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 ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Single chamber test tank</td>
<td>&nbsp;</td>
<td>Brock University Machine Shop</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>End-chamber test tank</td>
<td>&nbsp;</td>
<td>Brock University Machine Shop</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isolation container</td>
<td>&nbsp;</td>
<td>Rubbermaid</td>
<td>&nbsp;</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>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>48 cm x 25 cm x 13 cm</td>
</tr>
<tr>
<td>Social housing chamber</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>58 cm x 30 cm x 35 cm</td>
</tr>
<tr>
<td>small plastic flower pot</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to serve as shelter</td>
</tr>
<tr>
<td>Coolpix 4500 camera</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>T120 video cassette recorder</td>
<td>&nbsp;</td>
<td>RCA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Computer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Windows Movie Maker or other video acquiring software</td>
</tr>
<tr>
<td>webcam</td>
<td>&nbsp;</td>
<td>Logitech</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>modelling clay</td>
<td>&nbsp;</td>
<td>Sculptee</td>
<td>&nbsp;</td>
<td>to make control cube</td>
</tr>
<tr>
<td>red latex paint</td>
<td>&nbsp;</td>
<td>FolkArt</td>
<td>&nbsp;</td>
<td>to make control cube</td>
</tr>
<tr>
<td>waterbase varnish</td>
<td>&nbsp;</td>
<td>FolkArt</td>
<td>&nbsp;</td>
<td>to make control cube</td>
</tr>		</tbody>
		</table>
		</div>

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 ...

recording-behavioral-responses-to-reflection-in-crayfish

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10.6K 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

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 ...

Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar tasks. Participants' behavior during task performance in an fMRI scanner can then be correlated to the brain activity using the blood-oxygen-level-dependent signal. We measure behavior to be able to sort correct trials, where the subject performed the task correctly and then be able to examine the brain signals related to correct performance. Conversely, if subjects do not perform the task correctly, and these trials are included in the same analysis with the correct trials we would introduce trials that were not only for correct performance. Thus, in many cases these errors can be used themselves to then correlate brain activity to them. We describe two complementary tasks that are used in our lab to examine the brain during suppression of an automatic responses: the stroop1 and anti-saccade tasks. The emotional stroop paradigm instructs participants to either report the superimposed emotional 'word' across the affective faces or the facial 'expressions' of the face stimuli1,2. When the word and the facial expression refer to different emotions, a conflict between what must be said and what is automatically read occurs. The participant has to resolve the conflict between two simultaneously competing processes of word reading and facial expression. Our urge to read out a word leads to strong 'stimulus-response (SR)' associations; hence inhibiting these strong SR's is difficult and participants are prone to making errors. Overcoming this conflict and directing attention away from the face or the word requires the subject to inhibit bottom up processes which typically directs attention to the more salient stimulus. Similarly, in the anti-saccade task3,4,5,6, where an instruction cue is used to direct only attention to a peripheral stimulus location but then the eye movement is made to the mirror opposite position. Yet again we measure behavior by recording the eye movements of participants which allows for the sorting of the behavioral responses into correct and error trials7 which then can be correlated to brain activity. Neuroimaging now allows researchers to measure different behaviors of correct and error trials that are indicative of different cognitive processes and pinpoint the different neural networks involved.

The goal of our research is to correlate behavior to brain activity. Accurate behavioral measures and imaging techniques allow us to elucidate brain-behavior relationships.

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house single or multiple neurons inside, are considered to make the major contribution in responding to the chemical stimuli. By directly recording action potential in the olfactory sensillum after exposure to stimuli, single sensillum recording (SSR) technique provides a powerful approach for investigating the neural responses of insects to chemical stimuli. For the bed bug, which is a notorious human parasite, multiple types of olfactory sensillum have been characterized. In this study, we demonstrated neural responses of bed bug olfactory sensilla to two chemical stimuli and the dose-dependent responses to one of them using the SSR method. This approach enables researchers to conduct early screening for individual chemical stimuli on the bed bug olfactory sensilla, which would provide valuable information for the development of new bed bug attractants or repellents and benefits the bed bug control efforts.

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.
The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.
In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.
This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee&#39;s taste system.

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.