Name: physiological experimentation with the crayfish hindgut: a student laboratory exercise
Uploaded: 2011-01-18T17:30:00
Duration: 10 min 7 s
Description: Watch this Scientific Journal Video about Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise at JoVE.com

Supported by University of Kentucky, Department of Biology, Office of Undergraduate Studies and College of Arts &amp; Sciences.

1. Introduction

The most recent comprehensive overview of the crayfish hindgut was complied in a dissertation by Dr. Barbara E. Musolf (2007, Georgia State University, Atlanta, Georgia, USA), who focused primarily on serotonergic modulation and innervation (Musolf et al., 2009). Crustacean hindguts were first studied over 100 years ago by Alexandrowicz, a pioneer in comparative anatomy (Alexandrowicz, 1909), who described the various aspects of their innervation. Research continued over the years, identifying the types of muscle layers and architecture, addressing the overall function of the hindgut in osmoregulation for the whole animal, and examining modulatory control of hindgut contractility by various compounds (see review by Musolf, 2007). It is interesting to note that the anal portion of the hindgut acts not only to expel feces but also to take up the water from the environment for osmoregulation. This region of the gut can undergo forward or reverse peristalsis depending on the animal's needs.

Alexandrowicz (1909) identified two nerve plexuses innervating the crustacean hindgut, an inner plexus and an outer plexus, which later proved to be rich sources for identifying and characterizing neurotransmitters. In the 1950's Dr. Ernst Florey began a series of pharmacological studies on the crayfish hindgut and demonstrated that contractions are modulated by acetylcholine and its related compounds and also by epinephrine and norepinephrine (Florey, 1954). Florey, the discoverer of an inhibitory substance known as "Factor-I," studied the effects of this substance on various preparations, including the GI system in crayfish (Florey, 1961). Factor-I was later shown to be GABA. Thus, the crayfish hindgut played an important role in the early studies of synaptic inhibition.

Following the discovery of GABA, other putative transmitters were also shown to be associated with the hindgut plexuses and to elicit physiological responses. Such transmitters include dopamine (Elekes et al. 1988; Elofsson et al. 1968), proctolin (RYLPG-OH; Mercier et al. 1997) two orcokinin peptides (NFDEIDRSGFGFN and AFDEIDRSGFGFN; Bungart et al. 1994; Dircksen et al. 2000), orcomyotropin (FDAFTTGFamide; Dircksen et al. 2000) and a myosuppressin peptide (Mercier et al. 1997; most likely pQDLDHVFLRFamide, cf. Stemmler et al. 2007). Each of these substances modulates spontaneous hindgut contractions, and all except GABA appear to be excitatory. The hindgut also responds to glutamate and quisqualate (Jones, 1962; Wrong et al. 2003), but clear evidence for glutamatergic innervation has not been reported. Intracellular messengers that mediate the effects of the various transmitters are largely unexplored, but there is evidence for the involvement of cAMP in dopamine's ability to enhance contractions (Knotz &amp; Mercier, 1995).
Although the crustacean hindgut contracts spontaneously following denervation, such contractions are typically weak and disorganized (Wales, 1982; Winlow &amp; Laverack, 1972a). Peristaltic movement requires coordinated motor output from the central nervous system, apparently originating in the last abdominal ganglion (Winlow &amp; Laverack, 1972a, b, c). In crayfish, the motor output is carried to the hindgut through the 7th abdominal root, which contains 75 axons whose cell bodies are localized in the last abdominal ganglion (Kondoh &amp; Hisada, 1986). At least some of the peptides appear to be supplied to the hindgut plexus from neurons originating in the last abdominal ganglion (Dircksen et al. 2000; Mercier et al. 1991b; Siwicki &amp; Bishop, 1986), but dopamine is supplied by neurons in more anterior ganglia (Mercier et al. 1991a). Since motor output through the 7th abdominal root is necessary for large, coordinated contractions, it is likely that some or all of the putative transmitters listed above contribute in some way to peristalsis. Their relative contributions, however, are not known. Some insight may be gained by studying their effects on circular and longitudinal muscles separately (Mercier &amp; Lee, 2002).

