Methods of Research in Psychopharmacology

Research Methods for Evaluating the Brain and Behavior

Techniques in Behavioral Pharmacology

Evaluating Animal Behavior

• Techniques in behavioral pharmacology provide a means of quantifying animal behavior for drug testing, developing models for psychiatric disorders, and evaluating the neurochemical basis of behavior. Creating animal models of complex psychiatric disorders with uniquely human symptoms poses numerous hurtles.
• Advantages of animal testing include having a subject population with similar genetic background and history, maintaining highly controlled living environments, and being able to use invasive neurobiological techniques.
• Animal testing includes a wide range of measures that vary not only in validity and reliability, but also in complexity, time needed for completion, and cost.
• Some measures use simple quantitative observation of behaviors such as motor activity, response to noxious stimuli, and time spent in social interaction.
• Operant behaviors controlled by various schedules of reinforcement provide a sensitive measure of drug effects on patterns of behavior. Operant behaviors are also used in tests of addiction potential, anxiety, and analgesia.
• Other methods assess more complex behaviors such as learning and memory, using a variety of techniques such as the classic T-maze, as well as mazes modified to target spatial learning: the radial arm maze and Morris water maze. The delayed- response task assesses working memory.
• Many measures of anxiety use unconditioned animal reactions such as the tendency to avoid brightly lit places (light–dark crossing task, open field test), heights (elevated plus-maze, zero maze), electric shock (Vogel test), and novelty (novelty suppressed feeding).
• To assess fear a classically conditioned response is established by presenting a light or tone followed by an unavoidable foot shock. The light or tone becomes a cue associated with shock, and when presented alone, it produces physiological and behavioral responses of fearfulness. When the conditioned fear stimulus precedes a startleproducing stimulus, the startle is much greater.
• Common measures of depressive behaviors such as the forced swim test, tail suspension test, and learned helplessness utilize acute stress to create a sense of helplessness but have been criticized for responding to acute rather than chronic antidepressant treatment. Other models of depression use more prolonged stress (chronic mild unpredictable stress, chronic social defeat stress), which more closely resembles the human experience. These stress-induced depressive behaviors respond to chronic but not acute drug treatment. Early maternal separation models the impact of stress early in life on later biobehavioral outcomes.
• The sucrose preference test is a measure of anhedonia (i.e., loss of interest in normally reinforcing stimuli).
• The operant self-administration technique, in which animals lever press for drugs rather than food reward, is an accurate predictor of abuse potential in humans. Varying the schedule of reinforcement indicates how reinforcing a given drug is, because when the effort of lever pressing exceeds the reinforcement value, the animals fail to press further (the “breaking point”). Drug self-administration can also be used to study factors leading to relapse following extinction of the drug-taking response.
• In conditioned place preference, animals learn to associate a drug injection with one of two distinct compartments, and saline with the other. On test day, if the drug is rewarding, the animal spends more time in the environment associated with the drug. If aversive, the animal stays in the salineassociated environment.
• Drug effects act as discriminative stimuli in operant tasks, which means that the lever-pressing response of an animal depends on its recognizing internal cues produced by the drug. Novel drugs can be characterized by how similar their internal cues are to those of the known drug.
• Translational research is the interdisciplinary approach to improving the transfer of discoveries from molecular neuroscience, animal behavioral analysis, and clinical trials, with the goal of more quickly and inexpensively developing useful therapeutic drugs.
• To make animal research more predictive of therapeutic benefits in humans, animal models that create abnormal behaviors or neurobiological changes that closely resemble the pathophysiology and focal symptoms of the disorder are needed. Genetic manipulations and brain lesions are two approaches for achieving this goal.
• Developing parallel tasks for humans, non-human primates, and rodents will allow integration of neurochemical data from rodent studies with findings of imaging studies in humans performing the same task.

