I had not made a good electrocardiogram of a Daphnia or of any other kind of water flea, and I so informed Camougis. "Neither has anyone else," he replied, "but it's a lot of fun to try. All you need is a Daphnia plus a high-fidelity amplifier, a recorder of some sort and a few accessories. With this apparatus you can do the flea experiment and others involving the electrical effects associated with animal behavior, including those of various muscle actions that are otherwise hidden from the eye. I will mail a description of the general procedure in a few days and you can take it from there." He did, and I did.

Camougis pointed out that the electrical phenomena associated with the active tissues of animals have been known for a long time. Perhaps the most startling examples are the shocks of electric fishes, which Aristotle first described more than 2,000 years ago. It was not until the latter part of the 18th century, however, that the existence of animal electricity was definitely established. With the 19th century and the development of electrical instruments, many experiments were performed to investigate the physical basis of animal electricity, especially in nerve and muscle.

With the advent of modern electronic equipment, electrical phenomena associated with nerve and muscle action have come in for intensive study. The basic inquiries, according to Camougis, center on the functions of the receptive, integrative and response mechanisms underlying animal behavior. They include studies of vision and hearing, the functioning of the brain and the contraction of muscle. The behavior of an animal reflects the complex interaction of great numbers of nerve and muscle fibers. To isolate a single event the experimenter must often attach a number of electrodes and ingenious sensing devices to the animal. Indeed, many discoveries in physiology can be traced to the development and use of just such clever devices. It is possible, on the other hand, to study the electrical responses of the whole animal by means of a somewhat simpler and less delicate apparatus. Such an apparatus usually includes a mechanism that writes a record whenever the animal moves or shifts a limb. Of necessity some part of the apparatus must touch the animal and so to some extent restrict or impede its movements. This drawback is avoided 'in the very simple apparatus described by Camougis and used to study animals that live in water. The bioelectric potentials are picked up merely by placing a pair of electrodes in the water.

Camougis says that a number of materials can be used for electrodes. Heavy copper wire about l/8 inch in diameter works well for picking up currents from frogs and small fishes. (Brass, lead, soft iron and carbon can also be used.) The wire is cut into convenient lengths of about six inches. The ends can be flattened to provide a greater surface area. The wires are then insulated with several layers of quick-drying nail polish, except for the flattened part and about half an inch at the opposite end, where the leads are to be attached. Sleeves of plastic or rubber tubing are slipped over the wires as mechanical protection against the iron clamps used to support the electrodes in the water. A glass dish or other nonconducting vessel of appropriate size can be used as the container for the animal and water. The bare upper ends of the electrodes are connected by alligator clips to the input leads of the recording system, as shown in the illustration in Figure 1.

The electrical potentials normally picked up are so small that high-gain amplification is required. Stray fields set up by alternating-current power lines, by appliances and even by nearby radio stations can induce currents in the input leads almost as strong as the desired signal. Shielded cages that house the electrodes, the water container and a preamplifier have therefore become standard equipment for electrophysiological work. The shielded cage can be a plywood box covered on all sides with bronze or copper screening. It should have a hinged door on one side for access to the specimen and apparatus inside. It is essential that the door screening make good electrical contact with the rest of the shielding. Convenient dimensions are 30 by 20 by 20 inches, the door being 30 by 20 inches [see illustration above right]. The cage must be grounded, preferably to a nearby water pipe.

The characteristics of the amplifier and instrument required for displaying the electrical potentials are determined by the electrical phenomena to be investigated. When observing the action potentials of aquatic arthropods, a high-quality preamplifier capable of multiplying the input signal 10,000 times is essential. Capacitance-coupled, battery-operated preamplifiers are satisfactory, although direct-current preamplifiers must be used in the case of signals of very low frequency. A cathode-ray oscilloscope can be used to display the potentials and, along with the amplifier, should be grounded to the shielded cage. Any good oscilloscope will work, including the better kit models.

The technical problems of recording animal electricity vary inversely with the amplitude of the potential picked up by the electrodes. The potentials developed by some fishes, for example, can be fed directly into an oscilloscope of only moderate sensitivity, and the specimen need not be shielded.

