Modern Experimental Uses Of Rodent Behavioral Models

Having briefly described several of the basic rodent learning paradigms in common use, we now turn our attention to thinking about their application in cellular and molecular studies. By way of introduction to this topic, I think it is useful to step back and review the basics of experimental design. In the next section we will consider the basics of hypothesis testing. We then will proceed to considering how the fundamentals of experimental design are applied in the modern era in extending behavioral studies into the cellular and molecular realm.

A. The Four Basic Types of Experiments

In general there are four basic types of experiments that any scientist can perform. I refer to them as block, measure, mimic, and determine experiments. I have found this categorization a useful mnemonic device throughout my career as a scientist, and, at the risk of sounding overly pedantic, I strongly encourage any young scientist who reads this book to incorporate them into their thinking about experimental design. For example, every time I write or review a paper I ask whether the investigation has included all these different types of experiments. Especially when writing or reviewing grant applications, where multiyear projects are proposed to test a hypothesis comprehensively, I cross-check myself and others on whether all of these approaches (if technically possible) have been applied to the problem at hand. It is important because what we do as scientists is test hypotheses, and the testing of any hypothesis is much stronger if a variety of independent lines of evidence are available to support the conclusions reached.

What follows is a brief description of each of these four types of experiments.

The determine "experiment" is not really an experiment at all. The determine approach is to perform a basic characterization of the system or molecule at hand independent of any experimental manipulation whatsoever. Examples of this type of pursuit are determining the amino acid sequence of a protein, sequencing a genome, determining the crystal structure of an enzyme, or determining the structure of the DNA double helix. Determinations of this sort are not experiments in that no manipulation of the system is attempted— to do an experiment you tweak the system to see what happens. If you mutate a residue in a protein and see what effect that has on the structure, then you have done an experiment. The basic determination of the structure is not an experiment in and of itself.

Determinations are some of the most satisfying laboratory pursuits to undertake because these are the rare types of studies where definitive data can be obtained. An amino acid sequence is what it is—you get to use unambiguous words like "identical" (versus indistinguishable or similar) and "determined" (versus concluded or inferred) when describing gene and amino acid sequences. There's slightly more ambiguity in determining protein structures and anatomical structures, but in general this pales in comparison to the ambiguity of a conclusion made on the basis of an experimental manipulation. The down side of determinations is that, as a practical matter, they are viewed as boring unless they involve lots of expensive equipment. It's very difficult to get a grant review study section to recommend approval of a basic anatomical characterization, for example, because no experimental testing of a hypothesis is involved. In modern biomedical research, hypothesis testing is de rigueur. In rodent behavioral systems, which are the topic of this chapter, most of the basic behavioral characterization has already been done. However, there is a growing recognition that more sophisticated and detailed basic characterizations, and the development of new rodent behavioral models for human mental disorders, is necessary for the next stage of progress in this field.

Block, measure, and mimic are experiments, and they are all specific types of approaches to test different predictions of a hypothesis. For the following discussion we will take the simple case of testing the hypothesis "A causes C by activating B" (see Figure 13).

The mimic experiment tests the prediction that "if B causes C, then if I activate B artificially I should see C happen as a result." An example that we will return to later is: if I hypothesize that a particular protein kinase causes synaptic potentiation, then applying a drug that activates that protein kinase should elicit synaptic

Experiment Prediction

Experiment Prediction

• Determine

• None (A makes C happen)

• Block

• Blocking B should block A causing C

• Mimic

• Activating B should cause C

• Measure

• A makes B happen

FIGURE 13 The four basic types of experiments. See text for discussion.

FIGURE 13 The four basic types of experiments. See text for discussion.

potentiation. The mimic terminology arises from the fact that you are trying to mimic with a drug (etc.) an effect that occurs with some other stimulus, potentiation-inducing synaptic stimulation in this example. The principal limitation of the mimic experiment is that B may be able to cause C but that in reality A acts independently of B to cause the same effect. B causing C and A causing C may be true, true, and unrelated.

At the current state of understanding and experimental sophistication, mimic experiments are just about impossible to execute in the context of mammalian learning and memory. This is because an enormous amount of fundamental understanding of the system is necessary, along with the capacity for very subtle manipulation, in order for the experiment to work. For example, suppose I hypothesize that a synaptic potentiation underlies learning. In theory, the mimic experiment is to put an electrode in the brain, cause synaptic potentiation, and then the animal will have an altered behavior identical to that caused by a training session. Of course, doing this experiment requires that I know exactly which synapses to potentiate so that I can selectively achieve the right behavioral output—this is beyond the level of understanding for essentially all mammalian behaviors at this point.

The measure experiment tests the prediction that "A should cause activation of B." Using our example of kinases in synaptic potentiation, the measure experiment predicts that the potentiating stimulus should cause an increase in the activity of the kinase. This is, of course, determined by measuring the activity of the kinase as directly as possible, hence the measure terminology. The measure experiment has been applied in a variety of different ways in the memory field, ways that we will discuss at various points throughout the book including looking for anatomical, physiologic, and molecular changes in the nervous system in association with learning. The principal theoretical limitation of the measure experiment is that it is correlative. One can show that A causes activation of B, but that does not demonstrate that activation of B is necessary for C to occur.

Which brings us to the block experiment. The block experiment tests the prediction that "if I eliminate B, then A should not be able to cause C." In our working example, this means that a kinase inhibitor should block the ability of the potentiating stimulus to cause potentiation. At present, the vast majority of investigations into mechanisms of memory involve this approach, and we will make many references to this type of experiment throughout the book. Specific examples include anatomical lesions, drug infusion studies, and genetic manipulations. The principal theoretical limitation of the block experiment is that it does not distinguish whether activation of B is necessary for C, versus whether the activity of B is necessary for C. For example, suppose that B provides some tonic effect on C that is necessary for it to occur. Inhibiting B will block the production of effect C when in fact A never has any effect on B whatsoever. In behavioral terms for learning experiments, this is referred to as a performance deficit—the animal is simply unable to execute the behavioral read-out necessary to exhibit the fact that they have learned.

In summary, then, the mimic experiment tests sufficiency, the block experiment tests necessity, and the measure experiment tests whether the event does in fact occur. Each type of experiment has its strengths and weaknesses. Positive outcomes in testing each of these three predictions for any hypothesis makes for clear, strong support of the hypothesis.

B. Using Behavioral Paradigms in Block and Measure Experiments

The behavioral paradigms I have been discussing in this chapter have, by and large, been used in two ways in the modern era. The first application is as a stimulus in measure experiments. The second is as a read-out in blocking experiments. We will return to the specific results of several of these various experiments in Chapter 9. However, for our present purposes, I would like to describe briefly some examples of the use of behavioral paradigms in these two types of experiments. This is because the specific examples will help to introduce some refinements of the procedures that are necessary for some applications, and also to introduce some of the sorts of behavioral control experiments that are used to shore up the conclusions reached in executing the experiments.

Using Behavioral Paradigms as a Stimulus in Measure Experiments

In measure experiments, the behavioral paradigms we have reviewed are used to train the animal using a set of defined and optimized environmental signals that are known to elicit learning and memory. The behavioral read-out of the learned behavior is really only used as confirmation that the animal has learned—it is, in essence, control data that the procedure has been effective. What is really of interest in these types of experiments is determining what has gone on inside the animal's CNS while or after it learned. A variation that has great potential for future use is to monitor events occurring when the animal recalls a memory, but that type of experiment has received scant attention so far.

There are several prominent examples of great successes in measuring physiologic changes in the brain with behavioral training paradigms. The best-established paradigm is measuring alterations in hip-pocampal pyramidal neuron firing with rodent spatial learning. These elegant experiments use implanted recording electrodes to monitor neuronal responses in vivo in the behaving animal. As described previously, these experiments led to the identification of hippocampal "place" cells and variations thereof. The Eichenbaum, Wilson,

McNaughton, Tonegawa, and Kandel laboratories have all made significant use of this approach and I will review a number of their findings in Chapters 3 and 9.

Somewhat of a "holy grail" experiment in learning and memory has been to obtain data demonstrating that long-term potentia-tion of hippocampal synaptic transmission occurs with learning in rodents. To date, this approach has met with only limited success as pertains to the hippocampus. However, there have been landmark findings in this area from the LeDoux and Shinnick-Gallagher laboratories, utilizing fear conditioning and amygdala recordings of synaptic transmission. We will return to these observations later, in Chapter 9, with a review of the data supporting a role for long-term potentiation in learning.

Finally, while the search for alterations in hippocampal synaptic transmission with learning has been somewhat frustrated, there actually have been nice demonstrations of alterations of hippocampal neuron excitability that occur with learning. Both the Wilson and Disterhoft laboratories have found alterations in hippocampal pyramidal neuron excitability with spatial learning and trace eye-blink conditioning, respectively. These alterations, for which there is indirect and direct evidence suggesting involvement of altered potassium channel function, will be addressed in more detail in Chapter 3.

These studies all involve cellular changes, but what about molecular changes triggered in association with the behavioral paradigms we have been discussing? There have been fewer experiments looking at molecular changes in association with learning, and we will be discussing changes in kinase activity with learning in subsequent chapters. For our purposes here, I will limit my discussion to one set of studies in order to give a specific example of the approach.

In a sophisticated series of studies, Soren Impey and Dan Storm and co-workers produced a transgenic mouse line that allowed read-out of increased transcription

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