Techniques in Neuropharmacology

Multiple Neurobiological Techniques for Assessing the CNS

• Using a stereotaxic device, lesioning destroys brain cells in selected areas with high-frequency radio current. More selective lesions are made by injecting an excitatory neurotoxin that destroys cell bodies in the region without damaging axons passing through, or by injecting a neurotoxin selective for a given neurotransmitter.
• Using a specialized cannula, microdialysis allows researchers to collect material from extracellular fluid from deep within the brain in a freely moving animal, so corresponding changes in behavior can be monitored simultaneously. The material is analyzed and quantified by high-pressure liquid chromatography. In vivo voltammetry is a second way to measure the neurotransmitter released into the synapse.
• Macroelectrodes that are stereotaxically implanted are used to electrically stimulate deep brain regions while monitoring behavioral changes. They can also record electrical response following drug treatment or other experimental manipulation. Microelectrodes can record electrical activity from either inside a cell (intracellular) or near a single cell (extracellular).
• The function of individual ion channels is monitored with patch clamp electrophysiology.
• The radioligand binding method evaluates the number and affinity of specific receptor molecules in tissue homogenates. Verification of binding requires proof of selectivity, saturability, reversibility, high affinity, and biological relevance.
• To visualize the location of receptors in the brain, receptor autoradiography, both in vitro and in vivo, is used.
• The ability to make antibodies to proteins allows more precise cellular localization of receptors or other protein cell components such as enzymes with ICC. Antibodies are also used to very sensitively measure proteins in tissue homogenates with Western blot. Radioimmunoassays and ELISA measure important molecules in body fluids or tissue extracts with competitive binding of an antibody to its antigen.
• ICC identifies cells that contain a given protein; the complementary technique ISH is used to locate cells in a tissue slice that are manufacturing a particular protein by detecting the corresponding mRNA. It can also determine changes in mRNA levels, which provide an estimate of the rate of synthesis of that protein. Although ISH measures a single mRNA, microarrays (or gene chips) screen up to 20,000 genes on a single support (chip). The entire genome can be screened in one experiment with the use of just a few chips.
• Although they use two distinct scanning technologies, CT and MRI both create extremely detailed “slices” through the brains of living individuals that can also be displayed as three-dimensional images. Their excellent detail of brain structure depends on computer analysis. MRS uses MRI-generated data to calculate brain chemical concentration. Diffusion tensor imaging is one of several methods available to show structural connectivity among brain structures.
• PET maps the distribution of a radioactively labeled substance that has been injected into an individual. A labeled drug or ligand is administered to identify where drugs bind and to localize neurotransmitter receptors. It can also allow visualization of regional brain activity during task performance, which is reflected in increased glucose utilization, oxygen use, and blood flow, depending on the reagent labeled. SPECT depends on similar technology at a lower cost but with less resolution.
• Functional MRI (fMRI) provides both anatomical and functional imaging of the human brain by allowing visualization of changes in regional blood oxygenation caused by cell firing. It monitors brain activity as it is occurring and, unlike PET, requires no radioactivity. Resting-state fMRI shows correlated patterns of brain activity when the individual is not engaged in a task. Pharmacological MRI utilizes fMRI to image brain function following drug administration, provides information on the location of drug action and pharmacokinetic data, and potentially can predict patient treatment response.
• EEG utilizes electrodes placed on the scalp of humans to measure the electrical activity of populations of neurons. Quantitative EEG requires computer analysis of the data to produce color-coded maps of brain activity. EEG recording of event related potentials (ERPs) shows the processing of the cognitive response to momentary sensory stimulation in real time.
• Deleting a specific gene in mice produces an animal model that lacks a particular protein (knockout) in order to evaluate postlesion behavior and drug effects. Knockin mice have a gene inserted, so they produce a slightly different protein than is produced by wild-type mice. Transgenic mice are mice in which one gene is replaced by another.
• Optogenetics provides the opportunity to temporally and spatially excite or inhibit genetically specific cells while evaluating the animal’s behavior. Chemogenetics (DREADD) uses orally or systemically administered chemicals to stimulate implanted genetically engineered receptors in specific brain cells to alter animal behavior. Chemogenetics is slower, but longer lasting, than optogenetic effects. CRISPR is a technique that provides a more rapid and less costly way to genetically engineer mice.
• Magnetogenetics uses genetic manipulations to make target cells (even deep within the brain) sensitive to remote magnetic stimulation in order to see how specific cell activation alters behavior, sensory processing, and cognitive function.
• DART attaches a drug to a limited number of biochemically identified cells to determine cell-specific drug mechanism of action.