In addition to controlling the movement of undigested food, the nerve plexus associated with the crustacean hindgut appears to play an important endocrine function associated with molting. The hindgut of the crab, Carcinus maenas, contains endocrine cells that release crustacean hyperglycemic hormone and its related precursor peptide during ecdysis (Webster et al. 2000), suggesting a role in water uptake and swelling associated with molting. Thus, the crustacean hindgut participates in several important physiological processes.

This report is primarily a teaching tool for advanced high school and undergraduate students in physiology courses that participate in experimentation. The significance as well as the potential mechanism behind how the hindgut contracts and functions can be addressed by the students. Pharmacological agents, neurotransmitters, and neurohormones will be used, and dose-response curves will be constructed, which will engage students in how to acquire and interpret physiological and pharmacological data. A general understanding of receptor function with regard to agonists and antagonists can also be achieved. The students will also learn to present data in graphical form for statistical analysis.

It is very easy to dissect and record contractions from the crayfish hindgut. In the dissected preparation, the gut is easily exposed to exogenous substances. The alteration in peristaltic activity can be monitored visually or with a force transducer, which can also monitor the force of contractions. Because of its simplicity and reliability as a bioassay preparation, the crayfish hindgut is still very useful for investigating many research questions about the mechanisms of peristalsis, modulation of motor output and muscle contraction, and receptor function. The hindgut is also useful for testing insecticides, crustaceancides, and pollutants for specific mechanisms of actions.


2. Methods
2.1) Materials
<ul>
<li>crayfish</li>
<li>crayfish saline</li>
<li>dissection instruments (coarse &amp; fine scissors, coarse &amp; fine forceps)</li>
<li>Sylgard-bottomed Petri dish</li>
<li>steel dissecting pins</li>
<li>beakers (to hold chemical solutions)</li>
<li>parafilm (for covering &amp; mixing solutions)</li>
</ul>
2.2) Dissection
<ol>
<li>Crayfish (Procambarus clarkii) measuring 6-10 cm in body length should be placed on ice for 5 to 10 minutes to anesthetize the animal before dissection begins.</li>
<li>Hold the anesthetized crayfish from behind the claws with one hand. Quickly, cut from the eye socket to the middle of the head on both sides, and then behead the crayfish (Note: the blood from the preparation will be sticky when it dries, so wash the tools when completed). 
<img src="/files/ftp_upload/2324/2324fig1.jpg" alt="Figure 1" /> 
Figure 1: Decapitation of crayfish</li>
<li>Cut off the chelipeds and walking legs.</li>
<li>Cut off the left and right tailfins, only leaving the middle tailfin (uropod).</li>
<li>On the dorsal side of the crayfish cut ventrally on both the left and right side of the dorsal cuticle. 
<img src="/files/ftp_upload/2324/2324fig2.jpg" alt="Figure 2" /> 
Figure 2: Removal of Dorsal Cuticle</li>
<li>Cut transversely on the dorsal side of the cuticle, making sure that the cut is shallow to prevent damage to the G.I. Then remove the lower section of the dorsal abdominal cuticle.</li>
<li>Place the dissected crayfish into a Sylgard-lined dish.</li>
<li>Pin the crayfish to the dish at the tip of the tailfin. One may use more pins on either side of the G.I. as necessary to hold the body down.</li>
<li>Fill the dish with crayfish saline covering the G.I. Make sure to continually douse the G.I. with saline using a pipette. Saline is a modified Van Harreveld's solution (1936), which is made with 205 mM NaCl; 5.3 mM KCl; 13.5 mM CaCl2 2H2O; 2.45 mM MgCl2 6H2O; 5 mM HEPES and adjusted to pH 7.4. The dissected crayfish should appear as shown in Figure 3. 
<img src="/files/ftp_upload/2324/2324fig3.jpg" alt="Figure 3" /> 
Figure 3: Dissected crayfish with GI intact.</li>
</ol>
2.3) Experiments

2.3.1) Contractions in the animal
<ol>
<li>Have solutions of the compound to be tested ready and at the same temperature as the bathing saline. On might use individual solutions of serotonin (100 nM, 1microM), glutamate (1microM), and dopamine (1microM) made in crayfish saline as starting substances to be tested.</li>
<li>Allow the dissected crayfish to sit in the crayfish solution for about ten minutes to allow it to adjust to the shock of dissection and saline exposure. The slow peristalsis contractions from the hindgut should start to occur.</li>
<li>Once the contractions begin, record the number of contractions that occur in thirty seconds (note the type of contractions that occur: i.e. peristalsis type, or spastic, and the direction of any peristaltic waves). 
Table 1: Contractions in Crayfish Saline 
<table border="1">
<tbody>
 <tr>
 <td>Number of Contractions</td>
 <td>Type of Contractions</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table></li>
<li>Using a pipette, remove the saline within the cavity of the crayfish's exposed abdomen, and apply saline containing the substance to be examined directly onto the hindgut.</li>
<li>Immediately after adding the solution, record the number of contractions that occur after thirty seconds and note the type of contractions that occur (i.e. peristaltic, or spastic). 
Table 2: Contractions with exposure to compounds 
<table border="1">
<tbody>
 <tr>
 <td>Compound tested</td>
 <td>Concentration</td>
 <td>Number of Contractions</td>
 <td>Type of Contractions</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table></li>
<li>Immediately after recording the response to the test substance, rinse the G.I. several times with normal crayfish saline. Let the preparation stand for 5 minutes, while rinsing about every 30 seconds with crayfish saline.</li>
<li>Using a pipette, place some saline containing the next compound to be examined or a varied concentration of the last substance tested directly onto the hindgut. It is best to start with a lower concentration and work one's way to higher concentrations.</li>
<li>Immediately after adding each new substance or each new concentration, record the number of contractions that occur after thirty seconds and note the type of contractions.</li>
</ol>
2.3.2) Recording forces of contraction in excised preparations
<img src="/files/ftp_upload/2324/2324fig4.jpg" alt="Figure 4" /> 
Figure 4: Setup
<ol>
<li>Attach the transducer to the bridge pod.</li>
<li>Attach the bridge pod to the PowerLab 26T.</li>
<li>Attach the PowerLab 26T to the USB port on the computer.</li>
<li>Open LabChart7, by clicking on the labchart7 icon on the desktop.
<ul>
<li>The LabChart Welcome Center box will pop open. Close it.</li>
<li>Click on Setup</li>
<li>Click on channel settings. Change the number of channels to 1 (bottom left of 
box) push OK.</li>
<li>At the top left of the chart set the cycles per second to about 2k. Set the volts (y-
axis) to about 500 or 200 mV.</li>
<li>Click on Channel 1 on the right of the chart. Click on Input Amplifier. Ensure 
that the settings: single-ended, ac coupled, and invert (inverts the signal 
if needed), and anti-alias , are checked.</li>
<li>To begin recording press start.</li>
</ul></li>
<li>Pick up the preparation and cut the GI tract at the juncture between the thorax and the abdomen. Then carefully cut around the telson part of the tail. Remove the GI tract from the dissected crayfish and place it into the Sylgard dish. There is a blood vessel that runs along the dorsal aspect of the GI tract. Carefully pull this away from the GI tract. Pour fresh crayfish saline onto the preparation.</li>
<li>Place the force transducer in the clamp near the dissected crayfish.</li>
<li>Hook the force transducer in the crayfish G.I. as shown in Figure 5. 
<img src="/files/ftp_upload/2324/2324fig5.jpg" alt="Figure 5" /> 
Figure 5: Isolated hindgut of the crayfish in saline attached to the hook of the force transducer</li>
<li>Wait until the G.I. starts to contract, and push start on the LabChart7.</li>
<li>Let the program run for about 20 seconds. Push "stop," and add a comment labeled, 
 "Saline only". Fill out the Table 4. 
Table 4: Contractions with compound of interest 
<table border="1">
 <tbody>
 <tr>
 <td>Number of Contractions</td>
 <td>Amplitude of Contractions (mV)</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table></li>
<li>Add the test compounds of interest, and immediately push "start" on the LabChart7.</li>
<li>Let the program run for about 20 seconds. Push "stop" and add a comment directly on the chart file as to what substance was added and its concentration. Fill out the table below. 
Table 5: Contractions with _________ compound 
<table border="1">
 <tbody>
 <tr>
 <td>Number of Contractions</td>
 <td>Amplitude of Contractions (mV)</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table></li>
<li>Rinse off the preparation with crayfish saline using a pipette.</li>
<li>Add other substances or various concentrations in a similar manner. Immediately push "start" on the LabChart7.</li>
<li>Let the program run for about 20 seconds. Push "stop," and add a comment and label directly on the chart file indicating the compound used and the concentration.</li>
</ol>
3. Data Analysis
<ol>
<li>From 2.3.1 of the experiment, graph the number of contractions from each solution (saline, serotonin, glutamate) vs. time. Explain any trends.</li>
<li>From 2.3.2 of the experiment, graph the number of contractions from each solution (saline, serotonin, glutamate) vs. the amplitude of the contractions in mV. Explain any trends.</li>
</ol>
3.1 ) Measuring the rate and force of contractions
To index the rates of contractions on can measure the time from the beginning on one deflection to the beginning of the next or count the total deflections over a minute or two. The values can then be put in terms of contractions per minute or per second depending on how rapid they are occurring.
To index the force of contractions a relative measure could be used to compare different conditions. On the saved file one can use the marker "M" and move to the baseline. Then use the cursor and move to the peak of the deflection and take note of the value listed on the top right of the screen as a delta value (the difference from M to the peak). Note the cursor needs to be kept stationary for the measure of the change. Then move the M to the next wave form of interest and repeat the measure.