When the experimental animal in the container remains still, the horizontal trace of the oscilloscope is a steady, straight line. When the animal moves, peaks of varying form and amplitude appear that correspond to variations in the electrical field induced in the water by the animal. These potentials are caused mainly by muscle action. When the crayfish is used as an experimental animal, for example, the amplitude of the action potentials is on the order of 50 to 100 microvolts. The amplitude of the deflections increases when an animal moves close to the recording electrodes. Surface oscillations of the water also induce spurious excursions in the trace, but these are normally of low amplitude and do not confuse the observations, particularly in the case of animals that move slowly. Since most of the electrical phenomena recorded are fast transients that span no more than a few thousandths of a second, ordinarily no difficulty arises from electrochemical reactions between the electrodes and the water. When observing animals that develop substantial direct currents, however, it is advisable to use silver electrodes that have been immersed in salt water for a few minutes while connected to a six-volt source. The process coats the electrodes with a protective film of silver chloride.

The voltage that appears across the electrodes varies with the resistance, and hence the purity, of the water. Impure water, which contains ions, provides a good path for the currents and tends to "short-circuit" the animal. This presents no problem in the case of large potentials, such as the electric discharges from knife fishes. But if one is to record muscle action potentials from crayfish and smaller creatures, the specimen must be in distilled or deionized water.

A variety of bioelectrical phenomena can be recorded by this method. Muscle action potentials are readily detected. Crayfish, for example, react as if startled when a bright light is suddenly turned on them. How much time elapses between the instant when the light comes on and the response of the crayfish? The interval is difficult to time accurately by eye. But it shows plainly on the oscilloscope. Experiments of this sort are made easier and still more interesting by the addition of an audio system so that the action potentials can be heard as well as seen. Just hook any good audio amplifier across the output of the preamplifier (in parallel with the input to the oscilloscope) and feed the amplified signal into a loudspeaker.

Aquatic insects such as the corixids (water boatmen) also make interesting subjects. Periodic bursts of electrical activity are clearly noted, synchronized with readily observable movements of the locomotory appendages. Such swimming movements have not been extensively investigated because heretofore the available techniques of observing the movements required bright lights, mechanical attachments and related apparatus that tended to modify natural behavior. Any muscle that contracts in the water generates a signal that can be detected with the aid of an amplifier having sufficiently high gain and not so noisy that the signal is masked. Incidentally, convenient test signals for checking the apparatus can be generated merely by submerging your hand in the water and clenching your fist.

Electrocardiograms can be made by arranging the electrodes in a special configuration for each kind of animal. It is not always possible to detect every component of the complex curve. For example, sometimes the so-called P wave may be small or missing. The method has not yet been developed to the point where it is suitable for making a detailed study of the whole electrocardiogram complex, but it is a highly accurate method of recording the frequency of the heartbeat. In the case of larger animals, such as frogs, potentials generated by limb movements and other major parts of the body are superimposed on the electrocardiogram. This is not a serious problem; even the most active animals are still for brief intervals. Movements can be eliminated by anesthetics, of course, but then the technique of using remote electrodes does not offer much advantage over surface electrodes. Other expedients to compensate for movements, or to confine the observations to a part of the animal, include specially shaped containers, electrical wave filters and multiple electrode arrangements connected in compensating arrays. Some have been tried, but Camougis feels that all merit further investigation and refinement.

The nerve and muscle cells of animals the size of a lamprey and even much smaller normally develop potential differences on the order of a tenth of a volt, a potential substantially greater than the output of a high-quality phonograph pickup. Offhand, it might seem reasonable to suppose that an amplifier of relatively low gain could boost signals of this amplitude enough for display on an oscilloscope. A high-gain amplifier must ordinarily be used, however, because an electrical field that exists in a conducting medium such as water is strongest along the path of least resistance-the path closest to the animal. As the field curves outward from the animal it grows progressively weaker, much as the field that surrounds a horseshoe magnet weakens with distance. As a result electrodes in contact with the medium at points remote from the specimen pick up only a small fraction of the signal voltage. For this reason an electric field that amounts to as much as a tenth of a volt in the immediate vicinity of an excited cell can fall to as little as a few millionths of a volt between the remote electrodes.

Animal tissue is capable of developing potentials much greater than a tenth of a volt. A potential difference of about .06 volt normally exists across a layer of skin fresh]y removed from a frog. But when two dozen such layers are stacked with the inside of one layer in contact with the outside of the next, a potential of 1.5 volts is measured across the stack. Through an analogous process one species of electric fish can develop a potential of 500 volts and more. The cells of electric fishes have evolved in a pattern of alternate positive-to-negative interconnections that is precisely comparable to the battery that emerged from Alessandro Volta's experiments with frogs and led to man's harnessing of electricity Certain South American knife fishes that develop potentials of several volts are often available from dealers in tropical fishes. The discharges from these animals can be observed easily without amplification or shielding. Just touch the tips of the oscilloscope probes to the surface of the water in the aquarium or use the probes for the input of a hearing aid.

In Camougis' opinion the range of animals that can be investigated by the remote electrode technique and the kind of information it can reveal appear to be limited only by the ingenuity and imagination of the experimenter. He mentions one investigator, working at a medical school, who has looked into the matter of recording the electrocardiogram of an alligator. There seems to be no theoretical reason why the project should not succeed if he can catch the animal when it is not moving or learn how to make it remain motionless. At another extreme in animal size, Camougis suggests, it should be possible to pick up the potentials that accompany the beating movements of the antennnae of the water flea Daphnia. "This has, in fact, been tried," he writes, "but with only limited success. The problem appears to be merely one of refining techniques. High-gain, low-noise recording methods, as well as an appropriate container and the right electrodes, should result in recordings free of distortion but with good resolution. In the case of minute organisms, remotely placed electrodes appear to represent the only practical method of picking up the action potentials. It is difficult to imagine how one could attach electrodes directly to a delicate creature of microscopic size without injuring it or modifying its natural behavior."

The oscilloscope and loudspeaker limit the investigator to instantaneous visual and auditory monitoring of the electrical potentials. As one progresses with a problem, permanent records for leisurely study are usually wanted. One apparatus for making permanent records is the tape recorder. Signals from the preamplifier can be fed simultaneously to the tape machine, oscilloscope and audio system. Tapes can then be played back and displayed on the oscilloscope or other apparatus at will. A number of photographic techniques are also available for making permanent records. Various cameras can be easily adapted for use in photographing the screen of a cathode-ray tube.

Automatic pen recorders are also available that write on a rotating drum or paper strip. The paper moves at a constant speed and therefore provides a horizontal time axis, with voltages or currents appearing as vertical excursions of the pen. The inertia of these pens limits their frequency response. Accordingly they can distort or even fail to record the fast transients. Within limits, fast events can be recorded on magnetic tape at high speed and then, at low tape speed, reproduced as a graph by a pen recorder. Camougis often finds it desirable to integrate the bioelectric energy for driving an ink writer. He accomplishes the integration by feeding the muscle action potentials into a large capacitor, which then powers the pen recorder. The smoother excursions of the pen are thereby brought well within the frequency limits of the recording system, although at some sacrifice of fine detail in the variations of the signal.

"In summary," Camougis writes, "the method of observing the bioelectric effects associated with aquatic animals has the obvious advantage of simplicity and provides perhaps the only practical method of studying motor responses of animals that must not be mechanically impeded because of their behavior, delicate structure or small size. A disadvantage of the method is that it doesn't localize the electrically active site. In this respect it is not intended to replace the techniques that involve inserting a fine electrode directly into the tissue being studied. It is a method of investigation at an entirely different level -studying the behavioral responses of intact animals by means of simple apparatus.

The stagnant pond in back of my home teems with Daphnia, and last summer our hi-fi set was not fully engaged. So I dug up a packing box big enough to hold a preamplifier and a microscope and covered it with old copper screening. A hole was made in the screening to admit a microscope eyepiece for examining specimens through a 90-degree prism. My plan was to place a single Daphnia in a drop of water on a microscope slide and pick up the action potentials by inserting a pair of small copper wires into the drop from opposite sides. These electrodes would feed the battery-powered preamplifier inside the cage. I decided to try out the arrangement by means of a loudspeaker before connecting the oscilloscope. The construction went smoothly enough, and when the amplifiers were switched on, ample power appeared at the output-all noise, consisting mostly of 60-cycle hum.

