Psychotropics such as psychostimulants, narcotics (morphine), hallucinogenics, and anxiolytics (benzodiazepine), and nicotine of tobacco and alcohol are dependent (addictive) drugs. Dependence is state that hard to suppress repetitive craving for a substance (psychological dependence) and induced physical symptoms (physical dependence) such as headache after a long-term intake and physical disappearance by disruption of the intake (discontinuation or withdrawal syndrome). Dependent drugs potent to the extent that psychological and physical dependences can cause social problems are socially regulated. Palatable foods, to which we have potent preference, can induce craving for repetitive intakes like dependent drugs and lack of the foods causes physical symptoms in absence of nutritional substitution. Effects inducing craving for the repetitive intakes are called as reinforcing effects (rewarding effects), shown by the all dependent drugs, and observed in the palatable foods. The intakes of dependent drugs are social problems and we can keep abstinence if we are not dependent. However, we must continue to take foods, which is different in the dependent drugs. Therefore, reinforcing effects of foods are reasonable but their over intake are problematic in "age of plenty". We must take fats and sweet taste substances e.g. sugar as energy and their reinforcing effects are confirmed in rodents. We must take them even if little for sustaining our life but their over intake is afraid to induce metabolic syndrome. We know adverse effects by the over intake but cannot keep abstinence, which means similar effects to psychological dependence by the dependent drugs.

7-1 Dependence

7-1-1 Self-administration

Positive reinforcing effects (those are also simply called as reinforcing effects) are effects that promote operant behaviors such as lever pressing in rodents, namely give positive motivation to the behaviors. Stimuli to give positive motivation and negative motivation such as electric shocks, which suppresses the operant behaviors, are called as a positive reinforcer and a negative reinforcer, respectively. Reinforcing effects also called as rewarding effects and the positive and negative reinforcer is also called as reward and punishment, respectively. Reinforcing effects are necessary to dependent drugs and those induce strong desire for repetitive intake. These effects are measured by a self-administration method in rats. Some drugs cause lever pressing and other do not under the condition that an intravenously cannulated rat is given drug solution from an infusion pump connected to the cannulation if the rat presses a lever. The drugs that could cause the lever pressing are judged to have reinforcing effects and the other are not to have. Rats are considered as feel pleasant by the administered drug and press the lever to get this pleasant stimulus. Therefore, we can evaluate reinforcing effects by the lever press in the self-administration method. The stronger desire for the drug makes rats press the lever more and take the drug. The stronger pleasant stimulus makes the rats press the lever even if the rats must press the lever more to get the stimuli.

A number pressing the lever until the rat can get one administration of the drug solution is called as FR (fixed ratio). First, rats are trained to press lever at FR=1, namely every one response is reinforced. We can evaluate reinforcing potency of the drugs by gradually increasing FR.

If rats cannot connect the lever-pressing to pleasant sensation, it does not become motivation to press the lever. If it take a long time from injection of the drug solution to appearance of drug effects (pleasant sensation), rats hard to press the lever. Therefore, the drug is generally administered by an intravenous injection and only solution is available different from cases of subcutaneous or intraperitoneal injections. For purpose of free moving measurement, the cannulation is led and projected from a head in the rat and settled to a cage by a cannula swivel. Such operation and procedures to train the lever-pressing take a long time and are not easy.

Drugs are intravenously administered in the self-administration but oral intake of foods can cause pressing the lever in hungry rats. It is considered as self-administration of foods instead of the drugs. We consider that the rats can soon feel pleasant stimuli (good tastes) by oral intake and the rats easily become aware the rewards because food intake is a subjective behavior different from the drug administration, which might make oral administration is effective to press the lever in foods different from the drugs. The intake of the drug in the self-administration method become aware in rats by an oral administration but is conscious of unpleasant stimuli by its bitter taste, which makes the spontaneous intake difficult unless the rats soon feel pleasant sensation after the intake. Alcohol (ethanol) is self-administered via an oral route in rats but the lever-pressing is trained by sweetened ethanol as a reward in beginnings. Because later the rats become to press the lever for oral non-sweet ethanol intake, the rats are considered as could learn association between the lever-pressing and pleasant sensation by the ethanol intake until that time.