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-mass-resolution+direct-tissue+MALDI-FTMS+reveals+broad+conservation+of+three+neuropeptides+(APSGFLGMRamide,+GYRKPPFNGSIFamide+and+pQDLDHVFLRFamide)+across+members+of+seven+decapod+crustaean+infraorders.">High-mass-resolution direct-tissue MALDI-FTMS reveals broad conservation of three neuropeptides (APSGFLGMRamide, GYRKPPFNGSIFamide and pQDLDHVFLRFamide) across members of seven decapod crustaean infraorders.</a> Peptides. 28, 2104-2115 (2007).</li><li>Wales, W., Sandeman, D. C., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+mouthparts+and+gut.">Control of mouthparts and gut.</a> The Biology of Crustacea. 4, 165-191 (1982).</li><li>Webster, S. G., Dircksen, H., Chung, J. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+hindgut+motility+in+the+lobster+Homarus+gammarus+(L.).+2.+Motor+output.">The control of hindgut motility in the lobster Homarus gammarus (L.). 2. Motor output.</a> Mar. Behav. Physiol. 1, 29-47 (1972).</li><li>Winlow, W., Laverack, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+hindgut+motility+in+the+lobster+Homarus+gammarus+(L.).+3.+Structure+of+the+sixth+abdominal+ganglion+(6+A.G.)+and+associated+ablation+and+microelectrode+studies.">The control of hindgut motility in the lobster Homarus gammarus (L.). 3. Structure of the sixth abdominal ganglion (6 A.G.) and associated ablation and microelectrode studies.</a> Mar. Behav. Physiol. 1, 93-121 (1972).</li><li>Wrong, A. D., Sammahin, M., Richardson, R., 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=Pharmacological+properties+of+glutamate+receptors+associated+with+the+crayfish+hindgut.">Pharmacological properties of glutamate receptors associated with the crayfish hindgut.</a> J. Comp. Physiol. 189, 371-378 (2003).</li></ol>

No conflicts of interest declared.

The details provided in the associated movie and text describe the key steps needed to record the activity in the hindgut of the crayfish in situ as well as in vitro. One goal of our report is to increase the awareness to the potential of this preparation in student-run investigative laboratories that teach fundamental concepts in physiology and pharmacology.

These preparations can be used to investigate a number of experimental questions that will lead to a better understanding of the physiological functions of the hindgut. The mechanisms underlying regulation of peristaltic waves and their reversal are still not known. The mechanisms for higher control of the entire GI tract are also not fully understood. The question of how higher centers integrate their activity with the autonomic output that directly controls the GI system remains an open area of investigation (Shuranova et al., 2006). In addition, the osmoregulatory capabilities of the crustacean hindgut and the functions of osmoregulation during molting and environmental stress have not been fully elucidated. There are still many questions awaiting answers in this preparation.