The cage leaked noise like a sieve. Still more was picked up by the circuits because I had neglected to ground the main amplifier and had failed to solder the screening where it came together at the corners. The tarnished wire would not take solder, so I re-covered the box with new screening. I found that the door screening must make good electrical contact with the box screening every few inches around the edge, and that all ground connections must come together at a single point. These corrections eliminated the 60-cycle hum, but the character of the random noise did not change although I tried every available kind of water-distilled, deionized, tap, rain and pond.

Finally I hit on the idea of reducing the size and shape of the specimen container. I softened a glass tube in a gas flame and pulled it into a capillary with a bore just slightly larger than the animal. Then I put a drop of distilled water containing a single Daphnia on a microscope slide and "speared" it with the glass tubing. Capillary attraction made the drop rush into the tube and carry the specimen along with it. Snapping off a half-inch length of tubing containing the specimen, I pushed fine wires into the ends far enough to make contact with the water. The junctions between the wires and the ends of the glass were sealed by dabs of silicone grease and the assembly was pressed against the sticky side of Scotch tape to secure the leads. It worked! The oscilloscope displayed the heartbeats clearly during instants when they were not obscured by other muscle action.

I wanted to make a permanent record of the oscilloscope display. Like most other amateurs, however, I do not own a pen recorder. It occurred to me that perhaps a loudspeaker could be modified to act as a pen motor if a lightly hinged arm was simply attached to the driving coil of the cone. Roger Hayward, who illustrates this department, fashioned a pen lever from heavy aluminum foil and equipped it with a hinge in the form of a steel reed cut from shim stock about .001 inch thick, as shown in Figure 3. The reed was clamped between the head of a machine screw and a piece of strap aluminum that was bolted across the face of the metal frame that supports the cone assembly. The pen lever was driven from the side opposite the hinge by a second length of shim stock that was attached by epoxy cement to the apex of a cone made from heavy aluminum foil. The base of the aluminum cone was cemented to the paper cone of the loudspeaker over the driving coil.

The first model was promptly wrecked. The violent vibration of the cone broke the hinge attachment. A full-wave rectifier and a smoothing filter were then inserted between the output of the amplifier and the loudspeaker pen recorder [see Figure 4]. The arrangement worked nicely at frequencies between 20 and 30 cycles per second, the lower frequency limit of the amplifier.

To record still lower frequencies, I then inserted a chopper-a motor-driven switch-between the output of the preamplifier and the hi-fi amplifier. The chopper was a modified motor generator of the type used to step up the voltage of storage batteries for powering radio sets used in military aircraft. The armature windings of the generator were cut loose from the commutator, and alternate bars of the commutator were connected together. The commutator of my unit, which was picked up on the surplus market for a dollar, had f39 bars. The odd bar was not connected. The circuit to be chopped was simply connected in series with the brushes on the generator side. I found that copper-impregnated brushes introduced less noise in the chopper circuit than the graphite brushes with which the unit was originally equipped. At its normal operating speed of 6,400 revolutions per minute, the chopper converted direct current to alternating current at a frequency of about two kilocycles.

The end of the pen lever was bent at a right angle to act as a stylus and was supported against the smoked cylinder of a motor-driven drum recorder, or kymograph, by an apparatus stand. (For data on constructing a homemade kymograph see The Amateur Scientist; SCIENTIFIC AMERICAN, April, 1960.)

With this apparatus I succeeded in making a number of electrocardiograms of Daphnia and of other small animals, including frogs. At least I call them electrocardiograms. Actually they are rather smooth waves that appear in step with the heartbeats of the animal. I do not know what a really good electrocardiogram of a Daphnia should look like, and it is probable that a true one would require greater refinements in technique than this department managed to contrive. But, as Camougis predicted, the attempt was good fun.

Bibliography

BIOELECTRICITY. E. E. Suckling. McGraw-Hill Book Company, Inc., 1961.

AN INTRODUCTION TO ELECTRONICS FOR PHYSIOLOGICAL WORKERS I. C. Whitfield. St. Martin's Press, 1953.

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