint so that it is fully flexed (~90&#176;). In this position the muscle fibers of the opener are fully stretched. There may be some force measured by the transducer due to this passive stretch of the muscle.</li>
	<li>Stimulate the motor nerve at 100 Hz for 250 msec and measure the force as well as the firing frequency of the tension nerve.</li>
	<li>After measuring a force, proceed in a series of various frequencies: 20, 40, 60, 80, and 100 Hz for 8-10 sec&#160;in each joint position.</li>
	<li>Measure the response with a quick tension release. Arrange the preparation so that the hook on the force transducer can easily be pushed out of the hole in the dactyl.</li>
	<li>Bend/flex the joint so that it is fully flexed (~90&#176;). Now stimulate the motor nerve at 100 Hz with a continuous stimulation of 5 sec.</li>
	<li>After the first second or less, as tension has built up on the opener muscle, push the pin out of the hole in the dactyl.</li>
	<li>Examine the tension nerve recording before and after release of the pin holding the dactyl.</li>
	<li>Modulatory effects of neuroactive substances such as octopamine, serotonin, and proctolin, which alter the output of the tension neurons, can be determined by adding these molecules to the bathing media over the exposed opener muscle and repeating the experiments.</li>
	<li>Use a pipette and drip 1-2 ml over the preparation.</li>
</ol>
3. Staining Peripheral Nervous System Structures in Crustacean Walking Legs
<ol>
	<li>Methylene blue technique
	<ol>
		<li>Dilute one part methylene blue chloride stock solution (0.25%) with two parts of distilled water.</li>
		<li>Add this mixture to five parts of buffered saline. Thoroughly irrigate the area of interest.</li>
		<li>Dissect a leg according to the protocol in section 1. Incubate the preparation in the methylene blue solution at 12-13 &#176;C.</li>
		<li>Examine the preparation every 10-15 min&#160;with the dissecting microscope using low intensity illumination to follow the progress of the staining. In some cases staining will be completed in an hour, in others it may take overnight.</li>
	</ol>
	</li>
	<li>4-di-2-ASP technique
	<ol>
		<li>Fluorescent dyes can be used to back-fill the PD neuron or directly stain the preparation from the bath. However a suitable microscope with abilities to view the fluorescent stain is required.</li>
		<li>Dissect a leg according to a protocol in section 1.</li>
		<li>Incubate the preparation in a 10 &#181;M concentration of 4-Di-2-ASP solution and leave the preparation in the refrigerator for 15 min. Use just enough solution to cover the preparation.</li>
		<li>Photograph the preparations quickly and avoid over exposure to the mercury light. This fluorescent dye fades away relatively quickly.</li>
	</ol>
	</li>
	<li>Cobalt chloride technique
	<ol>
		<li>Expose the PD nerve according to the protocol in section 1 and keep it immersed in cold saline.</li>
		<li>Make a petroleum jelly well to hold the CoCl2. If any CoCl2 spills into the saline bath the entire preparation will stain black, and the preparation should be discarded.</li>
		<li>Slip a small cut of a polystyrene sheet, to make a plastic platform, and pin it in such a manner that it will not float away or become immersed in the saline bath (Figure 7). 
		<img alt="Figure 7" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig7.jpg" width="500px" /> 
		Figure 7. Polystyrene sheet with pins to hold in place in dish. <a href="https://www.jove.com/files/ftp_upload/51050/51050fig7highres.jpg" target="_blank">Click here to view larger image</a>.</li>
		<li>Eject petroleum jelly from a fine hypodermic needle fastened to a disposable syringe, then make a barrier (a circle might work well), which should be about 1-1.5 mm high except for a shallow &#34;V&#34; at midpoint where the nerve will be draped across, on top of the slip of polystyrene.</li>
		<li>Make a small puddle of saline on either side of the barrier near the &#34;V&#34;. Taking care not to stretch or pinch the nerve, lift the nerve carefully from the dish and place it in the saline puddle in the petroleum jelly well.</li>
		<li>Work quickly so that the section of nerve does not dry out, carefully eject petroleum jelly to cover the exposed nerve. Now test the barrier with saline and make sure it is not leaking.</li>
		<li>Blot away saline on the inside of the barrier and see if it fills up when the bath saline is high around the wall of petroleum jelly to be sure that the two sides are isolated from one another (Figure 8). 
		<img alt="Figure 8" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig8.jpg" width="500px" /> 
		Figure 8. Petroleum jelly well with saline and PD nerve spanning the well. The PD nerve has not been cut yet. <a href="https://www.jove.com/files/ftp_upload/51050/51050fig8highres.jpg" target="_blank">Click here to view larger image</a>.</li>
		<li>Wick away the saline in the well using a piece of tissue paper. Avoid letting the paper wrap up the nerve.</li>
		<li>Make a new puddle using a few small drops of distilled water, and then cut the nerve end. Be very careful not to pull the nerve through the barrier when making the cut. The osmotic shock of the distilled water will &#34;balloon&#34; the axons.</li>
		<li>Within 30 sec&#160;add a small drop of CoCl2 to the water, soak up this solution, and then add enough CoCl2 to form a puddle over this shortened section of nerve (Figure 9). Preparations are best kept refrigerated at 13&#160;&#176;C for 12-24 hr. 
		<img alt="Figure 9" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig9.jpg" width="500px" /> 
		Figure 9. The cut PD nerve being exposed to CoCl2. The cut nerve swells up in the presence of water that facilitates and accelerates the movement of dye molecules through the axons into the neuron cell bodies. <a href="https://www.jove.com/files/ftp_upload/51050/51050fig9highres.jpg" target="_blank">Click here to view larger image</a>.</li>
		<li>Remove the humidifying puddles. Blot away the cobalt solution using a tissue and wash away the remnants of cobalt with several changes of saline.</li>
		<li>Transfer the isolated preparation to a small glass Petri dish containing about 10 ml of crab saline. The neurons are washed and the following steps are performed in situ. Good metal tools are not to be used to handle the preparation after this step (you should use specific tools that are not to be used at a later time for physiology).</li>
		<li>Add 1-2 drops of ammonium sulfide (NH4)2S to the saline. Cap the (NH4)2S bottle tightly and place back into the hood. Observe the reaction in the preparation under a dissecting microscope</li>
		<li>Within 1-2 min, cobalt-filled neurons and their processes should begin to appear because they will stain black.</li>
		<li>After 5-10 min, replace the development solution with fresh saline.</li>
		<li>Make certain that the development solution you have poured into the sink drain is followed by running tap water for a few minutes or in a waste bottle with a lid.</li>
		<li>Pour out the saline and fix the nerve preparation for about 15 min with two changes of Bouin&#8217;s solution fixative. For larger tissues like the opener muscle (to stain tension neurons), increase the duration of fixation to 30 min.</li>
		<li>Dehydrate in a series of ascending order of ethanol concentration beginning at 70% (i.e. 70%, 80%, 90%, 100%). About 10 min at each concentration is sufficient for small tissues.</li>
		<li>After about 10-15 min in two changes of 100% ethanol, clear the tissue by replacing ethanol with 100% methyl salicylate. The preparation will stay in this solution permanently for repeated viewing. With time the filled cells will become more apparent because the surrounding tissue will become clearer. Intensification methods can be used to help prevent fading over time25.</li>
		<li>Use the same process to fill the tension nerve with CoCl2. However, change ethanol solution multiple times in each ethanol dehydration step and incubate the preparation inside alcohol for about 20 min for each step to thoroughly dehydrate the muscles and allow them to clear well.</li>
	</ol>
	</li>
</ol>

