Introduction
Organisms must exhibit a wide range of behaviors to respond to threatening stimuli and changes in the environment. The autonomic response is well studied in vertebrates as the fight or flight response in which the sympathetic nervous system physiologically readies the internal environment (Carpenter, 1976; Nicholls et al., 2001). It is through physiological changes that an organism can regulate the stress response and alter behavior to react to an impending attack or environmental change. Evidence shows that the cardio-vascular system is tightly connected with the respiratory system thus enabling the internal environment to respond and implement a response. It is well known that the autonomic control of the respiratory and cardiovascular systems can regulate oxygen availability and nutrients to specific target tissues needed for an impending behavioral response.
In vertebrates, the autonomic and physiological response is so well evolved; it would seem likely that complex invertebrates would similarly possess a developed response system (Schapker et al., 2002; Zavarzin, 1941). Even though vertebrates and invertebrates are very different systematically; highly developed invertebrates require the rapid cardiovascular and respiratory response to respond in a fight or flight manner. For many invertebrates, a sympathetic-like physiological response can be quantified. Dating back to 1927, many of the very early studies have been conducted in invertebrates, specifically using arthropods in general (Alexandrowicz, 1932; Orlov, 1927; Zavarzin, 1941).
Crayfish are known to exhibit a wide range of rapid behaviors as well as the ability to assess and respond to environmental stimuli. Very early studies have noted a sympathetic-like response of an immediate and rapid response of defense posturing (Bethe, 1897; Huxley, 1880; Shuranova et al., 2006; Wiersma, 1961). The adult crayfish heart is neurogenic since the beat and rhythm are controlled by the central nervous system (Alexandrowicz, 1932; Yamagishi and Hirose, 1997; Yamagishi et al., 1997; Wilkens, 1999). Routinely, measurement of an animal's heart rate (HR) provides a direct measure of excitability and readiness of the internal environment. In crayfish, an increase in HR is noted during defense posturing when presented with a perceived threatening stimulus (Listerman et al., 2000). This type of nervous system response was also shown in spiny lobsters (Yazawa and Katsuyama, 2001).
The ventilatory system is also neuronally controlled with a ventilatory central pattern generator (VPG) responsible for oxygen uptake across the gills by the pumping action of two specialized appendages termed scaphognathites (Mendelson, 1971). A single scaphognathite resides in each branchial chamber at the anterior ends and draws water across the gills by a rhythmic movement (Pasztor, 1968). The VPG, like with the heart, can be modulated by many factors. Previous work in the crab, Cancer magister shows cardiac and ventilatory rhythm can be altered by central control through command neurons (Wilkens et al., 1974). In addition, activity of the VPG is known to change with changes in the internal response and with social interactions or environmental changes in crustaceans (Burmistrov and Shuranova, 1996; Cuadras, 1979, 1980; Li et al., 2000; Listerman et al., 2000; McMahon and Wilkens, 1983; Schapker et al., 2002; Shuranova et al., 2002; Wilkens, 1976). As seen in the crayfish, ventilatory activity (VR) can vary depending on the internal state and changes in VR can be recorded during unexpected external stimuli (Shuranova et al., 1993; Shuranova et al., 2002).
Due to the dependability and feasibility of HR and VR measures, the future questions to be asked are endless. As noted previously, work has been done to examine the autonomic response during social interactions and environmental disturbances. Interestingly, many areas are left to be explored such as examination of the autonomic response during natural hormonal changes and/or other physiological processes occurring in the organism. Possible future directions may also examine sympathetic-like responses during mating and olfactory chemical reception. Most importantly, physiological responses may provide greater insights into an organism response to the environment when compared to behavioral observations since animals do not always respond behaviorally when they are readying themselves internally. Thus, the physiological measurement provides a biological index for assessing an organism's internal state which may explain overall changes in an organism. 
Methods
Electrocardiograms recording (ECGs) procedures for heart and ventilation rates
Before beginning to wire a crayfish, preparation of wires is required. Cut two insulated stainless steel wires (diameter 0.005 inches and with the coating 0.008 inches; A-M Systems, Carlsburg, WA) long enough to insert into the crayfish and adequately reach an impedance detector (UFI, model 2991). Remove insulation (~ 0.5 mm) by fire on the ends of both wires which will be inserted into the carapace. Be careful not remove too much insulation as a bare wire on the outside of the animal must be recoated to prevent short circuiting impedance recordings. Then, using forceps bend the burned ends of the wires in a 90 angle for placement into the carapace. Be sure that the inserted portion of the wire is not too long because this could do damage to internal organs. For feasibility of inserting the wires into a plastic tube (which protects the wires), it is best to loosely twist the wires together. If recording heart and ventilation rates, repeat the above steps a second time and the two sets of wires can be loosely braided together before placement into the plastic tubing. Be sure to designate the wires for heart and ventilation rate so as to not confuse which wires attach to which impedance detector for recording. It is best to label the wires or cut one pair shorter than the other in the beginning.
To feasibly and safely wire the crayfish, it is best to wrap the claws and legs (leaving the back and one side exposed) in wet cloth or paper towels to eliminate injury to the person and/or crayfish (Figure 1). 
<img src="/files/ftp_upload/1594/1594fig1.jpg" style="float:left;" alt="Figure 1" />Figure 1. Wrapping of the chelipeds and legs of the crayfish. Wrapping the crayfish in a wet paper towel will prevent injury to the person and the crayfish. The wrapping will hinder the ability of the crayfish to move the chelipeds and legs.
 To begin the wiring process, it is best to begin in the gill chamber since carapace thickness varies with each crayfish and excess force can cause damage to internal organs, thus by beginning in the gill chamber, excessive pressure is less likely to kill the crayfish as compared to the thoracic chamber. The first hole is made by using a fine point scalpel and just large enough for a wire to be inserted under the cuticle in the rostral area of the gill chamber (i.e., prebranchial chamber to monitor ventilatory rate; Schapker et al., 2002). A large hole will have excessive hemolymph loss and will not seal properly and this will increase the probability of the wires not remaining in the designated hole. Once the wire is in place, drop a small amount of glue (cyanoacrylate ester) and accelerator (HobbyTown USA, Lexington, KY) onto the wire. The use of the fast drying glue reduces handling stress of the animals. Be careful to place only a small amount of accelerator onto the cuticle since this is toxic to the crayfish and will cause death. It is best to immediately wipe any excess accelerator off with paper towel. Repeat these steps for the second wire in the gill chamber. These two wires are placed to span the scaphognathite (respiratory organ) to insure a strong accurate impedance measure during each scaphognathite movement (Figure 2). To get an accurate recording, be sure that the second wire is not placed outside the gill chamber and into the thoracic chamber. 
<img src="/files/ftp_upload/1594/1594fig2.jpg" style="float:left;" alt="Figure 2" />Figure 2. Placement of stainless steel wires in the gill chamber. The white dots on the carapace represent points for the stainless steel wires to be placed under the cuticle. The wires span the scaphognathite and measure movement through dynamic impedance.
The steps for wiring heart rate in the thoracic chamber is the same as above except the wires are placed under the dorsal carapace directly over the heart and span the heart in a rostral-caudal arrangement (Figure 3). This placement will ensure a strong accurate impedance measure during each heart contraction (Listerman et al., 2001).
 <img src="/files/ftp_upload/1594/1594fig3.jpg" style="float:left;" alt="Figure 3" />Figure 3. Placement of stainless steel wires in the thoracic chamber. The white dots on the carapace represent points for the stainless steel wires to be placed under the cuticle. The wires span the heart in a rostral-caudal arrangement. This will allow for measures of heart contraction through dynamic impedance. 
 Once placement of all four wires is complete, mix an instant adhesive (Eastman, 5-min drying epoxy) and coat the holes for each location as well as the wires. Once the wires and drilled holes are adequately covered, use the remaining epoxy to hold the plastic tubing to the back of the crayfish. This will ensure that the wires will not be pulled out of the holes in future handlings. Since the epoxy takes a few minutes to dry, hold or place the crayfish in a container without water until the epoxy is no longer sticky to the touch. Once the epoxy is dry, wash the crayfish in water to remove excess chemicals and place them back into an aquatic environment. To ensure the health of the crayfish, it is best to change the water in approximately one hour to remove any chemicals not previously removed. As noted from previous investigators, we found that prolonged handling alters the physiological measurements due stress for a time duration of 24 h to 3 days (Wilkens et al., 1985); therefore, animals should be left for 2 to 3 days before any experimentation.
To record ECGs, connect the wires from the crayfish to impedance detectors. The two wires spanning the heart (measure HR) should be attached to a single impedance detector and likewise for the two wires spanning the scaphognathite (measure VR). The impedance detector measures dynamic resistance between the two stainless steel wires. These signals are recorded by connecting the detector on-line to a PowerLab via a PowerLab/4SP interface (AD Instruments). All events are measured and calibrated with the PowerLab Chart software version 5.5.6 (AD Instruments, Australia). To begin, set an acquisition rate at 4 kHz. The large deflections will represent heartbeat and ventilation rate. For ease in counting each HR/VR, a smoothing function can applied to the data set. If 100 points were smoothed (i.e. averaged) the resultant trace will depict only the heartbeats. The heart rates can be determined by direct counts of each beat over 10-s intervals and then converted to beats per min (BPM).