<ol>
 <li>Crayfish (Procambarus clarkii). Atchafalaya Biological Supply Co., Raceland, LA., USA.</li>
 <li> Standard crayfish saline: Modified from Van Harreveld's solution (1936). (in mM) 205 NaCl; 5.3 KCl; 13.5 CaCl22H2O; 2.45 MgCl26H2O; 5 HEPES and adjusted to pH 7.4. Serotonin, glutamate and dopamine are made in crayfish saline. All chemicals are obtained from Sigma chemical company (St. Louis, MO).</li>
 <li> Dissection tools: Fine #5 tweezers, fine scissors, knife blade holder, #26002-20 insect pins (all obtained from Fine Science Tools (USA), Inc., 373-G Vintage Park Drive, Foster City, CA 94404-1139)</li>
 <li> Sylgard-bottomed Petri dish</li>
 <li> Beakers (to hold chemical solutions)</li>
 <li> Electrical signals are recorded on line to a <a href="http://www.adinstruments.com/products/hardware/corporate/product/ML856/" target="_blank">PowerLab 26T</a> interface to a computer (<a href="http://www.adinstruments.com" target="_blank">ADInstruments</a>, Colorado Springs, CO, USA). We use standard software from ADInstruments named Chart or Scope.</li>
 <li> <a href="http://www.adinstruments.com/products/hardware/research/Signal_Conditioners/Blood_Pressure,_Force,_Displacement/" target="_blank">Bridge pod</a> for the force transducer (different Bridge pods are required for different types of force transducers).</li>
 <li> <a href="http://www.adinstruments.com/products/hardware/corporate/product/MLT0402/" target="_blank">MLT0402 (2 grams) force transducer</a> is the research grade as shown in the video. <a href="http://www.adinstruments.com/products/mlt0210d#overview" target="_blank">MLT 0210/D (10 mg-25 grams)</a> and the <a href="http://www.adinstruments.com/products/MLTF500ST#overview" target="_blank">MLT 500/A (0-500g)</a> are the educational grades transducers (<a href="http://www.adinstruments.com/" target="_blank">ADInstruments</a>).</li>
</ol>

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.

Watch this Scientific Journal Video about Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise at JoVE.com

Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise

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 ...

physiological-experimentation-with-crayfish-hindgut-student

Research

JoVE Journal

Neuroscience

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).

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 ...

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 ...

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.

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 ...

VisualEyes: A Modular Software System for Oculomotor Experimentation

Eye movement studies have provided a strong foundation forming an understanding of how the brain acquires visual information in both the normal and dysfunctional brain.1 However, development of a platform to stimulate and store eye movements can require substantial programming, time and costs. Many systems do not offer the flexibility to program numerous stimuli for a variety of experimental needs. However, the VisualEyes System has a flexible architecture, allowing the operator to choose any background and foreground stimulus, program one or two screens for tandem or opposing eye movements and stimulate the left and right eye independently. This system can significantly reduce the programming development time needed to conduct an oculomotor study. The VisualEyes System will be discussed in three parts: 1) the oculomotor recording device to acquire eye movement responses, 2) the VisualEyes software written in LabView, to generate an array of stimuli and store responses as text files and 3) offline data analysis. Eye movements can be recorded by several types of instrumentation such as: a limbus tracking system, a sclera search coil, or a video image system. Typical eye movement stimuli such as saccadic steps, vergent ramps and vergent steps with the corresponding responses will be shown. In this video report, we demonstrate the flexibility of a system to create numerous visual stimuli and record eye movements that can be utilized by basic scientists and clinicians to study healthy as well as clinical populations.

Neural control and cognitive processes can be studied through eye movements. The VisualEyes software allows an operator to program stimuli on two computer screens independently using a simple, custom scripting language. The system can stimulate tandem eye movements (saccades and smooth pursuit) or opposing eye movements (vergence) or any combination.

Eye movement studies have provided a strong foundation forming an understanding of how the brain acquires visual information in both the normal and ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

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 ...

Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty acid receptors housed on taste cells. Previous research has shown that an individual&#39;s oral sensitivity to fatty acid, specifically oleic acid (C18:1) is associated with body mass index (BMI), dietary fat consumption, and the ability to identify fat in foods. We have developed a reliable and reproducible method to assess oral chemoreception of fatty acids, using a milk and C18:1 emulsion, together with an ascending forced choice triangle procedure. In parallel, a food matrix has been developed to assess an individual&#39;s ability to perceive fat, in addition to a simple method to assess fatty food liking. As an added measure tongue photography is used to assess papillae density, with higher density often being associated with increased taste sensitivity.

Oral chemoreception of fatty acids and the association with diet and fatty food preferences may enable the identification of mechanisms involved with the development of obesity and why dietary changes may be difficult for many individuals.

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...