7-1-2 Conditioned Place Preference Test

A simple and widely-used method for evaluation of reinforcing effects in rodents is a conditioned place preference (CPP) test. This method literally measures preference which is not equal to the reinforcing effects. However, rats and mice show preference in this test to dependent drugs with the reinforcing effects and do not show preference or show aversion to the drugs without the reinforcing effects. Therefore, the CPP test in rodents is used as a simple method to predict reinforcing effects of drugs.

A measurement cage consists of two compartments different in environment (color and texture) are connected each other. We confine the rodents in one compartment for a certain time e.g. 30 min after administration of a drug and confine similarly in the other compartment after a vehicle injection in the next day. These treatments are repeated and effects of the drug are conditioned to the environment of the one compartment in the animals. If the drug has pleasant stimuli, the rodents feel pleasant sensation in the drug-conditioned compartment and prefer to this compartment in which they will spend more time when they can freely move between the both compartments. The CPP test can be conducted in mice as well as rats by easy procedures. The conditioning (confinement in the compartment) generally conducted for 30 min and repeated alternately 3 times per each. Since time spent in the each compartment must be measured (for 20 min) a day before and after the conditioning in the CPP test, it totally takes 8 days at minimum. We often repeatedly measure time spent for several days before the conditioning to screen the rats and mice by preference to the each compartment and acclimate the animals to the both compartments, a measurement cage. In such cases, we need more than 8 days for the overall CPP test. Although some conditioning is conducted two times a day for shortening the test term, an experimenter should confirm and establish experimental conditions in which preference to dependent drugs as positive controls can be detected before the test drugs.

Two compartments are different in color as white and black and a texture of floors as rough and smooth in an original method. Thus, the rodents can discriminate the two compartments from two cues. Because the rodents are difficult to discriminate different colors, two clearly different colors, white and black or light and dark, are used. However, the rodents usually prefer a dark place and spend time in dark or black compartments compared with light or white ones. If preference to the each compartment is largely different, preference by the conditioning becomes difficult to be detected. Various floors such as mesh and rods as well as rough and smooth boards are used in combination with different colors for countermeasures to compensate preference to the color. Avoiding preference to compartments by light and dark, the two compartments in some experiments are discriminated with vertical or horizontal stripes instead of white and black. Further, in some experiments colors of the both compartments are same and only floor textures are different. However, because the rodents have a few cues to discriminate the compartments in this case, they may not discriminate the preferred compartment in the measurement of time spent in the both compartments after conditioning.

Preference to the compartment is only measured by time spent in the each compartment by the animals under they can freely select the each. Mice and rats may spend in the non-preferred compartment before the conditioning (baseline) to explore novel environment. Therefore, time spent in the baseline without acclimation to the both compartments is considered as baseline preference, it may not show preference to the compartment. Since confinement in the compartment during conditioning makes the rodents acclimate to it and time spent in the non-preferred compartment by exploratory behaviors diminishes, apparent preference may change regardless of conditioning. Enough acclimation to the both compartments before measuring baseline preference and multiple measurements (e.g. 3 times) of time spent in the baseline become countermeasures to it. In the latter case we can select subjects exception the mice or rats in which preference is largely different between multiple baseline measurements and between the compartments.

Although time spent in the compartment is measured as the time which the animal is present in it, the judgment which compartment the animal is present may often be difficult in a center place. Further, rats and mice may not discriminate the compartments around the center place. To reflect the animal's preference more correctly in his time spent avoiding such case, we can make a small neutral zone in center and use 3 compartments. A tunnel neutral zone and 3 compartments with a shape of T-maze are also reported and the CPP test with 3 compartments is recently increasing. Although an automatic measuring device the time spent in the each compartment by infrared beam sensors is available, we can manually measure the time spent by a stopwatch with an appropriate cage for the CPP test. Because we generally use one cage to measure multiple rats or mice, we should clean the cage with cloth or cotton alcohol soaked before measurement in the next animal to wipe effects by the previous animal such as smells. Since we only confine the animal in one compartment after a drug administration in conditioning, we may simultaneously confine two animals in each. However, the simultaneous conditioning of two animals with one cage is difficult because the rodents may be affected by a neighbor in smell, sound, vibration, or etc. in the conditioning. Some drugs significantly decrease time spent in the drug-conditioned compartment, which means the drugs have aversive effects and rodents showed aversion. Thus, the CPP test can evaluate aversion as well as preference although the self-administration method can discriminate aversion from non-preference.

7-1-3 Physical Dependence

Physical dependence is generally evaluated from physical symptoms shown by discontinuation of a dependent drug after a long-term intake. Narcotic morphine can induce withdrawal syndromes in the short term after 10 mg/kg s.c. injections 2 times a day for 5 days (total 10 times) in mice by a subcutaneous administration of naloxone, an opioid m antagonist, at a dose of 2 mg/kg on 6th day (1). Several schedules that a dose of morphine gradually increases as 20, 40, 60, 80, and 100 mg/kg, i.p. 2 times a day for 5 days (naloxone 0.1 mg/kg, s.c.) (2), and as 20, 40, 60, 80, 100, 100, and 100 mg/kg, i.p. 3 times a day with 8-hr interval (naloxone 1 mg/kg, s.c. on 3rd day 2 hrs. after the last morphine injection) (3) are reported. Physical symptoms such as diarrhea, a decrease in a body weight, leg tremor, jumping, wet dog shake, and so on are observed as withdrawal syndrome of morphine.

Alcohol (ethanol) can also induce physical symptoms after the long-term intake by oral intake mixed with foods (4), p.o. administration (5), (6), and aspiration by vapor (7). Physical symptoms such as irritability to touch (vocalization), tail rigidity, abnormal posture, and tremor are observed as withdrawal syndrome of ethanol and seizure is induced in some severe cases.

Because central and peripheral effects by the drugs other than reinforcing effects are different in each drug, symptoms by discontinuation of the drugs are also different. Therefore, evaluation methods for dependent drugs regarding slight discontinuation symptoms or severe withdrawal syndromes by which promotes repeated intake to avoid them are different in each drug.

7-1-4 The Other Test for Dependence
7-1-4-1 Self-stimulation

Similarly to a self-administration method, rats press levers in a self-stimulation method to receive stimuli to central nervous systems via an electrode placed in brains. Dopaminergic systems projecting to cerebral cortex from ventral tegmental area (VTA) in midbrain (A10 neuronal systems) are called as rewarding systems in the brain. Dependent drugs elicit pleasant effects by stimulating the rewarding systems. Therefore, direct electric stimuli can induce pleasant sensation and make rats press the lever. Such tests are called as self-stimulation which served a potent tool to clarify neuronal circuits in the rewarding systems.

7-1-4-2 Drug Discrimination

We can be aware of effects of drugs if those have pleasant or aversive stimuli. We become conscious of pleasant stimuli by the dependent drugs and crave intake again for these stimuli. Many people easily know pleasant sensation when drink alcohol beverages. Ethanol and anxiolytic (hypnotic) benzodiazepines induce relief and ease at a small dose and induce hypnotic effects at large. Someone says that psychostimulants have stimulating effects literally, LSD is hallucinogenic, and morphine induces euphoric effects. We can express various subjective effects of the drugs by words but cannot use words in rodents. A drug discrimination method is used to evaluate difference in the subjective effects in the rodents by making the animals compare the subjective effects of the drug to others.

We generally trained rats to press different levers with food rewards in a 2-lever operant chamber after drug or vehicle (saline) administrations in this method. If the drug (training drug) elicits subjective effects, rats can become to discriminate the drug injection from the vehicle injection. The other than the training drug is administered in trained rats after establishment of the drug discrimination, and the rats are observed which lever they press. If the rats respond to the drug-associated lever, we say that stimuli of the tested drug generalize to trained drug and consider the stimuli are similar to those of the trained drug.

7-2 Preference

7-2-2 Preference Test

A preference test evaluates preference in mice or rats measuring which options they select. Two or more than 2 options of foods or beverages are presented to the animals and their intakes are measured. It is a simple test, in which the option they consumed more is preferred. This test is based on assumption that mice and rats consume more the preferred one. If this assumption is not true, we cannot correctly evaluate the test results and we must conduct the test to let the assumption be true. Mice and rats may drink one beverage placed a preferred side by chance in two simultaneous presentations of a two-bottle choice test. In a short-term consumption test they may take from one bottle they first contacted. We should change the side the bottles are placed after a certain time for a countermeasure to it. Novel foods in color, smell, shape, taste, and so on are alerted and not easily taken, which is called as neophobia. Therefore, we should make the appearances of the options be similar to a maximum extent or acclimate the options before the test.

Solid foods are also available as well as liquid in the test. Although the intakes can be measured manually with scales as weights, a drinkometer (drip detection or weight measurement) for liquid and a food intake monitor for solid are also available for the preference test. A lickometer is also used for a short-time test, which measures a number of licks to a spout feeding beverages.

Too many options may be hard to select a preferred one appropriately. The preference test shows relative preference and we cannot know like or dislike of non-selected one. We may evaluate preference from the intake of an each object sequentially presented but a presenting order may affect results. We should sequentially increase in a concentration of the object in the test of the same object, and repeat by changing the presenting order and evaluate from the average in the test of the multiple different objects.

We described the assumption the rodents consume more the preferred one but we can also consider that they do not consume the least favorite one. In the latter case the selected option may not be preferred although it is not disliked. It will induce to decrease total intake of the both and the both may be few consumed in a single presentation. Since high fat foods are palatable and highly preferred, those are consumed more in the choice of high and low fat foods. However, in the single presentation the consumed weight of the high fat foods is not so high because those have high energy density per weight. We can think that comparisons on the energy basis are appropriate because we know the energy density. Similarly comparisons on the weight basis may interfered by a water concentration in foods. We can easily evaluate appropriate the results in such cases because we know factors affecting the weight-basis comparison. However, if substances having anorectic effects exist in foods, can we evaluate the results appropriately? It induces decrease in food intake but we cannot judge it as decrease in preference to the foods. In the choice test, the relative preference may be shown in the small consumption but we may consider the both foods (options) as least favorite from a few intakes. Thus, the comparison in the preference test (the choice test) only shows differences in intakes in the experimental condition and we must carefully discuss the results before judgment as preference.

7-2-3 Measurement of Reinforcing Effects

Foods can be also evaluated on their reinforcing effects in similar manner of dependent drugs by self-administration and the CPP test. Rats and mice press lever to get foods, which shows the foods have positive reinforcing (rewarding) effects in the hungry ones. Similarly, water has reinforcing effects in the thirsty rats and mice. Oil and sweet taste foods such as sugar solution show reinforcing effects in rodents without their abstinence of foods and water, namely preference in the CPP test and self-intake by lever pressing are shown.

Foods having reinforcing effects are evaluated that those elicit pleasant effects and easily judged as highly preferred (palatable) different from the preference test.

7-3 Closing

Reinforcing effects of food components such as oil and acupuncture are reported since the CPP test is developed. Reinforcing effects are studied as the effects of dependent drugs inducing psychological dependence. Because the reinforcing effects of subjects are the effects reinforce the behavior to take the subjects such as lever pressing in rats as an operant behavior, it is rational that palatable foods promoting repeated intake have the reinforcing effects as well as the dependent drugs. We can easily know that acupuncture eliciting pleasant stimuli also shows reinforcing effects in the CPP test.

Someone shows preference to intakes of tobacco and coffee, which includes ingredients having reinforcing effects, nicotine and caffeine, respectively. Differences in use of terms of "preference" and "dependence" are obscure. We generally definite a psychiatric disorder as a disease to be needed its therapy when the psychiatric disorder becomes severe to disturb their social life. To extent fact that we cannot stop intake the subject disturbs the social life, we call it as "dependence" on the subject but not "preference" to the subject and the "dependence" is needed to a therapy. Recently, phrases of "dependence on pachinko" or "dependence on shopping" are used from problems that we cannot stop to do them. We easily accept a sentence that nicotine (tobacco) and alcohol are dependent substances. However, we say "preference" to a high fat diet but not "dependence" on it. Although intakes of nicotine and alcohol are not necessary different from fat (essential fatty acids) for our health, "dependence" on the high fat food may become a common sentence when the fact will be widely known that we cannot stop the intake of the high fat diet in spite of deleterious effects of its overintake such as inducing metabolic syndrome.