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<table><tbody><tr><td>Scalpel</td><td></td><td></td><td>Stainless steel surgical blades No. 11 for No. 3 handle</td></tr><tr><td>Stainless steel wire</td><td>A-M Systems, Carlsburg, WA</td><td></td><td>diameter 0.005 inches with the coating 0.008 inches</td></tr><tr><td>Impedance detector</td><td>UFI</td><td>model 2991</td><td></td></tr><tr><td>Plastic tubing</td><td>Cole-Parmer</td><td></td><td>Tygon Tubing ID .0812, OD .1492</td></tr><tr><td>Fast-drying glue (cyanoacrylate ester)</td><td>HobbyTown USA, Lexington, KY</td><td></td><td></td></tr><tr><td>Accelerator</td><td>HobbyTown USA, Lexington, KY</td><td></td><td></td></tr><tr><td>Adhesive</td><td>Eastman</td><td></td><td>5-min drying epoxy</td></tr><tr><td>PowerLab via a PowerLab/4SP interface</td><td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML866/" target="_blank"><img src="/img/new_window.png" /> ADInstruments</a></td><td></td><td></td></tr><tr><td>PowerLab Chart</td><td><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank"><img src="/img/new_window.png" /> AD Instruments, Australia</a></td><td></td><td>software version 5.5.6</td></tr><tr><td>PowerLab Chart</td><td><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank"><img src="/img/new_window.png" /> AD Instruments, Australia</a></td><td></td><td>software version 3.5.6</td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Measures of Heart and Ventilatory Rates in Freely Moving Crayfish at JoVE.com

