Animal Models of Depression                                       Hong CJ, Oct 15, 2006

The diagnostic criteria of major depression is ‘at least five of the following nine symptoms for at least two weeks: (1) depressed mood, (2) loss of interest, (3) sleep disturbance, (4) 5% body weight change in a month, (5) retarded behavior, (6) feeling fatigue, (7) thoughts of worthlessness, (8) impaired attention and (9) thoughts of suicide. The first two are the most important and fundamental symptoms. If a patient has five of above symptoms which do not include either depressed mood or loss of interest, he does not have a case of major depression.


Most people think it is impossible to tell if a mouse has a depressed mood or loses its interest. However, when I was testing the model of amphetamine withdrawal model of depression, I found some mice would injure themselves. This mouse injured himself to such a degree that it finally died (fig). It actually died by committing suicide. This kind of phenomenon has been reported before. It is called amphetamine-induced self injury.


The theory of learned helplessness was developed by Seligmen and his colleagues in 1965. He found when an animal was put in such an inescapable environment and given repeated stressors, it would have a post-traumatic stress disorder (PTST) or it would learn that trying to escape is futile. The behavior of giving up escaping from a dangerous or uncomfortable situation is called learned helplessness. Learned helplessness may be evaluated with a behavioral method called the escape test. When a tested animal is put in this kind of box with electricity on the floor, it will try actively to escape from the dangerous or uncomfortable environment.  This is an example of a successful escape.(movie) As soon as this mouse felt the electrical shock it rushed to the other side of the box. When the mouse crossed over the fence and touched off the infrared sensors, the shocking electricity was cut off. The procedure is usually repeated for 10 or 20 times. The failure rate in 10 or 20 escape tests is counted as an index of the severity of helplessness. If mice are given inescapable stressors, some of them may become helpless. These three mice were unable to escape because their tails were stuck with a tape on the floor. After this kind of inescapable stress, the mouse in the previous video became helpless . It looked frightened and withdrawn.(movie) It might have had a PTSD.


Though the learned helplessness model is the most convincing model of depression, it has a major drawback. The depression-like symptoms caused by inescapable stress seldom lasts for more than two or three days. That’s not good for antidepressant studies. We modified the conditions of inescapable stress and set up several protocols that had successfully extended the duration of learned helplessness. The blue bars indicated the success rates of the mice before the stress of our protocol.(figure) The red bars were for the mice five days after the stress. The yellow bars were 10 days after the stress. A success rate of greater than 70% was regarded as normal, while lower than 30% was regarded as helpless. The effect of our protocol was demonstrated in this figure. Five of the eight mice remained helpless 10 days after the stress protocol.  We use the mice with learned helplessness to study the pathology of depression in the brain. The expression of mRNA and proteins in the brains are studied using gene expression arrays and immunohistochemistry. 


In order to obtain genetic models of depression, we used a battery of behavioral tests to screened specific patterns of behavior in mutagenized mice. This table lists the diseases we are interested in, and depression is highlighted here. The patterns of hypo-activity and social withdrawal were screened using the open field test and the sociability test. In the open field test, a mouse is put in such an open space. It is allowed to move freely. Its movements and locations are traced, recorded and calculated by a digital camera with software. These are the figures of the sociability test. A mouse who is unfamiliar to the tested mouse is secluded here, while the tested mouse is allowed to move freely among these rooms. On the separator there are many holes which are large enough to let the mouse poke its nose through. The two mice may touch each other, if they want to. If the tested mouse is depressed or has a case of social phobia, it will stay a shorter period of time in this room (which is next to the stranger). We found that several mice not only displayed depression-like behavior but some of them also had a problem of obesity. They are from the same pedigree. These are the pictures of the two obese mice. They were as heavy as 42 or 47 grams. The average body weight of this kind of mouse is around 25 grams. The normal mouse beside this obese mouse is actually one of its sibling. They were the same age. If the trait is inherited in a dominant patter, a half of the offspring of this mouse will display the same trait. If the trait is inherited in a recessive patter, a crossover among its offspring is required. A quarter of its grandchildren is expected to have the same trait. Mice with inheritable patterns of depression-like behavior will be the best animal models for localizing the genes of depression.


Dr. Porsolt thought out a very simple method in 1978 to determine if a mouse tended to become despaired. When a mouse gives up struggling in the water of a constrained cylinder earlier than other mice, it is regarded as a sign of despair, which is further interpreted as having a depressed mood. It is called ‘forced swimming test (FST)’. Though the FST was criticized by several researchers, this method has lasted for more than 20 years and has been accepted widely. Some of the critics argued that those early quitters did not drown themselves; on the contrary, they gave up useless struggling to save energy. They said, ‘When mice stop struggling to float on the water, they do not suffer. They look peaceful and seem to enjoy the situation. They never choke or drown in the FST.’ However, most researchers do not agree with this kind of observation and explanation. When a mouse stops struggling in the water, they display a freezing posture, this is a sign of anything but peace, relaxation or enjoyment. Debates as they were in the 1980s, along with the use of the FST several new effective antidepressants have been developed in the past two decades. 


According to the concept of the FST, an Italian researcher Dr. Steru invented an even simpler method, the tail suspension test (TST), to measure the tendency of despair in a mouse in 1985. In the TST a mouse is suspended from its tail. Normally, a suspended mouse will struggle to escape or fight against the dangerous or uncomfortable situation. However, most mice will stop struggling in 2-4 minutes. Have they tired in that short period of time? No, a mouse in normal physical condition is able to struggle for more than 10 or even 30 minutes. If a mouse stops struggling in 2-4 minutes, it’s more likely that the mouse cannot sustain its ‘willpower’, rather than ‘physical power’, to fight against the dangerous situation. In other words, the mouse’s spirit or psyche has come down to a level as low as a state of despair.


Though the significance of the FST and the TST has been confirmed and both tests have the advantage of simplicity, they are not really easy to use. How do you decide the behavior in between; between obvious agitation and coming to a standstill? The degree of despair is decided on the duration of how long the mouse stands still or freezes in a six-minute test. Most researchers use a stopwatch to count the amount of time when a mouse comes to a standstill. However it’s usually difficult to decide when to click on the stopwatch. What is the starting point of a freezing posture? Mice seldom slow down their struggling in a regular way. They may suddenly stop struggling for ten or twenty seconds and then become agitated again; or they may slow down their struggle in a gradual way, with intermittent bursts of kicking one leg, rotating the head, waving the tail or racing his forelegs. Even if an experimenter has been trained to identify a freezing posture, he or she may not be able to respond to every change of mouse’s activity efficiently. Even if an experimenter can respond to mouse’s activity to click the stopwatch quickly and correctly, he or she may not be able to maintain the same level of alertness and attention to observe 20 or 50 mice every day. However, the duration of each test is so short that any incorrect click on the stopwatch will result in a different result, so I invented devices to standardize and automate the FST and the TST.


I use a computer mouse to communicate with the tested real mouse. In the water of the FST I put a buoy which will fluctuates with the water which is disturbed by a struggling mouse. On the top of the buoy I attach a light pad which is constrained, but freely movable, to the bottom of a standing-up computer mouse. When the pad moves on the bottom surface of the computer mouse, the cursor of the computer mouse moves on the screen. I use a simple piece of software to record the coordinates of the cursor every 0.2 second, so the amplitude of activity of the tested mouse in the water can be recorded in the computer automatically.(figure; movie, P) Not only did the device save me a lot of time, but it also standardized the measurement of the FST. Even inexperienced students or assistants can quickly learn to use this method. This device has been approved with an invention patent from the USA(US 6,799,535 B2) and Taiwan.


With a similar concept, I invented a device to perform the TST. I used a spring to suspend the tail of a tested mouse. When a tested mouse struggles the spring is stretched up and down. Between the spring and the mouse’s tail the similar design of a pad with a computer mouse is inserted. So not only the movements, but also the amplitude of movements, of a tested mouse can be recorded automatically.(figure) This device also has been granted as an invention patent of the USA(US 6,955,139 B2) and Taiwan.


The third method that is commonly used to detect despair in animals is called escape test. In the test a mouse is put in a box with two connected rooms. The floor of the box can be charged with low electricity. Normally, a mouse will escape away from the room where it suffers an electrical shock very soon. If a mouse is despaired it usually takes a longer period of time to escape from the uncomfortable environment, or even fails to escape. The mean latency of escape or the number of failures in 20 escape tests is usually used as an index of the degree of despair or depressed mood in a mouse. I used a PLC and a piece of software to control and automate the whole procedure of the escape test. However, the device is still too complicated for new users. The time they spend in trouble shooting seems unbalanced with the time they save from the automation of the device.


The FST, TST and escape test are used to measure the tendency of despair which is taken as one of the two core symptoms of depression: a depressed mood and a loss of interest. In order to know if a mouse has a problem of loss of interest, I have to find a method to measure a mouse’s interest. What are the common interests of mice? Most people know mice like to run on a wheel, but no-one considers that natural instinct or ‘habit’ as an interest which is an important component of mood. I did it and I measured it. I used magnetic switchers with counters to automatically count the wheel cycle a mouse runs in a specific period of time. When a mouse has a problem of loss of interest in running on a wheel, the number of cycles will be lower than that of its previous performance or that of normal mice. This new but simple idea may not be intriguing enough and it’s usually very difficult to get an idea approved as a patent, so I tried to combine it with a method called sucrose preference test into a device. Most mice like to drink sweat water. If a bottle of plain water and a bottle of sucrose water are put side by side in a mouse cage, normal mice in the cage will drink sucrose water much more than plain water. Previous studies said depressed mice had a lower preference for sucrose water, which the data of my repeated experiments didn’t agree with. My depressed mice still drank more sucrose water than plain water when the two types of water were put together. I thought from my clinical experience: a depressed patient tended to give up his or her activities of interest especially when the activities required extra attention or exertion, so I moved up the sucrose water to a higher level where the tested mouse could not reach it easily. However, sucrose water should be placed on a place that was not too difficult to reach. I had proved that normal mice would climb up to the ‘difficult’ place and drink more sucrose water than plain water which was placed in an ‘easy’ place, in front of the mouse’s face.


I used a sensor to detect the levels of the sucrose water and the plain water, so the amount of both types of water that is consumed every hour or every day can be recorded automatically. When a mouse is kept in this kind of cage, with a countable wheel, its interests of running on a wheel and drinking sucrose water can be measured precisely. I called the cage ‘interest box’. An invention patent for the interest box has been approved by the United States Patent and Trademark Office (US 7,121,229 B2).


In order to prove those despaired mice do not have physical or neurological problems, we always examine the mice in an open field where they can move freely. The examined mouse is tracked by a digital camera up on the ceiling. The total distance, speed and location of the examined mouse are automatically recorded and calculated. A mouse with physical or neurological problems cannot move as much as a normal mouse. During the open field test, the tested mouse is also observed by the experimenter using the naked eye.


We used these methods to screen drugs that have a potential to become antidepressants. Many studies have shown that animals under stress have a lower level of brain-derived neurotrophic factor (BDNF) in the hippocampus (a brain region that is closely related to emotion and memory), while antidepressant like Prozac can increase the level. Based on this evidence, we are trying to find drugs that are logically able to increase BDNF level, but they are currently used as anything but antidepressants. No matter how convincing or how logical the mechanism is, the drugs of our interest will never be allowed to be tested on humans until they are demonstrated with the following two characters: First, BDNF level is increased in the brain of mice which have been given the drug of our interest; Second, those mice that have taken the drug display more struggling time in the FST or TST. If mice have been inflicted with some kind of stressor in advance, those with drug treatment should have a lower percentage of failure in the escape test, or have higher percentage of sucrose water consumption.