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Sci. 245 (725), 291-325 (1962).</li><li>Whitear, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fine+structure+of+crustacean+proprioceptors.+II.+The+thoracico-coxal+organs+in+Carcinus,+Pagurus+and+Astacus.">The fine structure of crustacean proprioceptors. II. The thoracico-coxal organs in Carcinus, Pagurus and Astacus.</a> Philos. Trans. R. Soc. Lond. B Biol. Sci. 248 (752), 437-456 (1965).</li><li>Leksrisawat, B., Cooper, A. S., Gilberts, A. B., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+Receptor+Organs+in+the+Crayfish+Abdomen:+A+Student+Laboratory+Exercise+in+Proprioception.">Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception.</a> J. Vis. Exp. (45), e2323 (2010).</li><li>Baierlein, B., Thurow, A. L., Atwood, H. L., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+Potentials,+Synaptic+Responses,+Neuronal+Circuitry,+Neuromodulation+and+Muscle+Histology+Using+the+Crayfish:+Student+Laboratory+Exercises.">Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises.</a> J. Vis. Exp. (47), e2322 (2011).</li><li>Delaney, K., Gelperin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+interneurons+controlling+fictive+feeding+in+Limaxmaximus;+I.+Anatomy+and+criteria+for+re-identification.">Cerebral interneurons controlling fictive feeding in Limaxmaximus; I. Anatomy and criteria for re-identification.</a> J. Comp. Physiol. A. 166, 297-310 (1990).</li><li>Hartman, H. B., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+and+molting+effects+on+a+proprioceptor+organ+in+the+Dungeness+crab,+Cancer+magister.">Regeneration and molting effects on a proprioceptor organ in the Dungeness crab, Cancer magister.</a> J. Neurobiol. 25 (5), 461-471 (1994).</li><li>Hartman, H. B., Austin, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proprioceptor+organs+in+the+antennae+of+Decapoda+Crustacea.">Proprioceptor organs in the antennae of Decapoda Crustacea.</a> J. Comp. Physiol. 81 (2), 187-202 (1972).</li><li>Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+sensory+processes+during+limb+regeneration+in+adult+crayfish.">Development of sensory processes during limb regeneration in adult crayfish.</a> J. Exp. Biol. 201 (Pt 11), 1745-1752 (1998).</li><li>Chung, Y. -. S., Cooper, R. M., Graff, J., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+and+chronic+effect+of+low+temperature+on+survival,+heart+rate+and+neural+function+in+crayfish+(Procambarus+clarkii)+and+prawn+(Macrobrachium+rosenbergii)+species.">The acute and chronic effect of low temperature on survival, heart rate and neural function in crayfish (Procambarus clarkii) and prawn (Macrobrachium rosenbergii) species.</a> Open J. Mol. Int. Physiol. 2 (3), 75-86 (2012).</li><li>Blundon, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+temperature+and+thermal+history+on+neuromuscular+properties+of+two+crustacean+species.">Effects of temperature and thermal history on neuromuscular properties of two crustacean species.</a> J. Comp. Physiol. B. 158 (6), 689-696 (1989).</li><li>Cooper, R. M., Schapker, H., Adami, H., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+and+ventilatory+measures+in+crayfish+during+copulation.">Heart and ventilatory measures in crayfish during copulation.</a> Open J. Mol. Int. Physiol. 1 (3), 36-42 (2011).</li><li>Bierbower, S. M., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanistic+action+of+carbon+dioxide+on+a+neural+circuit+and+NMJ+communication.">The mechanistic action of carbon dioxide on a neural circuit and NMJ communication.</a> Journal of Experimental Zoology. , (2013).</li><li>Bierbower, S. M., Shuranova, Z. P., Viele, K., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+systems+and+environmental+change+on+behavior+during+social+interactions.">Sensory systems and environmental change on behavior during social interactions.</a> Brain Behav. 3 (1), 4-13 (2013).</li></ol>

The authors declare that they have no competing financial interests.

The purpose of this set of experiments is 1) to teach and exhibit the fundamental principles of extracellular recordings from an identifiable proprioceptive organ and tension nerve and 2) to stress the importance of anatomical mapping in relation to physiological function of particular sensory systems. This experimental approach and the animal models utilized are inexpensive and relatively easy to conduct in neurophysiology teaching laboratories.
The neurons of chordotonal organs are of two specific functional types, those that respond to movement and those responding to static positions. Single cell recordings from a variety of chordotonal organs, no matter which joint is examined, have shown this to be the case3,5. Indeed, chordotonal organs associated with the antennal joints of lobsters reveal the same two sensory types and basic anatomy27.&#160;In addition to there being two neuron types (movement and position), the neurons share the same anatomical arrangement on their respective elastic strands. The large somata located proximally on the strand tend to belong to the dynamic movement sensitive neurons. Neurons that signal static positions have small somata and are located distally. These cells are tonically active. The PD joint only contains a single chordotonal organ while there are two chordotonal organs in the carpus-propodus (CP) and merus-carpus (MC) joints.
The dissection to expose proprioceptive structures in blue crabs (C. sapidus) for electrophysiological recording requires a strategy that allows joint movements to take place in the natural positions while recording from sensory neurons. The tension nerve for the opener muscle in the walking leg is a very fine nerve made up of several neurons. Unless care is taken, the tension nerve as well as the motor nerve innervating the muscle to be stimulated, can be damaged during this dissection. For optimal recordings the suction electrodes need to be tailored to the size of the nerve. Recordings are readily accessible in a student laboratory using a 30-40X magnification dissecting microscope and low-end micromanipulators.
Future experiments that would be interesting to pursue with the joint chordotonal organs would be to examine the structural and physiological profiles during leg regeneration in various species at different stages in the life cycle as a follow up to an initial study that used Cancer magister19,26. Questions remaining to be addressed are 1) does the distribution and organization of regenerated neurons depend on the age of the animal when regenerating a limb, 2) are the axonal projections to the CNS (ventral nerve cord) in a regenerating limb functional or does it take time and joint use to establish functional connections, and 3) what happens to the severed axons proximal to the autotomy plane when the limb is autotomized?28
Crustaceans conform to environmental conditions and their surrounding temperature, but it is unclear how they maintain coordination within a neural circuit as neurons alter their activity in response to temperature changes. A slow rate of change might allow the animal some time for acclimatization whereas a rapid change may not29,30.&#160;Physiological changes in pH or osmolarity due to metabolism, behavior31, or environmental impact may present similar challenges to neural circuits involved in proprioception. These crustacean preparations are ideal for addressing these types of problems because their function is well characterized at a single cell level.
In this protocol we have demonstrated the physiological importance of tension neurons in monitoring force generated by the opener muscle. These tension receptors can be traced to their location within the apodeme by using staining procedures. These neurons, as in mammals, detect force at various levels and recruit additional neurons as the force increases. The frequency in activity is related to the stimulation frequency of the motor neuron until saturation in reception is reached. Using a quick release protocol with the flexed dactylus joint, tension activity quickly disappears but then returns upon regaining tension in a fully extended joint. This is a classic experimental procedure to illustrate the force measured by tension receptors. Various neuromodulators can be applied to the preparation to see how it effects the development of force and neuronal response. One of the important aspects is how the neural responses are processed and integrated in the central nervous system and their impact on activity of motor neurons. The techniques we have shown allow one to start to address more information about the tension (sensory) nerve-motor neuron circuit function, i.e.&#160;signal in an intact leg to the ganglion and back to the muscle.
The staining procedures demonstrated are key to understanding the physiology of sensory neurons that innervate proprioceptive organs. Anatomical arrangement of the neurons based on function and the size of the soma are similar in the various chordotonal organs within the crab legs. It is not known if similar neuronal arrangements are also found in other crustacean species or insects. Combining physiological recordings from single cells and mapping the location allows direct structure function relationships. The long term preservation of the anatomical arrangement with CoCl2 staining and fixation allows one to repetitively make measures and assess the structural arrangement.
Proprioception and tension reception of skeletal muscles are sensory modalities that enable coordinated behaviors and responses to external and internal environment for articulated animals in a variety of skeletal muscle configurations. The muscle receptor organ in the abdomen of the crayfish is another well-documented preparation (see the Crawdad Project; <a href="http://www.crawdad.cornell.edu/">http://www.crawdad.cornell.edu/</a>) for teaching purposes of proprioception with only two neurons per abdominal hemi-segment23.&#160;Being able to record from single neurons to sensory nerve bundles provides further details that aid in understanding the basic principles of sensory reception. These relatively simple crustacean preparations allow one to address fundamental aspects of proprioception and tension monitoring, with the potential to determine the neural circuits that enable central integration of proprioceptive and other sensory inputs9-12, 32, 33.

