Behavioral Test Questions

How to choose among the different tests for a particular phenotype?

By July 25, 2018 No Comments

Over the last decade, the neurobehavioral field has experienced an explosion of new behavioral tasks targeting the vast and complex network of behavioral phenotypes in many laboratory animals. From rats and mice to even zebrafish, the number of available behavioral tasks is rapidly increasing in an effort to clearly depict the behavioral repertoire of each species. This expanding list of behavioral protocols can easily complicate and muddle the process of designing your experiments and choosing the appropriate task to perform.

Laboratory rodents display a remarkably diverse repertoire of behavior and are among the most commonly used animal models in behavioral neuroscience research. The diversity of their behaviors is mirrored on the diversity of the available tasks. Some tasks evoke and measure a specific behavior, whereas others simply involve the observation of spontaneously expressed behaviors. Some tests are designed to examine exclusively a particular phenotype, whereas others simply involve the observation of a stream of behaviors.

Given all this, there is a wide variety of behavioral tests available for laboratory rodents, from tests of basic locomotor and sensory function to analyses of more complex behavior related to cognition and emotionality.

Categories of phenotypes studied

Behavioral tests can be divided based on the phenotypes that they focus on, thus creating four broad categories of tests; basic motor and sensory function, anxiety, learning and memory, social behavior, and reward.

Motor and sensory function

There are many tests focusing on the motor and sensory capacities of laboratory animals; their aim is to validate the normal movement and senses of the animal. They are essential in animal models of ataxia and asymmetrical brain function, but also as an index of recovery after exposure to therapeutic drugs.

Some of the motor function tests are the motor activity, rotarod, beam walking and grip strength tests. Rotarod measures motor coordination and balance and is often used during early development to screen new drugs for possible side effects on motor coordination. Bear in mind that animals improve with repeated testing through motor learning.

Beam walking is another test focusing on the animal’s motor coordination and balance, commonly used to model neurodegenerative diseases and neurological dysfunctions. It should be preferred when the detection of fine motor deficits is needed.

The grip strength is a simple test assessing the neuromuscular functions of animals. Any alterations in grip strength could be a sign of motor neurotoxicity. Although it can accurately screen for neurobehavioral toxicity, the results can be influenced by body weight. As an advantage, for the majority of the motor and sensory tests, no training is required. However, most of them require automated equipment and software for video analysis.

The sensory function of the animals can be tested using simple tests (such as olfaction tests) and tests generally used for other phenotypes. Morris water maze and cued fear conditioning, for example, rely on intact visual and auditory systems respectively. Moreover, even though the pre-pulse inhibition and the acoustic startle response are commonly used to test anxiety, they are based on the reflexive response to loud auditory stimuli. These two tests can clearly detect any defects in the auditory senses but to do so they require sophisticated equipment to present stimuli and record responses. One should consider that they require little training of animals and permit the retesting of the same animals.

Anxiety

There are plenty of tasks focusing on the anxiety and the emotionality of the animals. Given that anxiety cannot be measured directly, these tests quantify specific behaviors which are altered as the animal becomes more or less anxious.

Anxiety-like behaviors are mainly studied in conjunction with the animal’s exploratory behavior in different novel environments. This could explain the popularity of the open field test in anxiety-related studies. Given that this task generates a considerably large volume of data, the use of an automated behavioral analysis system is needed. Another factor to consider is that the open field test is susceptible to habituation, therefore each animal should only be exposed to this task once.

Other tests in this category are the elevated plus maze, the light/dark exploration, the Vogel conflict test, the burying test, forced swim test, the prepulse inhibition (used mainly for the study of schizophrenia) and the acoustic startle response. The elevated plus maze is a popular test for the evaluation of anxiety-like behavior in rodents, especially when the evaluation is needed to screen potential anxiolytic agents. Nonetheless, keep in mind that certain factors such as the age of the animal[1], the familiarity with the experimenter[2] and the lighting conditions[3] can seriously affect the outcome of this task.

