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

No conflicts of interest declared.

Membrane Potential
As early as 1902, Bernstein was dealing with the issues of a resting potential in the axon of a squid. It is intriguing to consider how these early ideas and observations of Berstein (1902) and Nernst (1888) later influenced research in membrane physiology. (See review by Malmivuo and Plonsey, 1995; also available on the www <a href="http://www.bem.fi/book/">http://www.bem.fi/book/</a>). There are still, to this day, breakthroughs being made about ion channel function and properties of biological membranes that are very significant in understanding the cellular physiology which relates to the function of tissues, organs and systems.
The comparison of the experimental and theoretically derived effects of external [K+] on the resting membrane potential indicates the influence of ions on the membrane potential. Additional experiments using this same preparation remain to be performed to address fundamental physiological questions. Some were highlighted back in 1968 by Atwood and Parnas and have yet to be fully tackled. With the techniques obtained in this exercise, one can proceed 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 a model invertebrate preparation to address fundamental questions pertinent to all animals.
With the knowledge gained on the electrochemical gradients of ions in this above exercise, you can now advance to the excitability of membranes by examining synaptic transmission at neuromuscular preparations in the crayfish.

Measuring Synaptic Responses
The details provided for the first part of this laboratory, and the associated movie, have provided key steps for recording membrane potentials and investigating muscle structure. In the second part of this laboratory, the demonstration of dissection and recording synaptic transmission at the NMJs of phasic and tonic motor units provided an exposure to fundamental concepts in physiology. The exposure to a neural circuit, which can in part can be used to explain associated behaviors, in the intact animal has potential not only for students to investigate various open ended questions within their laboratory exercise but also for future research on neuronal circuits in a well established invertebrate preparation (Kennedy et al., 1969; Antonsen and Edwards, 2003)
These preparations can also be used to investigate synaptic facilitation, depression and long-term plasticity (not investigated in this laboratory study). Even within some species of crayfish, neuronal plasticity depends on the experimental stimulation conditions (Mercier and Atwood, 1989; Cooper et al., 1998) as well as their natural environment. To what extent the ability to alter synaptic efficacy and muscle dynamics serves the animal remains to be investigated. Since crayfish do alter their behavior in relation to seasonal variation and the molt cycle, there are relatively long-term activity differences in their neuromuscular systems. It has been shown that the phasic motor nerve terminals of claw closer muscles exhibit the classic phasic morphology during the winter, but swell and become more varicose along the length of the terminal during the summer months (Lnenicka 1993; Lnenicka and Zhao, 1991).
Some early studies conducted in crayfish lateral giant (LG) interneurons within the ventral nerve cord demonstrated the presence of gap junctions (Johnson, 1924; Watanabe and Grundfest, 1961). It is well known that CO2 has an effect on electrical communication by uncoupling gap junctions (Arellano et al, 1990). It was recently shown that the nerve cord and communication within the sensory-CNS-motor-muscle circuit, as described in this report, is also sensitive to CO2 exposure, indicating the presence of gap junctions (Bierbower, 2010; Bierbower and Cooper, 2010)
The spontaneous activity of the 3rd motor root has been a topic since the 1960's when Eckert (1961) examined if the tonic firing static muscle receptor organ (MRO) within the same or neighboring segment could account for the spontaneous motor drive. In these earlier studies it became apparent that the activity was driven within the ventral nerve cord (VNC) possibly from higher centers (Eckert, 1961; Kennedy and Takeda, 1965a,b; Strawn et al., 2000). Since the presence of CO2 stopped the spontaneous activity, one can assume somewhere in the drive to the motor neurons there might be gap junctions or glutamatergic excitatory drive. The NMJs are blocked or exhibit decreased sensitivity to glutamate in the presence of CO2, and they may be blocked as well within the CNS (Bierbower, 2010; Bierbower and Cooper, 2010; see also Badre et al., 2005).