Proprioception is the sensation of limb position and movement that enables coordinated motor behavior. Proprioceptors consist of position (static) and movement (kinesthetic) receptors. In insects and crustaceans, chordotonal organs are the structures that provide that information to the CNS1. Not all chordotonal organs span a joint but they can still monitor joint movements due to their attachment on the apodemes (tendon like structures) which span the joint and move in association with the skeletal muscle and joint articulation. Crab legs have six joints, each having one or two chordotonal organs2. Typically a chordotonal organ has 60-100 or more sensory neurons embedded within an elastic strand, neurons that signal static joint position, direction and speed of movement3-6. The input from chordotonal organs at each joint and leg is then centrally processed allowing coordinated movements by the animal.
The forces that leg muscles produce during isometric and isotonic contractions are detected by tension receptors associated with muscle fibers and their attachments to apodemes7-9.&#160;In the crustacean walking leg protocols that follow we present methodology for recordings from primary sensory neurons that monitor proprioception and the neurons that respond to forces generated by muscle fibers. A technique for activating leg movements and quantifying force generation is also presented, as well as anatomical techniques that can be used to characterize the arrangement of these peripheral nervous system structures.
The procedures demonstrated below enable structural and functional analysis of the neurons that innervate both types of receptors relative to their location on a chordotonal elastic strand and apodeme. To illustrate, we use the propodite-dactylopodite (PD) chordotonal organ, the organ that spans the distal most segment of the crab leg3. Though detailed electrophysiological studies began in the 1930s and are still being carried out today, some aspects have become known about the segmental connections of proprioceptors in the various joints and their roles in coordinated control of muscles10-16.&#160;Establishing the structure-function relationship between the proprioceptive organs, muscles and the nervous system will further help define these roles. For instance, labeling the somata and distal endings of tension neurons inserted into the apodeme will reveal their location relative to muscle fibers8,17-21.
We present three staining techniques for crustacean legs that can be used in research or academic laboratories. Methylene blue staining provides suitable contrast for muscles and nerves and is recommended as a simple technique for students to learn anatomy. Labs that have fluorescence microscopy setups can accomplish more selective neuronal staining by briefly exposing the nerves to the vital dye 4-di-2-ASP. The third alternative is CoCl2 backfill, which stains and fixes the neurons, and does not require fluorescence imaging. Though it is labor and time intensive, this staining process gives high contrast and specificity for the nerves that are filled. Together these techniques can be used for comparing various chordotonal organs, not only within a limb or between limbs, but also among other crustacean and insect species20-22. Blue crabs (C. sapidus) used in physiological recordings and for anatomical staining are readily available all around the Southern and Southeastern border of the United States. This species serves as a representative of the chordotonal and tension nerve arrangements found in most crabs. Laboratories on the west coast will prefer to use the much larger Dungeness crab (Cancer magister) for these experiments.

