Mentor: J. Timothy Cannon, Ph.D,
Director, Neuroscience Program, University of Scranton

    A.) The global objective of this project is to better understand the properties of human nociceptive pathways by studying nociceptive characteristics in the terrestrial snail Helix aspersa (also known as the edible snail, escargot).

    B.) Pain Pathways in General, and the importance of this snail:
    The two broad categories of nerve fibers responsible for nociception in the human and other mammals are known as the A-Delta Nociceptor and the C-Fiber Nociceptor. These fibers are responsible for two painful phenomena familiar to most humans known as the "first pain" and the "second pain." The first pain occurs immediately after physical trauma, such as stubbing the toe. Characterized by a sharp localized pain, the first pain allows the mammal to locate the injured area of the body immediately. The second pain occurs moments after and is often perceived as a dull burning pain. This pain remains for a longer time period than the first pain and offers the animal "incentive" to immobilize the injured body part and to allow sufficient time for it to heal.
    In an article titled "Mechanosensory Neurons Innervating Aplysia Siphon Encode Noxious Stimuli and Display Nociceptive Sensitization" (The Journal of Neuroscience, Vol. 17, No.1, Jan. 1, 1997, pp. 459-469), Paul A. Illich and Edgar T. Walters (University of Texas-Houston Medical School) demonstrated similarities between the vertebrate A-Delta nociceptors and the nociceptive nerve fibers of the aquatic snail Aplysia californica. According to Illich and Walters, properties of the A. californica nociceptors included a relatively high frequency of peak responses (greater than 30 Hz.), relatively large RFs, and lack of activation by most chemical activators which would normally activate C polymodal nociceptors. These characteristics, in addition to the display of sensitization, meet the accepted criteria for the A-Delta nociceptor which has been described in the vertebrate.
    Given the degree of similarity between vertebrate nociceptors and the nociceptors of A. californica, I believe it of benefit to pursue studies illucidating similarities between nociceptors of a related snail (H. aspersa) and the nociceptors of the vertebrate. The H. aspersa snail is easily maintained in a terrestrial environment consisting of several clear plastic ventilated rectangular containers, a constant temperature, 12:12 inverse light:dark cycles, and is allowed ad libitum access to food which is maintained in a shallow rectangular glass dish located in the center of the terraria. The H. aspersa snail requires far less maintenance than snails which require an aquatic environment.

II. General Methodology:
    Snails are tested during the dark phase of the light:dark cycle under red lighting conditions on an apparatus consisting of a horizontal sheet of transparency film tightly secured along the edges of a rectangular wooden frame of adjustable height. This setup allows four snails to be placed on the plastic at a time and is of sufficient size to allow a large roaming area. Therefore the snails travel outside of the testing region infrequently and the snails' courses of travel must only occasionally be altered. If necessary, a snail's course of travel can be modified either by gently picking the snail up by the shell with the thumb and forefinger and relocating it or by gently sliding the snail to the desired location in such a manner that its foot never leaves the plastic.
    Heat is administered in the form of light. The light source consists of a halogen photo optic lamp (Osram ENX 360W 82V J768) designed for use in overhead projection units. The reflective glass parabolic dish has been carefully removed from the lamp so that the lamp bulb can be fitted into a rectangular plastic housing ("light box") over which is a metal plate containing a 1.6 mm-diameter hole. The lamp is wired to a transformer of adjustable voltage with a digital readout display. The light box is movable and is maneuvered directly underneath the caudal foot. After some time period of applied stimulus, the snail will react by invaginating its body inward, thus attempting to withdraw from the stimulus.

III. Specific Objectives:
    A. Determination of the snail's noxious temperature threshold.
    B. Determination of the receptor type (opiate or non-opiate) which would be involved in blocking the nocifensive response with narcotics.
    C. Identification of the specific neurons which mediate the nocifensive withdraw reflex.

IV. Experimental Protocol:

    A. Phase One:

        1. Voltage Response Curve.

    Experimental  devices consist of a transformer with a digital readout which is also connected to an electronic timing device. A graph of voltage (X-axis) with respect to time (Y-axis) will illustrate a "behavioral range" of voltages which approach but do not exceed the basement and ceiling voltages. In other words, the voltage which causes the fastest withdrawal response time, and the voltage which causes the slowest withdrawal response time will be determined for subsequent experimentation. This range of voltages will be determined by a preliminary bioassay in which snail response times to light at a series of six voltages (50V, 60V, 70V, 80V, 90V, and 100V) will be tested. I predict that the results will resemble a dose-response curve.
    During the bioassay, all testing will begin with a "cold" light box, i.e., at room temperature. Temperature readings will be taken once per trial by placing the tip of the thermosensor onto the plastic film at a location directly above the 1.6mm diameter hole. Temperature readings will illustrate the increases in temperature of the light box which I expect to occur throughout the 30 min. testing period. The temperature curve eventually levels off because the rate of increase in heat is countered by the rate of cooling during the 30 sec. cool-off period in between trials. This temperature at which the curve levels off will be called the "operating temperature." In the bioassay, the light box will be cold in the beginning of the experiment and will heat up to the operating temperature. Testing for the voltage response curve, however, will begin only after the light box has been "pre-heated" to the operating temperature. The pre-heating step will be executed by a computer-driven program in which the light box will be light in a cycle 3 sec. on, followed by 27 sec. off, for a 15 min. time period. In this manner, the light box will be brought to operating temperature prior to experimentation.

        2. Temperature-Time Curve.
Because the electronic devices which measure voltage and time do not include a measure of temperature, the temperature at a particular voltage at a particular time must be determined by means of a separate device. In order to study the heat conduction properties of the snail, several snails must be rendered unconscious (in order to prevent locomotion). A thermosensor will then be placed both cutaneously and sub-cutaneously. The voltage will be varied and the time will be recorded. In this manner, Temperature (Y-axis) with respect to Time (X-axis) can be measured at any particular voltage. Computer analysis with the SPSS-x statistical package will determine the heat threshold of the snail, and will attempt to correlate experimental data with known data of the vertebrate A-Delta heat threshold properties.

        3. Response to Morphine and Naloxone.
The voltage which causes the fastest withdrawal response time, and the voltage which causes the slowest withdrawal response time will be administered to snails under the influence of the following:
                    1.) Control, Molluscan Saline
                    2.) Saline and Morphine (0.1 mg/ml)
                    3.) Saline and Morphine (1.0 mg/ml)
                    4.) Saline and Morphine (10.0 mg/ml)

                    5.) Naloxone (1.0 mg/ml) and Saline
                    6.) Naloxone (1.0 mg/ml) and Morphine (0.1 mg/ml)
                    7.) Naloxone (1.0 mg/ml) and Morphine (1.0 mg/ml)
                    8.) Naloxone (1.0 mg/ml) and Morphine (10.0 mg/ml)

This step seeks to determine whether or not the fast and the slow withdrawal nociceptive response can be blocked equally by morphine. If the nociceptive response can be blocked by opiates, then doses of Saline with increasing doses of morphine would be expected to cause increased response times to heat stimuli. Likewise, naloxone, which reverses the effects of morphine, would be expected to decrease the length of time for the snail to initiate the withdrawal response. Such behavior is expected to occur in the H. aspersa snail, because it has been demonstrated in a related terrestrial snial, Cepaea nemoralis.

    Phase Two: Surgery

    The snail's withdrawal response to radiant light occurs as a semi-circular invagination of the foot. Digital photographic technology will be used to record images of this invagination response at particular voltages. Computerized analysis through the SigmaScan package will calculate the area of the invagination in the healthy snail.
    A large bundle of nerves connected to the caudal foot from the pedal ganglion will be severed.
    The snails will then be tested again at particular voltages, and the invagination response will again be calculated. Comparisons will be made between the healthy and lesioned snails. If the invagination response still occurs, this would indicate that the response is mediated entirely or mostly by local factors. If the invagination does not occur, this would indicate that the response is mediated entirely or mostly by central factors.