Measures of Heart and Ventilatory Rates in Freely Moving Crayfish

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 ...

measures-of-heart-and-ventilatory-rates-in-freely-moving-crayfish

Research

JoVE Journal

Biology

15.2K Views.  University of Kentucky. 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.

Video: Measures of Heart and Ventilatory Rates in Freely Moving Crayfish

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 ...

Neuroscience

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

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

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

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

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

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive information for an animal. An additional objective of this experiment is to learn anatomy of the preparation by staining, dissection and viewing of neurons and sensory structures under a dissecting microscope. This is performed by using basic neurophysiological equipment to record the electrical activity from a joint receptor organ and staining techniques. The muscle receptor organ (MRO) system in the crayfish is analogous to the intrafusal muscle spindle in mammals, which aids in serving as a comparative model that is more readily accessible for electrophysiological recordings. In addition, these are identifiable sensory neurons among preparations. The preparation is viable in a minimal saline for hours which is amenable for student laboratory exercises. The MRO is also susceptible to neuromodulation which encourages intriguing questions in the sites of modulatory action and integration of dynamic signals of movements and static position along with a gain that can be changed in the system.

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

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

Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise

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

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

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

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

Impedance Pneumography for Minimally Invasive Measurement of Heart Rate in Late Stage Invertebrates

Temperatures in oceans are increasing rapidly as a consequence of widespread changes in world climates. As organismal physiology is heavily influenced by environmental temperature, this has the potential to alter thermal physiological performance in a variety of marine organisms. Using the American lobster (Homarus americanus) as a model organism, this protocol describes the use of impedance pneumography to understand how cardiac performance in late stage invertebrates changes under acute thermal stress. The protocol presents a minimally invasive technique that allows for real-time collection of heart rate during a temperature ramping experiment. Data are easily manipulated to generate an Arrhenius plot that is used to calculate Arrhenius break temperature (ABT), the temperature at which heart rate begins to decline with increasing temperatures. This technique can be used in a variety of late stage invertebrates (i.e., crabs, mussels, or shrimps). Although the protocol focuses solely on the impact of temperature on cardiac performance, it can be modified to understand the potential for additional stressors (e.g., hypoxia or hypercapnia) to interact with temperature to influence physiological performance. Thus, the method has potential for wide-ranging applications to further understand how marine invertebrates respond to acute changes in the environment.

Measuring heart rate during a thermal challenge provides insight into physiological responses of organisms as a consequence of acute environmental change. Using the American lobster (Homarus americanus) as a model organism, this protocol describes the use of impedance pneumography as a relatively noninvasive and nonlethal approach to measure heart rate in late stage invertebrates.

Temperatures in oceans are increasing rapidly as a consequence of widespread changes in world climates. As organismal physiology is heavily influenced by ...

Environment

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. ...

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 ...

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor cells in isolated eyestalks using the discontinuous single electrode-switched voltage-clamp configuration. First, with a razor blade we made an opening in the dorsal cornea to get access to the retina. Thereafter, we inserted a glass electrode through the opening, and penetrated a cell as reported by the recording of a negative potential. Membrane potential was clamped at the photoreceptor's resting potential and a light-pulse was applied to activate currents. Finally, the two light-flash protocol was employed to measure current desensitization and recovery. The first light-flash triggers, after a lag period, the transduction ionic current, which after reaching a peak amplitude decays towards a desensitized state; the second flash, applied at varying time intervals, assesses the state of the light-activated conductance. To characterize the light-elicited current, three parameters were measured: 1) latency (the time elapsed between light flash delivery and the moment in which current achieves 10% of its maximum value); 2) peak current; and 3) desensitization time constant (exponential time constant of the current decay phase). All parameters are affected by the first pulse.
To quantify recovery from desensitization, the ratio p2/p1 was employed versus time between pulses. p1 is the peak current evoked by the first light-pulse, and p2 is the peak current evoked by the second pulse. These data were fitted to a sum of exponential functions. Finally, these measurements were carried out as function of circadian time.

A protocol for the study of desensitization and sensitivity recovery of crayfish photoreceptors as a function of circadian time is presented.

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor ...