Research supported by NSERC.

1. Socialization
<ol>
	<li>To extinguish existing social ranks, each crayfish is housed for 7 - 14 days in a separate plastic container (30 cm long x 17.5 cm wide x 13 cm deep), filled with filtered, aerated water and a small plastic flower pot which serves as a shelter. If transparent containers are used, a divider (e.g. paper towel) is placed in between containers to ensure crayfish are visually isolated.</li>
	<li>Crayfish in the "Isolated" group remain in isolation for an additional 3 - 14 days. Water is changed and crayfish are fed 3 times weekly.</li>
	<li>To establish a social rank, crayfish are placed in same-sex pairs following the initial isolation. Crayfish are weighed, and body length is measured (rostrum to telson); pairs are size-matched to within 10% weight and length. White acrylic paint is applied to the carapace of each crayfish to identify individuals. (Usually a dot is applied to one member and a line to the other.)</li>
	<li>After a 30 min resting period, each crayfish pair is allowed to interact in a clean, white plastic container (48 cm long x 25 cm wide x 13 cm deep), filled half-way with filtered, aerated water (room temperature). This encounter is either recorded onto VHS tape with a camera connected to a video cassette recorder, or it is recorded onto a computer with a webcam. This interaction allows dominance ranks to develop.</li>
	<li>Following the initial interaction, each crayfish pair is housed together in a larger plastic container (58 cm long x 30 cm wide x 35 cm deep) for an additional 3 - 14 days. This container receives circulating water from a filtration system. A single PVC tube (approx. 10 cm length) is provided for shelter.</li>
</ol>
2. Construction of Test Aquarium and Controls
Method I
<ol>
	<li>To examine responses to a reflective environment, a glass or Plexiglas aquarium (52 cm long x 25 cm wide x 30 cm high) is used (Figure 1).</li>
	<li>One mirror (25 cm long x 20 cm high) is placed at one end of the aquarium and secured with either glue or mounting wax. Two mirrors (26 cm long x 20 cm high) are placed on the sides, abutting the end mirror, and are secured with glue or wax.</li>
	<li>The remaining walls are covered in a matte finish window film. This film removes the reflection but does not interfere with lighting conditions. A light sanding to the film may be required to ensure that the film is completely non-reflective.</li>
</ol>
Method II
<ol start="4">
	<li>To examine responses to visual stimuli provided by a live crayfish ("source crayfish"), a Plexiglas aquarium with two end chambers is used (Figure 2). The aquarium contains a large central compartment (48 cm long x 26 cm wide x 26 cm high) and two smaller end compartments of equal size (12 cm long x 26 cm wide and 26 cm high). The end compartments are separated from the center compartment by transparent Plexiglas, which is sealed to prevent water and chemical cues from passing between compartments.</li>
	<li>The outer perimeter of the entire aquarium is coated in a non-reflective matte plastic, leaving the two barriers at each end transparent.</li>
	<li>As a control, a cube constructed of modeling clay (e.g. Sculptee) is used to provide visual input from an inert object. The cube (3.5 cm x 3.0 cm x 3.3 cm) is the approximate volume of the source crayfish and is painted a similar color red.</li>
</ol>
3. Visual Training
Method I
<ol>
	<li>Crayfish are videotaped in the mirror / matte tank 3 - 14 days following the fight to determine dominance status.</li>
	<li>Crayfish are placed, one each, into a Tupperware container containing aerated, filtered water and a plastic flower pot used for shelter. Crayfish are taken to the testing room and given 30 min to acclimate before testing.</li>
	<li>The test aquarium, half-lined with mirrors and half-lined with non-reflective plastic, is filled with aerated, filtered water to a depth of approximately 20 cm.</li>
	<li>A digital camera is connected via audio/video cables to a VHS videocassette recorder and is placed above the aquarium high enough to view the entire aquarium. Videos are recorded onto VHS tape. Alternatively, a webcam connected to a computer can be used, and videos can be recorded with a movie-making program and stored on DVD.</li>
	<li>One crayfish of the pair is placed gently into the center of the aquarium using a flower pot, and its activity is recorded for 20 min. No person is present in the room during recording, to reduce interference from extraneous visual and auditory inputs. Every effort is made to ensure that the recording environment is quiet.</li>
	<li>Following testing, the crayfish is removed, and the tank is emptied, rinsed and refilled with water.</li>
	<li>To counter-balance experimental conditions, the aquarium is turned around to place the mirrors on the opposite side of the room. This helps to compensate for unknown factors that might cause crayfish to prefer one side of the aquarium.</li>
	<li>The second member of the pair is then tested in the same manner as the first.</li>
</ol>
Method II
<ol start="9">
	<li>As described in Method I, crayfish are brought to the testing room and are videotaped using the same recording equipment.</li>
	<li>All chambers (the middle testing chamber and the two end chambers) are filled with aerated, filtered water to a height of approximately 20 cm.</li>
	<li>A crayfish, approximately the same size as the crayfish being tested, is placed in one end chamber, and the control cube is placed in the other.</li>
	<li>Using the flower pot shelter, the crayfish being tested is gently placed into the center of the aquarium and is videotaped for 20 min.</li>
	<li>Following testing, the aquarium is emptied, rinsed and refilled with water.</li>
	<li>Before testing the second member of a pair, the source crayfish and control cube are moved to the opposite end chambers, to counter balance conditions.</li>
</ol>
4. Analysis of Responses to Visual Stimuli
Method I
<ol>
	<li>So that the experimenter is blind to the social status of the crayfish, behavior in the mirror / matte aquarium is analyzed before analyzing fighting behavior trials.</li>
	<li>The amount of time the crayfish spends on each side of the aquarium is recorded. The crayfish is considered to pass from one side to the next when the coxae of the chelipeds pass the midline of the tank (Figure 3).</li>
	<li>The frequency of the behaviors cornering, turning, crossing, freezing and reverse walking (see Table 1 for descriptions) are also recorded.</li>
	<li>Behaviors exhibited on the reflective side of the tank are compared to behaviors exhibited on the non-reflective side using a paired t-Test for each social group.</li>
</ol>
Method II
<ol start="5">
	<li>As in Method I, videotapes of crayfish behavior are analyzed before analyzing fight trials.</li>
	<li>The amount of time the crayfish spends on either side of the aquarium is recorded as described in Method I.</li>
	<li>The frequency of cornering, end facing, turning, crossing, rearing up and reverse walking (Table 1) are recorded.</li>
	<li>Behaviors performed on the source crayfish side are compared to behaviors performed on the control cube side using a paired t-Test for each social group.</li>
</ol>
5. Analysis of Fights
<ol>
	<li>Recordings of fight behavior during the initial 30 min of pairing are viewed and scored. Each 30 min recording session includes several encounters in which the crayfish make contact using their claws and/or antennae. Within these encounters, the chelae are usually used for pushing, striking or grasping the opponent, and the antennae are sometimes used for whipping the opponent. One crayfish may attempt to flip its opponent and hold him to the floor. Each encounter ends when one crayfish retreats by reverse walking or by flipping the tail to achieve "escape swimming."</li>
	<li>The number of agonistic encounters is recorded, and the identity of the crayfish (determined by the paint marking on the thorax) that retreats from each encounter is also recorded. The crayfish that loses the majority of encounters in each 30 min session is deemed the loser, and the other crayfish is deemed the winner.</li>
</ol>
6. Representative Results
During the 30 minute fighting period, all pairs exhibit agonistic behaviors. Such behaviors include antenna tapping, lunging, pushing, grasping, chelae striking, and flipping the opponent, as reported elsewhere (e.g. May &amp; Mercier 2006, 2007). Eventually one crayfish in each pair consistently retreats and avoids further contact. The retreating crayfish is deemed the loser and is ranked as subordinate; the winning crayfish is ranked as dominant. 
Rank can also be monitored throughout the paired housing period by observing crayfish behavior in the holding tank, which contains only one plastic tube for shelter. The dominant crayfish invariably occupies the shelter, and the subordinate crayfish does not. The subordinate crayfish often walks with the chelae held close together and out in front, and the dominant crayfish typically walks with the chelae raised and further apart (Guiasu &amp; Dunham, 1998). When new food is added to the holding tank, the dominant crayfish usually takes the food first. In our studies with P. clarkii (e.g. May &amp; Mercier 2006, 2007), there are typically no rank reversals after three days of continuous pairing.
In rare cases, one member of the pair dies before the pairing period is complete. This is more likely to occur if the pairing period is long (e.g. 14 days) and if the crayfish are too closely matched by size or weight. Thus, deaths can be avoided by allowing approximately a 10% difference in weight. If one member of a pair dies, data from the other member are not included in the study.
Dominant and subordinate crayfish typically show different responses to the reflective environment. Dominant crayfish consistently spend more time on the reflective side, but subordinate and isolated crayfish do not (May &amp; Mercier, 2006, 2007). Dominant crayfish also exhibit cornering and turning more frequently on the reflective side than on the non-reflective side, but isolates and subordinates typically do not. Such differences between dominant and subordinate crayfish, however, are not established immediately but develop over the 3-14 day pairing period (May &amp; Mercier, 2007).
Preliminary evidence indicates that dominant and subordinate crayfish respond differently to visual cues provided by a live crayfish (source crayfish) after 3 days of pairing (May et al., 2008). Dominant crayfish spend more time on the side of the aquarium with the source crayfish and corner for longer on that side, but subordinate crayfish do not exhibit such differences between the two sides. 
<table border="1">
	<tbody>
		<tr>
 	<td>Behavior</td>
			<td>Description </td>
	 </tr>
 	 <tr>
			<td>cornering</td>
			<td>facing the corner of the aquarium and remaining there for at least 5 s with the tips of both claws touching different walls (Figure 4)</td>
	 </tr>
 	 <tr>
			<td>end facing</td>
			<td>facing the wall at one end of the tank for at least 5 s with the tip of at least one claw touching the end wall (Figure 5)</td>
	 </tr>
 	 <tr>
			<td>turning </td>
			<td>changing the walking path direction from clockwise to counter-clockwise or vice versa (Figure 6)</td>
	 </tr>
 	 <tr>
			<td>crossing</td>
			<td>leaving the perimeter of the aquarium and walking at least one body length to any other wall (Figure 7)</td>
	 </tr>
 	 <tr>
			<td>reverse walking</td>
			<td>walking backwards for at least one body length</td>
	 </tr>
 	 <tr>
			<td>freezing</td>
			<td>abrupt cessation of all visible movement, including that of appendages and antennae, for at least 5 s</td>
 	 </tr>
 <tr>
			<td>rearing up</td>
			<td>standing on the fourth and fifth walking legs while lifting the ventral surface of the thorax against a wall and placing at least one walking leg on the wall</td>
 	 </tr>
 </tbody>
</table>
Table 1. Behaviors exhibited by P. clarkii in the central compartment of the test tank.
<img src="/files/ftp_upload/1956/1956fig1.jpg" alt="Figure 1" /> Figure 1. Schematic of aquarium used in Method I. Half of the aquarium is lined with mirrors, while the other half is lined with non-reflective plastic.
<img src="/files/ftp_upload/1956/1956fig2.jpg" alt="Figure 2" /> Figure 2. Schematic of aquarium used in Method II. This tank is comprised of a center area, where the test crayfish is placed, and two end chambers, divided by an intact Plexiglas barrier. A live crayfish is placed in one end chamber, and a cube is placed in the other end chamber as a control.
<img src="/files/ftp_upload/1956/1956fig3.jpg" alt="Figure 3" /> Figure 3. Schematic of test aquarium with the midline depicted as a vertical dotted line. The crayfish passes from one side to the other when the coxae of the chelipeds pass the midline.
<img src="/files/ftp_upload/1956/1956fig4.jpg" alt="Figure 4" /> Figure 4. Diagram indicating the position of the crayfish while cornering. Note that the tips of both chelae are touching different walls.
<img src="/files/ftp_upload/1956/1956fig5.jpg" alt="Figure 5" /> Figure 5. Diagram indicating the position of the crayfish while facing the end wall. Note that both chelae are touching the same wall. 
<img src="/files/ftp_upload/1956/1956fig6.jpg" alt="Figure 6" /> Figure 6. Schematic diagram indicating turning. Reversing the direction of movement constitutes a turn. Turns can occur in a corner (e.g. direction 1 to direction 2 and from direction 4 to direction 5) or at the side (not shown). Entering the cornering and leaving it in the same direction (e.g. direction 1 to direction 3) is not a turn.
<img src="/files/ftp_upload/1956/1956fig7.jpg" alt="Figure 7" /> Figure 7. Schematic showing crossing behavior. Crossing occurs when the crayfish leaves one wall and travels to another wall, at a distance of at least one body length from the nearest wall (arrow). 
<img src="/files/ftp_upload/1956/1956fig8.jpg" alt="Figure 8" /> Figure 8. Photograph depicting rearing up behavior. The crayfish stands on the last two walking legs and lifts the body against a wall, making contact with at least one walking leg.

<ol>
	<li>Bruski, C. A., Dunham, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+vision+in+agonistic+communication+of+the+crayfish+Orconectes+rusticus.+I:+an+analysis+of+bout+dynamics.">The importance of vision in agonistic communication of the crayfish Orconectes rusticus. I: an analysis of bout dynamics.</a> Behav. 103, 83-107 (1987).</li><li>Delgado-Morales, G., Hernandez-Falcon, J., Ramon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agnostic+behavior+in+crayfish:the+importance+of+sensory+inputs.">Agnostic behavior in crayfish:the importance of sensory inputs.</a> Crustaceana. 77 (1), 1-24 (2004).</li><li>Guiasu, R. C., Dunham, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inter-form+agonistic+contests+in+male+crayfishes,+Cambarus+robustus+(Decapoda,+Cambaridae).">Inter-form agonistic contests in male crayfishes, Cambarus robustus (Decapoda, Cambaridae).</a> Invert. Biol.. 117 (2), 144-154 (1998).</li><li>May, H. Y., Mercier, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+crayfish+to+a+reflective+environment+depend+on+dominance+status.">Responses of crayfish to a reflective environment depend on dominance status.</a> Can. J. Zool. 84 (8), 1104-1111 (2006).</li><li>May, H. Y., Mercier, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Joffre+Duration+of+socialization+influences+responses+to+a+mirror:+responses+of+dominant+and+subordinate+crayfish+diverge+with+time+of+pairing.">Joffre Duration of socialization influences responses to a mirror: responses of dominant and subordinate crayfish diverge with time of pairing.</a> J. Exp. Biol. 210, 4428-4436 (2007).</li><li>May, H. Y., Luebbert, J., Mercier, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+Crayfish+to+a+Visual+Stimulus+Provided+by+a+Conspecific.">Responses of Crayfish to a Visual Stimulus Provided by a Conspecific.</a> , (2008).</li></ol>

No conflicts of interest declared.

The observation that dominant crayfish spend more time in a reflective environment suggests that they recognize a reflected image as a conspecific, and that visual cues are sufficient to elicit approach behavior. Similar results using a live crayfish instead of a reflected image support such an interpretation. Subordinate and isolated crayfish do not show such a preference for the reflective environment, nor do subordinate crayfish appear to prefer a live crayfish over a cube. Thus, responses to visual cues depend on ...

<table><tbody><tr><td>Single chamber test tank</td><td>Brock University Machine Shop</td><td></td><td></td></tr><tr><td>End-chamber test tank</td><td>Brock University Machine Shop</td><td></td><td></td></tr><tr><td>Isolation container</td><td>Rubbermaid</td><td></td><td>30 cm x 17.5 cm x 13 cm holes in lid to allow air and light to pass through when lid is on</td></tr><tr><td>Fighting chamber</td><td></td><td></td><td>48 cm x 25 cm x 13 cm</td></tr><tr><td>Social housing chamber</td><td></td><td></td><td>58 cm x 30 cm x 35 cm</td></tr><tr><td>small plastic flower pot</td><td></td><td></td><td>to serve as shelter</td></tr><tr><td>Coolpix 4500 camera</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>T120 video cassette recorder</td><td>RCA</td><td></td><td></td></tr><tr><td>Computer</td><td></td><td></td><td>Windows Movie Maker or other video acquiring software</td></tr><tr><td>webcam</td><td>Logitech</td><td></td><td></td></tr><tr><td>modelling clay</td><td>Sculptee</td><td></td><td>to make control cube</td></tr><tr><td>red latex paint</td><td>FolkArt</td><td></td><td>to make control cube</td></tr><tr><td>waterbase varnish</td><td>FolkArt</td><td></td><td>to make control cube</td></tr></tbody></table>

recording behavioral responses to reflection in crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

Watch this Scientific Journal Video about Recording Behavioral Responses to Reflection in Crayfish at JoVE.com

Recording Behavioral Responses to Reflection in Crayfish

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

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Research

JoVE Journal

Neuroscience

10.7K Views.  Brock University. We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition. 

Video: Recording Behavioral Responses to Reflection in Crayfish

Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor neurons innervating the extensor muscle in the walking leg of a crayfish. Distinct differences are present in the physiology and morphology of the phasic and tonic nerve terminals. The tonic axon contains many more mitochondria, enabling it to take a vital stain more intensely than the phasic axon. The tonic terminals have varicosities, and the phasic terminal is filiform. The tonic terminals are low in synaptic efficacy but show dramatic facilitated responses. In contrast, the phasic terminals are high in quantal efficacy but show synaptic depression with high frequency stimulation. The quantal output is measured with a focal macropatch electrode placed directly over the visualized nerve terminals. Both phasic and tonic terminals innervate the same muscle fibers, which suggests that inherent differences in the neurons, rather than differential retrograde feedback from the muscle, account for the morphological and physiological differentiation.

This article demonstrates how to conduct electrophysiological recordings of synaptic responses on the extensor muscle in the walking leg of a crayfish and how the nerve terminals are visualized to show the gross morphological differences of high- and low-output nerve terminals.

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor ...

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.

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

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 ...

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

Biology

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as a useful biological indicator of water quality. Even though crayfish cannot directly specify the substances that cause water quality deterioration, they can immediately (within a few seconds) warn humans of water quality deterioration via acute changes in their cardiac and behavioral activities.
In this study, we present a noninvasive method that is simple enough to be implemented under various conditions due to a combination of simplicity and reliability in one model.
This approach, in which the biological organisms are implemented into environmental evaluation processes, provides a reliable and timely alarm for warning of and preventing acute water deterioration in an ambient environment. Therefore, this noninvasive system based on crayfish physiological and ethological parameter recordings was investigated for the detection of changes in an aquatic environment. This system is now applied at a local brewery for controlling&#160;quality of the water used for beverage production, but it can be used at any water treatment and supply facility for continuous, real-time water quality evaluation and for regular laboratory investigations of crayfish cardiac physiology and behavior.

This article presents a noninvasive biomonitoring system for the continuous recording and analyses of crayfish cardiac and locomotor activities. This system consists of a near-infrared optical sensor, a video-tracking module, and software for evaluating crayfish heartbeats that reflects its physiological condition and characterizes crayfish behavior during heartbeat fluctuations.

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as ...

Behavior

Recordings of Neural Circuit Activation in Freely Behaving Animals

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. Traditional non-invasive methods require at least partially restrained research subjects, and they only allow identification of large numbers of simultaneously activated neurons. On the other hand, small ensembles of neurons or individual neurons can only be measured using single-cell recordings obtained from largely reduced preparations. Since the expression of natural behavior is limited in restrained and dissected animals, the underlying neural mechanisms that control such behavior are difficult to identify. 
Here, I present a non-invasive physiological technique that allows measuring neural circuit activation in freely behaving animals. Using a pair of wire electrodes inside a water-filled chamber, the bath electrodes record neural and muscular field potentials generated by juvenile crayfish during natural or experimentally evoked escape responses. The primary escape responses of crayfish are mediated by three different types of tail-flips which move the animals away from the point of stimulation. Each type of tail-flip is controlled by its own neural circuit; the two fastest and most powerful escape responses require activation of different sets of large &#8220;command&#8221; neurons. In combination with behavioral observations, the bath electrode recordings allow unambiguous identification of these neurons and the associated neural circuits. Thus activity of neural circuitry underlying naturally occurring behavior can be measured in unrestrained animals and in different behavioral contexts.

Non-invasive measurements of neural activity patterns in freely behaving animals are obtained by combining neurophysiological recordings with high speed videography.

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. ...

Measures of Heart and Ventilatory Rates in Freely Moving Crayfish

The fear, flight or fight response serves as the fundamental physiological basis for examining an organism's awareness of its environment under an impending predator attack.  Although it is not known whether invertebrates posses an autonomic nervous system identical to that of vertebrates, evidence shows invertebrates have a  sympathetic-like  response to regulate the internal environment and ready the organism to act behaviorally to a given stimuli.  Furthermore, this physiological response can be feasibly measured and it acts as a biological index for the animal's internal state.  Measurements of the physiological response can be directly related to internal and external stressors through changes in the central nervous system controlled coordination of the cardio-vascular and respiratory systems.   More specifically, monitoring heart and ventilation rates provide quantifiable measures of the stress response not always behaviorally observed. Crayfish are good model organisms for heart and ventilatory rate measurements due to the feasibility of recording, as well as the rich history known of the morphology of the crayfish, dating back to Huxley in 1888, and the well-studied typical behaviors.

Invertebrates show an autonomic sympathetic-like response similar to that described for vertebrates. The coordination of the cardio-vascular and ventilatory systems allows for measurement of a biological index in which to assess an organism internal state.

The fear, flight or fight response serves as the fundamental physiological basis for examining an organism's awareness of its environment under an ...

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is still much to learn from this model preparation. In understanding the history one can appreciate why even today this NMJ still offers a rich playground to address questions regarding pre- and post-synaptic function and plasticity. The viability and ease of access to the terminal for intracellular as well as extracellular electrophysiology and imaging are significant advantages. The mechanisms behind the modulation of vesicular kinetics and fusion within the high- and low-output terminals are begging for investigation. The preparation also offers a testable model system for computational assessments and manipulations to examine key variables in theoretical models of synaptic function, for example calcium dynamics during short-term facilitation. The synaptic complexity of active zone and statistical nature of quantal release is also an open area for future investigation both experimentally and computationally.

The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity.

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is ...

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.

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 ...

Recording Behavioral Responses to Reflection in Crayfish

Summary

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Chapters in this video

Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle

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

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

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities

Recordings of Neural Circuit Activation in Freely Behaving Animals

Measures of Heart and Ventilatory Rates in Freely Moving Crayfish

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises