"For my science fair project during my senior year in high school," Miss Southwick writes, "I set up a controlled experiment to test the effect on rats of chlorpromazine, one of the new tranquilizing drugs. Tranquilizers were making news at the time, particularly in the treatment of mental disease, and this caught my interest. According to medical reports, the side effects of the tranquilizers had not been fully catalogued, and it seemed likely that a science fair project based on one of them would have a good chance of scoring high on originality.

"Chlorpromazine has the property of quieting mental patients who are restless, overactive and abnormally elated. Would the drug have a similarly depressing effect on normal animals? If so, how would it affect other aspects of their functioning?

"As subjects for the experiment I obtained six white rats, all males from the same litter. During the first phase of the experiment three rats were selected at random for treatment and the remaining three were reserved as controls. Each animal was tagged for identification. Midway through the experiment the treatment was switched; the controls were put on the drug, and the group previously treated became the controls. I called this the 'crossover' phase. It served as a check against previous results. Otherwise all animals were maintained under identical conditions as closely as possible, and each was given a standard ration of food and water on a fixed schedule. The experiment continued for five months as a spare-time activity.

"A thorough physical examination was made of each animal at the beginning of the experiment, both to assure that the animals were in good health and to provide comparison data for subsequent use. The effects of the drug were then observed by measuring changes in activity, intelligence, blood composition, pulse rate, body temperature, weight, respiratory rate, external features, sexual behavior, internal organs and metabolism. The animals were treated by administering; chlorpromazine along with their food, initially at the rate of five milligrams of the drug per kilogram of body weight. After one week the dose was increased to 15 milligrams; somewhat later, to 20 milligrams.

"Reaction to the drug was immediate. The treated rats became quieter than the control group. Their movements were slower and more deliberate. To measure the difference I designed a 'jiggle cage' consisting of a box of quarter-inch-wire mesh covered at the bottom with a sheet of heavy aluminum foil of the kind used by bakers. The cage was suspended by a weak spring from the bottom of a small table consisting of a seven-inch square of half-inch plywood fitted with legs of wooden dowel stock. The movements of the caged rat caused the cage to jiggle up and down and actuate the handle of a telegraph key in contact with the bottom of the cage. The key closed an electrical circuit between a battery and the coils of a modified buzzer. The buzzer contacts were closed; a wire stylus was attached to the armature. The stylus pressed against the smoked drum of a kymograph on which the movements of the rat registered as sharp vertical pips in an otherwise smooth trace. I borrowed the kymograph from the biology department of the Midland High School. It is not too difficult, however, to make such a drum recording-device. One is described, in fact, in 'The Amateur Scientist' for July, 1957. A recording speed of some three inches per second, equivalent to the speed of a six-inch drum turning at about one revolution per minute, is adequate for this experiment. "The activity of each rat was measured daily for one hour in darkness (when rats are commonly most active). Copies of two recordings are shown in the accompanying illustration [above]. These show the activity of the same rat before and after the drug had been administered.

"The effect of chlorpromazine on intelligence was tested by means of a changeable maze in which both the pattern of the paths and the obstacles (rectangular partitions ) could be altered. The animal was required to crawl under a partition or jump over it, depending on whether the partition was turned so that its opening was at the top or at the bottom. The maze was covered so the animals would not be distracted during the run. To test the adjustment of the rats to change the maze was altered four times for each group of animals during the course of the experiment. In the beginning the control rats required about seven runs to learn the maze, during which the time of the run dropped from eight minutes to 30 seconds. The treated group required substantially more practice to achieve comparable performance; at first these rats actually lost ground. The reaction of the rats was even more significant during the crossover phase of the experiment.

"During the crossover phase the rats in the control group learned to run the maze, with practice, in five seconds. When they were under the influence of chlorpromazine, however, none of these rats could do better than one minute. In contrast, the previously treated rats, after recovering from the drug, learned to run a new ,pattern in five seconds. Furthermore, when the crossover phase was started, the drug caused the controls to forget a maze pattern they had mastered. From this it would appear that the drug has a depressing effect on memory as well as on intelligence.

"Does the drug similarly depress organic functions? This was investigated in part by examining changes in the blood of the rats. The blood was taken by clipping the tip of the rat's tail. Incidentally, I quickly learned that rats are not as cooperative in all parts of the experiment as one could wish; blood sampling is a case in point. I first tried to hold the animals in one hand while taking the specimen with the other, but soon adopted the technique of wrapping them in a towel. Later I borrowed a glass vessel especially designed for the purpose. Standard clinical pipettes were used for withdrawing two specimens of blood from each rat: one specimen to ascertain the number of red blood cells; the other to test for the number of white blood cells. The pipettes are fitted with a short length of suction tubing; one simply places the glass tip in the fluid and withdraws enough to reach the .5 mark etched in the glass. Sufficient diluting fluid is then drawn into the pipette to reach the top mark. When one is sampling white cells, the top mark is 11; in the case of red cells the mark is 101. The diluting fluid for white cells (which causes the red cells to disintegrate) consists of one part by volume of hydrochloric acid to 100 parts of distilled water. The red-cell specimen is diluted by a fluid consisting of .5 gram of mercuric chloride, five grams of sodium sulfate and one gram of table salt dissolved in 200 cubic centimeters of distilled water. This solution causes the white cells to disintegrate.

"Blood cells are counted with the aid of a special chamber that divides the field of view into a pattern of uniform squares, somewhat like a sheet of graph paper. If one encounters difficulty in procuring a counting chamber, a rough estimate of change in the relative number of white cells and red cells can be made by comparing stained specimens. I borrowed a chamber.

"The red-cell count remained constant throughout the experiment for both treated and untreated rats. But the white-cell count increased substantially during the time the animals were on the drug, averaging 22,000 cells per cubic millimeter during 'treatment as against a normal count of 14,000. Counts were taken of all rats once each week for the first three weeks after the beginning of treatment. Measurements made during the crossover phase were identical with those recorded at the beginning of the experiment. In one exceptional case, however, both groups yielded identical counts. This occurred after the experiment had been running two months and suggests that the rats may have developed some tolerance for the drug.

"Stained blood-specimens were examined with the aid of a microscope equipped with a 10-power eyepiece, an oil-immersion objective of 97 power, and dry objectives of 10 power and 43 power. The combinations of eyepiece and objectives gave magnifying powers of 970, 100 and 430 diameters. The instrument was borrowed from the Midland Hospital.

"The technique of making differential smears is not difficult if one carefully follows a standard procedure. The microscope slides must be cleaned thoroughly. Ordinary household detergents, particularly those containing a soft abrasive powder, make a satisfactory cleaning agent. A drop of blood is first placed near the end of a freshly cleaned slide. The end of a second slide is then held at an angle of about 45 degrees at a point between the drop and the center of the second slide, so that the drop wets the lower surface of the upper slide. The fluid will immediately spread by capillary attraction across the line of contact between the two slides. The second slide is then quickly pushed forward toward the far end of the first. This distributes the specimen behind the top slide in a film which adheres to the lower slide. Do not place the drop in front of the top slide and push it with the end of the glass; the cells will be forced to flow between the two slides and may be broken.

"The smear is allowed to dry until it becomes tacky, and then is stained. I used Wright's stain, which can be procured through most drugstores. It is made into a solution consisting of .8 gram of powdered stain mixed with three grams of glycerin and 97 cubic centimeters of methyl alcohol. A drop of the solution is applied to the slide and allowed to stand for three minutes. A drop of buffer solution is then added. This consists of 1.63 grams of potassium phosphate and 3.2 grams of sodium phosphate dissolved in a liter of distilled water. The ingredients of both formulas, incidentally, may be cut in proportion if smaller quantities are desired. After the buffer has worked five minutes the slide is washed gently with distilled water, permitted to dry in open air and then placed under the microscope for examination.

"The differential smears from rats under treatment showed a 20-per-cent increase in the white cells known as lymphocytes, and an equal decrease in neutrophil white cells. Again, however, one measurement made two months after the beginning of the experiment proved exceptional and suggested the development of tolerance to the drug.

"As another index of reaction to the drug the pulse rate of all animals was taken twice during each phase of the experiment. This proved somewhat difficult because the pulse rate of a healthy rat is about 375 beats per minute, and, as I discovered, this rate is almost doubled by the administration of chlorpromazine. Accordingly accurate counts could not be made by listening to the rats' heartbeats with a stethoscope. I solved the problem with the aid of an ordinary magnetic-tape recorder. The rat was held against the microphone and a recording of the heart sounds made at a tape speed of 7.5 inches per second. The record was next played back at 3.25 inches per second and the beats counted by ear against a stop watch. The count was then multiplied by two. (The absolute tape speed, which varies with the make of the machine, is not important. One is only concerned with the ratio of speeds.) Rats tend to become excited when placed against the microphone, so they should be permitted to settle down for a few minutes before the recording is made. The pulse rate of tranquilized rats averaged 639.6 beats per minute, as against 389 beats per minute for the controls. The slightly higher-than-normal rate of the controls is explained by the excitement of the rats at being handled.

"The body temperature of tranquilized rats was also found to be abnormally high, averaging 100.1 degrees Fahrenheit as against an average of 99.7 degrees for the controls. The average difference is not great. But in every instance the lowest temperature observed in a treated animal was above the highest temperature among the controls.

This fact, when considered together with the elevated lymphocyte and neutrophil counts, suggests that the tranquilized rats had contracted an infection of some sort. This was further indicated by their behavior. Some appeared to be sick part of the time. The temperatures were taken with a conventional rectal thermometer.

"A careful record of body weight was made daily, along with the weight of food and water consumed. Early in the experiment the ratio of water to total body weight changed in a direction that suggested that the drug was causing dehydration, but this was not supported by the crossover observations. Both groups made comparable gains in weight throughout the experiment, as indicated by the accompanying graph [Figure 4].

"Chlorpromazine lowers the respiratory rate of rats substantially. Tranquilized rats average 72 inhalations per minute as against 95 for the controls. The rates coordinate well with the relative activity of the two groups. Counts were taken three times during each phase of the experiment.

"A close check was made throughout the experiment of the external appearance of the rats in both groups. I observed a number of obvious differences. Minor skin eruptions developed within a few days after each animal was put on the drug. In addition, the hair of the treated rats became rough and shed readily. In the case of the first group to receive the drug, violent muscle tremors occurred when the animals awakened from sleep. This, however, was not observed in the group treated during the crossover phase. The behavior of the groups also suggested that, at least in rats, chlorpromazine acts as a sexual stimulant.

"Tendencies to jaundice sometimes follow the administration of chlorpromazine, according to reports in the professional journals. This reaction appeared in both groups of rats approximately two weeks after treatment was started. Their eyes became pale and their feces lost color. To check possible damage to the liver an autopsy was performed on one control and one treated rat 17 days after the beginning of the crossover phase. Sections of liver tissue were taken from both rats and preserved in xylol. Both specimens showed abnormality. The damage appeared more extensive in the animal that was undergoing treatment at the time the autopsy was performed. This part of the experiment was interesting, but because the observations were limited to two animals the result could not be considered conclusive.

"No diabetic effect was observed. The qualitative test for this reaction was made by a procedure which I learned at the Midland Hospital that requires the use of chemically treated test-strips that one must either purchase or borrow. The strip is dipped in the urine of the animal; if sugar is present, the tip of the stick turns blue within a minute.

"Metabolism was measured by a variation of the method devised in 1890 by the noted British physiologist J. S. Haldane see "The Amateur Scientist"; August, 1957]. Essentially the test consists in supplying the animal for a known interval with air containing a minimum of water vapor and carbon dioxide and then subtracting the weight lost by the animal from the weight of the water vapor and carbon dioxide exhaled during the test interval. This gives the weight of oxygen absorbed by the animal, and when this figure is divided into the weight of exhaled carbon dioxide (adjusted for the molecular weights of oxygen and carbon dioxide), the result is equal to the respiratory quotient of the animal. My apparatus consisted of an air pump (a water-powered aspirator) and five flasks of one liter each connected in series [see illustration in Figure 7]. The initial flask in the series contained approximately 600 cubic centimeters of 'Ascarite,' a commercial preparation that has the property of absorbing carbon dioxide. The second flask held a comparable amount of anhydrous calcium chloride, which absorbs water vapor. The intake of the second flask was coupled to the exhaust of the third, a wide-mouthed jar capped with a close-fitting stopper, which served as the animal chamber. The intake of the animal chamber led respectively to flasks of calcium chloride, Ascarite and calcium chloride. All containers and the animal were weighed individually before and after a test interval of one hour. The weight (in grams) lost by the rat was then divided into the product of the weight gained by the fourth and sixth flasks multiplied by .7282 (the ratio of the molecular weights of oxygen and carbon dioxide).

"Only two animals remained in each group at the time the respiratory quotient was measured, the other pair having been used for the autopsy. The respiratory quotients of the rats then under treatment were .56 and .58, whereas those of the control rats were .82 and .65. Here again the sample was too small to yield reliable figures. Differences in the individual determinations show a spread, however, which suggests that the lower activity of the tranquilized rats is accompanied by a correspondingly low respiratory rate.

"From this experiment it would seem that a rat is tranquilized by the steady administration of chlorpromazine, but with at least temporary cost to its health. The drug depresses the animal's memory and intelligence, alters the composition of its blood, invites infection, increases its pulse and body temperature, lowers its metabolism, induces abnormal sexual stimulation and damages its skin, hair and liver."

Many readers of this magazine have undertaken to repeat some of the experiments in color vision recently described by Edwin H. Land [see "Experiments in Color Vision"; SCIENTIFIC AMERICAN, May]. In one of these experiments Land made two black-and-white photographs of a colored scene, one photograph through a red filter and the other through a green filter. Positive transparencies were then made of both photographs. When the "red" transparency was projected on a screen through a red filter, and the "green" transparency projected without a filter, the superimposed images reproduced the original scene in full color.

Most amateurs who have repeated this experiment have used two 85-millimeter slide projectors-one borrowed from a friend. The experiment was then prematurely terminated by the necessity of returning the borrowed projector. Jeremy M. Palmer of North Hollywood, Calif., solved this problem by means of a gadget that can be built in 1S minutes.

On the face of a block of wood half an inch thick and 2 1/2 inches square Palmer makes two saw cuts a quarter of an inch deep; one cut is parallel to one edge of the block and the other cut is parallel to an edge at a right angle to the first. A third cut is then made diagonally across the face of the block. This bisects the right angle made by the previous cuts. Two 35-millimeter slides made by Land's technique are placed in the right-angle cuts. A microscope cover-glass, mounted in a cardboard slide holder, is inserted in the diagonal cut. The cover glass functions as an optical beam-splitter. When one looks through it directly at one slide, the other is seen simultaneously by reflected light and in register if the two slides are properly positioned. One slide is lighted by reflection from a colored card and the other by reflection from a white card.

Roger Hayward, who illustrates this department, recommends a more elaborate version of Palmer's device. He makes a pair of wooden brackets with faces at an angle of 45 degrees and sandwiches a half-silvered mirror between them. The slides are clamped to the outer surfaces of the assembly (which meet at a right angle) by means of spring clothespins, as shown in the illustration in Figure 8. "Palmer's gadget works," writes Hayward, "but the slides tend to jiggle out of register in the saw kerfs. Clothespins, my favorite laboratory clamps, provide solid support. The substitution of a half-silvered mirror for the cover glass increases the brilliance of the image greatly because plain glass is a poor reflector."

William Tyrrel of Hempstead, N. Y., submits a solution for another problem that confronts many amateurs: the construction of a safe and inexpensive source of high voltage required for the operation of Crookes tubes and similar apparatus. "In the years before sources of alpha radiation became available," he writes, "I constructed a high-voltage, low-current supply for neutralizing the static charge that builds up on the paraffin ribbon from a microtome. It was based on a modified spark-coil from a Model-T Ford. The coil was driven by a 110-volt, alternating-current source, and the output was rectified by a vacuum tube diode. The Model-T coil gave better results than any other.

"The coil was taken apart and the windings immersed for an hour in a mixture of melted paraffin containing about 3 per cent of beeswax at a temperature of 130 degrees Fahrenheit. The primary and secondary windings were next separated and fitted with leads of flexible lamp cord. The assembly was then re-potted in the wax, with the leads extending from the box.

"A vibrator of the type used in the power supply of automobile radios was substituted for the one with which the coil is fitted and was connected with the primary winding [see Figure 9]. For maximum output the vibrator contact must close at the instant a four-microfarad line-capacitor reaches full charge. The time of closure is determined by the value of all iron-core inductance of the type used in 'B' battery eliminators, which is connected in series with the capacitor and is adjusted either by removing iron laminations from the core of the inductance or by adding capacitance. The proper adjustment must be determined experimentally. The coil that drives the armature of the vibrator is energized by the line voltage through a 10-watt lamp. Hence the device operates at 60 cycles per second, and a unidirectional voltage pulse appears at the output every half cycle. The quality of the output is improved by the addition of a rectifying tube of the high-voltage type used in power supply of television sets. The cathode of the tube operates at 30,000 volts above ground potential, so it must be energized by an insulated source. I used a flashlight battery. The current drain is low, however, so a cell will give many hours of service.

"Ford coils can still be procured from at least two of the large mail-order firms and from some of the suppliers who cater to those who make a hobby of restoring antique automobiles."

Bibliography

THE NEW PSYCHIATRIC DRUGS. Harold E. Himwich in Scientific American; October, 1955.

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