The action of various neuromodulators is also readily studied at the various types of NMJs (Cooper and Cooper, 2009; Griffis et al., 2000; Southard et al., 2000; Strawn et al., 2000). In addition, various influences are exerted by neuromodulators on the CNS circuitry. It has been suggested that the 5-HT and octopaminergic neurons may function as 'gain-setters' in altering the output of neuronal circuits (Ma et al., 1992; Schneider et al., 1996; H&ouml;rner et al., 1997; Edwards et al., 2002). Much work remains to be done before we can fully understand the effects of neuromodulators on individual target cells. Given that different neuromodulators may work in concert with one another, analysis of their mixed action is an area for future research (Djokaj et al., 2001). In addition, few studies, particularly in the vertebrates, address the effects of neuromodulators on entire pathways which can regulate a specific behavior. In this sensory-CNS-motor unit preparation one can examine the influence of both sensory input and neuromodulators on the activity of the motor neurons (Kennedy et al., 1969).
Since it has been postulated that 5-HT plays a role in regulating the behavioral state of crayfish, lobsters, and crabs (Livingstone et al.,1980; Sneddon et al., 2000), several attempts have been made to determine its concentration in the VNC, the hemolymph, and in isolated ganglia of lobsters (Livingstone et al., 1980; Harris-Warrick and Kravitz 1984; Fadool et al., 1988). However, there has been considerable variation in the recorded measurements, precluding specific dose-response relationships which could account for behavioral actions.
A crayfish with the claws held in a raised position and with the tail tucked under its abdomen has been thought to exhibit a dominate posture (Livingstone et al., 1980). The state of abdominal flexion in crayfish does not appear to be the posture that dominant crayfish, within a pair, exhibit during the social interactions or while maintaining a dominant hierarchical status (Listerman et al., 2000). Submissive crayfish will even tuck their abdomens under themselves as they retreat from an opponent. Such tail tucking is also seen as a defense posture (Listerman et al., 2000). These behaviors have been readily observed in the field and in laboratory settings (Bovbjerg, 1953, 1956; Bruski and Dunham, 1987; Li et al., 2000; Listerman et al., 2000). Interestingly, the behavioral postures noted in lobsters (Livingstone et al., 1980) are reversed for 5-HT and octopamine injections in the Australian crayfish, Cherax destructor (McRae, 1996). Possibly, entirely different responses would be observed in the superficial flexor preparation in the Australian crayfish. In addition, since dominance is generally size related among crayfish, one would expect a very plastic response system for rapidly altered social conditions (Strawn et al., 2000). The plasticity in responsiveness to neuromodulators in invertebrates is an open area of investigation.
Wyttenbach, Johnson, and Hoy (1999) have produced digital media and a laboratory manual for various crayfish experimentations involving the same muscular presented in this report in addition to other crayfish preparations. This is an excellent resource for student exercises.

<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. Bouin's fixative solution was used directly and methylene blue is 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 glass 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 (ADInstruments, Colorado Springs, CO, USA). We use standard <a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">software from ADInstruments named Chart or Scope</a>.</li>
 <li> Model 3000 AC/DC amplifier for intracellular as well as extracellular recordings can be used.</li>
 <li>Dissecting microscope with zoom function for intracellular recordings. For focal recording on visualized terminals a compound microscope with upright objectives (4 x and 20X) is used. One needs a Hg light source.</li>
 <li>For intracellular recordings we use glass capillary tubing (catalogue # 30-31-0 from FHC, Brunswick, ME, 04011, USA). The intracellular electrode should have a resistance of 20 to 30 mOhm. Intracellular electrodes were filled with 3 M KCl.</li>
 <li>A suction electrode is used to record extracellular signals.</li>
 <li>Faraday cage.</li>
 <li>High Intensity Illuminator (light source).</li>
 <li>Micromanipulator</li>
</ol>

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.

Watch this Scientific Journal Video about Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises at JoVE.com

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

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 ...

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58.4K Views.  University of Kentucky. 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.

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