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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sylgard</td>
			<td style="width:184px;">Dow Corning</td>
			<td style="width:119px;">182 silicone kit</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7653</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9333</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">C5670</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES acid</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;">3375</td>
			<td style="width:167px;">Free acid, crystalline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES base</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D-Glucose</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;">G7021</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">MgSO4&#8226;7H2O</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;">M2643</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Na2SO4&#8226;10H2O</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;">246980</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bouin&#8217;s solution fixative</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;">HT10-1-32</td>
			<td style="width:167px;">Caution: Hazardous material (Special shipping cost required)</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">CoCl2</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Caution: Hazardous material. Please follow proper disposal according to local and federal regulations.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Methylene blue chloride</td>
			<td style="width:184px;">Matheson Co., Inc</td>
			<td style="width:119px;">Basic Blue 9, C.I. 52015</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">4-Di-2-ASP</td>
			<td style="width:184px;">Molecular Probes</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">4-(4-diethylaminostyryl)-N-methylpyridinium iodide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bleach</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">To chloride silver wire</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaOH</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">221465</td>
			<td style="width:167px;">To adjust pH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HCl</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">H1758</td>
			<td style="width:167px;">To adjust pH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Materials</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting tools</td>
			<td style="width:184px;">World Precision Instruments</td>
			<td style="width:119px;">assortment</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Intracellular electrode probe</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Faraday cage</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insect Pins</td>
			<td style="width:184px;">Fine Science Tools, Inc</td>
			<td style="width:119px;">26001-60</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting microscope (100X)</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fiber optic lamp</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Small adjustable mirror</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">To direct light beam toward the preparation.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass electrodes</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">CLS7095B5X</td>
			<td style="width:167px;">Box of 200, suction electrodes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micromanipulator</td>
			<td style="width:184px;">World Precision Instruments</td>
			<td style="width:119px;">MD4-M3-R</td>
			<td style="width:167px;">Can fix for base or on a metal rod</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Raised preparation stand</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Silver wire (10/1,000 inch)</td>
			<td style="width:184px;">A-M Systems</td>
			<td style="width:119px;">782500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Computer</td>
			<td style="width:184px;">Any company</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AC/DC differential amplifier</td>
			<td style="width:184px;">A-M Systems</td>
			<td style="width:119px;">Model 3000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PowerLab 26T</td>
			<td style="width:184px;">AD Instruments</td>
			<td style="width:119px;">27T</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Force transducer</td>
			<td style="width:184px;">AD Instruments</td>
			<td style="width:119px;">0-50g</td>
			<td style="width:167px;">MLTF050/ST</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Head stage</td>
			<td style="width:184px;">AD Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Comes with AC/DC amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LabChart7</td>
			<td style="width:184px;">AD Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electrical leads</td>
			<td style="width:184px;">Any company</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass tools</td>
			<td style="width:184px;">Make yourself</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">For manipulating nerves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cable and connectors</td>
			<td style="width:184px;">Any company</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipettes with bulbs</td>
			<td style="width:184px;">Fisher Scientific</td>
			<td style="width:119px;">13-711-7</td>
			<td style="width:167px;">Box of 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Beakers</td>
			<td style="width:184px;">Any company</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Wax or modeling clay</td>
			<td style="width:184px;">Any company or local stores</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stimulator</td>
			<td style="width:184px;">Grass Instruments</td>
			<td style="width:119px;">SD9 or S88</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="84" rowspan="2" style="height:84px;width:216px;">Plastic tip for suction electrode</td>
			<td rowspan="2" style="width:184px;">Local hardware store (Watt&#8217;s brand)</td>
			<td rowspan="2" style="width:119px;">&#188; inch OD x 0.170 inch ID</td>
			<td style="width:167px;">Cut in small pieces. Pull out over a flame and cut back the tip to the correct size</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:167px;"></td>
		</tr>
	</tbody>
</table>

proprioception and tension receptors in crab limbs: student laboratory exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

When the PD organ is stretched by fully extending the joint, activity in the PD nerve is robust during the movement as shown for the first second in Figure 10. Some activity remains while it is held still in the open position. This activity is from the static position sensitive neurons (second half of the recording shown in Figure 10). Movement evokes a response during displacement, and the firing is mostly present during the stretching of the chordotonal strand (Figure 11).
Further analysis of the spikes can be readily approached by sorting the relative amplitudes. This is an approach to demonstrate different populations of sensory neurons being recruited for positions or types of movements5.&#160;Typical amplitudes range from 0.25-1.5 mV, but these values are dependent on the resistance (i.e.&#160;tightness) of the suction electrode seal. Frequency of spikes in the various size ranges can also be graphically represented for analysis.
Forces generated by the opener muscle with respect to the stimulation frequency can be compared by superimposing the respective voltage-time traces on top of each other (Figures 13A and&#160;B). This can also be performed for each joint position at each given stimulation frequency. Activity of the tension nerve can then be correlated with the amount of relative force generated at each stimulation frequency, and for each joint position. As in the PD nerve, a variety of spike amplitudes are seen in response to contraction of the opener muscle (Figure 13C).
Anatomical arrangement of neurons in the walking leg is clearly observed with methylene blue staining (Figure 14). Note the elastic chordotonal strand and tension neuron that is close to the apodeme. Several somata with different diameters and specific locations are also visible in this figure. The entire course of the tension nerve and stretcher motor nerve are shown in Figure 15. Individual neurons of the PD nerve are shown with higher contrast using 4-Di-2-ASP (Figure 16) and CoCl2 (Figure 17) backfill techniques. At high magnification the sensory endings can be seen inside of the supportive scolopales (Figures 16B)21,22,26.
<img alt="Figure 10" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig10.jpg" width="500px" /> 
Figure 10. Move and hold at 0&#176;. The dynamic neurons are robust in firing during the movement and spikes from static sensitive neurons are present while the joint is held open. <a href="https://www.jove.com/files/ftp_upload/51050/51050fig10highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 11" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig11.jpg" width="500px" /> 
Figure 11. Rapid open and closing from fully flexed to extended (90-0&#176;) position. <a href="https://www.jove.com/files/ftp_upload/51050/51050fig11highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 12" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig12.jpg" width="500px" /> 
Figure 12. Extracellular spikes recorded from the PD nerve. The joint is fully extended and then quickly moved to a &#189; flexed position and then held still. Notice the activity during the movement and decreased activity when static. <a href="https://www.jove.com/files/ftp_upload/51050/51050fig12highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 13" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig13.jpg" width="500px" /> 
Figure 13. The relative forces that are developed with the joint fully flexed and stimulated at the various frequencies. (A) Voltage-time traces from the force transducer are shown with each stimulation frequency. (B) The traces in panel A are superimposed in different colors for ease in comparison. (C) Voltage-time traces of electrical activity recorded from the tension nerve when the motor nerve was stimulated at 80 Hz. Note the regular pattern of the stimulus artifacts as compared to the neural activity. Also, note the various amplitudes of the neural responses. <a href="https://www.jove.com/files/ftp_upload/51050/51050fig13highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 14" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig14.jpg" width="500px" /> 
Figure 14. Methylene blue stain of the walking leg preparation. Individual somas are shown with their sensory endings projecting into the elastic strand. Close to the apodeme a tension neuron is shown. <a href="https://www.jove.com/files/ftp_upload/51050/51050fig14highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 15" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig15.jpg" width="500px" /> 
Figure 15. The tension nerve arising from distal end (red arrows) and joining the motor nerve (green arrow). <a href="https://www.jove.com/files/ftp_upload/51050/51050fig15highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 16" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig16.jpg" width="500px" /> 
Figure 16. (A) A back-fill of the PD nerve in&#160;Cancer magister with 4-Di-2-ASP. (B) Higher magnification of sensory endings.&#160;<a href="https://www.jove.com/files/ftp_upload/51050/51050fig16highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 17" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig17.jpg" width="500px" /> 
Figure 17.&#160;Neurons that were filled with CoCl2 and processed (A). Traced outline of the stained preparation shown (B). <a href="https://www.jove.com/files/ftp_upload/51050/51050fig17highres.jpg" target="_blank">Click here to view larger image</a>.
<table border="0" cellpadding="0" cellspacing="0" width="392">
	<colgroup>
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Saline</td>
			<td style="width:157px;">g/L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:235px;">NaCl</td>
			<td style="width:157px;">27.29</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:235px;">KCl</td>
			<td style="width:157px;">0.81</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">MgSO4&#8226;7H2O</td>
			<td style="width:157px;">4.81</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">CaCl2&#8226;2H2O</td>
			<td style="width:157px;">1.85</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">Na2SO4&#8226;10H2O</td>
			<td style="width:157px;">0.97</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:235px;">Dextrose (D-Glucose)</td>
			<td style="width:157px;">1.982</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:235px;">HEPES acid</td>
			<td style="width:157px;">0.476</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:235px;">HEPES salt</td>
			<td style="width:157px;">2.08</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:235px;">Adjust to pH 8.1 with NaOH or HCl</td>
			<td style="width:157px;"></td>
		</tr>
	</tbody>
</table>
Table 1. Recipes for crab saline.

Watch this Scientific Journal Video about Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises at JoVE.com

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

proprioception-tension-receptors-crab-limbs-student-laboratory

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SCIENTISTS IN ACTION

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats

The most insidious aspect of drug addiction is the high propensity for relapse. Animal models of relapse, known as reinstatement procedures, have been used extensively to study the neurobiology and phenomenology of relapse to drug use. Although procedural variations have emerged over the past several decades, the most conventional reinstatement procedures are based on the drug self-administration (SA) model. In this model, an animal is trained to perform an operant response to obtain drug. Subsequently, the behavior is extinguished by withholding response-contingent reinforcement. Reinstatement of drug seeking is then triggered by a discrete event, such as an injection of the training drug, re-exposure to drug-associated cues, or exposure to a stressor 1.
Reinstatement procedures were originally developed to study the ability of acute non-contingent exposure to the training drug to reinstate drug seeking in rats and monkeys 1, 2. Reinstatement procedures have since been modified to study the role of environmental stimuli, including drug-associated cues and exposure to various forms of stress, in relapse to drug seeking 1, 3, 4.
Over the past 15 years, a major focus of the reinstatement literature has been on the role of stress in drug relapse. One of the most commonly used forms of stress for studying this relationship is acute exposures to mild, intermittent, electric footshocks. The ability of footshock stress to induce reinstatement of drug seeking was originally demonstrated by Shaham and colleagues (1995) in rats with a history of intravenous heroin SA5. Subsequently, the effect was generalized to rats with histories of intravenous cocaine, methamphetamine, and nicotine SA, as well as oral ethanol SA 3, 6.
Although footshock-induced reinstatement of drug seeking can be achieved reliably and robustly, it is an effect that tends to be sensitive to certain parametrical variables. These include the arrangement of extinction and reinstatement test sessions, the intensity and duration of footshock stress, and the presence of drug-associated cues during extinction and testing for reinstatement. Here we present a protocol for footshock-induced reinstatement of cocaine seeking that we have used with consistent success to study the relationship between stress and cocaine seeking.

Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.

The most insidious aspect of drug addiction is the high propensity for relapse.  Animal models of relapse, known as reinstatement procedures, have been ...

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.

The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception

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.

The primary purpose of this experiment is to understand how primary sensory neurons convey information of joint movements and positions as proprioceptive information for an animal. An additional objective of this report is present the anatomy of the preparation by dissection and viewing of neurons under a dissecting microscope.

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive ...

Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings with a force transducer to monitor the strength of contraction. In addition, we demonstrate how to visually monitor peristaltic activity, which can be used as a bioassay for various peptides, biogenic amines and neurotransmitters. This preparation is amenable to student laboratories in physiology and for demonstrating pharmacological concepts to students. This preparation has been in use for over 100 years, and it still offers much as a model for investigating the generation and regulation of peristaltic rhythms and for describing the mechanisms underlying their modulation. The pharmacological assays and receptor sub-typing that were started over 50 years ago on the hindgut still contribute to research today. This robust preparation is well suited to training students in physiology and pharmacology.

In this report we demonstrate techniques that can be used to investigate the biology of the crayfish hindgut. We show how to dissect a crayfish abdomen and study the associated anatomy, physiology and modulation of activity. The peristaltic activity and strength of contractions are measured using a force transducer.

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings ...

Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors (GPCRs) 1 that are postulated to bind volatile odorant molecules and converting the extracellular signal into an intracellular signal by coupling with G protein G&alpha;olf. Binding of the odorants to the receptors is thought to follow a combinatorial rule, that is, one odorant may bind several receptors and one receptor may bind several odorants to varying degrees 2. Biochemical, signaling and ligand binding studies have been conveniently carried out for most GPCRs using heterologous cells. However use of heterologous cells for study of odorant receptors, was precluded for a long time since on transfection they failed to export to the surface. Saito et al have demonstrated single membrane pass Receptor Transporting Protein (RTP) family chaperones show enhanced expression in the olfactory sensory neurons and act as chaperones to traffic odorant receptors to the surface in heterologous cells, when co transfected together 3. To carry out biochemical assays for receptors using heterologous cells, one must first determine if the receptor shows robust surface expression in the cell line. This can be assayed by overexpressing the receptors with the chaperone RTP1S followed by live cell staining to fluorescently label the extracellular domain or a tag in the extracellular domain exclusively. Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.

Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors ...

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping.
Tissue specimens are harvested and stored at -80 &#176;C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in 125I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: &#39;non-specific binding&#39; (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and &#39;total binding&#39; with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 &#181;M) is also present in the incubation buffer to limit 125I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 &#176;C, NSP slides are exposed to 10 &#181;M PD123319 and losartan, while &#39;total binding&#39; slides are exposed to 10 &#181;M PD123319. Slides are then incubated with 125I-SI-Ang II in the presence of PD123319 for &#39;total binding&#39;, and PD123319 and losartan for NSP in assay buffer, followed by several &#39;washes&#39; in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons.
The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to ...