Although all these tests seem to measure the same thing, there are critical differences between the behaviors assessed. The Vogel conflict test is based on the assumption that anxiety mainly develops if an aversive situation has to be approached. The burying test focuses on the defensive behavior of the animal in response to a threatening object. It is a test which is commonly used to examine the effects of aging on emotionality and also to examine the effects of potential antianxiety agents. The forced swim test should be used when investigating the coping strategies of animals towards an intensely stressful situation. The prepulse inhibition and acoustic startle response constitute tasks which study the stimulus response neural plasticity. They are considered an important neurobehavioral tool for studying the effect of lesions or pharmacological manipulations in the response circuit of the animal. Finally, the light/dark exploration task is used to study the effect of various pharmacological manipulations on unconditioned anxiety responses. It is a test that does not require any previous training, however, there is some controversy around it. The results can often present serious variability depending on the strain, age and weight of the animal.

Tasks focused on anxiety basically belong to the wider category of emotionality. Depressive-like behaviors are studied through the forced swim and tail suspension tests which both recognize immobility as a measure of behavioral despair.

Learning & Memory

The long use of laboratory rodents in the study of learning and memory has faced a resurgence after the development of methods to manipulate the genetic material and create targeted mutations. The list of cognitive tests for this domain is vast and diverse. Some tests entail appetitive motivation that stimulates a mouse to look for some kind of positive reward, whereas others impose aversive stimuli that cause pain or distress and promote attempts to escape or avoid them.

All these diverse tasks study different neurobiological mechanisms. Based on the fact that different neural systems underlie the memory processes, different cognitive tasks have been designed to focus on each distinct neural system. At least five different neural systems are involved in the memory process:

  • Hippocampus
  • Amygdala
  • Dorsal striatum
  • Rhinal cortex
  • Cerebellum
  • Prefrontal cortex
different tests for a particular phenotype

The choice of the appropriate test will depend on the specific system that someone wants to investigate.

More specifically, hippocampal functioning has been typically examined with tests of spatial learning such as the Morris water maze and the radial arm maze. However, you should keep in mind that the learning process incorporated in the Morris water maze can equally depend on the amygdala as well since it is a process which can easily trigger anxiety and panicky feelings. Additionally, hippocampal formation can be studied through relearning processes.

Assuming that the amygdala is essential for the formation of emotional memories, its role in learning and memory is studied through tests of conditioned fear responses. Fear conditioning is widely used to study cellular and molecular correlates of consolidation and extinction of aversive memories. Tests examining amygdala’s implication are the place avoidance tests; achieved through either a passive avoidance or an active avoidance conditioning. Passive avoidance tasks require a very limited number of training trials and even from the first trial, the passive avoidance is acquired. This makes it possible to define the time-point of memory acquisition, which can be valuable if you want to conduct gene and protein expression analyses. On the contrary, active avoidance tasks are more laborious and require repeated training sessions. They have been commonly used when studying categorical learning in gerbils [4].

Operant conditioning tasks, such as the cued win-stay radial maze, investigate the involvement of the dorsal striatum on memory and learning as in stimulus-response habit learning.

The rhinal cortex’s involvement can be tested through the object recognition test. Recognition tasks are much simpler since they do not require reinforcement and a training period. They can be repeatedly performed on the same animal, as they are less stressful to the subjects.

The cerebellum is implicated in eyelid conditioning as well as in motor learning. Finally, the prefrontal cortex is studied in tasks requiring an intact working memory and in memory extinction.

Furthermore, spatial learning is studied in complex maze-like structures with behavioral protocols which require an array of training trials over a number of days. More specifically, the radial arm maze can test an animal’s ability to set different memory categories; procedural memory, working memory and reference memory. Morris water maze traditionally assesses hippocampal formation and spatial memory, and is a test with significant advantages, such as its ease to be performed, no olfactory problems present in the dry mazes and animals do not need to be food deprived. Nonetheless, data obtained from these tasks require an automated behavioral analysis system.

Social behavior

Tests studying the sociability behavior mainly examine the mouse’s preference for a novel social target. This category includes parental, sexual and reproductive behaviors and of course aggression. There is a wide variety of tests in this category, from groups of tests which observe and study the social interaction of animals to tests which investigate the social hierarchies formed in groups of mice.

These social interaction tests should be chosen if the researcher needs to assess any dysfunction in the limbic system which is known to control aggression and complex neural circuits involved in the regulation of endocrine hormones and pheromonal cues. The duration of the mutual exploration of the cage is indicative of anxiety-related behavior. These tests are rapid and simple without any need for previous training or equipment.

Reward

Finally, another broad category of tests is that of reward and addictive behaviors, which include the commonly used conditioned place preference task. The simpler sucrose preference test measures the sensitivity to reward and should be used in conjunction with measures of aggressive-like behavior, as a reduction in sucrose preference is associated with other signs of depression in rodents. In a similar context, apparatuses for self-administration measure the motivation of the animal to obtain a reward.

General considerations

Although the tasks can be categorized in basic domains based on the nature of the behavior studied, many tests are obviously pertinent to more than one domain. A common example is that of the open field test, which is characterized as a task assessing the exploratory activity but to some extent it explores the motor capabilities (rearing, grooming), the anxiety-related behavior and memory (habituation).

Given the complexity of the phenotypes studied and the multifactorial nature of behavior, many scientists have suggested to use a combination of behavioral tasks in order to receive more accurate results. Balietti et al. (2018) showed that a combination of two tests (Morris water maze and the step through passive avoidance test – STPA) is needed to assess the hippocampal function. The Morris water maze is used to estimate core aspects of spatial learning and memory, while the STPA is the best for studying post-training processes of hippocampal cellular memory consolidation.

Apart from the phenotype targeted, the choice of the behavioral task is influenced by the genotypes of the subjects, any special treatments involved in the experiment and of course the sample size available. The number and type of applied tests determine the structure of the experiment, so you should study all these elements while designing your experiment. The time available to complete your experiments is another important factor since some tests require several days of training or acclimatizing the animals to the new environments before acquiring any behavioral data. Additionally, you should not underestimate the species-specific behaviors which require an extensive literature research before choosing the appropriate task.

Another factor influencing the decision on the appropriate behavioral task is the detailed training needed for each test. It is crucial to perform a test in a correct and ethically approved manner. To do so, you should strictly follow a protocol which best describes the standard operating procedures. Official guidelines suggest that a behavioral task should be performed only after the experimenter receives extensive training by specialized personnel.

Therefore, when choosing the appropriate test, one should consider the factors influencing the results, from the traits of the animals (specific for the strain or even the species), the time and place of the testing and the housing conditions to the characteristics of the experimenter (his experience and training and his possible bias).

References

  1. M. Bessa, M. Oliveira, J. J. Cerqueira, O. F. Almeida, and N. Sousa, “Age-related qualitative shift in emotional behaviour: paradoxical findings after re-exposure of rats in the elevated-plus maze.,” Behav. Brain Res., vol. 162, no. 1, pp. 135–42, 2005.
  2. S. van Driel and J. C. Talling, “Familiarity increases consistency in animal tests.,” Behav. Brain Res., vol. 159, pp. 243–245, 2005.
  3. O. Pereira, I. C. da Cunha, J. M. Neto, M. A. Paschoalini, and M. S. Faria, “The gradient of luminosity between open/ enclosed arms, and not the absolute level of Lux, predicts the behaviour of rats in the plus maze.,” Behav. Brain Res., vol. 159, pp. 55–61, 2005.
  4. W. Ohl, M. Deliano, H. Scheich, and W. J. Freeman, “Analysis of evoked and emergent patterns of stimulus-related auditory cortical activity.,” Rev. Neurosci., vol. 14, pp. 35–42, 2003.
  5. H. Kogan, P. W. Frankland, and A. J. Silva, “Long-term memory underlying hippocampus-dependent social recognition in mice.,” Hippocampus2, vol. 10, pp. 47–56, 2000.
  6. Balietti, G. Fattorini, A. Pugliese, D. Marcotulli, L. Bragina, and F. Conti, “Two behavioral tests allow a better correlation between cognitive function and expression of synaptic proteins,” Front. Aging Neurosci., vol. 10, no. APR, pp. 1–5, 2018.

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