1-1 Anti-conflict test

Anxiolytic effects are historically evaluated to suppress an experimental conflict state. Conflict is the state in which we worry about ambivalent states simultaneously occurred, namely, the both positive and negative stimuli are given us. For example, we want to eat cake but not to gain body weights, dislike pains of dental therapy but cure bad teeth, and hungry but hard to ingest the foods due to a painful throat. We can understand to behave by overcoming some conflicts if we think our usual behaviors. In experimental conflicts, electric shocks are added in hungry or thirsty rodents if they get foods or water, respectively. In the both cases, negative stimuli (punishments) are simultaneously given with food or water intakes. Motivations to eat foods or to drink water are hungry and thirsty states in rats and mice, which induced by their foods or water deprivations. The water intake is directly measured by licking numbers to a nozzle feeding water but the food intake is substituted by numbers of lever-pressing and supplying foods which are fed as pellets or liquid diets associated by the lever-pressing in an operant chamber.

Geller and Seifter reported the conflict test in rats by the operant chamber in 1960 (1). The rats were trained the lever-pressing that causes to feed liquid diets and electric foot shocks were simultaneously given with the diets as the conflict in an alarm period when it was alarmed by a buzzer. In their report barbiturates having anxiolytic (anti-conflict) effects made the rats increase in the lever-pressing in the alarm period. Later, this method has been called as the Geller-type conflict test. We can think in this experiment that anxiolytics suppress the conflict between desire to foods and fear to electric foot shocks, namely decrease in anxiety to receive the shocks, and increase the lever-pressing. However, we needs a long time to train the lever-pressing in the rats and cannot easily conduct the experiment. In contrast, Vogel measured drinking behaviors without the training on the condition that the electric shocks are simultaneously given with the drinking (punished drinking) (2). Anxiolytics increased drinking water in this test and later, it is called as the Vogel-type conflict test. Previously, lever-pressing is only measured in rats and is difficult in mice but the Vogel-type conflict test can be measured in mice as well as rats, which have also provided a measure merit. Currently, a light lever that mice can press and measurement of a nose poke in substitution for the lever-pressing in mice are developed and the Geller-type conflict test can be conducted in mice (3), (4).

In the Geller- and Vogel-type conflict tests, are increases in eating and drinking behaviors anti-conflict effects as results anxiety suppressed? If the conflict suppresses the behaviors, suppressing the conflict will increase the behaviors but the experimental conflict can be suppressed by the other than anxiolytic effects. For example, analgesic effects inhibit to feel electric shocks and the shocks cannot induce the conflict. Because rodents do not know whether they pain or not until they receive the shocks, analgesic effects will not affect worry about pressing or not in the alarm period in which the shock is not yet given. However, the analgesic effects may affect drinking behaviors which are still observed under the electric shocks in the Vogel-type conflict test. Increases in motivation may increase the behavior suppressed by the conflict. Hungry and thirsty levels in rodents may increase to the extent they cannot be patient to intakes of foods and water in spite of the electric shocks. Although we should confirm whether drugs have such effects or not in the other than the conflict test, increasing effects in the intakes of foods and water cannot deny the anxiolytic effects of the drugs. Lever-pressing may also be increased by non-specific behavioral activation (e.g. stimulating effects of the drugs). However, if the lever-pressing increase in the alarm period but not in the safe period in which the lever-pressing does not cause the shocks, we can deny non-specific behavioral activation. Thus, it is important in behavioral pharmacology that we thoroughly and carefully discuss factors affecting behaviors in their change whether it is caused by the intended factor(s) or not. Although we can set up several experimental conditions in animals as similar, their behaviors observed are always ruled by multiple factors and we should consider the experimental results in the multiple points of view to understand them correctly.

Geller and Seifter1 reported that meprobamate, pentobarbital, and phenobarbital but not promazine and d-amphetamine increased the lever-pressing in the alarm period. In pharmacology we often evaluate drugs that different effects are known in clinical experiences and previous animal experiments, for example, anxiolytics, antidepressants, antipsychotics, and psychostimulants, in animal models and specificity of the models by their effects. If only anxiolytics among centrally acting drugs elicit effects in the animal model, it is highly possible to measure anxiety but we should carefully consider the drugs have only anxiolytic effects as described above. Studies in the animal models in which existent drugs are positive are tended to detect effects in the similar but not the different drugs (namely, revolutionary novel drug candidates) in mechanisms of their action. Barbiturates and benzodiazepine anxiolytics are told to respond well but not the drugs having different mechanisms in the Geller- and Vogel-type conflict tests even though they have anxiolytic activities.

1-2 Exploring Behavior in Novel Environment

Anxiety is also evaluated using by exploring behaviors in rodents without punishment like the shocks in the conflict test. We experimentally make ethologically similar environment in rodents as we feel anxiety and observe their exploring behaviors first in this environment, which is developed as the animal model for anxiety in rodents. In such methods we can also consider that rats and mice are in conflict between desire to exploration and fear of the environment experimentally set.

1-2-1 Elevated Plus-Maze

Lafayette elevated plus-maze
A photo cited from Lafayette Instrument

We generally feel much fear to walk on a balance beam. We will feel the fear to fall because we must move on a high and unstable place without any grip. The similar circumstances are set to measure anxiety in rodents, which is called as an elevated plus-maze test. The maze consisting of enclosed arms and open arms which are crossed as shown in the picture is placed high from a floor. Rodents are thought to feel fear on the open arms similar as we feel on the balance beam and actually they almost spend in the enclosed arms and sometime enter into the open arms. A number of entries into and time spent in the open arms are measured as indices of anxiety (5). This maze is remarkable at a point that enclosed arms as well as open arms like a balance beam are simultaneously set, by which we can evaluate anxiety by objective parameters such as time spent and a number of entries. Because rodents are thought to feel fear falling to a floor, the fear may decrease if open arms are widen and possibility to fall decreases (6). Therefore, we must prepare the different mazes in a size for mice and rats that are different in their body size.

Mice and rats must cross a central platform to move between enclosed arms and open arms in the elevated-plus maze. The platform has wall edges of enclosed arms and may decrease fear like that in open arms but time spent in the platform is ambiguously left for an analysis of results whether it is open or enclosed. Therefore, the platform was deleted and made an elevated "0"-maze (7). A circular arm like a "0" shape is divided into quarters. Walls are alternately set in two parts as enclosed arms and the others are open arms. In the "0"-maze rodents must cross the open arm to move the enclosed arm from another enclosed arm.

In the elevated-plus maze test we can manually measure the parameters, time spent and a number of entries, with a stopwatch. An experimenter should do the best not to disturb environments of an experimental room (always sit in the same place and observe quietly) for the better results. Video recording and remote observation of the video are possible. Video-tracking software for PC analysis of the elevated-plus maze and automatic measurement of parameters by infrared beam sensors are also available. Automatic measuring devices are useful but we must observe rodents in measurement because they may fall into a floor, especially by effects of anxiolytics such as benzodiazepines.

1-2-2 Light/dark Test

We feel fear in complete darkness and will look for a light place. Because mice and rats, nocturnal animals, are active in a dark period and sedative in a light period, they are thought to prefer a dark place and feel anxiety in a light place in contrast with our human. We prepare a light/dark box, a cage of which light and dark compartments are connected each other, and observe behaviors in rodents in the box to measure time spent and locomotion in an each compartment and crossings between the both compartments (shuttle crossings) as parameters of anxiety. It is called as a light/dark test and originally reported in mice (8). Mice are observed much exploration behaviors in their novel environment and the light/dark test are usually conducted in mice. The test in rats also reported (9) but there is a demerit that the light/dark box for rats becomes large size.

Time spent and locomotion in the each compartment and shuttle crossings are generally measured in rodents in the light/dark box in which they can freely move the both compartments. Anxiolytics increase exploring behaviors in the light compartment and therefore increase time spent and locomotion in the light compartment and shuttle crossings. Conversely, we can observe that anxiogenics decrease them. Because we can evaluate anxiety by time spent in the light compartment and shuttle crossings, theoretically can measure with a stopwatch same as the elevated plus-maze. However, correct manual measurement is difficult since mice move faster in the light/dark test than on the maze. We usually conduct the light/dark test with an automatic measuring device for time spent in the each compartment which can also measure locomotion and shuttle crossings automatically (10).

1-2-3 Social Interaction Test

In a social interaction test two individually-reared rodents are met in a measurement cage and observed their social interactions. We need brave for a social interaction to unknown person and will avoid the person if feel anxiety. We may possibly talk to unknown person if anxiety is suppressed by a drug. We can positively talk with alcohol drinking by anxiolytic effects of ethanol. Anxiolytics increase time spent in the social interaction in rodents (11). There are two important points for this test that rodents are individually-reared, namely unknown each other, and compared in the body weight. One point, unknown each other, may easily understand to make more anxiety in rodents for their interactions. The other point, similar body weights, are important for purposes to promote the interactions and prevent attacking behaviors. For the same purposes we do not put one rodent into a cage in which another rodent is reared. Putting into the reared cage means that an intruder violates a territory of a resident and easily induces attacking behaviors.

Sniffing, following, grooming, and so on are observed as social interactions by video recording and measured in their number and time spent. An observer should be blind to drug treatments for objective manual judgments. Anxiety levels in rodents may be changed during the social interaction with or without acclimation to a measurement cage before the interaction and different brightness of the cage in this test. Thus, the other anxiety to environments than that to the social interaction can be changed.

1-2-4 Marble Burying Test

We observe in a marble burying test marble burying behavior in rodents in which they bury and hide many marbles in a cage with nesting materials as an animal model of obsessive compulsive disorders. Obsessive compulsive disorders (OCD) are disorders that we can hardly stop to repeat unreasonable behaviors and burying safe objects (marbles) in rodents is thought to be resembled. For example, many people may confirm to be locked after they locked a house door with a key and I usually confirm it twice. The once or twice confirmation is a normal behavior and does not disturb our daily life but repeated confirmation by dozens blocks to leave the house for dozens of minutes, which will disturb your daily life. If we get our hands dirty by touching something outside of a home, we will wash the hands when returned to our home. This behavior is reasonable and recommended on public hygiene. However, if we wash the hands every time or cannot touch anything outside because we feel dirty with every touch of the hands outside, these behaviors may disturb our daily life and are often called as germophobia. If we are suffering from the germophobia too severe to disturb our daily life, it must be medically treated as the disorder. In these examples described above, our anxiety to a certain object as we are afraid whether a door is locked or something touched is dirty will compel us to repeat the behaviors. However, general anxiolytics such as benzodiazepines are not effective and antidepressants are clinically effective and active in the marble burying test (12).

Does a marble burying behavior reflect anxiety to marbles? Thomas studied relationship between a number of marbles buried in the marble burying test and shuttle crossings in the light/dark test or a ratio of distance traveled in the center to that in whole area in the open field test in 10 strains of inbred mice (13). Anxiolytics increase the parameter in the open field test as well as that in the light/dark test. Mice and rats feel anxiety and few explore in a central area rather than in a peripheral area, and the ratio is used to evaluate anxiety. Thomas found positive correlations between the parameters in the open field test and in the light/dark test but not the number of marbles buried and suggested that the marble burying behavior might not relevant to anxiety. Further, because the marble burying behavior does not decrease by repeated trials and acclimation to marbles, it is also not relevant to neophobia. Interestedly he told that this behavior is correlated to a digging behavior which is possibly thought to be innate behavior preparing a nest. If the marble burying behavior is caused by the digging behavior, it may be common to unreasonable behaviors in OCD because the rodents do not need to prepare the nest in their experimental condition.

1-3 Closing

Anxiolytic benzodiazepines suppress general behaviors at high doses and induce sleep. Rodents' behaviors intended in the all measurement models of anxiety described above diminish at such high doses and we cannot measure anxiety. Lower doses at which some behaviors remain may affect results in the models. We should consider sedative effects of the drugs by any methods such as observations of other than intended behaviors. Anxiety may be detected in some methods such as the Geller-type conflict test and the light/dark test in spite of sedation. Because many drugs have multiple effects and there is a case that different actions of the drugs cause the same behaviors, we should always discuss the results in multiple points of view.